This application is a continuation of US application serial no. 17/459,931, filed Aug. 27, 2021, which claims priority to international application PCT/US2020/020193, with an international filing date of Feb. 27, 2020, which claims the benefit of US provisional patents. Application no. 62/812,219, filed Feb. 28, 2019, U.S. Provisional Pat. Application no. 62/819,495, filed Mar. 15, 2019, U.S. Provisional Pat. Application no. 62/819,448, filed Mar. 15, 2019, U.S. Provisional Pat. Application no. 62/819,456, filed Mar. 15, 2019, U.S. Provisional Pat. Application no. 62/819,478, filed Mar. 15, 2019, U.S. Provisional Pat. Application no. 62/819,449, filed Mar. 15, 2019, U.S. Provisional Pat. Application no. 62/819,458, filed Mar. 15, 2019, U.S. Provisional Pat. Application no. 62/822,610, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,565, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,592, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,627, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,622, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,649, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,566, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,554, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,575, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,605, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,606, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,680, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/822,722, filed Mar. 22, 2019, U.S. Provisional Pat. Application no. 62/839,294, filed Apr. 26, 2019, U.S. Provisional Pat. Application no. 62/839,223, filed Apr. 26, 2019, U.S. Provisional Pat. Application no. 62/839,320, filed Apr. 26, 2019, U.S. Provisional Pat. Application no. 62/839,212, filed Apr. 26, 2019, U.S. Provisional Pat. Application no. 62/839,575, filed Apr. 26, 2019, U.S. Provisional Pat. Application no. 62/839,264, filed Apr. 26, 2019, U.S. Provisional Pat. Application no. 62/858,331, filed Jun. 7, 2019, U.S. Provisional Pat. Application no. 62/924,241, filed Oct. 22, 2019, U.S. Provisional Pat. Application no. 62/925,578, filed Oct. 24, 2019, U.S. Provisional Pat. Application no. 62/931,587, filed Nov. 6, 2019, U.S. Provisional Pat. Application no. 62/931,779, filed Nov. 6, 2019, U.S. Provisional Pat. Application no. 62/933,318, filed Nov. 8, 2019, U.S. Provisional Pat. Application no. 62/933,299, filed Nov. 8, 2019, U.S. Provisional Pat. Application no. 62/933,878, filed Nov. 11, 2019, U.S. Provisional Pat. Application no. 62/934,356, filed Nov. 12, 2019, U.S. Provisional Pat. Application no. 62/935,043, filed Nov. 13, 2019, U.S. Provisional Pat. Application no. 62/934,883, filed Nov. 13, 2019, U.S. Provisional Pat. Application no. 62/939,488, filed Nov. 22, 2019, U.S. Provisional Pat. Application no. 62/941,581 and US Provisional Patent, filed Nov. 27, 2019. Application no. 62/959,526, filed Jan. 10, 2020. The contents of these applications are incorporated by reference in their entirety.
sequence description
This application contains a sequence listing, which is electronically archived as an XML file named "47706-0074003_ST26.XML". This XML file was created on December 21, 2022 and is 11,673 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
background
Cells within a subject's tissue have differences in cellular morphology and/or function due to different levels of analytes (eg, gene and/or protein expression) in different cells. The specific location of a cell within a tissue (eg, the location of a cell relative to neighboring cells or the location of a cell relative to the tissue microenvironment) can influence, for example, cell morphology, differentiation, fate, survival, proliferation, behavior and signaling and interaction with the tissue .crosstalk with other stations.
Previous techniques used to study spatial heterogeneity provided data only for a small number of analytes in the context of intact or partial tissue, or for a large number of analytes from individual cells, but failed to provide information on the presence of individual cells in the parent biological samples (eg tissue sample). .
Genetic material and associated gene and protein expression influence the fate and behavior of a cell. Spatial heterogeneity in developmental systems is often investigated by RNA hybridization, immunohistochemistry, purification or induction of fluorescent reporters or predefined subpopulations, and subsequent genomic analysis such as RNA-seq. However, such methods rely on a small set of predefined markers, thereby introducing selection bias, limiting discovery, and increasing the cost and labor required to map the entire RNA transcriptome.
summarize
Provided herein are methods for identifying the location of a biological analyte in a biological sample, the method comprising: (a) providing a biological sample containing living cells; (b) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein the capture probe of the plurality of capture probes comprises (i) a spatial barcode and (ii) a biological sample capture domain that specifically binds the biological analyte; all or part of the analyte sequence or its complement, and (ii) all or part of the spatial barcode sequence or its complement, and using the determined sequence (i) and (ii) to identify the location of the biological analyte in the biological sample.
Provided herein are methods for identifying the location of a biological analyte in a biological sample, the method comprising: (a) providing a biological sample containing living cells; (b) contacting the plurality of analyte capture means with the biological sample deposited on the substrate, where: the analyte capture means in the plurality of analyte capture means containing (i) an analyte binding group that specifically binds the biological analyte, (ii) a barcode analyte binding units; (iii) an analyte capture sequence; [0030] and the Substrate comprises a plurality of capture probes, wherein the plurality of capture probes comprises (i) a spatial barcode and (ii) a capture domain that specifically binds to an analyte capture sequence; (c) determining (i) all or part of the spatial barcode or its complement, and (ii) all or part of the part of the barcode or its complement that binds to the analyte, and using the determined sequence (i) and (ii) to identify the location of the biological analyte in the biological sample.
Provided herein are methods for identifying the location of a biological analyte in a biological sample, the method comprising: (a) contacting the biological sample with a substrate comprising a plurality of capture probes, wherein one capture probe of the plurality of needle capture probes comprises (i) a spatial barcode and (ii) a capture domain that specifically binds to a biological analyte in a biological sample; (b) determining (i) the full or partial sequence of the biological analyte corresponding to the specifically bound capture domain or its complement, and (ii) all or part of the spatial barcode or its complement, and using the defined sequence (i) and (ii) to identify locations of the biological analyte in the biological sample.
In some embodiments, the biological sample is an organism. In some embodiments, the biological sample is an organoid. In some embodiments, the biological sample is an in vitro cell culture containing a plurality of living cells.
In some embodiments, the biological analyte is selected from the group consisting of: CD45, CD3, CD4, CD8, CD56, CD19, CD20, CD11c, CD14, CD33, CD66b, CD34, CD41, CD61, CD235a, CD146 and an epithelial cell adhesion molecule ( EpCAM).
In some embodiments, a living cell of a plurality of living cells is disposed on a substrate. In some embodiments, a living cell of a plurality of living cells is arrayed on a second substrate. In some embodiments, the second substrate is in contact with the substrate. In some embodiments, a living cell of the plurality of living cells is immobilized by the addition of a hydrogel. In some embodiments, a living cell of a plurality of living cells is immobilized by applying an electric field.
Provided herein are methods for determining the presence of a biological analyte in a living cell at a location on a substrate comprising: (a) allowing one of a plurality of living cells to migrate to a substrate comprising a plurality of capture probes, wherein the capture probes in the plurality of capture probes contains (i) a spatial barcode and (ii) a capture domain that specifically binds a biological analyte; (b) determine a biological analyte that (i) specifically binds to the capture domain or its complement, and (ii) all or part of the spatial barcode or its complement, and use the identified sequences (i) and (ii) to determine the presence of the biological analyte in the matrix in the living cell at the position of
Provided herein are methods for determining the presence of a biological analyte in a living cell at a site on a substrate, comprising: (a) combining a plurality of analyte capture means with one of a plurality of living cells disposed on the contacting living cell substrate, wherein: an analyte capture means of a plurality of analyte capture means contains (i) an analyte binding group that specifically binds a biological analyte, (ii) a barcode of the analyte binding unit; (iii) an analyte capture sequence (b) that enables a living cell of the plurality of living cells to migrate to a substrate comprising a plurality of capture probes, wherein the capture probe of the plurality of capture probes comprises a spatial barcode and a capture structure that specifically binds analyte capture domain order; (c) determine (i) all or part of the spatial barcode or its complement, and (ii) all or part of the part of the barcode or its complement that binds to the analytes, and determine using the established sequences (i) and (ii) Biological Analytes are present somewhere on the matrix in living cells.
In some embodiments, the migration is passive migration. In some embodiments, the migration is an active migration. In some embodiments, active migration is induced by a chemoattractant gradient. In some embodiments, the substrate comprises one or more channels. In some embodiments, one or more channels restrict cell migration along one axis of the substrate. In some embodiments, one or more channels restrict cell migration along more than one axis of the substrate. In some embodiments, one or more channels restrict cell migration along two substrate axes.
Provided herein are methods for identifying the location of a biological analyte in a biological sample, comprising: (a) contacting the biological sample containing cells with a substrate comprising a plurality of capture probes, wherein one of the plurality of capture probes comprises a spatial barcode; and capture domain; (b) releases the biological analyte from the subcellular region of the cell where the biological analyte is specifically bound by the capture probe; (c) determine the biological analyte specifically bound by (i) the capture domain All or part of the analyte sequence or its complement, and (ii) all or part of the spatial barcode sequence or its complement, and use the determined sequence (i) and (ii) to identification of the location of the biological analyte in the biological sample.
Provided herein are methods for identifying the location of a biological analyte in a biological sample, comprising: (a) contacting a plurality of analyte capture means with a biological sample containing living cells, wherein the biological sample is placed on a substrate, wherein: a plurality of Capture Agents analyte capture in the analyte capture means contains (i) an analyte binding moiety that specifically binds a biological analyte, (ii) a barcode of the analyte binding moiety; (iii) analyte capture sequence; and the substrate comprises a poly a capture probe, wherein the capture probes of the plurality of capture proteins comprise (i) a spatial barcode and (ii) a capture domain; (b) releasing the biological analyte from the subcellular region of the cell, wherein the analyte capture sequence of the capture domain specifically binds; (c) determining (i) all or part of the spatial barcode or its complement, and (ii) all or part of the barcode part or its complement that binds to analytes, and using the determined sequence (i ) and (ii) identifying the location of the biological analyte in a biological sample.
In some embodiments, the biological analytes are released from subcellular regions of the cell, wherein the release of other biological analytes from other subcellular regions of the cell is substantially prevented. In some embodiments, the subcellular region is the cytosol. In some embodiments, the subcellular region is the nucleus. In some embodiments, the subcellular region is a mitochondrion. In some embodiments, the subcellular region is a microsome.
Provided herein are methods for the temporal and spatial analysis of biological analytes present in a biological sample, comprising: (a) contacting the biological sample with a substrate containing a plurality of capture probes, wherein one of the plurality of capture probes captures the spatial barcode probe, and capture domain; (b) releases the biological analyte from the biological sample, wherein the capture domain specifically binds the released biological analyte; (c) determines a biological assay of (i) specific binding of the capture domain to all or part of the analyte sequence or its complement, and (ii) all or part of the spatial barcode or its complement, and use the defined sequence (i) and (ii) to identify locations of the biological analyte in the biological sample; (d) Repeat steps (a)-(c) after the specified time. Further provided herein are methods of temporal and spatial analysis of biological analytes present in a biological sample, comprising: (a) contacting a plurality of analyte capture means with a biological sample containing living cells, wherein the biological sample is placed on a substrate, wherein: an analyte capture means of a plurality of analyte capture means contains (i) an analyte binding group that specifically binds a biological analyte, (ii) a barcode of the analyte binding unit; (iii) an analyte capture sequence; and The substrate comprises a plurality of capture probes, wherein the capture probes of the plurality of capture proteins comprise (i) a spatial barcode and (ii) a capture domain; (b) release of the biological analyte from the living cell, wherein the assay (c) determines (i) all or part of the sequence of the analyte-binding barcode portion that specifically binds to the capture domain or its complement, and (ii) the sequence of the spatial barcode code or complement. sequence all or part thereof, and use the determined sequences (i) and (ii) to identify the location of the biological analyte in the biological sample; (d) repeat steps (a)-(c) after a specified time.
Provided herein are methods for the temporal and spatial analysis of biological analytes present in a biological sample, comprising: (a) contacting the biological sample with a substrate containing a plurality of capture probes, wherein one of the plurality of capture probes captures the spatial barcode probe, and capture domain; (b) releases the biological analyte from the biological sample, wherein the capture domain specifically binds the released biological analyte; (c) determines a biological assay of (i) specific binding of the capture domain to all or part of the analyte sequence or its complement, and (ii) all or part of the spatial barcode or its complement, and use the defined sequence (i) and (ii) to identify locations of the biological analyte in the biological sample; (d) Repeat steps (b)-(c) after the specified time. Further provided herein are methods of temporal and spatial analysis of biological analytes present in a biological sample, comprising: (a) contacting a plurality of analyte capture means with a biological sample containing living cells, wherein the biological sample is placed on a substrate, wherein: an analyte capture means of a plurality of analyte capture means contains (i) an analyte binding group that specifically binds a biological analyte, (ii) a barcode of the analyte binding unit; (iii) an analyte capture sequence; and The substrate comprises a plurality of capture probes, wherein the capture probes of the plurality of capture proteins comprise (i) a spatial barcode and (ii) a capture domain; (b) release of the biological analyte from the living cell, wherein the assay (c) determines (i) all or part of the sequence of the analyte-binding barcode portion that specifically binds to the capture domain or its complement, and (ii) the sequence of the spatial barcode code or complement. sequence all or part thereof, and use the determined sequences (i) and (ii) to identify the location of the biological analyte in the biological sample; (d) repeat steps (b)-(c) after a specified period of time.
In some embodiments, the biological sample includes a tissue, organ, organism, organoid, or cell culture sample. In some embodiments, the biological sample is subjected to one or more conditions prior to repeating the step. In some embodiments, the one or more conditions include applying one or more reagents, applying light, changing temperature, changing pressure, applying an electric field, applying a magnetic field, and combinations thereof. In some embodiments, one or more reagents comprise a DNA modification system. In some embodiments, the DNA modification system is a CRISPR system.
In some embodiments, steps (a) through (c) are repeated one, two, three or four times. In some embodiments, the substrate comprises a cell-permissive coating. In some embodiments, the releasing step includes permeabilizing one or more cells of the biological sample. In some embodiments, permeabilization involves contacting the biological sample with a hydrogel containing a permeabilizing reagent.
In some embodiments, the biological analyte is DNA. In some embodiments, the biological analyte is RNA. In some embodiments, the capture domain comprises a poly-dT sequence. In some embodiments, the capture probes also comprise a unique molecular identifier (UMI). In some embodiments, the capture probe further comprises a functional domain. In some embodiments, the capture probe further comprises a cleavage domain. In some embodiments, any of the methods described herein further includes washing the sample. In some embodiments, any of the methods described herein further includes imaging a biological sample.
Provided herein are methods for identifying the location of a biological analyte in a biological sample, comprising: (a) contacting a plurality of analyte capture means with a biological sample deposited on a substrate, wherein: one of the plurality of analyte capture means analyzes an analyte capture reagent comprising (i) binding group of the analyte that specifically binds to the biological analyte, (ii) bar code for the binding group of the analyte; (iii) an analyte capture sequence; the substrate comprises a plurality of capture probes, wherein the plurality of capture probes The capture probe in the needle consists of a spatial barcode and a capture domain that specifically binds the analyte capture sequence; (b) determines (i) all or part of the spatial barcode or its complement, and (ii) all or part of the barcode of the analyte-binding part or its complement, and using the established sequence to (i) and (ii) identify the location of the biological analyte in biological sample; (c) performing immunofluorescence microscopy on the biological sample to detect the presence of other biological analytes in the biological sample Location.
Provided herein are methods for identifying the location of a biological analyte in a biological sample, comprising: (a) contacting a plurality of analyte capture means with a biological sample deposited on a substrate, wherein: one of the plurality of analyte capture means analyzes an analyte capture reagent comprising (i) binding group of the analyte that specifically binds the biological analyte, (ii) barcode of the binding unit of the analyte; (iii) an analyte capture sequence, wherein the analyte capture sequence hybridizes to a blocking probe; and the substrate comprises a plurality of capture probes, wherein the plurality of capture probes comprises a spatial barcode and a capture domain; (b) releasing the blocking probe from the analyte capture sequence, thereby enabling the analyte capture sequence to specifically bind the capture agent (c) determining (i) all or part of the spatial barcode or its complement, and (ii) all or part of the barcode portion or its complement that binds to the analyte, and use the specified sequences (i) and (ii) to identify the biological site of the biological analyte sample.
In some embodiments, step (b) includes heating the analyte capture agent to release the blocked probes. In some embodiments, step (b) comprises contacting the analyte capture agent with the enzyme to release the blocking probe. In some embodiments, the enzyme that releases the blocking probe is RNAse H. In some embodiments, the method further includes facilitating the movement or migration of the analyte capture agent onto the substrate. In some embodiments, the method also includes imaging the biological sample.
In some embodiments, the substrate is an array of beads. In some embodiments, the analyte capture sequence comprises a nucleic acid sequence. In some embodiments, the nucleic acid sequence of the analyte capture sequence comprises a sequence substantially complementary to the blocking probe. In some embodiments, the nucleic acid sequence of the analyte capture sequence comprises a sequence that is completely complementary to the blocking probe. In some embodiments, the nucleic acid sequence of the analyte capture sequence comprises a polyA sequence. In some embodiments, the nucleic acid sequence of the analyte capture sequence has a GC content of at least 30%. In some embodiments, the analyte capture means comprises a label. In some embodiments, the label is a fluorophore.
In some embodiments, the biological analyte is a protein. In some embodiments, the analyte binding portion comprises an antibody or an antigen binding domain thereof. In some embodiments, the method further includes performing immunofluorescence microscopy on the biological sample to detect the location of additional biological analytes in the biological sample. In some embodiments, the additional biological analytes are proteins. In some embodiments, the method further includes generating an image of the location of the biological analyte in the biological sample and generating an image of the location of the additional biological analyte in the biological sample. In some embodiments, the method further includes comparing or overlaying an image of the location of the biological analyte in the biological sample with an image of the location of another biological analyte in the biological sample. In some embodiments, the biological analyte is a protein, and the method further comprises performing immunofluorescence microscopy on the biological sample to detect the location of the additional biological analyte in the biological sample, wherein the additional biological analyte is a nucleic acid. In some embodiments, the additional biological analyte is a nucleic acid encoding the biological analyte.
In some embodiments, the method further includes generating an image of the location of the biological analyte and an image of the location of an additional biological analyte in the biological sample. In some embodiments, the method further includes comparing or overlaying an image of the location of the biological analyte in the biological sample with an image of the location of the additional biological analyte. In some embodiments, the analyte capture agent comprises an analyte binding moiety that is linked to the barcode domain of the capture agent via a cleavable domain. In some embodiments, the method further comprises permeabilizing the biological sample prior to step (a). In some embodiments, any of the methods described herein further comprises recording the substrate. In some embodiments, any of the methods described herein further comprises performing a gene expression analysis. In some embodiments, the gene expression analysis includes performing high-throughput sequencing. In some embodiments, the imaging includes the use of fiducial markers.
In some embodiments, the biological sample includes a tissue, tissue portion, organ, organism, organoid, or cell culture sample. In some embodiments, the biological sample is fixed prior to step (a). In some embodiments, the biological sample is fixed with formaldehyde. In some embodiments, the biological sample is fixed with methanol.
All publications, patents, patent applications and information available on the Internet and mentioned in this specification are hereby incorporated by reference to the same extent as if each individual publication, patent, patent application or information was specifically and individually indicated to be incorporated by reference. To the extent that publications, patents, patent applications and information incorporated by reference conflict with a disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such conflicting material.
Where values are described in terms of ranges, such description is understood to include disclosure of all possible subranges within such ranges, as well as specific values falling within such ranges, whether the specific value or the specific subrange is expressly stated.
The term "each", when used to refer to a collection of items, is intended to identify one item in the collection, but does not necessarily refer to every item in the collection, unless expressly stated otherwise or the context of use clearly indicates otherwise.
Various embodiments of the disclosure features are described herein. However, it should be understood that these embodiments are given by way of example only, and that many variations, changes, and substitutions may occur to those skilled in the art without departing from the scope of this disclosure. It is also to be understood that various alternatives to the specific embodiments described herein are within the scope of this disclosure.
The following figures illustrate certain embodiments of the features and advantages of the present disclosure. These examples are not intended to limit the scope of the appended claims in any way. The same reference symbols in the figures indicate the same elements.
Detailed description 1. Introduction
This disclosure describes devices, systems, methods and compositions for the spatial analysis of biological samples. This section describes certain general terms, analytes, sample types, and preparation steps that are mentioned later in this publication.
(a) Spatial analysis
Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from unicellular or multicellular organisms such as mammals. Tissues and cells derived from mammals (eg, humans) often have different levels of analytes (eg, gene and/or protein expression), which can lead to differences in cell morphology and/or function. The location of a cell or subset of cells (eg, adjacent cells and/or non-adjacent cells) within a tissue can affect, for example, cell fate, behavior, morphology, and signaling and crosstalk with other cells in the tissue. Information on differences in levels (gene and/or protein expression) of various intracellular analytes in mammalian tissues may also assist physicians in selecting or administering effective treatments and allow researchers to identify and elucidate cell morphology and/or differences in analyte levels within different cells in tissues , cellular functions in unicellular or multicellular organisms (eg mammals). Differences in the levels of various intracellular analytes in mammalian tissues can also provide information about how tissues (eg, healthy and diseased tissues) function and/or develop. Differences in the levels of various intracellular analytes in mammalian tissues may also provide information on different mechanisms of disease pathogenesis in tissues and mechanisms of action of therapeutic treatments within tissues. Differences in levels of various intracellular analytes in mammalian tissues may also provide information on drug resistance mechanisms and their development in mammalian tissues. Differences in the presence or absence of various intracellular analytes in the tissues of multicellular organisms (eg, mammals) can provide information about drug resistance mechanisms and their development in the tissues of multicellular organisms.
The method of spatial analysis is used herein to detect differences in analyte levels (eg, gene and/or protein expression) within different cells in mammalian tissues or within a single mammalian cell. For example, spatial analysis methods can be used to detect differences in analyte levels (eg, gene and/or protein expression) in different cells in histological slide specimens, and data from these data can be reassembled to create three-dimensional maps of analytes from Levels (eg gene and/or protein expression) of tissue samples obtained from mammals, eg with a degree of spatial resolution (eg single cell resolution).
Spatial heterogeneity in developmental systems is often investigated by RNA hybridization, immunohistochemistry, purification or induction of fluorescent reporters or predefined subpopulations, and subsequent genomic analysis such as RNA-seq. However, such methods rely on a relatively small set of predefined markers, thus introducing a selection bias that limits detection. These prior methods also rely on prior knowledge. Traditionally, spatial analysis of RNA has relied on staining a limited number of RNA species. In contrast, single-cell RNA-sequencing enables in-depth analysis of cellular gene expression, including non-coding RNA, but established methods separate cells from their natural spatial environment.
The spatial analysis methods described here provide large amounts of analyte level and/or expression data for a wide array of analytes within a sample at high spatial resolution, eg while preserving the natural spatial context. Spatial analysis methods include, for example, the use of capture probes that contain spatial barcodes (e.g., nucleic acid sequences that provide information about the location of the capture probes in a cell or tissue sample (e.g., a mammalian cell or mammalian tissue sample)) and a domain for capture capable of binding an analyte (eg protein and/or nucleic acid) produced and/or present in the cell. As described herein, the spatial barcode can be a nucleic acid having a unique sequence, a unique fluorophore or a unique combination of fluorophores, a unique amino acid sequence, a unique heavy metal or a unique combination of heavy metals, or any other unique detectable reagent. The capture domain may be capable of binding an analyte produced by and/or present in the cell (e.g., a nucleic acid capable of hybridizing to a nucleic acid from the cell (e.g., mRNA, genomic DNA, mitochondrial DNA, or miRNA)), substrates including analyte assays, partners for analyte binding or antibodies that specifically bind to the analyte). Capture probes may also include nucleic acid sequences that are complementary to the sequences of the universal forward and/or universal reverse primers. Capture probes may also include cleavage sites (eg, cleavage recognition sites for restriction endonucleases), photolabile, thermosensitive, or chemically sensitive linkages.
Binding of analytes to capture probes can be detected using a number of different methods, such as nucleic acid sequencing, fluorophore detection, nucleic acid amplification, nucleic acid ligation detection, and/or nucleic acid cleavage product detection. In some examples, detection is used to associate a specific spatial barcode with a specific analyte produced and/or present in a cell (eg, a mammalian cell).
Capture probes can, for example, be attached to a surface, such as a solid array, bead, or coverslip. In some examples, the capture probes are not attached to the surface. In some examples, capture probes can be encapsulated, embedded within, or deposited on the surface of a permeable composition (eg, any of the substrates described herein). For example, capture probes can be encapsulated or placed inside permeable beads (eg, gel beads). In some examples, capture probes may be encapsulated, embedded, or laminated to the surface of a substrate (eg, any of the substrate examples described herein, such as hydrogels or porous membranes).
In some examples, cells or a tissue sample containing the cells are contacted with capture probes attached to a substrate (e.g., the surface of the substrate), and the cells or tissue sample are permeabilized to allow release of the analyte from the cells and binding to the substrate attached to capture probe substrate. In some examples, analytes released from cells can be actively directed to capture substrate-attached probes using various methods, such as electrophoresis, chemical gradients, pressure gradients, fluid flow, or magnetic fields.
In other examples, various methods can be used to direct the capture probe to interact with a cell or tissue sample, e.g. inclusion of a lipid anchor in the capture probe, inclusion of a reagent that specifically binds to or forms the lipid anchor. They bind covalently to membrane proteins in capture probes, fluid flow, pressure gradients, chemical gradients, or magnetic fields.
Non-limiting aspects of spatial analysis methods are described in WO 2011/127099, WO 2014/210233, WO 2014/210225, WO 2016/162309, WO 2018/091676, WO 2012/140224, WO 2014/060483, US Pat. Patent no. 10,002,316 US Patent No. 9,727,810. Application publication no. 2017/0016053, Rodriques et al.,science363(6434):1463-1467, 2019; WO 2018/045186, Lee et al.,Nat. Agreement10(3):442-458, 2015; WO 2016/007839, WO 2018/045181, WO 2014/163886, Trejo et al.,PLOS14(2):e0212031, 2019, US Pat. Application publication no. 2018/0245142, Chen et al.,science348(6233):aaa6090, 2015, Gao et al.BMC Biology.15:50, 2017., WO 2017/144338, WO 2018/107054, WO 2017/222453, WO 2019/068880, WO 2011/094669, US PAT. US patent bridge 7,709,198. U.S. patent br. 8,604,182. US patent br. 8,951,726. U.S. patent br. 9,783,841. NO 2016/057552, NO 2017/147483, NO 2018/022809, NO 2016/166128, NO 2017/027367, NO 2017/027368, NO 2018/136856, NO 2019/075 091, s AD Patent. br. 10,059,990, WO 2018/057999, WO 2015/161173 in Gupta et al.nature biotechnology36:1197-1202, 2018, and may be used in any combination herein. Other non-limiting aspects of spatial analysis methods are described herein.
(b) General Terms and Conditions
Specific terms are used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions. This subsection includes explanations of certain terms that appear later in this publication. In the event of an apparent conflict between the description in this section and usage elsewhere in this publication, the definitions in this section take precedence.
(i) bar code
A "bar code" is a label or identifier that conveys or may convey information, eg, about the analyte in a sample, bead, and/or capture probe. Barcodes can be part of the analyte or independent of the analyte. Barcodes can be attached to analytes. Certain barcodes may be unique to other barcodes.
Barcodes can be in many different formats. For example, barcodes may include non-random, semi-random and/or random nucleic acid and/or amino acid sequences, as well as synthetic nucleic acid and/or amino acid sequences. A barcode can be attached to an analyte or other group or structure in a reversible or irreversible manner. Barcodes can be added to sample fragments, for example, deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) before or during sample sequencing. A barcode may enable the identification and/or quantification of individual sequencing reads (eg, the barcode may be or may include a unique molecular identifier or "UMI").
A barcode can spatially resolve the molecular components found in a biological sample, for example, at single cell resolution (eg, the barcode can be or include a "spatial barcode"). In some embodiments, the barcodes include UMI and spatial barcodes. In some embodiments, the barcode includes two or more sub-barcodes that together act as a single barcode (eg, a polynucleotide barcode). For example, a polynucleotide barcode may comprise two or more polynucleotide sequences (eg, sub-barcodes), which may be separated by one or more non-barcode sequences.
(ii) Nucleic acids and nucleotides
The terms "nucleic acid" and "nucleotide" are as used in the art and include naturally occurring species or functional analogs thereof. Particularly useful functional analogs of nucleic acids are those that can hybridize to nucleic acids in a sequence-specific manner (eg, that can hybridize to two nucleic acids so that linkage can occur between the two hybridized nucleic acids) or that can be used to replication of specific nucleotides Sequence template. Naturally occurring nucleic acids usually have a backbone consisting of phosphodiester bonds. Similar structures may have alternative backbone linkages, including any of a variety of linkages known in the art. Naturally occurring nucleic acids usually have the sugar deoxyribose (as found in deoxyribonucleic acid (DNA)) or the sugar ribose (as found in ribonucleic acid (RNA)).
Nucleic acids may contain nucleotides having any of the various analogs of these sugar residues known in the art. Nucleic acids may include natural or non-natural nucleotides. In this sense, natural deoxyribonucleic acid can have one or more bases selected from adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acid can have one or more bases selected from uracil (U) , adenine (A), cytosine (C) or guanine (G). Useful unnatural bases that can be incorporated into nucleic acids or nucleotides are known in the art.
(iii) Detection and targeting
"Probe" or "target", when referring to a nucleic acid or nucleic acid sequence, is intended as a semantic identifier of the nucleic acid or sequence in the context of a method or composition and does not limit the structure or function of the nucleic acid. acid or sequence beyond what is expressly indicated.
(iv) Oligonucleotides and polynucleotides
The terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polymers of nucleotides from about 2 to about 500 nucleotides in length. Oligonucleotides can be synthetic, prepared enzymatically (for example, by polymerization) or using a "pooling" approach. An oligonucleotide may contain ribonucleotide monomers (ie, they may be oligoribonucleotides) and/or deoxyribonucleotide monomers (ie, oligodeoxyribonucleotides). In some examples, an oligonucleotide may include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in an oligonucleotide (eg, a combination of deoxyribonucleotide monomers and ribonucleotide monomers in a random or ordered combination). Oligonucleotides can be 4 to 10, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, 150 to 200, 200 to e.g. . , 250, 250 to 300, 300 to 350, 350 to 400 or 400-500 nucleotides in length. An oligonucleotide may include one or more functional units linked (eg, covalently or non-covalently) to a polymeric structure. For example, oligonucleotides may include one or more detectable labels (eg, radioisotopes or fluorophores).
(v) subject
A "subject" is an animal, such as a mammal (eg, a human or a non-human ape), or an avian (eg, a bird), or another organism, such as a plant. Examples of subjects include, but are not limited to, mammals such as rodents, mice, rats, rabbits, guinea pigs, ungulates, horses, sheep, pigs, goats, cows, cats, dogs, primates (ie, human or non-human primates); a plant such asArabidopsis, corn, sorghum, oats, wheat, rice, canola or soybeans; algae such asChlamydomonas reinhardtii; Nematodes such asCaenorhabditis elegans; an insect such asDrosophila, mosquitoes, wine flies or bees; arachnids like spiders; fish like zebrafish; reptiles; amphibians like frogs orAfrican clawed frog; ADictyostelium discoideum; mushroom as it isPneumocystis carinii, Takifugu rubripes, yeast,Saccharomyces cerevisiaeorSchizosaccharomyces pombe;orPlasmodium falciparum.
(vi) through
"Genome" generally refers to the genomic information of a subject, for example, it may be at least some or all of the genetic information encoded by the subject's genes. A genome can include coding regions (eg, protein-coding regions) as well as non-coding regions. A genome may include a sequence of some or all of a subject's chromosomes. For example, the human genome usually has a total of 46 chromosomes. Some or all of these sequences may constitute a genome.
(vii) Adapters, Adapters and Labels
"Adapter", "adaptor" and "tag" are terms used interchangeably in this publication to refer to any method that can be joined to a polynucleotide sequence (in a process known as "tagging") One of many different techniques including ( but not limited to) ligation, hybridization and labeling. Adapters can also be nucleic acid sequences that add functionality, such as spacer sequences, primer sequences/sites, barcode sequences, unique molecular identifier sequences.
(viii) hybridization, hybridization, annealing and annealing
The terms "hybridize", "hybridize", "anneal" and "anneal" are used interchangeably in this disclosure to refer to the pairing of substantially complementary or complementary nucleic acid sequences within two different molecules. Pairing can be accomplished by any method in which a nucleic acid sequence is base-paired with a substantially or completely complementary sequence to form a hybridization complex. For purposes of hybridization, two nucleic acid sequences are "substantially complementary" if at least 60% (eg, at least 70%, at least 80%, or at least 90%) of the individual bases of the two nucleic acid sequences are complementary. one to another.
(nine) first
A "primer" is a single-stranded nucleic acid sequence having a 3' end that serves as a chemical substrate for a nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed from RNA nucleotides and are used in RNA synthesis, while DNA primers are formed from DNA nucleotides and are used in DNA synthesis. Examples may also include RNA nucleotides and DNA nucleotides (eg, in random or designed patterns). Examples may also include other natural or synthetic nucleotides described herein that may have additional functions. In some examples, DNA primers can be used to initiate RNA synthesis, and vice versa (eg, RNA primers can be used to initiate DNA synthesis). Examples may vary in length. For example, a primer can be about 6 bases to about 120 bases. For example, an example may include up to about 25 bases.
(x) Example extension
"Primer extension" refers to the overlapping of two nucleic acid sequences (eg, constant regions from each of two different capture probes) with complementary nucleic acid sequences at their ends (ie, for eg the 3' end). Such ligation can be followed by nucleic acid extension (eg, enzymatic extension) of one or both ends, using another nucleic acid sequence as a template for extension. Enzymatic extension can be performed using enzymes, including but not limited to polymerases and/or reverse transcriptases.
(xi) Proximity Connections
"Proximity ligation" is a method of joining two (or more) nucleic acid sequences that are in close proximity to each other by enzymatic means (eg ligase). In some embodiments, the close ligation may include a "gap filling" step that involves incorporation of one or more nucleic acids by a polymerase based on the nucleic acid sequence of the template nucleic acid molecule, spanning the distance between the two nucleic acid molecules (See, e.g., US Patent No. 7,264,929, the entire contents of which are incorporated herein by reference).
A variety of different methods can be used to closely ligate nucleic acid molecules, including, but not limited to, "sticky end" and "blunt end" ligation. Additionally, single-stranded ligation can be used to effect close ligation of single-stranded nucleic acid molecules. Close sticky-end ligation involves the hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be ligated, prior to the ligation event itself. Proximate blunt-end ligation typically does not involve hybridization of complementary regions from each nucleic acid molecule because the two nucleic acid molecules do not have single-stranded overhangs at the joining site.
(xii) Nucleic acid extension
"Nucleic acid extension" generally involves the incorporation of one or more nucleic acids (eg, A, G, C, T, U, nucleotide analogs or derivatives thereof) into a molecule (such as, but not limited to, a nucleic acid sequence) depending on template In such a way that successive nucleic acids are incorporated by enzymes such as polymerase or reverse transcriptase, resulting in newly synthesized nucleic acid molecules. For example, primers that hybridize to a complementary nucleic acid sequence can be used to synthesize new nucleic acid molecules using the complementary nucleic acid sequence as a template for nucleic acid synthesis. Similarly, the 3' polyadenylation tail of an mRNA transcript hybridized to a poly (dT) sequence (such as a capture domain) can be used as a template for single-stranded synthesis of the corresponding cDNA molecule.
(xiii) PCR amplification
"PCR amplification" refers to the use of the polymerase chain reaction (PCR) to make copies of genetic material, including DNA and RNA sequences. Suitable reagents and conditions for performing PCR are described, for example, in U.S. Pat. patents no. 4,683,202, 4,683,195, 4,800,159, 4,965,188 and 5,512,462, the entire contents of each of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes the genetic material to be amplified, enzymes, one or more primers for the primer extension reaction, and reagents for the reaction. Oligonucleotide primers are of sufficient length to ensure hybridization of complementary genetic material under annealing conditions. The length of the primer usually depends on the length of the amplified domain, but is usually at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 bases right (bp ), at least 11 bp, at least 12 bp, at least 13 bp, at least 14 bp, at least 15 bp, at least 16 bp, at least 17 bp, at least 18 bp, at least 19 bp, at least 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, can be up to 40 bp or more Long, primer length is generally between 18-50 bp. The genetic material can be contacted with a single primer or a set of two primers (forward and reverse), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.
In some embodiments, the PCR amplification process uses DNA polymerase. DNA polymerase activity can be provided by one or more different DNA polymerases. In certain embodiments, the DNA polymerase is from bacteria, e.g. DNA polymerase is a bacterial DNA polymerase. For example, DNA polymerases can be derived from the genus BacteriaEscherichia coli, Bacillus, Thermus,orPyrococcus.
Exemplary DNA polymerases that may be used include, but are not limited to: E. coli DNA Polymerase I, Bsu DNA Polymerase, Bst DNA Polymerase, Taq DNA Polymerase, VENT™ DNA Polymerase, DEEPVENT™ DNA Polymerase, LongAmp® Taq DNA Polymerase, LongAmp® Hot Start Taq DNA Polymerase, Crimson LongAmp® Taq DNA Polymerase, Crimson Taq DNA Polymerase, OneTaq® DNA Polymerase, OneTaq® Quick-Load® DNA Polymerase, Hemo KlenTaq® DNA Polymerase, REDTaq® DNA Polymerase, Phusion® DNA Polymerase, Phusion® High-Fidelity DNA polymerase, Platinum Pfx DNA polymerase, AccuPrime Pfx DNA polymerase, Phi29 DNA polymerase, Klenow fragment, Pwo DNA polymerase, Pfu DNA polymerase, T4 DNA polymerase and T7 DNA polymerase.
The term "DNA polymerase" includes not only natural enzymes, but also all modified derivatives thereof, including derivatives of natural DNA polymerases. For example, in some embodiments, the DNA polymerase is modified to remove 5'-3' exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that may be used include, but are not limited to, mutants that retain at least some functionality, such as the DNA polymerase activity of the wild-type sequence. Under different reaction conditions, such as temperature, sample concentration, primer concentration, etc., mutations can affect the characteristics of enzyme activity, such as increasing or decreasing the rate of polymerization. Mutations or sequence modifications can also affect exonuclease activity and/or enzyme thermostability.
In some embodiments, PCR amplification may include reactions such as, but not limited to, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, isothermal amplification, and/or ring-mediated amplification. Guided amplification reaction.
In some embodiments, PCR amplification uses a single primer complementary to the 3' tag of the DNA fragment of interest. In some embodiments, the PCR amplification uses first and second primers, wherein at least a portion of the 3' end of the first primer is complementary to at least a portion of the 3' tag of the target nucleic acid fragment, and wherein at least the 3' end of the second primer partially displays the sequence of at least portion 5 ' tags of the target nucleic acid fragment. In some embodiments, the 5' end of the first primer is not complementary to the 3' tag of the target nucleic acid fragment, and the 5' end of the second primer does not display at least a portion of the 5' tag sequence of the target nucleic acid fragment. In some embodiments, the first primer includes a first universal sequence and/or the second primer includes a second universal sequence.
In some embodiments (eg, when PCR amplification amplifies the captured DNA), DNA ligase can be used to ligate the PCR amplification product to additional sequences. DNA ligase activity can be provided by one or more different DNA ligases. In some embodiments, the DNA ligase is from bacteria, e.g. DNA ligase is a bacterial DNA ligase. In some embodiments, the DNA ligase is from a virus (eg, a bacteriophage). For example, the DNA ligase may be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase,Thermococcussp. (strain 9oN) DNA ligase (9oN™ DNA ligase, available from New England Biolabs, Ipswich, MA) and Ampligase® (available from Lucigen, Middleton, WI). Derivatives, such as sequence-modified derivatives and/or mutants thereof, can also be used.
In some embodiments, the genetic material is amplified by reverse transcription polymerase chain reaction (RT-PCR). The desired reverse transcriptase activity can be provided by one or more different reverse transcriptases (ie, RNA-dependent DNA polymerases), suitable examples of which include, but are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScript™, MultiScribe™ Enzymes, ThermoScript ™ and SuperScript® I, II, III and IV. "Reverse transcriptase" includes not only the native enzyme, but also all such modified derivatives thereof, and also includes derivatives of native reverse transcriptase.
Additionally, sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptase can be used for reverse transcription, including mutants that retain at least some function, such as reverse transcriptase, of the wild-type active sequence. Reverse transcriptase can be provided as part of a composition containing other components, for example, stabilizing components that enhance or improve reverse transcriptase activity, such as ribonuclease inhibitors, inhibitors of DNA-dependent DNA synthesis, such as . , actinomycin D. Sequence-modified derivatives or mutants of many reverse transcriptases, such as M-MLV, and compositions including unmodified and modified enzymes, such as ArrayScript™, MultiScribe™, ThermoScript™ and SuperScript® I, II, III and IV enzymes.
Certain reverse transcriptases (for example, avian myeloblastosis virus (AMV) reverse transcriptase and Moloney murine leukemia virus (M-MuLV, MMLV) reverse transcriptase can use both RNA (cDNA synthesis) and single-stranded DNA (ssDNA) to synthesize the complementary strand of DNA) as template. Therefore, in some embodiments, the reverse transcription reaction may use an enzyme (reverse transcriptase) capable of using RNA and ssDNA as a template for the extension reaction, such as AMV or MMLV reverse transcriptase.
In some embodiments, quantification of RNA and/or DNA is performed by real-time PCR (also known as quantitative PCR or qPCR), using techniques well known in the art, such as, but not limited to "TAQMAN™", or dyes such as "SYBR®", or capillary ("LightCycler® Capillary"). In some embodiments, quantification of the genetic material is determined by absorbance and real-time PCR. In some embodiments, quantification of the genetic material is determined by digital PCR. In some embodiments, the analyzed gene can be compared to reference nucleic acid extracts (DNA and RNA) corresponding to expression (mRNA) and quantity (DNA) to compare expression levels of the target nucleic acid.
(xiv) Antibodies
An "antibody" is a polypeptide molecule that recognizes and binds a complementary target antigen. Antibodies usually have a form of molecular structure that resembles the Y shape or its polymers. Natural antibodies, called immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD and IgE. Antibodies can also be produced synthetically. For example, a recombinant antibody that is a monoclonal antibody can be synthesized using a synthetic gene by recovering the antibody gene from a cell of origin, cloning it into an appropriate vector, and introducing the vector into a host so that the host expresses the recombinant antibody. In general, recombinant antibodies can be cloned from any antibody-producing animal species using appropriate oligonucleotide primers and/or hybridization probes. Recombinant techniques can be used to generate antibodies and antibody fragments, including non-endogenous species.
Synthetic antibodies can be obtained from sources other than immunoglobulins. For example, antibodies can be generated from nucleic acids (eg, aptamers) and non-globulin scaffolds (eg, peptide aptamers) into which hypervariable loops have been inserted to form the antigen binding site. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than antibodies derived from immunoglobulins, resulting in greater tissue penetration.
Antibodies may also include Affibody proteins, which are affinity reagents, typically with a molecular weight of about 12-14 kDa. Affinity proteins typically bind a target (eg, a target protein) with high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive protein. In some embodiments, the affinity protein is derived from cystatin and includes a peptide loop and a variable N-terminal sequence that provides a binding site.
Antibodies may also include single-domain antibodies (VHH domain and VNAR domain), scFvs and Fab fragments.
(xv) Affinity groups
An "affinity group" is a molecule or part of a molecule that has a high affinity or tendency to bind or bind to another specific or particular molecule or part. Association or binding with another specific or specific molecule or residue can be through non-covalent interactions such as hydrogen bonds, ionic forces, and van der Waals interactions. The affinity group can be for example biotin, which has a high affinity or preferentially binds or binds to the protein avidin or streptavidin. The affinity group can also refer to, for example, avidin or streptavidin, which have an affinity for biotin. Other examples of affinity groups and specific or specific molecules or moieties to which they bind or associate include, but are not limited to, antibodies or antibody fragments and their respective antigens, such as digoxin and anti-digoxigenin antibodies, lectins, and carbohydrates (eg, sugars , monosaccharides, disaccharides or polysaccharides), as well as receptors and receptor ligands.
Each pair of affinity groups and the specific or specific molecules or parts to which they bind or connect can have opposite effects, for example, so that between a first molecule and a second molecule, in the first case, the first molecule is characterized as an affinity group for the second molecule, and in the second case, the second molecule is characterized as an affinity group for the first molecule.
(xvi) Labels, detectable labels and optical labels
The terms "detectable label", "optical label" and "label" are used interchangeably herein to refer to a directly or indirectly detectable moiety that is associated with (eg, conjugated to) a molecule to be detected, such as a capture probe or analyte. Detectable labels can be detected directly (eg, radioisotope or fluorescent labels) or, in the case of enzymatic labels, indirectly, eg, by catalyzing a chemical change in a directly detectable chemical substrate compound or composition. Detectable markers can be adapted for small-scale testing and/or high-throughput screening. Accordingly, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.
A detectable label can be detected qualitatively (eg, optically or spectroscopically) or can be quantified. Qualitative tests typically include tests that confirm the presence or presence of detectable markers, while quantifiable tests typically include tests that have measurable (eg, numerically reported) values, such as intensity, duration, polarization, and/or other properties. In some embodiments, the detectable label is bound to the feature or a capture probe associated with the feature. For example, detectable label features may include fluorescent, colorimetric, or chemiluminescent labels attached to beads (see, e.g., Rajeswari et al.,J. Microbiological methods139:22-28, 2017, Forcucci et al.,J. Biomedical Possibilities.10:105010, 2015, each article is hereby incorporated by reference in its entirety).
In some embodiments, multiple detectable labels may be attached to the feature, capture probe, or composition to be detected. For example, a detectable label (eg, Cy5®-labeled nucleotides, such as Cy5®-dCTP) can be incorporated during nucleic acid polymerization or amplification. Any suitable detectable label may be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be from the group consisting of: 7-AAD (7-Aminoactinomycin D), Acridine Orange (+DNA), Acridine Orange (+RNA), Alexa Fluor® 350, Alexa Fluor® 430, Alexa Fluor® 488, Alexa Fluor® 532, Alexa Fluor® 546, Alexa Fluor® 555, Alexa Fluor® 568, Alexa Fluor® 594, Alexa Fluor® 633, Alexa Fluor® 647, Alexa Fluor® 660, Alexa Fluor® 680, Alexa Fluor® 700, Alexa Fluor® 750, allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, aniline blue, ANS, APC-Cy7, ATTO-TAG ™ CBQCA, ATTO-TAG™ FQ, Auramine O-Feulgen, BCECF (High pH), BFP (Blue Fluorescent Protein), BFP / GFP FRET, BOBO™-1 / BO-PRO™-1, BOBO™-3 / BO -PRO™-3, BODIPY® FL, BODIPY® TMR, BODIPY® TR-X, BODIPY® 530/550, BODIPY® 558/568, BODIPY® 564/570, BODIPY® 581/591, BODIPY® 630/ 650 - X, BODIPY® 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson™, Calcium Green-1™, Calcium Orange™, Calcofluor® White, 5-Carboxyfluoroscein (5-FAM), 5-Carboxynaphthofluoroscein, 6 - Carboxyrodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), Carboxy-X-rhodamine (5-ROX), Cascade Blue®, Cascade Yellow™, CCF2 (GeneBLAzer™), CFP (cyan fluorescent protein), CFP / YFP FRET, Chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC Formazan, Cy2®, Cy3®, Cy3.5®, Cy5®, Cy5.5®, Cy7®, Cychrome (PE-Cy5), Dansylamine , Dansyl cadaverine, Dansyl chloride, DAPI, Dapoxil, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18(5)), DIDS, Dil (DilC18(3)), DiO (DiOC18(3) ) ), DiR (DilC18(7)), Di-4 ANEPPS, Di-8 ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, ELF® 97 alcohol, Eosin, erythrosin, ethidium bromide, ethidium homodimer-1 (EthD-1), europium(III) chloride, 5-FAM (5-carboxyfluorescein), Fast Blue, fluorescein-dT phosphoramidite, FITC, Fluo-3, Fluo -4, FluorX®, Fluoro-Gold (high pH), Fluoro-Gold™ (low pH), Fluoro-Jade, FM® 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red™ (high calcium), Fura Red™ / Fluo-3, GeneBLAzer™ (CCF2), GFP Red Shifted (rsGFP), GFP Wild Type, GFP / BFP FRET, GFP / DsRed FRET, Hoechst 33342 & 33258, 7-Hydroxy-4 -methylcoumarin ( pH 9), 1, 5 IAEDANS, Indo-1 (high calcium), Indo-1 (low calcium), indodicarbocyanine, indotricarbocyanine, JC-1, 6-JOE, JOJO™-1 / JO-PRO™- 1, LDS 751 (+DNA), LDS 751 (+RNA), LOLO™-1 / LO-PRO™-1, Lucifer Yellow, LysoSensor™ Blue (pH 5), LysoSensor™ Green (pH 5), LysoSensor™ Yellow /Blue ( pH 4.2), LysoTracker® Green, LysoTracker® Red, LysoTracker® Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green™, Marina Blue®, 4-Methylumbelliferone, Mithramycin, MitoTracker® Green , MitoTracker® Orange, MitoTracker® Red, NBD (amine), Nile Red, Oregon Green® 488, Oregon Green® 500, Oregon Green® 514, Pacific Blue, PBF1, PE (R-Phycoerythrin), PE-Cy5, PE- Cy7, PE-Texas Red, PerCP (peridinin chlorophyll protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide) , PKH26, PKH67, POPO™-1 / PO-PRO™-1, POPO™-3 / PO-PRO™-3, propidium iodide (PI), PyMPO, pyrene, pyronine Y, Quantam Red (PE-Cy5), quinacrine mustard, R670 (PE-Cy5), red 613 (PE-Texas Red), red fluorescent protein (DsRed), resorufin, RH 414, Rhod-2, rhodamine B, rhodamine green™, rhodamine red™, rhodamine phalloidin, rhodamine 110, rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, SNAFL®-1 (high pH), SNAFL®-2, SNARF®-1 (high pH pH ), SNARF®- 1 (low pH), Sodium Green™, SpectrumAqua®, SpectrumGreen® #1, SpectrumGreen® #2, SpectrumOrange®, SpectrumRed®, SYTO® 11, SYTO® 13, SYTO® 17, SYTO® 45, SYTOX® Blue, SYTOX ® Green, SYTOX® Orange, 5-TAMRA (5-carboxytetramethylrhodamine), Tetramethylrhodamine (TRITC), Texas Red® / Texas Red®-X, Texas Red®-X (NHS ester), Thiadicarbocyanine, Thiazole Orange , TOTO®-1 / TO-PRO®-1, TOTO®-3 / TO-PRO®-3, TO-PRO®-5, tricolor (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5. 5), WW 781 , X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), YOYO®-1 / YO-PRO®-1, YOYO®-3 / YO-PRO®-3, 6-FAM (fluorescein), 6-FAM (NHS ester), 6-FAM (azide), HEX, TAMRA (NHS ester), Yakima yellow, MAX, TET, TEX615, ATTO 488, ATTO 532, ATTO 550, ATTO 565 , ATTO Rho101, ATTO 590, ATTO 633, ATTO 647N, TYE 563, TYE 665, TYE 705, 5′ IRDye® 700, 5′ IRDye® 800, 5′ IRDye® 800CW (NHS ester), WellRED D4, WellRED D3 dye. dye, WellRED D2 dye, Lightcycler® 640 (NHS ester) and Dy 750 (NHS ester).
As noted above, in some embodiments the detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent/chemiluminescent units include, but are not limited to, peroxidases such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. With an appropriate chemical substrate (for example, an oxidant plus a chemiluminescent compound), these protein moieties can catalyze a chemiluminescent reaction. Many families of compounds are known to provide chemiluminescence under different conditions. Non-limiting examples of the family of chemiluminescent compounds include 2,3-dihydro-1,4-phthalazindione luminol, 5-amino-6,7,8-trimethoxy- and dimethylamino[ca]benzene analogs. These compounds can emit light in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of a family of chemiluminescent compounds include, for example, 2,4,5-triphenylimidazole, p-dimethylamino and -methoxy substituents, oxalates such as oxalyl active ester, p-nitrobenzene base, N-alkyl acridinium ester, luciferin, lucigenin or acridinium ester.
(xvii) Template switching oligonukleotidi
A "template-switching oligonucleotide" is an oligonucleotide that hybridizes to non-template nucleotides added by a reverse transcriptase (eg, an enzyme with terminal transferase activity) during reverse transcription. In some embodiments, the template-switching oligonucleotide hybridizes to template-free poly(C) nucleotides added by reverse transcriptase. In some embodiments, the template-switching oligonucleotides add the normal 5' sequence of the full-length cDNA for cDNA amplification.
In some embodiments, the template-switching oligonucleotide adds a consensus sequence to the 5' end of the reverse transcribed RNA. For example, a template-changing oligonucleotide can hybridize with non-template poly(C) nucleotides added to the end of a cDNA molecule and provide a template for reverse transcriptase to continue replication to the 5' end of the template-changing oligonucleotide, resulting in a full-length cDNA ready for further multiplication. In some embodiments, template-switching oligonucleotides can serve as primers in cDNA amplification reactions after full-length cDNA molecules have been produced.
In some embodiments, the template-switching oligonucleotide is added before, simultaneously with, or after reverse transcription or other terminal transferase-based reactions. In some embodiments, a template-switching oligonucleotide is included in the capture probe. In certain embodiments, a method of analyzing a sample using a template-switching oligonucleotide may include generating a nucleic acid product from a tissue sample analyte, followed by further processing the nucleic acid product with a template-switching oligonucleotide.
A template-changing oligonucleotide may include a hybridization region and a template region. A hybridizing region can include any sequence capable of hybridizing to a target. In some embodiments, the hybridization region may, for example, include a sequence of G bases that complement the overhanging C bases at the 3' end of the cDNA molecule. A series of G bases may include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. Template sequences can include any sequence that is incorporated into cDNA. In other embodiments, the hybridization region may include at least one base in addition to at least one G base. In other embodiments, the hybridization may include bases other than G bases. In some embodiments, the template region includes at least 1 (eg, at least 2, 3, 4, 5, or more) tag sequences and/or functional sequences. In some embodiments, the template region and the hybridization region are separated by a space.
In some embodiments, the template area includes a barcode string. A barcode array can serve as a spatial barcode and/or as a unique molecular identifier. Template-switching oligonucleotides may include deoxyribonucleic acid; ribonucleic acid; modified nucleic acids including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), inverted dT, 5-methyl dC, 2'-deoxyinosine, Super T (5 -hydroxybutynl-2'-deoxyuridine), Super G ( 8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, such as UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2' fluoro bases (eg, fluoro-C, fluoro-U, fluoro-A, and fluoro-G), or any combination of the above.
In some embodiments, a pattern-switching oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides or longer. In some embodiments, a template switching oligonucleotide can be up to about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides in length or more.
(xviii) Oligonucleotides udlaka
A "splint" is an oligonucleotide that, when hybridized to other polynucleotides, acts as a "splint" to place the polynucleotides next to each other so that they can be linked together. In some embodiments, the oligonucleotide splint is DNA or RNA. An inserted oligonucleotide may contain a nucleotide sequence that is partially complementary to the nucleotide sequence from two or more different oligonucleotides. In some embodiments, the oligonucleotide splint facilitates the ligation of the "donor" oligonucleotide and the "acceptor" oligonucleotide. Usually, RNA ligase, DNA ligase, or various other ligases are used to join the two nucleotide sequences
In some embodiments, the oligonucleotides of the splint are between 10 and 50 oligonucleotides long, for example, between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and between 20 oligonucleotides. In some embodiments, the oligonucleotides of the splint are between 15 and 50, 15 and 45, 15 and 40, 15 and 35, 15 and 30, 15 and 30, or 15 and 25 nucleotides in length.
(c) Analytes
The devices, systems, methods and compositions described in this disclosure can be used to detect and analyze a variety of different analytes. For purposes of this disclosure, an "analyte" may include any biological substance, structure, part, or component to be analyzed. The term "target" can similarly refer to an analyte of interest.
Analytes can be broadly classified into one of two groups: nucleic acid analytes and non-nucleic acid analytes. Examples of non-nucleic acid analytes include, but are not limited to, lipids, carbohydrates, peptides, proteins, glycoproteins (N-linked or O-linked), lipoproteins, phosphoproteins, specific phosphorylated or acetylated protein variants, protein amidated protein variants, hydroxylated variants proteins, methylated protein variants, ubiquitinated protein variants, sulfated protein variants, viral envelope proteins, extracellular and intracellular proteins, antibodies and antigens Match the fragments. In some embodiments, the analyte can be an organelle (eg, nucleus or mitochondrion).
Cell surface features suitable for analytes may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiated protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, intercellular interactions. protein complexes, antigen-presenting complexes, major histocompatibility complexes, engineered T-cell receptors, T-cell receptors, B-cell receptors, chimeric antigen receptors, extracellular matrix proteins, cell surface proteins Post-modification (eg, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation), the status of gaps and adherens junctions.
Analytes may originate from specific cell types and/or specific subcellular regions. For example, the analyte may originate from the cytosol, nucleus, mitochondria, microsomes, and generally, from any other compartment, organelle, or part of the cell. Permeability enhancers that specifically target specific cellular compartments and organelles can be used to selectively release analytes from cells for analysis.
Examples of nucleic acid analytes include DNA analytes such as genomic DNA, methylated DNA, specific methylated DNA sequences, fragmented DNA, mitochondrial DNA, in situ synthesized PCR products, and RNA/DNA hybrids.
Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Examples of different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and viral RNA. RNAs can be transcripts (eg, present in tissue sections). RNA can be small (eg, less than 200 nucleobases in length) or large (eg, RNA more than 200 nucleobases in length). Small RNA mainly includes 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snoRNA), Piwi-interacting RNA (piRNA), tRNA- derived small RNA (tsRNA) and small RNA derived from rDNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. The RNA may be bacterial rRNA (for example, 16 s rRNA or 23 s rRNA).
Additional examples of analytes include mRNA and cell surface features (e.g., use of labeling agents described herein), mRNA and intracellular proteins (e.g., transcription factors), mRNA and cellular methylation status, mRNA and available chromatin (e.g., ATAC-seq, DNase -seq, and/or MNase-seq), mRNA and metabolites (e.g., using labeling agents described herein), barcode labeling agents (e.g., oligonucleotide-labeled antibodies), and V(D)J sequences of immune antibodies Cell receptors ( e.g., T cell receptors), mRNA and perturbants (e.g., CRISPR crRNA/sgRNA, TALENs, zinc finger nucleases, and/or antisense oligonucleotides, as described herein). In some embodiments, the perturbing agent can be a small molecule, antibody, drug, aptamer, miRNA, physical environment (eg, temperature change), or any other known perturbing agent.
The analyte may include a nucleic acid molecule having a nucleic acid sequence encoding at least a portion of the V(D)J sequence of an immune cell receptor (eg, TCR or BCR). In some embodiments, the nucleic acid molecule is a cDNA first generated from reverse transcription of the corresponding mRNA using poly(T)-primers. The resulting cDNA can then be encoded using a capture probe having a barcode sequence (and optionally a UMI sequence) that hybridizes to at least a portion of the generated cDNA. In some embodiments, the template-switching oligonucleotide hybridizes to a poly(C) tail that has been added to the 3' end of the cDNA by reverse transcriptase. Crude mRNA templates and template-altering oligonucleotides can then be denatured from the cDNA, and barcode capture probes can then hybridize to the cDNA and generate the complementary cDNA sequence. Other methods and compositions suitable for barcoding cDNA generated from mRNA transcripts, including those encoding V(D)J regions of immune cell receptors and/or including templates, are described in PCT patent application PCT/US2017/057269 Barcoding Methods and Compositions for Switching Oligonucleotides , filed Oct. 18, 2017, US Pat. Application serial no. 15/825,740, filed Nov. 29, 2017, is hereby incorporated by reference in its entirety. V(D)J analysis can also be achieved using one or more markers that bind to specific surface features of immune cells and link to barcode sequences. One or more labeling agents may include MHC or MHC multimers.
As noted above, analytes may include nucleic acids capable of functioning as components of gene editing reactions, such as clustered regular interspersed short palindromic repeat (CRISPR)-based gene editing. Thus, capture probes can include nucleic acid sequences that are complementary to the analyte (eg, sequences that can hybridize to CRISPR RNA (crRNA), single-guide RNA (sgRNA), or linker sequences engineered into crRNA or sgRNA).
In certain embodiments, analytes can be extracted from living cells. Processing conditions can be adjusted to ensure that biological samples remain viable during analysis and that analytes are extracted (or released) from living sample cells. Analytes derived from living cells may be obtained only once from a sample or may be obtained at intervals from a sample that is still kept alive.
In general, the systems, devices, methods and compositions can be used to analyze any number of analytes. For example, the number of analytes analyzed may be at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 20, at least about 25, at least about 30, at least about 40, at least about 50, at least about 100, at least about 1,000, at least about 10,000, at least about 100,000 or multiple different analytes are present within a sample region or within a single substrate feature. Methods for performing multiplex assays for the analysis of two or more different analytes are discussed in the following sections of this disclosure.
(d) biological samples (i) Types of biological samples
A "biological sample" is obtained from a subject for analysis using any of a variety of techniques, including but not limited to biopsy, surgery, and laser microscopy (LCM), and generally includes the subject's cells and/or other biomaterial subjects. In addition to the subjects mentioned above, biological samples can also be obtained from non-mammalian organisms (e.g. plants, insects, spiders, nematodes (e.g.Caenorhabditis elegans), fungi, amphibians or fish (e.g. zebrafish)). Biological samples can be obtained from prokaryotes such as bacteria, e.g.Escherichia coli, Staphylococcus aureusMycoplasma pneumoniae; archaea; viruses such as hepatitis C virus or human immunodeficiency virus; or viroids. Biological samples can be obtained from eukaryotes, such as patient-derived organoids (PDO) or patient-derived xenografts (PDX). Biological samples can include organoids, miniaturized and simplified versions of organs that display realistic microanatomy in three dimensions in vitro. Organoids can be generated from one or more cells in a tissue, embryonic stem cells and/or induced pluripotent stem cells, which can self-organize in 3D culture due to their ability to self-renew and differentiate. In some embodiments, the organoid is a brain organoid, an intestinal organoid, a stomach organoid, a tongue organoid, a thyroid organoid, a thymus organoid, a testicular organoid, a liver organoid, a pancreatic organoid, an epithelial organoid, a lung organoid, a kidney organoid, a gastruloid, a cardiac organoid, or a retinal organoid. A subject from whom a biological sample may be obtained may be a healthy or asymptomatic individual, an individual suffering from or suspected of having a disease (eg, cancer) or susceptible to a disease, and/or an individual in need of treatment or treatment. suspected to require treatment.
A biological sample may be derived from a homogeneous culture or population of a subject or organism referred to herein, or alternatively from a collection of several different organisms, for example, in a colony or ecosystem.
A biological sample may include one or more diseased cells. Diseased cells may have altered metabolic properties, gene expression, protein expression and/or morphological characteristics. Examples of diseases include inflammatory disorders, metabolic disorders, neurological disorders, and cancer. Cancer cells can be derived from solid tumors, hematological malignancies, cell lines or obtained as circulating tumor cells.
Biological samples may also include fetal cells. For example, procedures such as amniocentesis may be performed to obtain a sample of fetal cells from the mother's circulation. Fetal cell sequencing can be used to identify any number of genetic disorders, including aneuploidies such as Down syndrome, Edwards syndrome and Patau syndrome. In addition, cell surface characteristics of fetal cells can be used to identify any number of disorders or diseases.
Biological samples may also include immune cells. Sequence analysis of the immune repertoire of such cells, including genomic, proteomic, and cell surface features, can provide a wealth of information for understanding the state and function of the immune system. For example, determination of minimal residual disease (MRD) status (e.g., negative or positive) after autologous stem cell transplantation in patients with multiple myeloma (MM) is considered a predictor of MRD in patients with MM (see, e.g., U.S. Patent Application Publication No. 2018 /0156784, which is hereby incorporated by reference in its entirety).
Examples of immune cells in a biological sample include, but are not limited to, B cells, T cells (eg, cytotoxic T cells, natural killer T cells, regulatory T cells, and T helper cells), natural killer cells, cytokine-induced killer cells (KIK) , bone marrow cells such as granulocytes (basophils, eosinophils, neutrophils/hypersegmented neutrophils), monocytes/macrophages, mast cells, platelets/megakaryocytes and dendritic cells.
A biological sample may include any number of macromolecules, eg, cellular macromolecules and organelles (eg, mitochondria and nuclei). A biological sample can be a nucleic acid sample and/or a protein sample. A biological sample can be a carbohydrate sample or a lipid sample. A biological sample may be obtained as a tissue sample, such as a tissue section, biopsy, core biopsy, needle aspiration, or fine needle aspiration. The sample may be a fluid sample, such as a blood sample, urine sample, or saliva sample. Samples may be skin samples, colon samples, cheek swabs, histology samples, histopathology samples, plasma or serum samples, tumor samples, living cells, cultured cells, clinical samples such as whole blood or blood products, blood cells or cultured tissue or cells, including cell suspensions.
A cell-free biological sample may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from body samples such as blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces and tears.
As noted above, a biological sample may include a single analyte of interest or more than one analyte of interest. Methods for performing multiplex assays for the analysis of two or more different analytes in a single biological sample are discussed in the following sections of this disclosure.
(ii) Preparation of biological samples
Various steps can be taken to prepare a biological sample for analysis. Unless otherwise noted, the preparation steps described below can generally be combined in any manner to conveniently prepare a particular sample for analysis.
Tissue sections
A biological sample may be collected from a subject (eg, by surgical biopsy, excised from a whole subject), cultured in vitro as a population of cells on a growth substrate or dish, or prepared as a section or section of tissue. Cultured samples can be thin enough to be analyzed without further processing steps. Alternatively, cultured and biopsy or sectioned specimens can be prepared into thin tissue sections using a mechanical cutting device such as a vibrating blade microtome. Alternatively, in some embodiments, thin sections of tissue can be prepared by applying a contact imprint of a biological sample to a suitable substrate material.
The thickness of the tissue section may be a fraction of the largest cross-sectional dimension of the cell (eg, less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0, 1). However, tissue sections whose thickness is greater than the largest cross-sectional cell size can also be used. For example, cryostat sections can be used, the thickness of which can be, for example, 10-20 μm.
More generally, the thickness of tissue sections generally depends on the method used to prepare the section and the physical properties of the tissue, so that sections of different thicknesses can be prepared and used. For example, the thickness of the tissue section may be at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1.0, 1.5, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 12, 13, 14, 15, 20, 30, 40 or 50 microns. Thicker sections such as at least 70, 80, 90 or 100 microns or larger may also be used if desired or convenient. Typically, tissue section thicknesses are 1-100 microns, 1-50 microns, 1-30 microns, 1-25 microns, 1-20 microns, 1-15 microns, 1-10 microns, 2-8 microns, 3 microns. -7 microns Microns or 4-6 microns, but as noted above, sections with thicknesses greater or less than these ranges can also be analyzed.
Multiple sections can also be obtained from a single biological sample. For example, multiple tissue sections can be obtained from a surgical biopsy specimen by serially sectioning the biopsy specimen with a cutting blade. In this way, spatial information can be preserved between successive slices, and slices can be analyzed sequentially to obtain three-dimensional information of biological samples.
freezing
In some embodiments, a biological sample (eg, a tissue section as described above) can be prepared by deep freezing at a temperature suitable to maintain or preserve the structural integrity (eg, physical properties) of the tissue. Such a temperature may be, for example, below -20°C, or below -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, 80°C, -90°C, -100°C, -110°C, -120°C, -130°C, -140°C, -150°C. , -160°C, -170°C, -180°C, -190°C or -200°C. Frozen tissue samples can be cut using any method, e.g. cut in the appropriate number of methods on the surface of the substrate. For example, a tissue sample can be prepared using a cryostat (eg, a cryostat) set at a temperature suitable to maintain the structural integrity of the tissue sample and the chemical nature of the nucleic acids in the sample. Such temperatures may be, for example, below -15°C, below -20°C or below -25°C. Samples can be flash frozen in isopentane and liquid nitrogen. Frozen samples can be stored in airtight containers before installation.
formalin fixed paraffin embedding
In some embodiments, biological samples can be prepared using formalin fixation and paraffin embedding (FFPE), which is an established method. In some embodiments, cell suspensions and other non-tissue samples can be prepared by formalin fixation and paraffin embedding. After the specimen is fixed and embedded in paraffin or a resin block, the specimen can be sectioned as described above. Tissue sections can be prepared by incubating the tissue sections in an appropriate solvent such as xylene, followed by washing (eg, 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, 70% ethanol for 2 minutes) prior to analysis.
fixed
As an alternative to the formalin fixation described above, biological samples can be fixed in any of a number of other fixatives to preserve the biological structure of the sample prior to analysis. For example, samples can be fixed by immersion in ethanol, methanol, acetone, formaldehyde (eg, 2% formaldehyde), paraformaldehyde-Triton, glutaraldehyde, or combinations thereof.
In some embodiments, acetone fixation is used with fresh frozen samples, which may include, but are not limited to, cortical tissue, mouse olfactory bulbs, human brain tumors, human postmortem brain, and breast cancer samples. In some embodiments, compatible fixation methods are selected and/or optimized based on the desired workflow. For example, formaldehyde fixation can be chosen to be compatible with protein visualization workflows using IHC/IF protocols. As another example, for workflows that emphasize the quality of RNA/DNA libraries, methanol fixation may be an option. Acetone fixation may be an option for tissue permeabilization in some applications. When performing acetone fixation, the pre-permeabilization step (described below) may not be performed. Alternatively, acetone fixation can be performed together with the permeabilization step.
to incorporate
As an alternative to the previously described paraffin embedding, biological samples can be embedded in any of a number of other embedding materials to provide a matrix for the sample prior to sectioning and other processing steps. Usually, the embedding material is removed before analyzing the tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (eg, methacrylic resins), epoxy resins, and agar.
dyeing
For easier visualization, biological samples can be stained with different colors and staining techniques. In some embodiments, samples can be stained with any number of biological stains including, but not limited to, Acridine Orange, Bismarck Brown, Carmine, Coomassie Blue, Cresyl Violet, DAPI, Eosin, Ethyl Bromide Tablet, Acid Fuchsin, Hematoxylin, Hoechst staining, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine or saffron.
Specimens can be stained using known staining techniques including Can-Grunwald, Giemsa, Hematoxylin and Eosin (H&E), Jenner's, Leishman, Masson's Trichrome, Papanicolaou, Romanowski, Silver, Sudan, Wright and/or Periodic Acid Schiff (PAS) staining techniques. PAS staining is usually performed after fixation with formalin or acetone.
In some embodiments, biological samples can be stained with detectable labels (eg, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes), as described elsewhere herein. In some embodiments, biological samples are stained using only one type of stain or technique. In some embodiments, staining includes biological staining techniques, such as H&E staining. In some embodiments, the staining includes the use of fluorescently conjugated antibodies to identify the analyte. In some embodiments, the biological sample is stained using two or more different types of dyes or two or more different staining techniques. For example, a biological sample can be prepared by staining and imaging with one technique (eg, H&E staining and bright field imaging), then stained and imaged with another technique (eg, IHC/IF staining and fluorescence microscopy) on the same biological sample.
In some embodiments, biological samples can be decolorized. Methods for decolorizing or decolorizing biological samples are known in the art and generally depend on the nature of the stain applied to the sample. For example, H&E staining can be depigmented by washing the sample in HCl or any other low pH acid (eg, selenic acid, sulfuric acid, hydroiodic acid, benzoic acid, carbonic acid, malic acid, phosphoric acid, oxalic acid, succinic acid) , salicylic acid, tartaric acid, sulfuric acid, trichloroacetic acid, hydrobromic acid, hydrochloric acid, nitric acid, orthophosphoric acid, arsenic acid, selenic acid, chromic acid, citric acid, hydrofluoric acid, nitric acid, isocyanic acid, formic acid, hydrogen selenide, molybdic acid, lactic acid, acetic acid, carbonic acid, hydrogen sulphide or their combinations). In some embodiments, decolorization may include 1, 2, 3, 4, 5 or more washes in a low pH acid (eg, HCl). In some embodiments, decolorization may involve adding HCl to a downstream solution (eg, a permeabilization solution). In some embodiments, decolorization may involve dissolving the enzyme (eg, pepsin) used in the described methods in an acidic solution with a low pH (eg, HCl). In some embodiments, after destaining the hematoxylin with a low pH acid, other reagents may be added to the destaining solution to increase the pH for other applications. For example, SDS can be added to a low pH acidic destaining solution to increase the pH compared to the low pH acidic destaining solution alone. As another example, in some embodiments, one or more immunofluorescent dyes are applied to the sample by antibody conjugation. Such stains can be removed using techniques such as cleavage of disulfide bonds by treatment with reducing agents and washing with detergents, treatment with chaotropic salts, treatment with antigen retrieval solutions, and treatment with acidic glycine buffer. Methods for multiple staining and destaining are described, for example, in Bolognesi et al.,J. Histochemistry. cytochemistry2017; 65(8): 431-444, Lin et al.,common land2015;6:8390, Pirici et al.,J. Histochemistry. cytochemistry2009;57:567-75, in Glass et al.,J. Histochemistry. cytochemistry2009;57:899-905, each of which is incorporated herein by reference in its entirety.
installation of hydrogel
In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (eg, a tissue section) is embedded in the hydrogel. In some embodiments, hydrogel subunits are injected into a biological sample, and polymerization of the hydrogel is triggered by an external or internal stimulus. A "hydrogel" as described herein may comprise a cross-linked 3D network of hydrophilic polymer chains. A "hydrogel subunit" can be a hydrophilic monomer, molecular precursor, or polymer that can polymerize (eg, cross-link) to form a three-dimensional (3D) hydrogel network.
Hydrogels can swell in the presence of water. In some embodiments, the hydrogel contains natural materials. In some embodiments, the hydrogel includes a synthetic material. In some embodiments, the hydrogel comprises a hybrid material, eg, the hydrogel material comprises components of synthetic and natural polymers. Any hydrogel material or hydrogel containing polypeptide-based material described herein can be used. Embedding the sample in this manner typically involves bringing the biological sample into contact with the hydrogel so that the biological sample is surrounded by the hydrogel. For example, the sample can be incorporated by contacting the sample with a suitable polymeric material and activating the polymeric material to form a hydrogel. In some embodiments, the hydrogel is formed by internalizing the hydrogel in a biological sample.
In some embodiments, the biological sample is immobilized in the hydrogel by cross-linking the polymeric material that forms the hydrogel. Cross-linking can be performed chemically and/or photochemically, or alternatively by any other method of hydrogel formation known in the art. For example, biological samples can be immobilized in hydrogels by polyacrylamide cross-linking. In addition, analytes of biological samples can be immobilized in hydrogels by cross-linking (eg, polyacrylamide cross-linking).
The composition and application of hydrogels to biological samples often depends on the nature and preparation of the biological sample (eg cut, uncut, fresh frozen tissue, type of fixation). The hydrogel can be any suitable hydrogel wherein, after the formation of the hydrogel on the biological sample, the biological sample becomes anchored or embedded in the hydrogel. Non-limiting examples of hydrogels are described herein or known in the art. As an example, where the biological sample is part of a tissue, the hydrogel may include a monomer solution and an ammonium persulfate (APS)/tetramethylethylenediamine (TEMED) accelerator solution. As another example, if the biological sample consists of cells (eg, cultured cells or cells isolated from a tissue sample), the cells can be incubated with the monomer solution and the APS/TEMED solution. For cells, hydrogels are formed in compartments, including but not limited to cell culture, maintenance, or transport devices. For example, hydrogels can be formed by adding monomer solutions plus APS/TEMED to compartments to depths ranging from about 0.1 µm to about 5 mm.
In some embodiments, the hydrogel includes linkers that allow the biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel contains a linker that allows anchoring of the biological analyte to the hydrogel. In this case, the linkers can be added to the hydrogel before, simultaneously with or after the formation of the hydrogel. Non-limiting examples of linkers that anchor nucleic acids to hydrogels may include 6-((acryloyl)amino)hexanoic acid (Acryloyl-X SE) (available from ThermoFisher, Waltham, MA), Label-IT amine (available from MirusBio, Madison, WI) and Label X (Chen et al., Nat. Methods 13:679-684, (2016)).
In some embodiments, functionalization chemistry may be used. In some embodiments, the functionalization chemistry includes hydrogel histochemistry (HTC). Any hydrogel tissue scaffold (eg, synthetic or natural) suitable for HTC can be used to anchor biomacromolecules and modulate functionalization. Non-limiting examples of methods using variants of the HTC backbone include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, the hydrogel formation within the biological sample is permanent. For example, biomacromolecules can be permanently attached to hydrogels, allowing multiple rounds of testing. In some embodiments, the formation of the hydrogel within the biological sample is reversible.
In some embodiments, additional reagents are added to the hydrogel subunits prior to, simultaneously with, and/or after polymerization. For example, additional reagents may include, but are not limited to, oligonucleotides (eg, capture probes), DNA fragmentation endonucleases, DNA fragmentation buffers, DNA polymerases, for amplifying nucleic acids, and attaching barcodes to amplicons. Addition of fragmented dNTPs. Other enzymes may be used including, but not limited to, RNA polymerase, transposase, ligase, proteinase K, and DNase. Additional reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers, and replacement oligonucleotides. In some embodiments, optical labels are added to hydrogel subunits before, during, and/or after polymerization.
In some embodiments, the HTC reagent is added to the hydrogel prior to, simultaneously with, and/or after polymerization. In some embodiments, the cell labeling agent is added to the hydrogel prior to, simultaneously with, and/or after polymerization. In some embodiments, cell penetrating agents are added to the hydrogel prior to, concurrently with, and/or after polymerization.
In some embodiments, the biological sample is embedded in the hydrogel to facilitate transfer of the sample to another location (eg, to an array). For example, archived biological samples such as FFPE tissue sections can be transferred from storage to spatial arrays to perform spatial analysis. In some embodiments, the biological sample on the substrate can be coated with any of the prepolymer solutions described herein. In some embodiments, the prepolymer solution can be polymerized to form a hydrogel on and/or around the biological sample. Hydrogel formation can occur in a manner sufficient to anchor (eg, embed) the biological sample into the hydrogel. After the formation of the hydrogel, the biological sample is anchored (eg embedded in) the hydrogel, whereby the separation of the hydrogel from the substrate (eg the glass slide) results in the separation of the biological sample from the hydrogel together with the substrate. The biological sample contained in the hydrogel can then be brought into contact with the spatial array, and the biological sample can be subjected to spatial analysis.
The properties of any species can determine the transfer conditions required for a particular biological sample. Non-limiting examples of characteristics that may affect transfer conditions include different sample conditions (eg, thickness, immobilization and cross-linking) and/or analytes of interest (preservation and/or transfer of various analytes (eg, DNA, RNA) and proteins)).
In some embodiments, the hydrogel is removed after the biological sample contacts the spatial array. For example, the methods described herein may include event-dependent (eg, photo or chemical) depolymerization of the hydrogel, wherein upon application of the event (eg, an external stimulus), the hydrogel depolymerizes. In one example, a biological sample can be anchored to a DTT-sensitive hydrogel, where the addition of DTT can cause the hydrogel to disaggregate and release the anchored biological sample.
Hydrogels embedded in biological samples can be removed by any suitable method. For example, electrophoretic tissue clearing methods can be used to remove biomacromolecules from hydrogel-embedded samples. In some embodiments, hydrogel-embedded samples are stored in a medium (eg, mounting medium, methylcellulose, or other semi-solid medium) before or after hydrogel clarification.
In some embodiments, the hydrogel chemistry can be tuned to specifically bind (eg, retain) a particular class of analyte (eg, RNA, DNA, protein, etc.). In some embodiments, the hydrogel includes linkers that allow the biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel contains a linker that enables anchoring of the biological analyte to the hydrogel. In this case, the linkers can be added to the hydrogel before, simultaneously with, or after the formation of the hydrogel. Non-limiting examples of linkers that anchor nucleic acids to hydrogels may include 6-((acryloyl)amino)hexanoic acid (acryloyl-X SE), Label-IT amine, and Label X (Chen et al., Nat. Methods 13: 679- 684, (2016)). Non-limiting examples of characteristics that may affect transfer conditions include different sample conditions (eg, thickness, immobilization and cross-linking) and/or analytes of interest (preservation and/or transfer of various analytes (eg, DNA, RNA) and proteins)).
Other methods and aspects of incorporating hydrogels into biological samples are described, for example, in Chen et al., Science 347(6221):543-548, 2015, the entire contents of which are incorporated herein by reference.
biological sample transfer
In some embodiments, the hydrogel is used to transfer a substrate-fixed biological sample (eg, a biological sample prepared using methanol fixation or formalin fixation and paraffin embedding (FFPE)) to a spatial array. In some embodiments, the hydrogel is formed over a biological sample on a substrate (eg, glass). For example, hydrogel formation can occur in a manner sufficient to anchor (eg, embed) the biological sample into the hydrogel. After the formation of the hydrogel, the biological sample is anchored (eg embedded in) the hydrogel, whereby the separation of the hydrogel from the substrate results in the separation of the biological sample from the substrate together with the hydrogel. The biological sample can then be brought into contact with the spatial array, enabling spatial analysis of the biological sample. In some embodiments, the hydrogel is removed after the biological sample contacts the spatial array. For example, the methods described herein may include event-dependent (eg, photo or chemical) depolymerization of the hydrogel, wherein upon application of the event (eg, an external stimulus), the hydrogel depolymerizes. In one example, a biological sample can be anchored to a DTT-sensitive hydrogel, where the addition of DTT can cause the hydrogel to disaggregate and release the anchored biological sample. The hydrogel can be any suitable hydrogel wherein, after the formation of the hydrogel on the biological sample, the biological sample becomes anchored or embedded in the hydrogel. Non-limiting examples of hydrogels are described herein or known in the art. In some embodiments, the hydrogel includes linkers that allow the biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel contains a linker that allows anchoring of the biological analyte to the hydrogel. In this case, the linkers can be added to the hydrogel before, simultaneously with or after the formation of the hydrogel. Non-limiting examples of linkers for anchoring nucleic acids to hydrogels may include 6-((acryloyl)amino)hexanoic acid (Acryloyl-X SE) (available from ThermoFisher, Waltham, MA), Label-IT amine (available from MirusBio, Madison, WI) and Label X (Chen et al., Nat. Methods 13:679-684, 2016). The properties of any species can determine the transfer conditions required for a particular biological sample. Non-limiting examples of characteristics that may affect transfer conditions include different conditions for samples (eg, thickness, immobilization and cross-linking) and/or analytes of interest (preservation and/or transfer of different analytes (eg, DNA, RNA) and proteins)). In some embodiments, hydrogel formation may occur in a manner sufficient to anchor (eg, embed) the analyte in the biological sample to the hydrogel. In some embodiments, the hydrogel can implode (eg, shrink) with anchored analytes present in the biological sample (eg, embedded in the hydrogel). In some embodiments, the hydrogel can swell (eg, expand isometrically) with anchor analytes present in the biological sample (eg, embedded in the hydrogel). In some embodiments, the hydrogel can implode (eg, shrink) and then expand with anchored analytes present in the biological sample (eg, embedded in the hydrogel).
Isometric expansion
In some embodiments, the biological sample embedded in the hydrogel can be spread isometrically. Isometric expansion methods that can be used include hydration, a preparatory step in expansion microscopy, as described in Chen et al., Science 347(6221):543-548, 2015; Asano et al.current agreement2018, 80:1, doi:10.1002/cpcb.56 i Gao et al.BMC Biology. 2017, 15:50, doi:10.1186/s12915-017-0393-3, Wassie, A.T., et al, Expansion microscopy: principles and applications in biological research,natural method, 16(1): 33-41 (2018), each of which is incorporated by reference in its entirety.
In general, the steps used to perform isometric expansion of biological samples may depend on the characteristics of the sample (e.g., thickness of tissue sections, fixation, cross-linking) and/or the analyte of interest (e.g., different conditions render RNA, DNA, and proteins anchored to the gel ).
Isometric swelling can be performed by anchoring one or more components of a biological sample to a gel, followed by gel formation, proteolysis, and swelling. Isometric spreading of the biological sample can occur before the biological sample is immobilized on the support or after the biological sample is immobilized on the support. In some embodiments, the isometrically expanded biological sample can be removed from the substrate prior to contacting the expanded biological sample with a spatially barcoded array (eg, spatially barcoded capture probes on the substrate).
In some embodiments, the proteins in the biological sample are anchored to a swelling gel, such as a polyelectrolyte gel. Antibodies can be directed against the protein before, after, or simultaneously with anchoring to the swelling gel. DNA and/or RNA from biological samples can also be immobilized on swellable gels via suitable linkers. Examples of such linkers include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (Acryloyl-X SE) (available from ThermoFisher, Waltham, MA), Label-IT Amine (available from MirusBio, Madison, WI), ) ) and X marks (e.g. in Chen et al.,Nat. method13:679-684, 2016, which is incorporated herein by reference in its entirety).
Isometric sample expansion can increase the spatial resolution of subsequent sample analysis. For example, isometric expansion of biological samples can improve the resolution of spatial analysis (eg, single cell analysis). Increased resolution in spatial analysis can be determined by comparing samples with equidistant spread and samples without equidistant spread.
Equidistant spreading provides three-dimensional spatial resolution for subsequent sample analysis. In some embodiments, isometric expansion of the biological sample can occur in the presence of a spatial analysis reagent (eg, an analyte capture agent or capture probe). For example, a swelling gel may include an analyte capture agent or a capture probe that is anchored to the swelling gel by a suitable linker. In some embodiments, spatial analysis reagents can be delivered to specific locations in an isometrically expanded biological sample.
In some embodiments, the biological sample is isometrically expanded to at least 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9×, 3×, 3.1× volume, 3, 2×, 3.3×, 3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4×, 4.1×, 4.2× , 4.3 ×, 4.4 ×, 4.5 ×, 4.6 ×, 4.7 ×, 4.8 × , or 4.9 times the unexpanded volume. In some embodiments, the sample is isometrically expanded to at least 2 times and less than 20 times its unexpanded volume.
In some embodiments, the biological sample embedded in the hydrogel is isometrically expanded to at least 2×, 2.1×, 2.2×, 2.3×, 2.4×, 2.5×, 2.6×, 2.7×, 2.8×, 2.9× ×, a volume of 3×, 3.1×, 3.2×, 3.3×, 3.4×, 3.5×, 3.6×, 3.7×, 3.8×, 3.9×, 4×, 4, 1 ×, 4.2 ×, 4.3 ×, 4.4 ×, 4.5 ×, 4.6 × , 4.7 ×, 4.8 × or 4.9 × its unexpanded volume. In some embodiments, the biological sample embedded in the hydrogel expands isometrically to at least 2 times and less than 20 times its unexpanded volume.
Substrate accessories
In some embodiments, the biological sample can be attached to a substrate. Examples of substrates suitable for this purpose are detailed below. Fixation of biological samples may be irreversible or irreversible, depending on the nature of the sample and the subsequent steps in the analytical method.
In certain embodiments, the pattern can be reversibly attached to the substrate by applying a suitable polymer coating to the substrate and bringing the pattern into contact with the polymer coating. The sample can then be separated from the substrate using an organic solvent that at least partially dissolves the polymer coating. Hydrogels are examples of polymers suitable for this purpose.
More generally, in some embodiments, the substrate may be coated or functionalized with one or more substances to facilitate attachment of the pattern to the substrate. Suitable substances that can be used to coat or functionalize the substrate include, but are not limited to, lectins, polylysine, antibodies, and polysaccharides.
unaggregated stations
In some embodiments, the biological sample corresponds to cells (eg, obtained from a cell culture or tissue sample). In a multicellular cell pattern, individual cells cannot naturally aggregate. For example, cells may be derived from cell suspensions and/or dissociated or disaggregated cells from tissues or tissue parts.
Alternatively, cells in a sample may aggregate and may be broken down into individual cells using, for example, enzymatic or mechanical techniques. Examples of enzymatic depolymerization enzymes include, but are not limited to, dispase, collagenase, trypsin, or combinations thereof. Mechanical disaggregation can be performed, for example, using a tissue homogenizer.
In some embodiments of unaggregated cells or disaggregated cells, the cells are arranged on the substrate such that at least one cell occupies a different spatial feature on the substrate. Cells can be immobilized on a matrix (eg to prevent lateral spread of cells). In some embodiments, cell immobilization agents can be used to immobilize unaggregated or disaggregated samples on spatially barcoded arrays prior to analyte capture. "Cell fixative" can refer to a matrix-bound antibody that binds cell surface markers. In some embodiments, the distribution of the plurality of cells on the substrate follows Poisson statistics.
In some embodiments, cells from a plurality of cells are immobilized on a substrate. In some embodiments, the cells are fixed to prevent lateral diffusion, eg, by adding a hydrogel and/or applying an electric field.
suspension and adherent cells
In some embodiments, the biological sample can be derived from a cell culture grown in vitro. A sample obtained from a cell culture may include one or more suspension cells, which are independent of anchorage in the cell culture. Examples of such cells include, but are not limited to, cell lines derived from hematopoietic cells and cell lines from the following: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI- H322M and MALME-3M.
A sample obtained from a cell culture may include one or more adherent cells growing on the surface of a container containing the medium. Non-limiting examples of adherent cells include DU145 (prostate carcinoma) cells, H295R (adrenocortical carcinoma) cells, HeLa (cervical carcinoma) cells, KBM-7 (chronic myelogenous leukemia) cells, LNCaP (prostate carcinoma) cells, MCF-7 (cancer cells breast, MDA-MB-468 cells (breast cancer), PC3 cells (prostate cancer), SaOS-2 cells (bone cancer), SH-SY5Y cells (neuroblastoma, cloned from myeloma), T-47D (breast) cancer cells, THP-1 cells (acute myeloid leukemia), U87 cells (glioblastoma), National Cancer Institute cell line panel of 60 (NCI60), vero cells (Chlorocebus African green monkey kidney epithelial cell line), MC3T3 (embryonic skull) cells, GH3 cells (pituitary tumor), PC12 cells (phaeochromocytoma), canine MDCK kidney epithelial cells, Xenopus laevis A6 kidney epithelial cells, zebrafish AB9 cells and insect Sf9 epithelial cells.
Additional examples of adherent cells are shown in Table 1 and cataloged, for example, in "Catalog of In Vitro Cell Lines, Transplantable Animal and Human Tumors, and Yeast", National Cancer Institute Division of Cancer Therapy and Diagnosis (DCTD) (2013), and Abaan et al., "The Exome of the NCI-60 Panel: A Genomic Resource for Cancer Biology and Systems Pharmacology,"cancer research73(14):4372-82, 2013, each of which is incorporated herein by reference in its entirety.
In some embodiments, the adherent cells are cells corresponding to one or more of the following cell lines: BT549, HS 578T, MCF7, MDA-MB-231, MDA-MB-468, T-47D, SF268, SF295, SF539, SNB-19, SNB-75, U251, Colo205, HCC 2998, HCT-116, HCT-15, HT29, KM12, SW620, 786-O, A498, ACHN, CAKI, RXF 393, SN12C, TK-10, UO-31 , A549, EKVX, HOP-62, HOP-92, NCI-H226, NCI-H23, NCI-H460, NCI-H522, LOX IMVI, M14, MALME-3M, MDA-MB-435, SK-, EL- 2 , SK- MEL-28, SK-MEL-5, UACC-257, UACC-62, IGROV1, OVCAR-3, OVCAR-4, OVCAR-5, OVCAR-8, SK-OV-3, NCI-ADR-RES , DU145, PC-3, DU145, H295R, HeLa, KBM-7, LNCaP, MCF-7, MDA-MB-468, PC3, SaOS-2, SH-SY5Y, T-47D, THP-1, U87, vero , MC3T3, GH3, PC12, MDCK dog kidney epithelial cells, Xenopus A6 kidney epithelial cells, zebrafish insect epithelial cell lines AB9 and Sf9.
tissue permeabilization
In some embodiments, the biological sample may be permeabilized to facilitate transfer of analytes from the sample and/or to facilitate transfer of substances (eg, capture probes) into the sample. If the sample is not sufficiently permeabilized, the amount of analyte taken from the sample may be too small for proper analysis. Conversely, if the tissue sample is too permeable, the relative spatial relationship of the analytes within the tissue sample may be lost. Therefore, a balance needs to be struck between sufficient penetration into the tissue sample to obtain good signal intensity while maintaining the spatial resolution of the analyte distribution in the sample.
Typically, biological samples can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable reagents for this purpose include, but are not limited to, organic solvents (such as acetone, ethanol, and methanol), cross-linking agents (such as paraformaldehyde), detergents (such as saponins, Triton X-100™, Tween- 20™, or ten Sodium dialkyl sulfate (SDS)) and enzymes such as trypsin, proteases such as proteinase K. In some embodiments, the detergent is an anionic detergent such as SDS or sodium salt solution N-lauroyl sarcosine). , the biological sample can be treated enzymatically (eg, with any enzyme described herein, eg, trypsin, protease (eg, pepsin and/or proteinase K)).
In some embodiments, the sample can be exposed to concentrations greater than about 1.0 w/v% (e.g., greater than about 2.0 w/v%, greater than about 3.0 w/v%, greater than about 4, 0 w/v%, greater than about 5.0 w/v%, greater than about 6.0 w/v%, greater than about 7.0 w/v%, greater than about 8.0 w/v%, greater than about 9.0 w/v%, greater than about 10.0 w/v %, greater than about 11.0 w/v %, greater than about 12.0 w/v %, or greater than about 13.0 w/v % sodium dodecyl sulfate (SDS) and/or N-lauroyl sarcosine or N-lauroyl sarcosine sodium salt In some embodiments, a biological sample can be obtained by exposing the sample (eg, about 5 minutes to about 1 hour, about 5 minutes to about 40 minutes, about 5 minutes to about 30 minutes, about 5 minutes to about 20 minutes, about 5 minutes to about 20 minutes, or about 5 minutes to about 10 minutes) to about 1.0 w/v% to about 14 .0 w/v% (e.g., about 2.0 w/v% to about 14.0 w/v%, about 2.0 w/v% to about 12.0w/v%, about 2.0w/v % to about 10.0w/v%, about 4.0w/v% to about 14.0w/v%, about 4.0w/v% to about 12.0w/v% , about 4.0 w/v% to about 10.0 w/v%, about 6.0 w/v% to about 14.0 w/v%, about 6.0 w/v% to about 12.0 w/v%, about 6.0 w/v % to about 10.0w/v%, about 8.0w/v% to about 14.0w/v%, about 8.0w/v% to about 12.0w/v%, about 8.0w/ v% to about 10.0w/v% , about 10.0% w/v% to about 14.0 w/v%, about 10.0 w/v% to about 12.0 w/v%, or about 12.0 w/v% to about 14.0 w/v%) SDS and/or N -lauryl acyl sarcosinate and/or proteinase K solution (for example, at about 4% to about 35°C, about 4°C up to about 25°C, about 4°C to about 20°C, about 4°C to about 10°C, about 10°C to about 25°C, about 10°C to about 20°C, about 10°C to about 15°C, about 35°C to about 50°C C., about 35°C to about 45°C, about 35°C to about 40°C, about 40°C to about 50°C, about 40 °C to about 45°C, or about 45°C to about 50°C).
In some embodiments, the biological sample may be incubated with a permeabilizing agent to facilitate permeabilization of the sample. For example, Jamur et al. describe other methods for sample permeabilization,Methods Molecular biology588:63-66, 2010, which is incorporated herein by reference in its entirety.
Reagents for lizu
In some embodiments, the biological sample can be permeabilized by adding one or more lysis reagents to the sample. Examples of suitable lysing agents include, but are not limited to, biologically active agents, such as lytic enzymes for lysing various cell types, such as Gram-positive or negative bacteria, plants, yeasts, mammals, such as lysozyme, leukopeptidase, lysostaphin, labiasis, kitalase, lyticase and various other commercially available lytic enzymes.
Other lysis agents may additionally or alternatively be added to biological samples to facilitate permeabilization. For example, surfactant-based lysis solutions can be used to lyse sample cells. The lysis solution may include ionic surfactants such as sarkosyl and sodium dodecyl sulfate (SDS). More generally, chemical lysis agents may include, but are not limited to, organic solvents, chelating agents, detergents, surfactants, and chaotropic agents.
In some embodiments, biological samples can be permeabilized by non-chemical permeabilization methods. Non-chemical permeabilization methods are known in the art. For example, non-chemical permeabilization methods that may be used include, but are not limited to, physical lysis techniques such as electroporation, mechanical permeabilization methods (eg, beating beads using homogenizers and grinding balls to mechanically disrupt the structure of the tissue sample), acoustic permeabilization (eg, sonication) and pyrolysis techniques, such as heating to induce thermal permeabilization of the sample.
protein
In some embodiments, the culture medium, solution, or permeabilization solution may contain one or more proteases. In some embodiments, treatment of a biological sample with a protease capable of degrading histones results in fragmented genomic DNA. Fragmented genomic DNA can be captured using the same capture domains used to capture mRNA (eg, capture domains with poly(T) sequences). In some embodiments, the biological sample is treated with a protease capable of degrading histones and an RNA protectant prior to spatial analysis to facilitate capture of genomic DNA and mRNA.
In some embodiments, the biological sample is permeabilized by exposing the sample to a protease capable of degrading histones. As used herein, the term "histones" generally refers to linker histones (eg, H1) and/or core histones (eg, H2A, H2B, H3, and H4). In some embodiments, the protease degrades the linker histone, the core histone, or both the linker histone and the core histone. Any suitable protease capable of degrading histones in a biological sample can be used. Non-limiting examples of proteases capable of degrading histones include leupeptin-inhibited proteases and TLCK (tosyl-L-lysyl-chloromethane hydrochloride), a protease encoded by the EUO gene ofChlamydia trachomatis serotype A,Granzim A, serine protease (e.g. trypsin or trypsin-like protease, neutral serine protease, elastase, kathepsin G), aspartyl protease (e.g. kathepsin D), enzyme C1 family peptidaza (e.g. kathepsin L), pepsin, proteinase K, protease inhibited by diazomethane inhibitor Z-Phe-Phe-CHN(2) or epoxy inhibitor E-64, lysosomal protease or azurinaza (e.g. kathepsin G, elastase, proteinase 3, neutral serine protease). In some embodiments, serine protease is trypsin, an enzyme similar to trypsin, or its functional variant or derivative (e.g. P00761; COHK48; Q8IYP2; Q8BW11; Q6IE06; P35035; P00760; P06871; Q90627; P16049; P07477; P007 62 P35031; P19799 ; P35036; Q29463; P06872; Q90628; P07478; P07146; P00763; P35032; 35 038; P12788; P29787; P35039; P35040; Q8NHM4; P35041; P35043; P 35044; P5 4624; P04814; P35045; P32821; P54625; P35004; P35046; P32822; 905; P83348; P00765; P35042; P81071; P35049; P51588; P35050; P35034; P35051; P24664; P35048; P00764; P00775; P54628; P4227 8; P546 29 P42279 Q91041 P54630 , P00760, Q29463 or a combination thereof. In some embodiments, the protease capable of degrading one or more histones contains an amino acid sequence that has at least 80% sequence identity to P00761, P00760, or Q29463. In some embodiments, the protease capable of degrading one or more histones contains at least 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% agree with P00761, P00760 or Q29463. If the protease has at least 50%, kao što je at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, u dsvu na activitie protease pod optimalnim za enzyme activity. In some cases, enzyme treatment with pepsin or enzymes similar to pepsin can include: P03954/PEPA1_MACFU; P28712/PEPA1_RABIT; P27677/PEPA2MACFU; P27821/PEPA2RABIT; UMAN P27678/PEPA4 MACFU; P28713/PEPA4 Zec; P0DJD9/PEPA 5 Man; Q9D106 /PEPA5_Miš; P27823/PEPAFRABIT; ; P11489/PEPA MACMU; P00791/PEPA pig; Q9GMY7/PEPA RHIFE; Q9GMY8/PEPA Solon; P81497/PEPA Sunwood; P13636/PEPA URSTH and their functional variants and derivatives or their combinations. U nekim izlizijama, pepsin može contre: P00791/PEPA_PIG; P00792/PEPA_BOVIN, functional variants, derivatives or their combinations.
In addition, proteases can be included in reaction mixtures (solutions) that also include other components (e.g. buffers, salts, chelating agents (e.g. EDTA) and/or detergents (e.g. SDS, N-lauroyl sarcosinic acid sodium salt solution ) The reaction mixture can be buffered, with a pH of about 6.5-8.5, such as about 7.0-8.0.Furthermore, the reaction mixture can be used at any suitable temperature, for example about 10-50 °C, for example around 10-44 °C, 11-43 °C, 12-42 °C, 13-41 °C, 14 °C -40 °C, 15-39 °C, 16-38 °C, 17-37 °C, eg around 10 °C, 12 °C, 15 °C, 18 °C, 20 °C, 22 °C 25 °C °C, 28 °C, 30 °C, 33 °C, 35°C or 37°C, preferably around 35-45°C, for example around 37°C.
other reagents
In some embodiments, the permeabilization solution may contain additional reagents or the biological sample may be treated with additional reagents to optimize permeabilization of the biological sample. In some embodiments, the additional agent is an RNA protecting agent. As used herein, the term "RNA protective agent" generally refers to an agent that protects RNA from RNA nucleases (eg, RNase). Any suitable RNA protectant that protects the RNA from degradation can be used. Non-limiting examples of RNA protecting agents include organic solvents (eg, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% v/v organic solvents), including but not limited to ethanol, methanol, 2-propanol, acetone, trichloroacetic acid, propanol, polyethylene glycol, acetic acid or combinations thereof. In some embodiments, the RNA protecting agent comprises ethanol, methanol and/or propan-2-ol or a combination thereof. In some embodiments, the RNA protection agent comprises RNAlater ICE (ThermoFisher Scientific). In some embodiments, the RNA protecting agent comprises at least about 60% ethanol. In some embodiments, the RNA protecting agent comprises about 60-95% ethanol, about 0-35% methanol, and about 0-35% propan-2-ol, wherein the total amount of organic solvent in the culture medium is no greater than about 95%. In some embodiments, the RNA protecting agent comprises about 60-95% ethanol, about 5-20% methanol, and about 5-20% propan-2-ol, wherein the total amount of organic solvent in the culture medium does not exceed about 95%.
In some embodiments, the RNA protecting agent comprises a salt. Such salts may include ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, iron cesium (II) sulfate, chromium (III) sulfate, cobalt (II) sulfate, copper (II) sulfate, lithium chloride, lithium acetate, lithium sulfate, magnesium sulfate, magnesium chloride, manganese sulfate, manganese chloride, potassium chloride, potassium sulfate, sodium chloride, sodium acetate, sodium sulfate, zinc chloride, zinc acetate, zinc sulfate. In some embodiments, the salt is a sulfate, for example, ammonium sulfate, ammonium bisulfate, cesium sulfate, cadmium sulfate, iron cesium(II) sulfate, chromium(III) sulfate, cobalt(II) sulfate, copper(II) sulfate, lithium sulfate , magnesium sulfate, manganese sulfate, potassium sulfate, sodium sulfate or zinc sulfate. In some embodiments, the salt is ammonium sulfate. The salt may be present at a concentration of about 20 g/100 ml of medium or less, such as about 15 g/100 ml, 10 g/100 ml, 9 g/100 ml, 8 g/100 ml, 7 g/100 ml , 6 g/100 ml, 5 g/100 ml or less, for example about 4 g, 3 g, 2 g or 1 g/100 ml.
Additionally, the RNA protecting agent may be contained in a chelating agent (eg, EDTA), a buffer (eg, sodium citrate, sodium acetate, potassium citrate or potassium acetate, preferably sodium acetate) and/or buffered to a pH between about Between 4-8 ( for example, about 5).
In some embodiments, the biological sample is treated with one or more RNA protective agents prior to, concurrently with, or after permeabilization. For example, the biological sample is treated with one or more RNA protectants prior to treatment with one or more permeabilizing agents (eg, one or more proteases). In another example, the biological sample is treated with a solution containing one or more RNA protecting agents and one or more permeabilizing agents (eg, one or more proteases). In yet another example, the biological sample is treated with one or more RNA protecting agents after the biological sample has been treated with one or more permeabilizing agents (eg, one or more proteases). In some embodiments, the biological sample is treated with one or more RNA protectants prior to fixation.
In some embodiments, identifying the location of the captured analyte in the biological sample involves a nucleic acid extension reaction. In some embodiments where capture probes capture fragmented genomic DNA molecules, the nucleic acid extension reaction involves a DNA polymerase. For example, a nucleic acid extension reaction involves using a DNA polymerase to extend a capture probe that hybridizes to a captured analyte (eg, fragmented genomic DNA) using the captured analyte (eg, fragmented genomic DNA) as a template. Extension reaction products include spatially barcoded analytes (eg, spatially barcoded fragmented genomic DNA). Spatially barcoded analytes (eg, spatially barcoded fragmented genomic DNA) can be used to identify the spatial location of an analyte in a biological sample. Any DNA polymerase capable of extending a capture probe using the captured analyte as a template can be used in the methods described herein. Non-limiting examples of DNA polymerases include T7 DNA polymerase; Bsu DNA polymerase; and E. coli DNA polymerase pol I.
antidiffusion medium
In some embodiments, the diffusion-resistant medium typically used to limit analyte diffusion may include at least one permeability-enhancing agent. For example, a diffusion-resistant medium (eg, a hydrogel) may include pores (eg, micropores, nanopores, or picopores or pores) that contain buffers or permeabilization reagents. In some embodiments, the anti-diffusion medium (eg, hydrogel) is soaked with a permeabilization buffer prior to contacting the hydrogel with the sample. In some embodiments, the hydrogel or other diffusion-resistant medium may contain dried reagents or monomers to deliver the permeabilization reagents after application of the diffusion-resistant medium to the biological sample. In some embodiments, the diffusion-resistant medium (eg, hydrogel) is covalently bound to a rigid support (eg, acrylic glass slides).
In some embodiments, the hydrogel can be modified to deliver both permeabilization reagents and capture probes. For example, hydrogel membranes can be modified to include spatially barcoded capture probes. The spatially barcoded hydrogel membrane is then soaked in permeabilization buffer, and then the spatially barcoded hydrogel membrane is contacted with the sample. In another example, hydrogels can be modified to include spatially barcoded capture probes and designed to function as porous membranes (eg, permeable hydrogels) when exposed to a permeabilization buffer or any other biological sample preparation reagent. glue). The permeation reagent diffuses through the spatially barcoded permeable hydrogel and penetrates the biological sample on the other side of the hydrogel. Analytes then diffuse into the spatially barcoded hydrogel upon exposure to a permeabilizing reagent. In this case, spatially barcoded hydrogels (eg, porous membranes) facilitate the diffusion of biological analytes from biological samples into the hydrogel. In some embodiments, the biological analyte diffuses into the hydrogel prior to exposure to the permeabilizing reagent (eg, when secreted analytes are present outside the biological sample or where the biological sample is lysed by other means prior to the addition of the permeabilizing reagent). or in case of permeabilization). In some embodiments, the permeabilizing reagent flows through the hydrogel at a variable flow rate (eg, any flow rate that facilitates diffusion of the permeabilizing reagent through the spatially barcoded hydrogel). In some embodiments, the permeabilization reagent flows through a microfluidic chamber or channel above the spatially barcoded hydrogel. In some embodiments, after flow is used to introduce permeabilization reagents into the biological sample, reagents for preparing the biological sample can flow through the hydrogel to further facilitate the diffusion of biological analytes into the spatially barcoded hydrogel. The spatially barcoded hydrogel membrane thus delivers permeabilization reagents to the sample surface in contact with the spatially barcoded hydrogel, enhancing analyte migration and capture. In some embodiments, the spatially barcoded hydrogel is applied to the sample and placed in a permeabilized solution. In some embodiments, a hydrogel membrane impregnated with a permeabilization reagent is located between the sample and the spatial barcode array. In some embodiments, target analytes can diffuse through the permeabilization reagent-soaked hydrogel and hybridize or bind to capture probes on the other side of the hydrogel. In some embodiments, the thickness of the hydrogel is proportional to the loss of resolution. In some embodiments, a well (eg, microwell, nanowell, or picopore) may contain a spatially barcoded capture probe and a permeabilization reagent and/or buffer. In some embodiments, spatially barcoded capture probes and permeabilization reagents are held between spacers. In some embodiments, the sample is punched, cut, or transferred into wells where the target analyte diffuses through the permeabilization reagent/buffer and reaches the spatially barcoded capture probe. In some embodiments, the loss of resolution may be proportional to the thickness of the gap (eg, the amount of permeabilization buffer between the sample and the capture probe). In some embodiments, the anti-diffusion medium (eg, hydrogel) is between about 50-500 microns thick, including 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 microns thick, or any thickness between 50 and 500 microns.
In some embodiments, the biological sample is exposed to a porous membrane (eg, a permeable hydrogel) to aid in permeabilization and limit diffusive loss of analyte while allowing permeabilization reagents to reach the sample. Membrane chemistry and pore volume can be controlled to minimize analyte loss. In some embodiments, porous membranes can be made of glass, silicon, paper, hydrogels, polymer monoliths, or other materials. In some embodiments, the material may be naturally porous. In some embodiments, the material may have pores or pores etched into the solid material. In some embodiments, the permeabilization reagent flows through the microfluidic chamber or channel above the porous membrane. In some embodiments, flow controls sample access to permeabilization reagents. In some embodiments, the porous membrane is a permeable hydrogel. For example, a hydrogel is permeable when permeabilization reagents and/or biological sample preparation reagents can pass through the hydrogel by diffusion. Any suitable permeabilization reagent and/or biological sample preparation reagent described herein can be used under conditions sufficient to release the analyte (eg, nucleic acid, protein, metabolite, lipid, etc.) from the biological sample. In some embodiments, the hydrogel is exposed to a biological sample on one side and a permeabilizing reagent on the other side. The permeation reagent diffuses through the permeable hydrogel and penetrates the biological sample on the other side of the hydrogel. In some embodiments, the permeabilizing reagent flows through the hydrogel at a variable flow rate (eg, any flow rate that facilitates diffusion of the permeabilizing reagent through the hydrogel). In some embodiments, the permeabilization reagent flows through the microfluidic chamber or channel above the hydrogel. Permeabilization reagents passing through the hydrogel can control reagent concentration. In some embodiments, hydrogel chemistry and pore volume can be adjusted to improve permeabilization and limit diffusional loss of analyte.
In some embodiments, a porous membrane is sandwiched between the barcode spatial array and the sample, with the permeabilization solution applied to the porous membrane. Permeabilization reagents diffuse through the membrane pores and enter the biological sample. In some embodiments, the biological sample can be placed on a substrate (eg, a glass slide). The biological analyte then diffuses through the porous membrane and into the space containing the capture probes. In some embodiments, the porous membrane is modified to include capture probes. For example, capture probes can be attached to the surface of a porous membrane by any of the methods described herein. In another example, the capture probes can be embedded in the porous membrane at any depth that allows interaction with the biological analyte. In some embodiments, the porous membrane is placed over the biological sample in a configuration that allows for interaction between capture probes on the porous membrane and a biological analyte from the biological sample. For example, capture probes are located on the side of the porous membrane that is close to the biological sample. In this case, the permeabilization reagent on the other side of the porous membrane diffuses through the porous membrane to the site containing the biological sample and capture probes to facilitate permeabilization of the biological sample (eg, it also facilitates capture of the biological analyte by the capture probe). In some embodiments, a porous membrane is located between the sample and the capture probes. In some embodiments, the permeabilization reagent flows through the microfluidic chamber or channel above the porous membrane.
Selective permeabilization/selective lysis
In some embodiments, biological samples can be treated according to established methods to selectively release analytes from subcellular regions of cells. In some embodiments, the methods provided herein may include detecting at least one biological analyte present in a subcellular region of cells in a biological sample. As used herein, "subcellular region" may refer to any subcellular region. For example, a subcellular region may refer to the cytosol, mitochondria, nucleus, nucleolus, endoplasmic reticulum, lysosomes, vesicles, Golgi apparatus, plastids, vacuoles, ribosomes, cytoskeleton, or combinations thereof. In some embodiments, the subcellular region includes at least one of cytosol, nucleus, mitochondria, and microsomes. In some embodiments, the subcellular region is the cytosol. In some embodiments, the subcellular region is the nucleus. In some embodiments, the subcellular region is a mitochondrion. In some embodiments, the subcellular region is a microsome.
For example, biological analytes can be selectively released from subcellular regions of cells by selective permeability or selective lysis. In some embodiments, "selective permeabilization" may refer to a permeabilization method that permeabilizes the membrane of a subcellular region while leaving another subcellular region essentially intact (eg, because of the permeabilization method employed, biological test substances are not released from the subcellular regions). Non-limiting examples of selective permeabilization methods include the use of electrophoresis and/or the use of permeabilization reagents. In some embodiments, "selective lysis" may refer to a lysis method that cleaves the membrane of a subcellular region while leaving another subcellular region essentially intact (eg, a biological analyte is not detected due to the lysis method employed). regions). Several methods for selective permeabilization or lysis are known to those skilled in the art, including those described in Lu et al.lab chip2005, January;5(1):23-9; Niklas et al.anal biochemistry2011 September 15; 416 (2): 218-27; Cox and Emily.natural agreement2006;1(4):1872-8;Chaing et al.J Biochem. biophysics. method2000. studenoga;46(1-2):53-68;Yamauchi i Herr et al.microsystem. nanoengineering2017; 3. pii: 16079; each of which is incorporated herein by reference in its entirety.
In some embodiments, "selective permeabilization" or "selective lysis" refers to selective permeabilization or selective lysis of a particular cell type. For example, "selective permeabilization" or "selective lysis" may refer to the lysis of one type of cell while leaving another type of cell largely intact (eg, biological analytes are not removed from the cells due to the permeabilization or lysis method used). A cell that is a "different cell type" from another cell may refer to a cell from a different taxonomic kingdom, a prokaryotic cell versus a eukaryotic cell, a cell from a different tissue type, and the like. Many procedures are known to those skilled in the art of selectively permeabilizing or lysing various cell types. Non-limiting examples include the use of permeabilization reagents, electroporation and/or sonication. See, e.g. International application No.WO 2012/168003; Han et al.Nanoengineering microsystems2019. 17 June; 5:30; Gould et al.tumor target2018 Mar 20;9(21):15606-15615; Oren and Shay.biochemistry1997 Feb 18;36(7):1826-35; Algueyer et al.molecular... 2019 May 31;24(11). Many: E2079; Hipp et al.leukemia2017 October; 31(10):2278; International application no. WO 2012/168003; and U.S. Pat. 7,785,869, all of which are incorporated herein by reference in their entirety. In some embodiments, administering the selective permeabilization or lysis reagent comprises contacting the biological sample with a hydrogel containing the permeabilization or lysis reagent.
In some embodiments, the biological sample is contacted with two or more arrays (eg, a flexible array as described herein). For example, after a subcellular region is permeabilized and biological analytes from the subcellular region are captured on a first array, the first array can be removed and biological analytes from a second subcellular region can be captured on a second array on the array.
Selective enrichment of RNA species
In some embodiments, one or more RNA analytes of interest can be selectively enriched when the RNA analyte (eg, Adiconis et al., Comparative analysis of RNA sequencing methods for degraded and low-input samples,nature, scroll. 10, July 2013, 623-632, which is hereby incorporated by reference in its entirety). For example, one or more RNAs can be selected by adding one or more oligonucleotides to the sample. In some embodiments, the additional oligonucleotide is a polymerase priming sequence. For example, one or more primer sequences having sequence complementarity to one or more RNAs of interest can be used to amplify one or more RNAs of interest, thereby selectively enriching for those RNAs. In some embodiments, oligonucleotides having a sequence complementary to the complementary strand of the captured RNA (eg, cDNA) can bind to the cDNA. For example, biotinylated oligonucleotides having a sequence complementary to one or more cDNAs of interest are bound to the cDNA and can be bound to the cDNA using any of a number of methods known in the art (eg, streptavidin beads) selected using biotinylation-affinity to streptavidin.
Alternatively, any of a number of methods can be used to select (eg, remove, deplete) one or more RNAs (eg, ribosomal and/or mitochondrial RNAs). Non-limiting examples of hybridization and capture methods to remove ribosomal RNA include RiboMinus™, RiboCop™, and Ribo-Zero™. Another non-limiting method of RNA removal involves hybridization of complementary DNA oligonucleotides to the unwanted RNA, followed by the use of RNase H to degrade the RNA/DNA hybrid. Non-limiting examples of hybridization and degradation methods include NEBNext® rRNA Depletion, NuGEN AnyDeplete, or RiboZero Plus. Another non-restrictive method of removing ribosomal RNA involves ZapR™ digestion, such as SMARTer. In the SMARTer method, random nucleic acid adapters are hybridized to RNA, first strand and tail synthesis by reverse transcriptase, followed by template switching and extension by reverse transcriptase. Additionally, the first round of PCR amplification adds Illumina adapters for full-length sequencing (eg Illumina Index). Ribosomal RNA is cleaved by ZapR v2 and R probe v2. A second round of PCR is performed to amplify non-rRNA molecules (such as cDNA). Portions or steps of these ribosome removal protocols/kits can be further combined with the methods described herein to optimize the protocol for a particular biological sample.
In depletion protocols, probes can be applied to samples that selectively hybridize to ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. Probes can be applied to biological samples that selectively hybridize to mitochondrial RNA (mtRNA), thus reducing the amount and concentration of mtRNA in the sample. In some embodiments, probes complementary to mitochondrial RNA can be added during cDNA synthesis, or probes complementary to ribosomal and mitochondrial RNA can be added during cDNA synthesis. Subsequent application of capture probes to the sample may improve the capture of other types of RNA due to the reduction of non-specific RNA (eg, reduced RNA) present in the sample. Additionally and alternatively, duplex-specific nuclease (DSN) treatment can deplete rRNA (see, e.g., Archer et al., Selective and Flexible Depletion of Problematic Sequences from an RNA-seq Library at the cDNA Stage,BMC Genomics, 15 401, (2014), which is hereby incorporated by reference in its entirety). In addition, hydroxyapatite chromatography can remove abundant species (e.g., rRNA) (see, e.g., Vandernoot, V.A., Normalization of cDNA by hydroxyapatite chromatography for transcriptome enrichment for a diversity of RNA-seq applications,Biotechnology, 53(6) 373-80, (2012), the entire contents of which are incorporated herein by reference).
other reagents
Additional reagents may be added to a biological sample to perform various functions prior to analysis of the biological sample. In some embodiments, nuclease inhibitors, such as DNase and RNase inactivators or protease inhibitors, and/or chelating agents, such as EDTA, can be added to the biological sample. In other embodiments, nucleases (such as DNase or RNAse) or proteases (such as pepsin or proteinase K) are added to the sample. In some embodiments, additional reagents may be dissolved in the solution or applied to the sample as a medium. In some embodiments, additional reagents (eg, pepsin) may be dissolved in HCl prior to application to the sample. For example, hematoxylin from H&E stains can be optionally removed from biological specimens by washing in dilute HCl (0.001 M to 0.1 M) before further processing. In some embodiments, the pepsin may be dissolved in dilute HCl (0.001 M to 0.1 M) prior to further processing. In some embodiments, the biological sample may be washed an additional number of times (e.g., 2, 3, 4, 5 or more) in dilute HCl prior to incubation with a protease (e.g., pepsin) but after proteinase K treatment.
In some embodiments, the biological sample may be treated with one or more enzymes. For example, one or more endonucleases to fragment DNA, DNA polymerases and dNTPs to amplify nucleic acids may be added. Other enzymes that may also be added to the biological sample include, but are not limited to, polymerases, transposases, ligases, and deoxyribonucleases and ribonucleases.
In some embodiments, reverse transcriptase enzymes, including enzymes with terminal transferase activity, primers, and template-switching oligonucleotides (TSOs), can be added to the sample. Template switching can be used to increase the length of cDNA, for example, by adding a predefined nucleic acid sequence to the cDNA. In some embodiments, the additional nucleic acid sequence comprises one or more ribonucleotides.
In some embodiments, additional reagents can be added to increase the recovery of one or more target molecules (eg, cDNA molecules, mRNA transcripts). For example, adding carrier RNA to the RNA sample workflow can increase the yield of RNA/DNA hybrids extracted from biological samples. In some embodiments, carrier molecules are useful when the concentration of input molecules or target molecules is low compared to the remaining molecules. Usually, a single target molecule cannot form a precipitate, and the addition of a carrier molecule can help to form a precipitate. Some target recovery protocols use a carrier RNA to prevent irreversible binding of the small amounts of target nucleic acid present in the sample. In some embodiments, the carrier RNA can be added just prior to the second strand synthesis step. In some embodiments, the carrier RNA can be added just prior to second strand cDNA synthesis on oligonucleotides released from the array. In some embodiments, the carrier RNA can be added immediately prior to the in vitro post-transcriptional cleanup step. In some embodiments, carrier RNA can be added prior to purification and quantification of the amplified RNA. In some embodiments, carrier RNA can be added prior to RNA quantification. In some embodiments, carrier RNA can be added immediately prior to second strand cDNA synthesis and post-transcriptional cleanup steps in vitro.
Preprocessing to capture probe interactions
In some embodiments, analytes in a biological sample may be pretreated prior to interaction with capture probes. For example, polymerase-catalyzed polymerization (eg, DNA polymerase or reverse transcriptase) occurs in the biological sample prior to interaction with the capture probe. In some embodiments, primers for polymerization reactions include functional groups that enhance hybridization to capture probes. Capture probes can include suitable capture domains for capturing biological analytes of interest (eg, poly(dT) sequences for capturing poly(A) mRNA).
In some embodiments, biological analytes are preprocessed to generate a library by next-generation sequencing. For example, analytes can be preprocessed by adding modifications (eg, linking sequences that allow interaction with capture probes). In some embodiments, the analyte (eg, DNA or RNA) is fragmented using a fragmentation technique (eg, using a transposase and/or a fragmentation buffer).
After fragmentation, the analyte can be modified. For example, modifications can be added to allow binding of adapter sequences that hybridize to capture probes. In some embodiments, the polyA tail is performed when the analyte of interest is RNA. Addition of a poly(A) tail to an RNA without a poly(A) tail can facilitate hybridization to a capture probe that includes a capture domain with a functional poly(dT) sequence.
In some embodiments, a ligase-catalyzed ligation reaction is performed in the biological sample prior to interaction with the capture probe. In some embodiments, the bonding may be by chemical bonding. In some embodiments, ligation can be performed using click chemistry as further described below. In some embodiments, the capture domain comprises a DNA sequence that is complementary to an RNA molecule, wherein the RNA molecule is complementary to another DNA sequence, and the RNA-DNA sequence complementarity is used to link the other DNA sequence to a sequence in the DNA capture domain. In these embodiments, direct detection of RNA molecules is possible.
In some embodiments, target-specific reactions are performed in a biological sample prior to interaction with capture probes. Examples of target-specific reactions include, but are not limited to, ligation of target-specific adapters, probes and/or other oligonucleotides, target-specific amplification using primers specific for one or more analytes, and the use of in situ hybridization, DNA microscopy for target-specific detection and/or antibody detection. In some embodiments, the capture probe includes a capture domain that targets a target-specific product (eg, amplification or ligation).
two. Generic analysis method based on spatial array
Methods, devices, systems and compositions for spatial array-based analysis of biological samples are provided herein.
(a) Spatial analysis methods
Array-based spatial analysis methods involve the transfer of one or more analytes from a biological sample to a series of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of transferred analytes involves determining the identity of the analyte and the spatial location of each analyte in the biological sample. The spatial position of each analyte in a biological sample is determined based on the features on the array to which each analyte binds and the relative spatial position of the features on the array.
There are at least two general approaches to associating a spatial barcode with one or more adjacent cells such that the spatial barcode identifies one or more cells and/or the contents of one or more cells, such as associating with a specific spatial location. A general approach is to elevate analytes from cells into spatially labeled arrays.
Another general approach is to cut the spatially barcoded capture probes from the array and push the spatially barcoded capture probes towards and/or into or onto the biological sample.
Capture probes can be selectively cut from the array405, and the captured analytes can be spatially barcoded by performing a first-strand cDNA reverse transcriptase reaction. The first strand cDNA reaction can optionally be performed using a template-switching oligonucleotide. For example, template-changing oligonucleotides can be hybridized by reverse transcriptase to poly(C) tails added to the 3' end of cDNA in a template-independent manner. Crude mRNA templates and template-altering oligonucleotides can then be denatured from cDNA, then spatially barcoded capture probes can be hybridized to cDNA, and the complement of cDNA can be generated. The first strand cDNA can then be purified and collected for downstream amplification steps. The first strand of cDNA can be amplified using PCR406, where forward and reverse primers flank the spatial barcode and the analyte region of interest, generating libraries associated with specific spatial barcodes407In some embodiments, library preparation can be quantified and/or quality controlled to confirm the success of the library preparation step408In some embodiments, the cDNA includes primer sequencing (SBS). Sequencing and analysis of library amplicons for decoding spatial information407.
RNA transcripts present in a biological sample (eg, a tissue sample) can be used for spatial transcriptome analysis using the methods, compositions, systems, kits, and devices described herein. In particular, in some cases, barcoded oligonucleotides can be configured to anneal, amplify, and thus generate products with barcoded products from RNA templates or derivatives thereof. For example, in some cases, the barcoded oligonucleotides may include mRNA-specific promoter sequences, such as poly-T primer fragments that allow initiation and replication of mRNA in reverse transcription reactions, or other targeted promoter sequences. Alternatively or additionally, random RNA priming can be performed using random segments of N-mer primers of barcoded oligonucleotides. Reverse transcriptase (RT) can direct the synthesis of the first strand of complementary DNA (cDNA) using an RNA template and a primer complementary to the 3' end of the RNA template. A number of RTs are available for this reverse transcription reaction, including, for example, avian myeloblastosis virus (AMV) reverse transcriptase, Moloney murine leukemia virus (M-MuLV or MMLV), and other variants. Some recombinant M-MuLV reverse transcriptases, such as PROTOSCRIPT® II reverse transcriptase, may have reduced RNase H activity and increased thermostability compared to their wild-type counterparts and offer higher specificity, higher cDNA yield, and more complete cDNA products up to 12 kilobases (kb ) in length. In some embodiments, the reverse transcriptase is a mutated reverse transcriptase, such as, but not limited to, a mutant MMLV reverse transcriptase. In another embodiment, the reverse transcriptase is a mutated MMLV reverse transcriptase, such as, but not limited to, one or more variants described in U.S. Pat. patent no. 5,777.37. Publication no. 20180312822 and U.S. Provisional Patent. Application no. 62/946,885, filed Dec. 11, 2019, both of which are hereby incorporated by reference in their entirety.
In one non-limiting example of the workflow described above, biological samples (eg, tissue sections) can be fixed with methanol, stained with hematoxylin and eosin, and then imaged. Optionally, samples can be destained prior to permeabilization. These images can be used to map spatial patterns of gene expression back into biological samples. Permeabilase can be used to permeabilize biological samples directly on slides. Analytes (eg, polyadenylated mRNA) released from the upper cells of the biological sample can be captured by capture probes within the capture region on the substrate. Reverse transcription (RT) reagents can be added to permeabilized biological samples. Incubation with RT reagents can generate spatially barcoded full-length cDNA from captured analytes (eg, polyadenylated mRNA). Second-strand reagents (eg, second-strand primers, enzymes) can be added to the biological sample on the slide to initiate second-strand synthesis. The resulting cDNA can be denatured from the capture probe template and transferred (eg, to a clean tube) for amplification and/or library construction. Spatially barcoded full-length cDNAs can be amplified by PCR prior to library construction. The amplicons can then be subjected to enzymatic fragmentation and/or size selection to obtain the desired amplicon size. In some embodiments, when using the Illumina® library preparation method, P5 and P7 sequences can be added to the amplicon, allowing capture of the library preparation on a sequencing flow cell (eg, an Illumina sequencing instrument). Additionally, the i7 and i5 can add index sequences as index samples if multiple libraries are to be pooled and sequenced together. In addition, read 1 and read 2 sequences can be added to library preparation for sequencing purposes. The above sequences can be added to library preparation samples, eg, by end repair, A-tailing, adapter ligation, and/or PCR. The cDNA fragments can then be sequenced using, for example, paired-end sequencing using TruSeq Read 1 and TruSeq Read 2 as starting sequencing sites.
In some embodiments, correlation analysis of data generated by this workflow and others described herein can yield correlations greater than 95% for genes expressed in two capture regions (e.g., 95% or greater, 96% or greater, 97% or greater), 98% or more, or 99% or more). When performing the described workflow using single-cell RNA sequencing, in some embodiments, correlation analysis of the data may yield greater than 90% (eg, greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%) %, 97%, 98% or 99%) correlation of genes expressed in the two captured regions.
In some embodiments, after the cDNA is generated (eg, by reverse transcription), the cDNA can be amplified directly on the substrate surface. Generating multiple copies of cDNA (eg, cDNA synthesized from captured analytes) by amplification directly on the substrate surface can increase the complexity of the final sequencing library. Thus, in some embodiments, the cDNA can be amplified directly on the substrate surface by isothermal nucleic acid amplification. In some embodiments, isothermal nucleic acid amplification can amplify RNA or DNA.
In some embodiments, isothermal amplification can be faster than standard PCR reactions. In some embodiments, isothermal amplification can be linear amplification (eg, asymmetric with one primer) or exponential amplification (eg, with two primers). In some embodiments, isothermal nucleic acid amplification can be performed using template-switched oligonucleotide primers. In some embodiments, the template-switching oligonucleotide adds a consensus sequence to the 5' end of the reverse transcribed RNA. For example, upon interaction of a capture probe with an analyte (eg, mRNA) and reverse transcription, additional nucleotides are added to the end of the cDNA, creating a 3' overhang as described herein. In some embodiments, the template-switching oligonucleotide hybridizes to template-free poly(C) nucleotides added by reverse transcriptase to continue replication to the 5' end of the template-switching oligonucleotide, resulting in a full-length cDNA ready for further amplification. In some embodiments, the template-switching oligonucleotide adds a common 5' sequence (eg, the reverse complement of the template-switching oligonucleotide) to the full-length cDNA used to amplify the cDNA.
In some embodiments, after a full-length cDNA molecule is generated, the template-switching oligonucleotide can serve as a primer in a cDNA amplification reaction (eg, with DNA polymerase). In some embodiments, a double-stranded cDNA (eg, a first-strand cDNA and a second-strand reverse complement cDNA) can be amplified by isothermal amplification using a helicase or recombinase, followed by DNA polymerase that displaces the strand. Strand-displacing DNA polymerases can generate a displaced second strand, resulting in an amplification product.
In any of the isothermal amplification methods described herein, barcode modification (eg, spatial barcoding) can occur after the first amplification cycle in which unused capture probes are present on the substrate surface. In some embodiments, the free 3'OH ends of unused capture probes can be blocked by any suitable 3'OH blocking method. In some embodiments, the 3'OH may be blocked by a hairpin junction.
Isothermal nucleic acid amplification can be used to supplement or replace standard PCR reactions (for example, PCR reactions that require heating to approximately 95°C to denature double-stranded DNA). Isothermal amplification of a nucleic acid generally does not require the use of a thermocycler, but in some embodiments, isothermal amplification can be performed in a thermocycler. In some embodiments, isothermal amplification can be performed at about 35°C to about 75°C. In some embodiments, isothermal amplification may be performed at about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, or about 70°C, or both. temperature between them, depending on the polymerase and auxiliary enzymes used.
Isothermal nucleic acid amplification techniques are known in the art and can be used alone or in combination with any of the spatial methods described herein. For example, non-limiting examples of suitable isothermal nucleic acid amplification techniques include transcription-mediated amplification, nucleic acid sequence-based amplification, signal-mediated RNA amplification techniques, strand displacement amplification, rolling circle amplification, circular amplification, DNA guided isothermal amplification (LAMP), isothermal amplification multiple displacement, recombinase polymerase amplification, helicase-dependent amplification, single-primer isothermal amplification, and circular helicase-dependent amplification (see, e.g., Gill and Ghaemi, Isothermal Nucleic Acid Amplification Techniques: An Overview,Nucleozides, nucleotides i nukleinske kiske, 27(3), 224-43, doi: 10.1080/15257770701845204 (2008), which is incorporated herein by reference in its entirety).
In some embodiments, the isothermal nucleic acid amplification is a helicase-dependent nucleic acid amplification. Vincent et al describe helicase-dependent isothermal amplification of nucleic acid. et al., Helicase-dependent isothermal amplification of DNA,EMBO representative., 795-800 (2004) and US Pat. LOUSE. Patent no. 7,282,328, which is incorporated herein by reference in its entirety. Furthermore, helicase-dependent nucleic acid amplification on a substrate (eg, on a chip) is described in Andresen et al. et al., Helicase-dependent amplification: potential for on-chip amplification and point-of-care diagnostics,Expert Rev Mol Diagn., 9, 645-650, doi: 10.1586/erm.09.46 (2009), which is incorporated herein by reference in its entirety. In some embodiments, the isothermal nucleic acid amplification is recombinase polymerase nucleic acid amplification. Amplification of nucleic acid by polymerase recombinase is described in Piepenburg et al., DNA Detection Using Recombinant Proteins,PLOS Biology., 4, 7 e204 (2006) i Li, et.analyst, 144, 31-67, doi: 10.1039/C8AN01621F (2019), both of which are incorporated herein by reference in their entirety.
In general, isothermal amplification techniques use standard PCR reagents (eg, buffers, dNTPs, etc.) known in the art. Some isothermal amplification techniques may require additional reagents. For example, helicase-dependent nucleic acid amplification uses single-stranded binding proteins and accessory proteins. In another example, polymerase recombinase nucleic acid amplification uses a recombinase (e.g., T4 UvsX), a recombinase loading factor (e.g., TF UvsY), a single-stranded binding protein (e.g., T4 gp32), a collection agent (e.g., PEG-35K), and adenosine triphosphate.
After isothermal amplification of full-length cDNA nucleic acid by any of the methods described herein, the isothermally amplified cDNA (eg, single-stranded or double-stranded) can be recovered from the substrate and optionally followed by typical cDNA PCR Amplify in microcentrifuge tubes. The patterns can then be used with any of the spatial methods described in this article.
(i) Immunohistochemistry and immunofluorescence
In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) can be performed as part of or in addition to the spatial example workflow presented herein. For example, tissue sections can be fixed according to the methods described herein. Biological samples can be transferred to an array (eg capture probe array) where analytes (eg proteins) are detected using an immunofluorescence protocol. For example, when using a fluorescent primary antibody (1:100 in 3XSSC, 2% BSA, 0.1% Triton X, 1 U/µl RNAse inhibitor, 4°C for 30 minutes). It can be washed according to the analyte capture or spatial workflows described here, coverslip (in glycerol + 1 U/µl RNAse inhibitor), image (eg using a confocal microscope or other equipment capable of detecting fluorescence), wash and process biological samples .
As used herein, "antigen retrieval buffer" enhances antibody capture in IF/IHC protocols. An example of an antigen retrieval protocol may be preheating the antigen retrieval buffer (eg, to 95°C), immersing the biological sample in the heated antigen retrieval buffer for a predetermined time, then removing the biological sample from the antigen retrieval buffer. and Wash biological samples.
In some embodiments, optimization of permeabilization can be used to identify intracellular analytes. Optimization of permeabilization may include selection of permeabilizing agent, concentration of permeabilizing agent, and duration of permeabilization. Tissue permeabilization is discussed elsewhere.
In some embodiments, blocking the array and/or biological sample during preparation of the labeled biological sample reduces non-specific binding of the antibody to the array and/or biological sample (reduces background). Some embodiments provide a blocking buffer/blocking solution that can be applied prior to and/or during labeling, wherein the blocking buffer may include a blocking agent and optionally a surfactant and/or salt solution. In some embodiments, the blocking agent can be bovine serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk (e.g., nonfat dry milk), casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polyvinylpyrrolidone (PVP), biotin blocker, peroxidase blocker, levamisole, Carnoy's solution, glycine, lysine, sodium borohydride, Punta sky blue, Sudan black, Taiwan pan blue, FITC blocker and/or acetic acid. A blocking buffer/blocking solution can be applied to the array and/or biological sample prior to and/or during labeling of the biological sample (eg, application of a fluorophore-conjugated antibody).
In some embodiments, additional steps or optimizations may be included in performing IF/IHC protocols in conjunction with spatial arrays. Additional steps or optimizations may be involved in performing the spatially labeled analyte capture workflow described herein.
In some embodiments, provided herein are methods for spatially detecting an analyte (eg, detecting the location of an analyte, eg, a biological analyte) from a biological sample (eg, an analyte present in a biological sample such as a tissue section). include: (a) applying the biological sample to the substrate; (b) staining the biological sample on the substrate, imaging the stained biological sample and selecting the biological sample or a portion of the biological sample (eg regions of interest) performing the analysis; (c) providing an array comprising one or more plurality of capture probes on the substrate; (d) contacting the biological sample with the array, thereby allowing one of the one or more capture probes to capture the sensor and the analyte of (e) analyzing the captured analyte for spatial detection of the analyte of interest. Any of a number of staining and imaging techniques described herein or known in the art may be used in accordance with the methods described herein. In some embodiments, the staining includes an optical label as described herein, including but not limited to a fluorescent, radioactive, chemiluminescent, calorimetric, or detectable colorimetric label. In some embodiments, the staining includes fluorescent antibodies directed against target analytes (eg, cell surface or intracellular proteins) in the biological sample. In some embodiments, the staining comprises immunohistochemical staining for an analyte of interest (eg, a cell surface or intracellular protein) in the biological sample. In some embodiments, the staining includes chemical stains such as hematoxylin and eosin (H&E) or periodic acid Schiff (PAS). In some embodiments, significant time (eg, days, months, or years) may elapse between staining and/or imaging of a biological sample and analysis. In some embodiments, reagents for performing the assay are added to the biological sample before, while, or after the array contacts the biological sample. In some embodiments, step (d) includes placing the array on the biological sample. In some embodiments, the array is a flexible array, wherein multiple spatial barcode features (eg, substrate with capture probes, beads with capture probes) are attached to a flexible substrate. In some embodiments, steps are taken to slow the reaction (eg, cooling the temperature of the biological sample or using an enzyme that preferentially performs its primary function at a lower or higher temperature than its optimal functional temperature) prior to contacting the array. sample. In some embodiments, step (e) is performed without removing the biological sample from contact with the array. In some embodiments, step (e) is performed after the biological sample is no longer in contact with the array. In some embodiments, the biological sample is labeled with an analyte capture agent before, concurrently with, or after staining and/or imaging the biological sample. In such cases, significant time (eg, days, months, or years) may elapse between staining and/or imaging and analysis. In some embodiments, the array is adapted to facilitate the migration of biological analytes from the stained and/or imaged biological sample onto the array (eg, using any of the materials or methods described herein). In some embodiments, the biological sample is permeabilized prior to contact with the array. In some embodiments, the rate of permeabilization is slowed prior to contacting the biological sample with the array (eg, to limit diffusion of the analyte from its original site in the biological sample). In some embodiments, modulating the rate of permeabilization (eg, modulating the activity of a permeabilizing reagent) can occur by modulating the conditions to which the biological sample is exposed (eg, modulating temperature, pH, and/or light). In some embodiments, modulating the rate of permeabilization includes modulating the rate of permeabilization using an external stimulus (eg, small molecules, enzymes and/or activators). For example, a biological sample may be provided with a permeabilizing agent prior to contact with an array that responds to changes in conditions (eg, temperature, pH, and/or light) or external stimuli (eg, provision of small molecules, enzymes, and/or an activating agent). .
In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting the location of an analyte, e.g., a biological analyte) from a biological sample (e.g., present in a biological sample such as a tissue section), comprising: (a) providing biological sample on the base; (b) staining the biological sample on the substrate, imaging the stained biological sample and selecting the biological sample or part of the biological sample (eg, regions of interest) for spatial Transcriptomic analysis; (c) providing an array containing one or more plurality of capture probes on the substrate; (d) contacting the biological sample with the array, thereby allowing one of the one or more capture probes to capture the biological analyte of interest; (e) analyzing the captured biological analyte for spatial detection of the biological analyte of interest.
(b) capture probe
"Capture probe" refers to any molecule that can capture (directly or indirectly) and/or label an analyte (eg, an analyte of interest) in a biological sample. In some embodiments, the capture probes are nucleic acids or polypeptides. In some embodiments, the capture probe is a conjugate (eg, an oligonucleotide-antibody conjugate). In some embodiments, the capture probe includes a barcode (eg, a spatial barcode and/or a unique molecular identifier (UMI)) and a capture domain.
In some embodiments, spatial barcodes605, functional sequence604(eg flow cell connection order) i606(eg sequences of sequencing primers) may be common to all probes associated with a given feature. Spatial barcodes can also include recording fields607To facilitate the capture of target analytes.
(i) recording domain
As noted above, each capture probe includes at least one capture domain. A "capture domain" can be an oligonucleotide, a polypeptide, a small molecule, or any combination thereof that specifically binds the desired analyte. In some embodiments, the capture domain can be used to capture or detect a desired analyte.
In some embodiments, the capture domain is a functional nucleic acid sequence configured to interact with one or more analytes, such as one or more different types of nucleic acids (eg, RNA molecules and DNA molecules). In some embodiments, a functional nucleic acid sequence may include an N-mer sequence (eg, a random N-mer sequence) configured to interact with multiple DNA molecules. In some embodiments, the functional sequence may include a poly(T) sequence configured to interact with a messenger RNA (mRNA) molecule via the poly(A) tail of the mRNA transcript. In some embodiments, the functional nucleic acid sequence is a binding target for a protein (eg, transcription factor, DNA-binding protein, or RNA-binding protein), wherein the analyte of interest is a protein.
Capture probes can contain ribonucleotides and/or deoxyribonucleotides as well as synthetic nucleotide residues that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the capture domain is capable of initiating a reverse transcription reaction to generate cDNA complementary to the captured RNA molecule. In some embodiments, the capture domain of the capture probe can initiate a DNA extension (polymerase) reaction to generate DNA complementary to the captured DNA molecule. In some embodiments, the capture domain can form a binding reaction between the captured DNA molecule and a surface probe immobilized directly or indirectly on the substrate. In some embodiments, the capture domain can be attached to a single strand of the captured DNA molecule. For example, SplintR ligase together with RNA or DNA sequences (eg, degenerate RNA) can be used to ligate single-stranded DNA or RNA to capture domains. In some embodiments, a ligase with RNA template ligase activity, such as SplintR ligase, T4 RNA ligase 2, or KOD ligase, can be used to ligate single-stranded DNA or RNA to the capture domain. In some embodiments, the capture domain includes a splinted oligonucleotide. In some embodiments, the capture domain captures oligonucleotide probes.
In some embodiments, the capture domain is located at the 3' end of the capture probe and includes a free 3' end that can be extended, for example, by template-dependent polymerization, to form an extended capture probe as described herein. In some embodiments, the capture domain includes a nucleotide sequence capable of hybridizing to a nucleic acid, e.g. RNA or other analyte present in the cells of the biological sample in contact with the array. In some embodiments, the capture domain can be selected or designed to selectively or specifically bind a target nucleic acid. For example, capture domains can be selected or designed to capture mRNA by hybridization to the mRNA poly(A) tail. Therefore, in some embodiments, the capture domain comprises a poly(T)DNA oligonucleotide, such as a series of contiguous deoxythymidine residues linked by phosphodiester bonds, capable of hybridizing to poly(A) tail mRNA. In some embodiments, the capture domain may include nucleotides that are functionally or structurally similar to a poly(T) tail. For example, poly(U) oligonucleotides or oligonucleotides containing deoxythymidine analogs. In some embodiments, the capture domain comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the capture domain comprises at least 25, 30, or 35 nucleotides.
In some embodiments, the capture probe includes a capture domain having a sequence capable of binding mRNA and/or genomic DNA. For example, a capture probe may comprise a capture domain comprising a nucleic acid sequence (eg, a poly(T) sequence) capable of binding to the poly(A) tail and/or poly(A) homopolymer of the mRNA genome. Sequences present in DNA. In some embodiments, a terminal transferase is used to add homopolymeric sequences to mRNA molecules or genomic DNA molecules to generate analytes with poly(A) or poly(T) sequences. For example, a poly(A) sequence can be added to an analyte (eg, a genomic DNA fragment) so that the analyte can be captured by a poly(T) capture domain.
In some embodiments, random sequences, such as random hexamers or similar sequences, can be used to form all or part of the capture domain. For example, random sequences can be used in combination with poly(T) (or poly(T) analog) sequences. Thus, when a capture domain contains poly(T) (or "poly(T)-like") oligonucleotides, it may also contain random oligonucleotide sequences (eg, "poly(T)-random sequence" probes). For example, it may be 5' or 3' to the poly(T) sequence, e.g. 3' relative to the capture domain. poly(T)-random sequence probes facilitate capture of mRNA poly(A) tails. In some embodiments, the capture fields can be a completely random sequence. In some embodiments, a degenerate capture domain may be used.
In some embodiments, a group of two or more capture probes form a mixture, wherein the capture domain of one or more capture probes comprises a poly(T) sequence and the capture domain of one or more capture probes comprises a random sequence. In some embodiments, a set of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly(T)-like sequence and the capture domain of one or more capture probes includes random sequences. In some embodiments, a collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes comprises a poly(T)-random sequence and a capture structure of one or more capture probes. The domain consists of random sequences. In some embodiments, probes with degenerate capture domains can be added to any of the above combinations listed herein. In some embodiments, a probe with a degenerate capture domain can replace one of the probes in each pair described herein.
Capture domains can be based on specific gene sequences or specific motif sequences or common/conserved sequences, which are designed for capture (ie, sequence-specific capture domains). Therefore, in some embodiments, the capture domain is capable of selectively binding a desired subtype or subset of nucleic acids, such as a particular type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tmRNA, snRNA, snoRNA, SmY RNA, scaRNA, gRNA, RNase P, RNase MRP, TERC, SL RNA, aRNA, cis-NAT, crRNA, lncRNA, miRNA, piRNA, siRNA, shRNA, tasiRNA, rasiRNA, 7SK, eRNA, ncRNA or other types of RNA. In a non-limiting example, the capture domain is capable of selectively binding a desired subset of ribonucleic acids, such as microbiome RNA, such as 16S rRNA.
In some embodiments, the capture domain includes an "anchor" or "capture sequence", which is a nucleotide sequence designed to ensure hybridization of the capture domain to the intended analyte. In some embodiments, the anchor sequence comprises a nucleotide sequence, including 1-mer, 2-mer, 3-mer, or longer sequences. In some embodiments, the short sequences are random. For example, capture domains containing poly(T) sequences can be designed to capture mRNA. In such embodiments, the anchor sequence may include a random 3-mer (eg, GGG) that helps ensure hybridization of the poly(T) capture domain to the mRNA. In some embodiments, the anchor sequence can be HV, N or LV. Alternatively, the sequence can be designed using a specific nucleotide sequence. In some embodiments, the anchor sequence is located 3' to the capture domain. In some embodiments, the anchor sequence is located 5' to the capture domain.
In some embodiments, the capture domain of the capture probe is blocked prior to contacting the biological sample with the array, and blocking probes are used when the nucleic acids in the biological sample are modified prior to their capture on the array. In some embodiments, a blocking probe is used to block or modify the free 3' end of the capture domain. In some embodiments, the blocking probe can be hybridized to a capture probe, such as a hairpin probe, a partially double-stranded probe, or a complementary sequence, to mask the free 3' end of the capture domain. In some embodiments, the free 3' end of the capture domain can be blocked by chemical modification, for example, by adding an azidomethyl group as a chemically reversible capping moiety so that the capture probe does not include the free 3' end. Blocking or modifying the capture probe, particularly at the free 3' end of the capture domain, before exposing the biological sample to the array prevents modification of the capture probe, for example, adding a poly(A) tail of the capture probe to the free 3' end.
Non-limiting examples of 3' modifications include dideoxy C-3' (3'-ddC), 3' inverted dT, 3' C3 spacer, 3' amino group, and 3' phosphorylation. In some embodiments, the nucleic acid in the biological sample can be modified so that it can be captured by a capture domain. For example, linker sequences (including binding domains capable of binding capture domains of capture probes) can be added to the ends of nucleic acids, such as fragmented genomic DNA. In some embodiments, this is accomplished by ligation of linker sequences or extension of nucleic acids. In some embodiments, enzymes are used to incorporate additional nucleotides, such as polyA tails, at the ends of nucleic acid sequences. In some embodiments, the capture probe can be reversibly masked or modified so that the capture domain of the capture probe does not include a free 3' end. In some embodiments, the 3' end is removed, modified, or rendered inaccessible so that the capture domain is not amenable to processes used to modify the nucleic acids of the biological sample, such as ligation or extension.
In some embodiments, the capture domain of the capture probe is modified to allow removal of any modification of the capture probe that occurs during modification of the nucleic acid molecule of the biological sample. In some embodiments, the capture probe may include additional sequences downstream of the capture domain, e.g., 3' of the capture domain, ie, the blocking domain.
In some embodiments, the capture domain of the capture probe can be a non-nucleic acid domain. Examples of suitable capture domains that are not entirely based on nucleic acid include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any of the capture domains described herein.
(ii) Cleavage domain
Each capture probe can optionally include at least one cleavage domain. The cleavage domain represents the portion of the probe that is used to reversibly bind the probe to the array features, as will be described further herein. Additionally, one or more segments or regions of the capture probe can be optionally released from the sequence features by cleaving the cleavage domain. For example, spatial barcodes and/or universal molecular identifiers (UMIs) can be released by cleavage of the cleavage domain.
In some embodiments, the cleavage domain linking the capture probe to the feature is an enzyme-cleavable linker. Enzymes can be added to cleave the cleavage domain, resulting in the release of the capture probe from the feature. As another example, heating can also result in degradation of the cleavage domain and release of attached capture probes from array features. In some embodiments, laser radiation is used to heat and degrade the cleaved domains of the capture probes at specific sites. In some embodiments, the cleavage domain is a photosensitive chemical bond (eg, a chemical bond that dissociates when exposed to light, such as ultraviolet light). In some embodiments, the cutting field may be an ultrasonic cutting field. For example, ultrasound cleavage may depend on nucleotide sequence, length, pH, ionic strength, temperature, and ultrasound frequency (eg, 22 kHz, 44 kHz) (Grokhovsky, S.L., Specificity of DNA cleavage by ultrasound,molecular biology, 40(2), 276-283 (2006)).
Oligonucleotides with photosensitive chemical bonds (eg, photocleavable linkers) offer several advantages. They can be cut efficiently and quickly (eg in nanoseconds and milliseconds). In some cases, photomasks can be used so that only certain regions of the array are exposed to the cleavable stimuli (eg, exposure to ultraviolet light, exposure to light, exposure to laser-induced heat). When a cleavable linker is used, the cleavable reaction is light-driven and highly selective for the linker and therefore biorthogonal. Typically, the absorption wavelength of photocleavable linkers is in the near ultraviolet region of the spectrum. In some embodiments, λmaximumThe length of the photocleavable linker is from about 300 nm to about 400 nm, or from about 310 nm to about 365 nm. In some embodiments, λmaximumThe photocleavable linker is about 300 nm, about 312 nm, about 325 nm, about 330 nm, about 340 nm, about 345 nm, about 355 nm, about 365 nm, or about 400 nm in length.
Non-limiting examples of photosensitive chemical bonds that can be used to cleave domains include those described in Leriche et al.biomedical chemistry2012 Jan 15;20(2):571-82 and U.S. publication no. For example, linkers containing photosensitive chemical bonds include 3-amino-3-(2-nitrophenyl)propionic acid (ANP), benzoyl ester derivatives, 8-quinolylbenzenesulfonate, dicumarin, 6-bromo-7-alkoxymarin-4-ylmethoxycarbonyl, dialkyl - bisarylhydrazone-based linkers. In some embodiments, the photosensitive linkage is part of a cleavable linker, such as the following ortho-nitrobenzyl (ONB) linkers:
u:
- X is selected from O and NH;
- R1of H and C1-3alkyl;
- R2of H and C1-3to begin with;
- n is 1, 2 or 3; and
- a and b represent the attachment point of the adapter to the substrate and the attachment point of the adapter to the capture probe, respectively.
In some embodiments, at least one spacer is included between the substrate and the ortho-nitrobenzyl (ONB) linker, and at least one spacer is included between the ortho-nitrobenzyl (ONB) linker and the capture probe. In some aspects of these embodiments, the spacer contains at least one member selected from the group consisting of C1-6 alkylene, C2-6 alkenylene, C2-6 alkynylene, C=O, O, S, NH, -(C= O )O-, -(C=O)NH-, -S-S-, ethylene glycol, polyethylene glycol, propylene glycol and polypropylene glycol, or any combination thereof. In some embodiments, X is O. In some embodiments, X is NH. In some embodiments, R1It is H. In some embodiments, R1is C1-3alkyl. In some embodiments, R1is methyl. In some embodiments, R2It is H. In some embodiments, R2is C1-3Alkoxy. In some embodiments, R2is methoxy. In some embodiments, R1is H and R2It is H. In some embodiments, R1is H and R2is methoxy. In some embodiments, R1is methyl and R2It is H. In some embodiments, R1is methyl and R2is methoxy.
In some embodiments, the photocleavable linker has the formula:
In some embodiments, the photocleavable linker has the formula:
In some embodiments, the photocleavable linker has the formula:
In some embodiments, the photocleavable linker has the formula:
In some embodiments, the photocleavable linker has the formula:
Without being bound by any particular theory, it is believed that excitation of the ortho-nitrobenzyl (ONB) linker results in a Norrish-type hydrogen abstraction reaction at the gamma position, with subsequent formation of a highly reactive azic acid compound rearranged to the nitroso group, resulting in complete cleavage of the linker, as is shown in the image below:
In some embodiments, the photocleavable linker is a 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker:
where X, R2, n, a and b are ortho-nitrobenzyl (ONB) linkers as described herein.
In some embodiments, the photocleavable linker has the formula:
In some embodiments, the photocleavable linker is a benzoate linker:
where a and b are as described herein for an ortho-nitrobenzyl (ONB) linker.
Other examples of photosensitive chemical bonds that can be used to cleave domains include halogenated nucleosides such as bromodeoxyuridine (BrdU). BrdU is a thymidine analog that can be easily incorporated into oligonucleotides (for example, in the cleavage domain of capture probes) and is sensitive to UVB light (280-320 nm range). After exposure to UVB light, photocleavage reactions occur (eg, on nucleosides immediately 5' from the Brdu binding site (Doddridge et al., Chem. Comm., 1998, 18:1997-1998 and Cook et al., Chemistry ). and Biology, 1999, 6:451-459), resulting in the release of the capture probe from the feature.
Other examples of cleavage domains include labile chemical bonds such as, but are not limited to, ester bonds (eg, cleavable by acids, bases, or hydroxylamine), vicinal diol bonds (eg, cleavable by sodium periodate), Diels -Alder bonds (e.g., cleaved by heat), sulfonic bonds (e.g., cleaved by base), silyl ether bonds (e.g., cleaved by acid), glycosidic bonds (e.g., cleaved by amylase), peptide bonds (on e.g., cleaved by proteases), abasic or apurinic/apyrimidinic (AP) sites (e.g., cleavable by base or AP endonucleases), or phosphodiester bonds (e.g., cleaved by cleavage nucleases (e.g., DNAases).
In some embodiments, the cleavage domain includes a sequence recognized by one or more enzymes capable of cleaving a nucleic acid molecule, eg, capable of cleaving a phosphodiester bond between two or more nucleotides. The bonds can be cleaved by other enzymes that target nucleic acid molecules, such as restriction enzymes (eg, restriction endonucleases). For example, the cleavage domain may include a restriction endonuclease (restriction enzyme) recognition sequence. Restriction enzymes cut double- or single-stranded DNA at specific recognition nucleotide sequences called restriction sites. In some embodiments, a restriction enzyme that cuts infrequently, such as one with a long recognition site (at least 8 base pairs in length), is used to reduce the likelihood of cleavage elsewhere in the capture probe.
In some embodiments, the cleavage domain comprises a polynucleotide cleavable by a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER™ enzyme).(U) sequences. Once released, releasable capture probes are available for reactions. So, for example, an activatable capture probe can be activated by releasing the capture probe from the feature.
In some embodiments, when the capture probe is attached to the substrate indirectly, such as via a surface probe, the cleavage domain includes one or more mismatched nucleotides such that the complementary portions of the surface probe and the capture probe are not 100% complementary (for example, the number of mismatches base pairs can be one, two or three base pairs). Such mismatches are recognized by enzymes such as MutY and T7 endonuclease I, resulting in cleavage of the nucleic acid molecule at the site of the mismatch. As used herein, a "surface probe" can be any moiety present on the surface of a substrate capable of binding a reagent (eg, a capture probe). In some embodiments, surface probes are oligonucleotides. In some embodiments, the surface probe is part of the capture probe.
In some embodiments, where the capture probe is indirectly attached (e.g., immobilized) to the feature, e.g., via a surface probe, the cleavage domain includes a nickase recognition site or sequence. Nicases are endonucleases that cleave only one strand of the DNA duplex. Thus, the cleavage domain may include a nickase recognition site near the 5' end of the surface probe (and/or the 5' end of the capture probe), such that cleavage of the surface probe or the capture probe results in a double cleavage between the body strand destabilizes the probe and the probe to capture, thereby releasing the capture probe from the feature).
Nickases can also be used in some embodiments where the capture probes are attached (eg, immobilized) directly to the features. For example, a substrate can be contacted with a nucleic acid molecule that hybridizes to the cleavage domain of a capture probe to provide or recreate the recognition site of a cutting enzyme, such as a cleavage helper probe. Therefore, contact with the cutting enzyme will result in cleavage of the cleavage domain, thereby releasing the capture probe from the feature. Such auxiliary cleavage probes can also be used to provide or recreate cleavage recognition sites for other cleavage enzymes, such as restriction enzymes.
Some nickases introduce single-stranded nicks only at specific locations on the DNA molecule by binding to and recognizing specific nucleotide recognition sequences. A number of natural nickases have been discovered, and the sequence recognition properties of at least four of them have now been determined. The cases are described in the U.S. patent no. LOUSE. patent no. 6,867,028, the entire contents of which are incorporated herein by reference. In general, any suitable nickase can be used in combination with a complementary nickase recognition site in the cleavage domain. After use, the cutting enzyme can be removed from the assay or deactivated after release of the capture probes to prevent unwanted cleavage of the capture probes.
Examples of suitable capture domains that are not entirely based on nucleic acid include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any of the capture domains described herein.
In some embodiments, the cleavage domain is absent from the capture probe. Examples of substrates to which capture probes lacking a cleavage domain are bound are described, for example, in Macosko et al., (2015) Cell 161, 1202-1214, the entire contents of which are incorporated herein by reference.
In some embodiments, the region of the capture probe corresponding to the cleavage domain may be used for some other function. For example, additional nucleic acid extension or amplification regions can be included where a cleavage domain would normally be located. In such embodiments, this region may complement the functional domain or even exist as an additional functional domain. In some embodiments, a cleavage domain is present, but its use is optional.
(iii) Functional domain
Each capture probe can optionally include at least one functional domain. Each functional domain typically includes a functional nucleotide sequence for use in downstream analysis steps during the assay.
In some embodiments, the capture probe may include a functional domain for attachment to a sequencing flow cell, such as a P5 sequencer for Illumina® sequencing. In some embodiments, the capture probe or derivative thereof may include another functional domain, such as a P7 sequence, for attachment to a sequencing flow cell for Illumina® sequencing. Functional domains can be selected for compatibility with different sequencing systems, eg 454 sequencing, Ion Torrent Proton or PGM, Illumina X10, etc., and their requirements.
In some embodiments, the functional domain includes a primer. Primers can include R1 sequences of Illumina® sequencing primers, and in some embodiments, R2 sequences of Illumina® sequencing primers. Examples of such capture probes and their use are described in US Pat. publication no. 2014/0378345 and 2015/0376609, the entire contents of which are incorporated herein by reference.
(iv) Spatial barcode
As noted above, capture probes may include one or more spatial barcodes (eg, two or more, three or more, four or more, five or more) spatial barcodes. A "spatial barcode" is a single contiguous nucleic acid segment or two or more unrelated nucleic acid segments that serve as tags or identifiers that convey or are capable of conveying spatial information. In some embodiments, the capture probes comprise a spatial barcode having a spatial aspect, wherein the barcode is associated with a specific location within the array or a specific location on the substrate.
Spatial barcodes can be part of the analyte or independent of the analyte (eg, part of the capture probe). The spatial barcode can be a tag attached to the analyte (eg nucleic acid molecule) or a combination of tags with an endogenous characteristic of the analyte (eg size or terminal sequence of the analyte). Spatial barcodes can be unique. In some embodiments where the spatial barcode is unique, the spatial barcode serves as both a spatial barcode and a unique molecular identifier (UMI) associated with one particular capture probe.
Spatial barcodes can be in many different formats. For example, spatial barcodes may include polynucleotide spatial barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences. In some embodiments, the spatial barcode is attached to the analyte in a reversible or irreversible manner. In some embodiments, spatial barcodes are added, e.g. DNA or RNA sample fragments, before, during and/or after sample sequencing. In some embodiments, spatial barcoding enables identification and/or quantification of individual sequencing reads. In some embodiments, the spatial barcode is used as a fluorescent barcode, for which purpose fluorescently labeled oligonucleotide probes are hybridized to the spatial barcode.
In some embodiments, the spatial barcode is a nucleic acid sequence that does not substantially hybridize to an analyte nucleic acid molecule in a biological sample. In some embodiments, the spatial barcode has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to the majority (e.g., 80%) of the nucleic acid sequence or more ) nucleic acid molecules in biological samples.
The spatial barcode sequence may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the barcode space sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the spatial barcode sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or more in length. In some embodiments, the spatial barcode sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or less.
Nucleotides may be completely contiguous, eg within a sequence of contiguous nucleotides, or may be divided into two or more separate subsequences separated by one or more nucleotides. An isolated barcode space subsequence can be about 4 to about 16 nucleotides in length. In some embodiments, the subsequence of the spatial barcode can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, a subsequence of a spatial barcode can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the subsequence of the spatial barcode can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
For multiple capture probes connected to a common array feature, one or more multiple capture probe spatial barcode strings may include the same sequence for all capture probes associated with that feature and/or be the same across all capture probes. Various capture probe sequences are associated with this feature.
Capture probes associated with a single array feature may include the same (or shared) spatial barcode sequence, different spatial barcode sequences, or a combination of both. Capture probes associated with a feature may include sets of capture probes. The probes for capturing a given set may include the same spatial barcode sequence. The same spatial barcode sequence may differ from the spatial barcode sequence of another set of capture probes.
A plurality of capture probes may include a spatial barcode sequence (eg, a nucleic acid barcode sequence) associated with a particular location on the spatial array. For example, a first plurality of capture probes may be associated with a first region based on a spatial barcode sequence shared by the capture probes within the first region, and a second plurality of capture probes may be associated with a second region based on a second Spatial Barcode Sequence they share recording probes within the region. The second area may or may not be connected to the first area. Additional multiples of capture probes can be linked to spatial barcode sequences shared by capture probes in other regions. In some embodiments, the spatial barcode sequence may be the same among multiple capture probe molecules.
In some embodiments, multiple different spatial barcodes are incorporated into a single array of capture probes. For example, a set of mixed but known spatial barcode sequences can provide stronger attribution of addresses or spatial barcodes to a given place or location by providing repeated or independent confirmation of the identity of the location. In some embodiments, the plurality of spatial barcodes represent increased specificity of the location of a particular array point.
(v) Unique molecular identifier
A capture probe may include one or more (eg, two or more, three or more, four or more, five or more) unique molecular identifiers (UMIs). A unique molecular identifier is a contiguous nucleic acid fragment or two or more discontinuous nucleic acid fragments that serve as a label or identifier for a specific analyte, or as a capture probe for binding a specific analyte (eg, a capture region).
UMIs can be unique. UMI may contain one or more specific polynucleotide sequences, one or more random sequences of nucleic acids and/or amino acids, and/or one or more synthetic sequences of nucleic acids and/or amino acids, or combinations thereof.
In some embodiments, the UMI is a nucleic acid sequence that does not substantially hybridize to an analyte nucleic acid molecule in a biological sample. In some embodiments, UMIs are associated with nucleic acid molecules that comprise a significant portion (eg, 80% or more) of a biological sample.
The UMI may contain from about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the UMI sequence can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the UMI sequence can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the UMI sequence is at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or less.
These nucleotides can be completely contiguous, i.e. in a series of contiguous nucleotides, or they can be divided into two or more separate subsequences separated by 1 or more nucleotides. Isolated UMI subsequences can be from about 4 to about 16 nucleotides in length. In some embodiments, the UMI subsequence can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequence can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.
In some embodiments, the UMI is bound to the analyte in a reversible or irreversible manner. In some embodiments, UMI is added to, for example, DNA or RNA sample fragments before, during, and/or after analyte sequencing. In some embodiments, UMIs allow identification and/or quantification of individual sequencing reads. In some embodiments, the UMI is used as a fluorescent barcode, for which fluorescently labeled oligonucleotide probes are hybridized to the UMI.
(vi) Other aspects of capture probes
For capture probes attached to array features, an individual array feature may include one or more capture probes. In some embodiments, individual array features include hundreds or thousands of capture probes. In some embodiments, the capture probe is associated with a specific individual characteristic, wherein the individual characteristic comprises a capture probe that contains a spatial barcode unique to a defined region or location on the array.
In some embodiments, a particular feature may comprise capture probes that include more than one spatial barcode (eg, one capture probe on a particular feature may comprise a different spatial barcode than another capture probe on the same particular feature). needle, and both capture probes contain another common spatial barcode), where each spatial barcode corresponds to a specific defined region or location on the array. For example, multiple spatial barcode arrays associated with a particular feature in the array can provide a stronger address or attribution to a given location by providing duplicate or independent confirmation of location. In some embodiments, the plurality of spatial barcodes represent increased specificity of the location of a particular array point. In a non-limiting example, a particular array point may be encoded with two different spatial barcodes, where each spatial barcode identifies a particular defined area within the array, and the two spatial barcode array point identifies a subdivision where two defined regions of the Region overlap, such as overlapping parts of the Venn diagram.
In another non-limiting example, a particular array point may be encoded with three different spatial barcodes, where the first spatial barcode identifies a first region within the array, and the second spatial barcode identifies a second region, where the second region is an entire subregion within the first region, and the third spatial barcode identifies the third region, wherein the third region is a subregion entirely within the first and second subregions.
In some embodiments, capture probes coupled to the array features are released from the array features for sequencing. Alternatively, in some embodiments, the capture probes remain attached to the array features, and the probes are sequenced (eg, by in situ sequencing) while they remain attached to the array features. Additional aspects of capture probe sequencing are described in the following sections of this disclosure.
In some embodiments, the array feature may include different types of capture probes attached to the feature. For example, an array feature may include a first type of capture probe having a capture domain designed to bind one type of analyte, and a second type of capture probe having a capture domain designed to bind another type of analyte. Needle. Typically, array features may include one or more (eg, two or more, three or more, four or more, five or more, six or more, eight or more, ten or more, 12 or more, 15 or more, 20 or more, 30 or more, 50 or more) of different types of capture probes connected to a single array feature.
In some embodiments, the capture probes are nucleic acids. In some embodiments, the capture probes are attached to the array elements via their 5' ends. In some embodiments, the capture probe comprises, from the 5' to the 3' end: one or more barcodes (eg, spatial barcodes and/or UMIs) and one or more capture domains. In some embodiments, the capture probe includes from the 5' to the 3' end: a barcode (eg, spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe comprises, from the 5' to the 3' end: a cleavage domain, a functional domain, one or more barcodes (eg, spatial barcodes and/or UMI), and a capture domain. In some embodiments, the capture probe comprises from the 5' to the 3' end: a cleavage domain, a functional domain, one or more barcodes (eg, spatial barcodes and/or UMIs), another functional domain, and a capture domain. In some embodiments, the capture probe comprises from the 5' to the 3' end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probes do not include spatial barcodes. In some embodiments, the capture probes do not include a UMI. In some embodiments, the capture probe includes a sequence to initiate a sequencing reaction.
In some embodiments, the capture probe is immobilized to the element via its 3' end. In some embodiments, the capture probe comprises, from the 3' to the 5' end: one or more barcodes (eg, spatial barcodes and/or UMIs) and one or more capture domains. In some embodiments, the capture probe includes from the 3' to the 5' end: a barcode (eg, spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe comprises from the 3' to the 5' end: a cleavage domain, a functional domain, one or more barcodes (eg, spatial barcodes and/or UMI), and a capture domain. In some embodiments, the capture probe comprises from the 3' to the 5' end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.
In some embodiments, capture probes include in situ synthesized oligonucleotides. Oligonucleotides synthesized in situ can be attached to the substrate or to elements on the substrate. In some embodiments, the oligonucleotide synthesized in situ includes one or more constant sequences, one or more of which serve as primer sequences (eg, primers for amplification of a target nucleic acid). An oligonucleotide synthesized in situ may, for example, include a constant sequence at the 3' end that is characteristic of the substrate or of the substrate. Additionally or alternatively, the in situ synthesized oligonucleotide may contain a constant sequence at the free 5' end. In some embodiments, one or more constant sequences may be cleavable sequences. In some embodiments, the in situ synthesized oligonucleotides include barcode sequences, e.g. variable barcode sequences. The barcode can be any of the barcodes described here. Barcodes can be approximately 8 to 16 nucleotides long (eg, 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides). In situ synthesized oligonucleotides can be shorter than 100 nucleotides (eg, less than 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, or 20 nucleotides). In some cases, the in situ synthesized oligonucleotides are about 20 to about 40 nucleotides in length. Exemplary in situ synthesized oligonucleotides are manufactured by Affymetrix. In some embodiments, in situ synthesized oligonucleotides are linked to array features.
Additional oligonucleotides can be ligated to in situ synthesized oligonucleotides to produce capture probes. For example, a primer complementary to a portion of an in situ synthesized oligonucleotide (e.g., a constant sequence in an oligonucleotide) can be used to hybridize to another oligonucleotide and extend (using the in situ synthesized oligonucleotide as a template, e.g., a primer extension reaction) to form double-stranded oligonucleotides still generate 3' overhangs. In some embodiments, the 3' overhang can be generated by a template-independent ligase (eg, terminal deoxynucleotidyl transferase (TdT) or poly(A) polymerase). Additional oligonucleotides containing one or more capture domains can be ligated to the 3' overhangs using a suitable enzyme (eg, ligase) and oligonucleotides that are cleaved to generate capture probes. Thus, in some embodiments, the capture probe is the product of two or more oligonucleotide sequences linked together (eg, an in situ synthesized oligonucleotide and an additional oligonucleotide). In some embodiments, one of the oligonucleotide sequences is an oligonucleotide synthesized in situ.
In some embodiments, a probe oligonucleotide (eg, any of the probe oligonucleotides described herein) can be used to prepare capture probes. Two or more oligonucleotides can be ligated together using a stranded oligonucleotide and any type of ligase known in the art or described herein (eg, SplintR ligase).
One of the oligonucleotides may include, for example, a constant sequence (eg, a sequence complementary to a portion of the target oligonucleotide), a degenerate sequence, and/or a capture domain (eg, as described herein). One of the oligonucleotides may also include a sequence compatible with ligation or hybridization of an analyte of interest in a biological sample. Analytes of interest (eg, mRNA) can also be used as oligonucleotide splints to attach multiple oligonucleotides to capture probes. In some embodiments, capture probes are generated by enzymatic addition of polynucleotides to the ends of oligonucleotide sequences. Capture probes can include degenerate sequences that can be used as unique molecular identifiers.
A degenerate sequence refers to a sequence in which certain positions of the nucleotide sequence contain multiple possible bases. The degenerate sequence may be a degenerate nucleotide sequence comprising about or at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45 or 50 nucleotides. In some embodiments, the nucleotide sequence comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or more degenerate positions within the nucleotide sequence. In some embodiments, degenerate sequences are used as UMIs.
In some embodiments, the capture probe includes a restriction endonuclease recognition sequence or a nucleotide sequence that can be cleaved by a specific enzymatic activity. For example, uracil sequences can be enzymatically cleaved from nucleotide sequences using uracil DNA glycosylase (UDG) or uracil-specific excision reagent (USER). As another example, other modified bases (eg, modified by methylation) can be recognized and cleaved by specific endonucleases. Enzymatic cleavage of the capture probe can remove the blocking domain and any additional nucleotides added to the 3' end of the capture probe during modification. Removal of the blocking domain exposes and/or restores the free 3' end of the capture domain to the capture probe. In some embodiments, additional nucleotides can be removed to expose and/or restore the 3' end of the capture domain of the capture probe.
In some embodiments, the blocking domain can be incorporated into the capture probe at the time of synthesis of the capture probe or after its synthesis. The terminal nucleotides of the capture domain are reversible terminator nucleotides (eg, 3'-O-blocked reversible terminator and 3'-unblocked reversible terminator), and can be included during or after probe synthesis in the capture probe.
(vii) Extended capture probes
An "extended capture probe" is a capture probe that has an amplified nucleic acid sequence. For example, when a capture probe comprises a nucleic acid, "extended 3' end" means that further nucleotides are added to the top 3' nucleotide of the capture probe to extend the length of the capture probe, e.g. standard polymerization reactions are used to extend nucleic acid molecules and involve template polymerization catalyzed by polymerases such as DNA polymerase or reverse transcriptase.
In some embodiments, extending the capture probe involves generating cDNA from the captured (hybridized) RNA. The process involves the synthesis of complementary strands of hybridized nucleic acids, e.g. generation of cDNA based on the captured RNA template (RNA that hybridizes to the capture domain of the capture probe). Therefore, in the initial step of extending the capture probe, e.g. generation of cDNA, captured (hybridized) nucleic acid, e.g. RNA, serves as a template for the extension step (eg, reverse transcription).
In some embodiments, capture probes are amplified using reverse transcription. For example, reverse transcription involves the synthesis of cDNA (complementary or copied DNA) from RNA such as (messenger RNA) by reverse transcriptase. In some embodiments, reverse transcription is performed while the tissue is still in situ, generating an analyte library, wherein the analyte library includes spatial barcodes from adjacent capture probes. In some embodiments, the capture probe is extended by one or more DNA polymerases.
In some embodiments, the capture domain of the capture probe includes a primer to generate the complementary strand of the nucleic acid to which the capture probe hybridizes, such as a DNA polymerase and/or reverse transcription primer. Nucleic acids, eg, DNA and/or cDNA molecules, which are formed by the extension reaction, include capture probe sequences. Extension of capture probes, eg, DNA polymerase reaction and/or reverse transcription, can be performed using a variety of suitable enzymes and protocols.
In some embodiments, full-length DNA is produced, e.g. cDNA molecules. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if a nucleic acid, such as RNA, is partially degraded in a tissue sample, the captured nucleic acid molecule will not be the same length as the original RNA in the tissue sample. In some embodiments, the 3' end of the extended probe (eg, the first strand of the cDNA molecule) is modified. For example, linkers or adapters can be attached to the 3' ends of extension probes. This can be accomplished using a single-stranded ligase such as T4 RNA ligase or Circligase™ (available from Lucigen, Middleton, WI). In some embodiments, template-switching oligonucleotides are used to extend cDNA to generate full-length cDNA (or as close to full-length cDNA as possible). In some embodiments, a second-strand synthesis helper probe (a partially double-stranded DNA molecule capable of hybridizing to the 3' end of the extension capture probe) can be ligated to the 3' end of the extension probe, such as first-strand cDNA, using double-stranded ligase molecules as which is T4 DNA ligase. Other enzymes suitable for the ligation step are known in the art and include, for example, Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9°N) DNA Ligase (9°N™ DNA Ligase, New England Biolabs), Ampligase™ (available from Lucigen, Middleton, WI) and SplintR (available from New England Biolabs, Ipswich, MA). In some embodiments, the polynucleotide tail, e.g. poly(A) tail, is incorporated into the 3' end of the extended probe molecule. In some embodiments, the polynucleotide tail is incorporated by a terminally active transferase enzyme.
In some embodiments, double-stranded extended capture probes are treated to remove any unextended capture probes prior to amplification and/or analysis, e.g. sequence analysis. This can be achieved in various ways, for example, by using enzymes to degrade unextended probes, such as exonucleases or purification columns.
In some embodiments, the extended capture probes are amplified to obtain sufficient amounts for analysis, eg, by DNA sequencing. In some embodiments, the first strand of an extended capture probe (eg, DNA and/or cDNA molecule) serves as a template for an amplification reaction (eg, polymerase chain reaction).
In some embodiments, the amplification reaction includes an affinity group to an extended capture probe (eg, an RNA-cDNA hybrid) using a primer containing the affinity group. In some embodiments, the primer includes an affinity group and the extended capture probe includes an affinity group. The affinity group may correspond to any of the previously described affinity groups.
In some embodiments, an extended capture probe containing an affinity group can be linked to an affinity group-specific array feature. In some embodiments, the substrate may include an antibody or antibody fragment. In some embodiments, the array feature includes avidin or streptavidin and the affinity group includes biotin. In some embodiments, the sequence feature comprises maltose and the affinity group comprises a maltose-binding protein. In some embodiments, the array feature includes a maltose-binding protein and the affinity group includes maltose. In some embodiments, amplification of the extended capture probes can act to release the extended probes from the array features, until copies of the extended probes are bound to the array features.
In some embodiments, the extended capture probe or its complement or amplicon is free from array features. The step of releasing the extended capture probes or their complements or amplicons from the array features can be accomplished in a number of ways. In some embodiments, the extended capture probe or its complement is released from the feature by nucleic acid cleavage and/or denaturation (eg, heating to denature double-stranded molecules).
In some embodiments, the extended capture probes or their complements or amplicons are physically released from the array features. For example, methods of inducing physical release include denaturation of double-stranded nucleic acid molecules. Another way to release probes with extended capture is to use a solution that interferes with the hydrogen bonding of the double-stranded molecules. In some embodiments, the extended capture probes are released by applying hot water, such as water or a buffer at at least 85°C, such as at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 °C. In some embodiments, a solution is added that contains salts, surfactants, etc. that can further disrupt the interactions between the nucleic acid molecules to free the extended capture probes from the array features. In some embodiments, formamide solutions can be used to destabilize interactions between nucleic acid molecules to release extended capture probes from array features.
(viii) Amplification of capture probes
In some embodiments, methods are provided herein for amplifying capture probes immobilized on a spatial array, wherein amplifying the capture probes increases the number of capture domains and spatial barcodes on the spatial array. In some embodiments where the pickup probes are amplified, the amplification is by circular amplification. In some embodiments, the capture probes that are amplified include sequences capable of rolling circle amplification (eg, docking sequences, functional sequences, and/or primer sequences). In one example, the capture probe can include a functional sequence capable of binding an amplification primer. In another example, the capture probe can include one or more splice sequences (e.g., a first splice sequence and a second splice sequence), which can be combined with one or more rolling circle hybridization oligonucleotides (for example, probes with padlock). In some embodiments, additional probes are immobilized to the substrate, wherein the additional probes include sequences capable of rolling circle amplification (eg, docking sequences, functional sequences, and/or primer sequences). In some embodiments, the spacer array is contacted with oligonucleotides (eg, padlock probes). As used herein, a "padlock probe" refers to an oligonucleotide that has at its 5' and 3' ends a sequence complementary to a target sequence (eg, a docking sequence) adjacent to or adjacent to a capture probe. After hybridization to a target sequence (eg, docking sequence), the two ends of the padlock probe are either touched or one end is extended until the two ends touch, thereby circularizing the padlock probe by ligation (eg, using any of the methods described herein). In some embodiments, after circularizing the oligonucleotide, rolling circle amplification can be used to amplify ligation products that contain at least the capture domain and the spatial barcode from the capture probe. In some embodiments, the use of padlock oligonucleotides and amplification in a capture probe amplification circuit increases the number of capture domains and the number of spatial barcodes on the spatial array.
In some embodiments, the method of increasing the capture efficiency of the spatial array includes amplifying all or a portion of the capture probes immobilized on the substrate. For example, amplification of all or part of the substrate-immobilized capture probes can increase the capture efficiency of spatial arrays by increasing the number of capture domains and spatial barcodes. In some embodiments, methods of determining the location of an analyte in a biological sample include the use of spatial arrays with enhanced capture efficiency (eg, spatial arrays in which the capture probes are amplified as described herein). For example, the capture efficiency of spatial arrays can be increased by amplifying all or part of the capture probes prior to contact with the biological sample. Amplification results in an increased number of capture domains, allowing the capture of more analytes, compared to spatial arrays where capture probes are not amplified prior to exposure to the biological sample. In some embodiments, methods of generating spatial arrays with increased capture efficiency include amplifying all or a portion of the capture probes. In some embodiments where a spatial array with increased capture efficiency is generated by amplifying all or a portion of the capture probes, the amplification increases the number of capture domains and the number of spatial barcodes on the spatial array. In some embodiments, the method of determining the location of capture probes (ie, capture probes on a feature) on a spatial array includes amplifying all or a portion of the capture probes. For example, amplification of capture probes immobilized on a substrate can increase the number of spatial barcodes detected for direct decoding (eg, using any of the methods described herein, including but not limited to in situ sequencing) of capture positions.
(ix) Analyte capture reagent
The present disclosure also provides methods and materials for the spatial analysis of biological analytes (eg, mRNA, genomic DNA, available chromatin and cell surface or intracellular proteins and/or metabolites) using analyte capture means. As used herein, an "analyte capture agent" (formerly sometimes referred to as a "cell labeling agent") refers to a reagent that interacts with an analyte (eg, in a sample) and a capture probe (eg, attached to a substrate). capture probe) for analyte identification. In some embodiments, the analyte capture agent includes an analyte binding group and a barcode domain of the capture agent.
As used herein, the term "analyte binding unit" refers to a molecule or group capable of binding a macromolecular component, e.g. analyte, e.g. biological analyte. In some embodiments of any of the spatial analysis methods described herein, the analyte-binding portion of the analyte capture agent may include, but is not limited to, an antibody or fragment thereof that binds to an epitope, a molecule that binds a cell surface receptor. , receptor ligands, small molecules, bispecific antibodies, bispecific involved T-cells, involved T-cell receptors, involved B-cell receptors, precursors, aptamers, monomers, aphibodies, darpins and protein carriers, or any combination thereof. An analyte-binding moiety can bind a macromolecular component (eg, an analyte) with high affinity and/or high specificity. An analyte-binding unit may comprise a nucleotide sequence (eg, an oligonucleotide), which may correspond to at least part or all of an analyte-binding moiety. Analyte binding moieties may include polypeptides and/or aptamers (eg, polypeptides and/or aptamers that bind a specific target molecule (eg, analyte)). Analyte-binding moieties may include antibodies or antibody fragments (eg, antigen-binding fragments) that bind a particular analyte (eg, polypeptide).
In some embodiments, the analyte-binding portion of the analyte capture agent comprises one or more antibodies or antigen-binding fragments thereof. An antibody or antigen-binding fragment that includes an analyte-binding moiety can specifically bind the target analyte. In some embodiments, the analyte is a protein (eg, a protein on the surface of a biological sample (eg, a cell) or a protein within a cell). In some embodiments, multiple analyte capture agents comprising multiple analyte binding moieties bind multiple analytes present in a biological sample. In some embodiments, the plurality of analytes includes a single type of analyte (eg, a single type of polypeptide). In some embodiments where the multiple analytes include a single type of analyte, the analyte binding moieties of the multiple analyte capture agents are the same. In some embodiments where the plurality of analytes includes a single type of analyte, the analyte binding moieties in the array of analyte capture means are different (e.g., members of the plurality of analyte capture means may have two or more analyte binding moieties, wherein each of the two or more binding moieties analyte binds one type of analyte, e.g. at different binding sites). In some embodiments, the plurality of analytes includes multiple different types of analytes (eg, multiple different types of polypeptides).
Analyte capture means may include analyte binding moieties. The analyte binding moiety can be an antibody. Exemplary, non-limiting antibodies that can be used as an analyte binding moiety in an analyte capture reagent or that can be used in IHC/IF applications disclosed herein include any of the following antibodies, including variants thereof: A-ACT, A-AT , ACTH, specific for actin-muscle, actin-smooth muscle (SMA), AE1, AE1/AE3, AE3, AFP, AKT phosphate, ALK-1, amyloid A, androgen receptor, annexin A1, B72.3, BCA-225 , BCL-1 (cyclin D1), BCL-⅟CD20, BCL-2, BCL-2/BCL-6, BCL-6, Ber-EP4, β-amyloid, β-catenin, BG8 (Lewis Y), BOB-1 , CA 19.9, CA 125, CAIX, calcitonin, caldesmon, calponin, calretinin, CAM 5.2, CAM 5.2/AE1, CD1a, CD2, CD3 (M), CD3 (P), CD3/CD20, CD4, CD5, CD7, CD8 , CD10, CD14, CD15, CD20, CD21, CD22, CD23, CD25, CD30, CD31, CD33, CD34, CD35, CD43, CD45 (LCA), CD45RA, CD56, CD57, CD61, CD68, CD71, CD74, CD79a, CD99, CD117 (c-KIT), CD123, CD138, CD163, CDX-2, CDX-2/CK-7, CEA (M), CEA (P), chromogranin A, chymotrypsin, CK-5, CK-⅚, CK-7, CK-7/TTF-1, CK-14, CK-17, CK-18, CK-19, CK- 20. CK-HMW, CK-LMW, CMV-IH, COLL-IV, COX- 2, D2-40, DBA44, Desmin, DOG1, EBER-ISH, EBV (LMP1), E-cadherin, EGFR, EMA, ER, ERCC1, Factor VIII (vWF), Factor XIIIa, Fascin, FLI-1, FHS, Galectin-3, Gastrin, GCDFP-15, GFAP, Glucagon, Glycoprotein A, Glypican-3, Granzyme B, Growth Hormone (GH), GST, HAM 56, HMBE-1, HBP, HCAg, HCG, Hemoglobin A, HEP B Core (HBcAg), HEP B SURF, (HBsAg), HepPar1, HER2, Herpes I, Herpes II, HHV-8, HLA-DR, HMB 45, HPL, HPV-IHC, HPV (6/11)-ISH, HPV (16/18)-ISH, HPV (31/33)-ISH, HPV WSS -ISH, HPV high ISH, HPV low ISH, HPV high and low ISH, IgA, IgD, IgG, IgG4, IgM, Inhibin, Insulin, JC Virus-ISH, Kappa-ISH, KER PAN, Ki-67, Lambda-IHC, Lambda-ISH, LH, Lipase, Lysozyme (MURA), Mammaglobin, MART-1, MBP, M-cell tryptase, MEL -5, Melan-A, Melan-A/Ki-67, Meta Cortin, MiTF, MLH-1, MOC-31, MPO, MSH-2, MSH-6, MUC1, MUC2, MUC4, MUC5AC, MUM-1, MYO D1, myogenin, myoglobin, myosin heavy chain, Napsin A, NB84a, NEW-N, NF, NK1-C3, NPM, NSE, OCT-2, OCT-¾, OSCAR, p16, p21, p27/Kip1, p53, p57, p63 , p120, P504S, pan melanoma, PANC.POLY, parvovirus B19, PAX-2, PAX-5, PAX-5/CD43, PAX=5/CD5, PAX-8, PC, PD1, perforin, PGP 9.5 , PLAP, PMS-2, PR, prolactin, PSA, PSAP, PSMA, PTEN, PTH, PTS, RB, RCC, S6, S100, serotonin, somatostatin, surfactant (SP-A), synaptophysin, synuclein, TAU, TCL-1, TCR beta, TdT, Thrombomodulin, Thyroglobulin, TIA-1, TOXO, TRAP, TriView™ Breast, TriView™ Prostate, Trypsin, TS, TSH, TTF-1, Tyrosinase, Ubiquitin, Uroplakin, VEGF, Villin, Vimentin (VIM ), VIP , VZV, WT1 (M) N-terminus, WT1 (P) C-terminus, ZAP-70.
Additionally, examples of non-limiting antibodies that can be used as analyte binding moieties in analyte capture reagents or that can be used in IHC/IF applications disclosed herein include any of the following antibodies (and variants thereof): cell surface proteins , intracellular proteins, kinases (e.g. AGC kinase family (e.g. AKT1, AKT2, PDK1, protein kinase C, ROCK1, ROCK2, SGK3), CAMK kinase family (e.g. AMPK1, AMPK2, CAMK, Chk1, Chk2, Zip), CK1 kinase family, TK kinase family (eg Abl2, AXL, CD167, CD246/ALK, c-Met, CSK, c-Src, EGFR, ErbB2 (HER2/neu), ErbB3, ErbB4, FAK, Fyn, LCK, Lyn , PKT7, Syk , Zap70), STE kinase family (eg ASK1, MAPK, MEK1, MEK2, MEK3 MEK4, MEK5, PAK1, PAK2, PAK4, PAK6), CMGC kinase family (eg Cdk2, Cdk4, Cdk5, Cdk6, Cdk7, Cdk9, Erk1, GSK3, Jnk/MAPK8, Jnk2/MAPK9, JNK3/MAPK10, p38/MAPK) and TKL kinase families (eg, ALK1, ILK1, IRAK1, IRAK2, IRAK3, IRAK4, LIMK1, LIMK2, M3K11, RAF1). , RIP1, RIP3 , VEGFR1, VEGFR2, VEGFR3), Aurora A kinase, Aurora B kinase, IKK, Nemo-like kinase, PINK, PLK3, ULK2, WEE1, transcription factors (e.g. FOXP3, ATF3, BACH1, EGR, ELF3 , FOXA1, FOXA2, FOX01 , GATA), growth factor receptors, tumor suppressors (eg anti-p53, anti-BLM, anti-Cdk2, anti-Chk2, anti-BRCA-1, anti-NBS1, anti-BRCA-2, anti-WRN, anti-PTEN, anti-WT1, anti-p38).
In some embodiments, the analyte capture agent is capable of binding an analyte present within a cell. In some embodiments, the analyte capture agent is capable of binding an analyte on a cell surface, which may include, but is not limited to, receptors, antigens, surface proteins, transmembrane proteins, differentiation protein clusters, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, protein complexes in intercellular interaction, antigen presentation complexes, major histocompatibility complexes, engineered T-cell receptors, T-cell receptors, B-cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modification (eg .phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation or lipidation) status of cell surface proteins, gap junctions and adherens junctions. In some embodiments, the analyte capture agent is capable of binding the analyte to a post-translationally modified cell surface. In such embodiments, based on a given state of post-translational modification (eg, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation), the analyte agent may be specific for a cell surface analyte such that the cell surface analyte profile may include modification information that post-translationally modifies one or more analytes.
In some embodiments, the analyte capture agent includes a barcode domain of the capture agent conjugated or otherwise linked to an analyte binding moiety. In some embodiments, the barcode domain of the capture agent is covalently linked to the analyte binding moiety. In some embodiments, the barcode domain of the capture agent is a nucleic acid sequence. In some embodiments, the barcode domain of the capture agent includes an analyte binding moiety barcode and an analyte capture sequence.
As used herein, the term "analyte binding moiety barcode" refers to a barcode associated with or otherwise identifying an analyte binding moiety. In some embodiments, an analyte bound by an analyte-binding group can also be identified by identifying the analyte-binding group and its associated analyte-binding group barcode. The barcode for the analyte binding group can be a nucleic acid sequence of a given length and/or a sequence associated with the analyte binding group. Barcodes for an analyte binding group can generally comprise any of the various aspects of the barcodes described herein. For example, an analyte capture agent specific for one type of analyte may have a first capture agent barcode domain associated with it (e.g., that includes a first analyte binding moiety barcode), while another analyte has a specific analyte capture agent may have a different barcode domain. code of the capture agent coupled to it (eg includes a second barcode of the analyte binding part). In some aspects, such a capture agent barcode domain may include an analyte binding moiety barcode that enables identification of an analyte binding moiety coupled to the capture agent barcode domain. Selection of the barcode domain of the capture agent can allow significant diversity in the array, while also readily binding to most analyte-binding moieties (eg, antibodies or aptamers) and being easily detected (eg, using sequencing or array technology).
In some embodiments, the capture agent barcode domain of the analyte capture agent includes an analyte capture sequence. As used herein, the term "analyte capture sequence" refers to a region or portion configured to hybridize, bind, splice, or otherwise interact with the capture domain of a capture probe. In some embodiments, the analyte capture sequence comprises a nucleic acid sequence that is complementary or substantially complementary to the capture domain of the capture probe such that the analyte capture sequence hybridizes to the capture domain of the capture probe. In some embodiments, the analyte capture sequence comprises a poly(A) nucleic acid sequence that hybridizes to a capture domain comprising a poly(T) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a poly(T) nucleic acid sequence that hybridizes to a capture domain comprising a poly(A) nucleic acid sequence. In some embodiments, the analyte capture sequence comprises a non-homopolar nucleic acid sequence hybridized to a capture domain comprising a non-homopolar nucleic acid sequence that is complementary (or substantially complementary) to the non-homopolar nucleic acid sequence of the analyte capture region.
In some embodiments of any of the spatial analysis methods described herein using analyte capture agents, the barcode domains of the capture agent can be linked directly to analyte binding moieties or can be attached to beads, molecular lattices, e.g., linear, spherical, cross linkages or other polymers, or other frameworks linked or otherwise linked to an analyte-binding moiety, which allow multiple capture agent barcode domains to be linked to a single analyte-binding moiety. Attachment (coupling) of the barcode domain of the capture agent to the binding moiety of the analyte can be accomplished by any number of direct or indirect, covalent or non-covalent associations or linkages. For example, where the barcode domain of the capture agent is fused to an analyte-binding portion comprising an antibody or antigen-binding fragment, such barcode domain of the capture agent may be covalently linked to the antibody portion or antigen-binding fragment using chemical coupling techniques ( eg Lightning-Link® Antibody Labeling Kit available from Innova Biosciences). In some embodiments, the barcode domain of the capture agent can be linked to an antibody or antigen-binding fragment using a non-covalent linkage mechanism (eg, using a biotinylated antibody and an oligonucleotide or an oligonucleotide containing one or more biotinylated linkers). beads, coupled to oligonucleotides with avidin or streptavidin linkers.) Antibody and oligonucleotide biotinylation techniques can be used, eg Fang et al.,nucleic acid research(2003), 31(2):708-715, the entire contents of which are incorporated herein by reference. Similarly, protein and peptide biotinylation techniques have been developed and are available, for example, in the U.S. patent no. LOUSE. patent no. 6,265,552, the entire contents of which are incorporated herein by reference. Additionally, chemical click reactions such as methyltetrazine-PEG5-NHS ester reaction, TCO-PEG4-NHS ester reaction, etc. can be used to couple the barcode domain of the capture agent to the analyte linker. Reactive moieties on the analyte binding group can also include amines for targeting aldehydes, amines for targeting maleimides (e.g. free thiols), click chemistry targeting compounds (e.g. alkyne azide for streptavidin targeting, phosphate for EDC targeting which in turn targets active esters (e.g., NH2). The reactive group on the analyte-binding group can be a compound or a group that binds to the reactive group on the analyte-binding group. Examples of strategies for conjugating analyte-binding moieties to capture barcode domains include the use of commercial kits (e.g. . Solulink, Thunder link), slight reduction of the hinge region and incorporation of maleimide labels, dye-assisted click chemical reactions, conjugation with labeled amides (e.g. copper-free), as well as periodic oxidation and amine conjugation of sugar chains. When the analyte binding moiety is an antibody , the antibody can be modified prior to or simultaneously with oligonucleotide conjugation. For example, antibodies can be used with chemical substrate-permissive mutants of β-1,4-galactosyltransferase, GalT (Y289L), and the uridine diphosphate-GalNAz analog carrying azide uridine diphosphate- N-acetylgalactosamine is glycosylated. Modified antibodies can be conjugated to oligonucleotides bearing dibenzocyclooctyne-PEG4-NHS groups. In some embodiments, certain steps (eg, COOH activation (eg, EDC) and homobifunctional cross-linkers) can be avoided to prevent conjugation of the analyte linking group to itself. In some embodiments of any of the spatial analysis methods described herein, the analyte capture agent (e.g., an analyte-binding moiety linked to an oligonucleotide) can be obtained, for example, by transfection (e.g., using transfectamine, cationic polymerization (e.g., calcium phosphate) in cells or by electroporation), transduction (eg, using bacteriophage or recombinant viral vectors), mechanical delivery (eg, magnetic beads), lipids (eg, 1,2-diol acyl-sn-glycero-3-phosphocholine (DOPC)), or through the conveyor. Exosomes can be used to deliver analyte capture agents into cells. For example, a first cell can be generated that releases exosomes containing an analyte capture agent. Analyte capture agents can be attached to exosomal membranes. The analyte capture agent may reside within the cytosol of the exosome. The released exosomes can be collected and delivered to another cell, thereby delivering the analyte capture agent to the other cell. The analyte capture agent can be released from the exosome membrane before, during, or after delivery to the cell. In some embodiments, the cells are permeabilized to allow coupling of the analyte capture agents to intracellular components such as, but not limited to, intracellular proteins, metabolites, and nuclear membrane proteins. After intracellular delivery, analyte capture agents can be used to analyze intracellular components as described herein.
In some embodiments of any of the spatial analysis methods described herein, the capture agent barcode domain associated with the analyte capture agent may include a modification that renders it non-amplifiable by polymerase. In some embodiments, the barcode domain of the capture agent can be used as a template, instead of a primer, when binding to the capture domain of a capture probe or nucleic acid in a sample for a primer extension reaction. When the barcode domain of the capture agent also includes a barcode (eg, a barcode with an analyte binding portion), such a design can increase the efficiency of molecular barcoding by increasing the affinity between the barcode domain of the capture agent and non-encoded sample nucleic acids and eliminate the potential formation of adapter artifacts. In some embodiments, the barcode domain of the capture agent may include a random N-mer sequence that has been modified to be restricted so that it cannot be extended by the polymerase. In some cases, the composition of random N-mer sequences can be engineered to maximize binding efficiency to free, non-coding ssDNA molecules. The design may include random sequence composition with higher GC content, partially randomized sequences with G or C fixed at specific positions, use of guanosine, use of locked nucleic acids, or any combination thereof.
Modifications used to block polymerase bud extension can be carbon spacers or dideoxynucleotides of various lengths. In some embodiments, the modification can be an abasic site with an abasic or pyrimidine structure, a base analog, or a phosphate backbone analog, such as an amide-linked N-(2-aminoethyl)-glycine, tetrahydrofuran, or 1',2'-dideoxyribose backbone. Modifications can also be uracil bases, 2'OMe modified RNA, C3-18 spacers (eg, structures with 3-18 consecutive carbon atoms, such as C3 spacers), glycol polymer spacers (eg, spacer 18 (hexaethylene glycol spacer) , biotin, dideoxynucleotide triphosphate, ethylene glycol, amine or phosphate.
In some embodiments of any of the spatial analysis methods described herein, the barcode domain of the capture agent fused to the analyte binding moiety comprises a cleavable domain. For example, upon binding of an analyte capture agent to an analyte (eg, a cell surface analyte), the barcoded domain of the capture agent can be cleaved off and collected for downstream analysis according to the methods described herein. In some embodiments, the cleavable domain of the capture agent barcode domain includes a U-cleavage element that enables release of the substance from the bead. In some embodiments, the U-resection element can comprise a single-stranded DNA (ssDNA) sequence that contains at least one uracil. Species can be attached to beads via ssDNA sequences. This species can be released by a combination of uracil-DNA glycosylases (eg, removes uracil) and endonucleases (eg, induces ssDNA fragmentation). If the endonuclease generates a 5' phosphate group from the cleavage, an additional enzymatic treatment to remove the phosphate group can be included in downstream processing, for example, prior to binding of additional sequencing processing elements such as Illumina full P5sequence, part P5row, all R1sequence and/or part of R1order.
In some embodiments, a plurality of different classes of analytes (eg, polypeptides) from a biological sample can then be associated with one or more physical properties of the biological sample. For example, multiple different analyte classes can be associated with the location of the analyte in a biological sample. Such information (eg, proteomic information when an analyte binds a partially recognized polypeptide) can be combined with other spatial information (eg, genetic information from biological samples such as DNA sequence information, transcriptome information (ie, transcript sequences) or both). For example, a cell surface protein may be associated with one or more physical properties of the cell (eg, shape, size, activity, or cell type). One or more physical properties can be characterized by a cell image. Cells can be bound with an analyte capture agent comprising an analyte binding moiety that binds a protein on the surface of the cell and an analyte binding unit barcode that recognizes the analyte binding moiety, and the cells can be spatially analyzed (eg, by any of the various methods described herein). analysis). For example, an analyte capture agent bound to a cell surface protein can bind to a capture probe (eg, an array capture probe) that includes a capture structure that interacts with an analyte capture sequence present on the barcode domain of the capture agent. capture reagent to capture the analyte domain. All or part of the capture agent barcode domain (including the barcode of the analyte-binding portion) can be replicated by polymerase using the 3' end of the capture domain as a starting site, thereby generating extended capture probes that include all or part of the complementary copy corresponding to of the capture probe sequence (including the spatial barcode present on the capture probe) and the analyte binding group barcode. In some embodiments, an analyte capture agent having an extended capture agent barcode domain that includes a sequence complementary to the spatial barcode of the capture probe is referred to as a "sterically labeled analyte capture agent".
In some embodiments, a spatial array of spatially labeled analyte capture means may contact the sample, wherein the analyte capture means associated with the spatial array capture the analyte of interest. Analyte capture agents containing extended capture probes, including sequences complementary to the capture probe spatial barcodes and analyte binding moiety barcodes, can then be extracted from the spatially distributed capture probes. This allows reuse of spatial arrays. Samples can be disaggregated into non-aggregated cells (eg single cells) and analyzed using the single cell/droplet method described here. The spatially labeled analyte capture agent can be sequenced to obtain the nucleic acid sequence of the spatial barcode of the capture probe and the barcode of the analyte binding portion of the analyte capture agent. The nucleic acid sequence of the extended capture probe can therefore bind to an analyte (eg, a cell surface protein) and, in turn, to one or more physical properties of the cell (eg, cell shape or type). In some embodiments, the nucleic acid sequence of the extended capture probe can be linked to an intracellular analyte of a nearby cell, wherein the intracellular analyte is released using any of the cell permeabilization or analyte mobilization techniques described herein.
In some embodiments of any of the spatial distribution methods described herein, the capture agent barcode domain released from the analyte capture agent can then be subjected to sequence analysis to identify which analyte capture agents bind the analyte. An analyte profile can be generated for a biological sample based on the presence of capture agent barcode domains and analyte barcode sequences that bind to features associated with a spatial array (eg, features at specific locations). The profile of an individual cell or population of cells can be compared to that of other cells (eg, "normal" cells) to identify changes in analytes that provide diagnostically relevant information. In some embodiments, these profiles are useful for diagnosing various disorders characterized by variations in cell surface receptors, such as cancer and other disorders.
In some embodiments of any of the spatial analysis methods described herein, the method is used to identify immune cell profiles. Immune cells express various adaptive immune receptors associated with immune function, such as T-cell receptor (TCR) and B-cell receptor (BCR). T-cell receptors and B-cell receptors play a role in the immune response by specifically recognizing and binding antigens and helping to destroy them.
The T-cell receptor, or TCR, is a molecule found on the surface of T-cells that is normally responsible for recognizing antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules. TCRs are typically heterodimers of two chains, each a member of the immunoglobulin superfamily, with an N-terminal variable (V) domain and a C-terminal constant domain. In humans, TCRs consist of alpha (α) and beta (β) chains in 95% of T cells and gamma and delta (γ/δ) chains in 5% of T cells. This ratio varies depending on ontogeny and disease state, as well as between species. T lymphocytes are activated via signal transduction when the TCR binds antigenic peptides and MHC (peptide/MHC or pMHC).
Each of the two chains of the TCR contains multiple copies of a gene segment—the variable "V" gene segment, the "D" diversity gene segment, and the junctional "J" gene segment. TCR alpha chain (TCRa) is formed by recombination of V and J segments, while beta chain (TCRb) is formed by recombination of V, D and J segments. Similarly, production of the TCR gamma chain involves recombination of gene segments V and J, while production of the TCR delta chain occurs through recombination of gene segments V, D, and J. The intersections of these specific regions (V and J for α or γ chains, or V, D and J for β or δ chains) correspond to regions of CDR3 that are important for MHC antigen recognition. Complementarity determining regions (eg, CDR1, CDR2 and CDR3) or hypervariable regions are sequences in the variable domains of antigen receptors (eg, T-cell receptors and immunoglobulins) that can be complementary to an antigen. Most of the CDR diversity is present in CDR3, and the diversity is generated by somatic recombination events during T lymphocyte development. The unique nucleotide sequences that arise during gene alignment can be called clonotypes.
B cell receptor or BCR is a molecule found on the surface of B cells. The antigen-binding portion of the BCR consists of a membrane-bound antibody, which, like most antibodies such as immunoglobulins, has a unique and randomly determined antigen-binding site. The antigen-binding portion of the BCR includes membrane-bound immunoglobulin molecules of one isotype (such as IgD, IgM, IgA, IgG, or IgE). When a B cell is activated by its first encounter with a cognate antigen, the cell proliferates and differentiates, giving rise to a population of antibody-secreting plasma B cells and memory B cells. Different immunoglobulin isotypes differ in their biological characteristics, structure, target specificity and distribution. There are multiple molecular mechanisms for generating initial diversity, including genetic recombination at multiple sites.
The BCR consists of two genes, IgH and IgK (or IgL), which encode the heavy and light chains of the antibody. Immunoglobulins are produced by recombination between gene segments, sequence diversification at the junctions of these segments and point mutations throughout the genes. Each heavy chain gene contains multiple copies of three different gene segments — the variable "V" gene segment, the "D" diversity gene segment, and the "J" splicing gene segment. Each light chain gene contains multiple copies of two different gene segments of the variable region of the protein - a variable "V" gene segment and a junctional "J" gene segment.
Recombination produces molecules with one V, D and J segment each. In addition, several bases can be deleted and others added (called N and P nucleotides) at each of the two junctions, resulting in greater diversity. After the activation of B cells, the process of affinity maturation occurs by somatic hypermutation. During this process, progeny of activated B cells accumulate various somatic mutations in the gene, with a higher concentration of mutations in the CDR regions, leading to the production of antibodies with higher affinity for the antigen.
In addition to somatic hypermutation, activated B cells undergo a process of isotype switching. Antibodies with the same variable segment can have different forms (isotypes), depending on the constant segment. While all naïve B cells express IgM (or IgD), activated B cells express primarily IgG, but also IgM, IgA, and IgE. This change in expression from IgM (and/or IgD) to IgG, IgA or IgE occurs through recombination events, resulting in a cell specialized to produce a particular isotype. The unique nucleotide sequences that emerge during gene alignment can similarly be called clonotypes.
Certain methods described herein are used to analyze different sequences, such as different clonotypes, of TCRs and BCRs from immune cells. In some embodiments, the method is used to analyze TCR alpha chain, TCR beta chain, TCR delta chain, TCR gamma chain, or any fragment thereof (e.g., variable regions, including V(D)J or VJ region, constant region, transmembrane regions , their fragments, their combinations and combinations of their fragments). In some embodiments, the methods described herein can be used to analyze a B cell receptor heavy chain, a B cell receptor light chain, or any fragment thereof (e.g., a variable region including a V(D)J or VJ region, a constant region, regions spanning on the membrane, their fragments, their combinations and combinations of their fragments).
Where immune cells are to be analyzed, exemplary sequences that can be used in any of a variety of barcode sequence attachment manipulations and/or amplification reactions may include gene-specific sequences that target genes or gene regions of immune cell proteins, such as immune receptors. Such gene sequences include, but are not limited to, various T-cell receptor alpha variable genes (TRAV genes), T-cell receptor alpha junction genes (TRAJ genes), T-cell receptor alpha constant genes (TRAC genes), receptor alpha sequence T-cell β variable gene (TRBV gene), T-cell receptor β diversity gene (TRBD gene), T-cell receptor β junction gene (TRBJ gene), T-cell receptor β constant gene (TRBC gene), T-cell receptor gamma variable gene (TRGV gene) ), T-cell receptor gamma junction gene (TRGJ gene), T-cell receptor gamma constant gene (TRGC gene), T-cell receptor delta variable gene (TRDV gene), T-cell receptor delta diversity gene (TRDD gene), T cell receptor delta junction gene (TRDJ gene) and T cell receptor delta constant gene (TRDC gene).
In some embodiments, the analyte binding moiety is based on a major histocompatibility complex (MHC) class I or class II. In some embodiments, the analyte-binding moiety is an MHC multimer, including, but not limited to, MHC dextromers, MHC tetramers, and MHC pentamers (see, e.g., US Patent Application Publication Nos. US 2018/0180601 and US 2017/0343545, MHC (e.g., soluble MHC monomeric molecules), including whole or partial MHC peptides, useful as part of an analyte-binding capture reagent linked to the barcode domain of the capture reagent, including recognition of the associated MHC assay (thus, for example, TCR binding partners In some embodiments, the MHC is used to analyze one or more cell surface features of the T cell, for example, in some cases, multiple MHC in the MHC bind together into a larger complex (MHC multimer) to increase the binding affinity of the MHC to the TCR through the synergistic effect of multiple ligand binding.
(c) Substrates
For the spatial array-based assay methods described here, the substrate serves as a support for direct or indirect attachment of capture probes to the array features. Additionally, in some embodiments, a substrate (eg, the same substrate or a different substrate) can be used to provide support for a biological sample, such as particularly thin sections of tissue. Accordingly, a "substrate" is a support that is insoluble in aqueous liquids and that allows positioning of biological samples, analytes, signatures and/or capture probes on the substrate.
Furthermore, as used herein, when not preceded by the modifier "chemical", "substrate" refers to a member having at least one surface that typically serves to provide physical support for a biological sample, analyte, and/or any other role of the substance . Chemical and/or physical moieties, reagents and structures described herein. Substrates can be phase-changed or transitioned from a variety of solid materials, gel-based materials, colloidal materials, semi-solid materials (eg, at least partially cross-linked materials), fully or partially cured materials, and materials that have been cured to provide physical support. Examples of substrates that can be used in the methods and systems described herein include, but are not limited to, glass slides (e.g., glass slides made of various glasses, glass slides made of various polymers), hydrogels, layers and/or membranes, membranes (e.g. . porous membranes), flow cells, cuvettes, wafers, plates or combinations thereof. In some embodiments, the substrate may optionally include functional elements such as indentations, protrusions, microfluidic elements (eg, channels, reservoirs, electrodes, valves, seals), and various markings, as discussed in more detail below.
(i) Properties of the substrate
The substrate may generally be of any suitable shape or format. For example, the substrate may be flat, curved, eg convex or concavely curved towards the area where the interaction between the biological sample (eg tissue sample) and the substrate occurs. In some embodiments, the substrate is flat, such as flat, chip or glass. The substrate may contain one or more patterned surfaces (eg, channels, holes, ridges, ridges, pits, etc.) within the substrate.
The base can be of the desired shape. For example, the substrate may generally be thin, flat (eg, square or rectangular). In some embodiments, the substrate structure has rounded corners (eg, for added security or robustness). In some embodiments, the base structure has one or more cut corners (eg, for use with sliders or cross tables). In some embodiments, when the base structure is planar, the base structure may be any suitable type of support with a flat surface (eg, a chip or an object slide, such as a microscope slide).
The substrate may optionally include various structures such as, but not limited to, ridges, ridges, and channels. The substrate may be micropatterned to limit lateral diffusion (eg, to prevent spatial overlap of the bar code). Substrates modified with such structures can be modified to allow binding of analytes, features (eg beads) or probes at different sites. For example, substrate modification sites with different structures may or may not be adjacent to other sites.
In some embodiments, the surface of the substrate can be modified to form discrete sites that can have or accept only one feature. In some embodiments, the surface of the substrate can be modified to hold features at random locations.
In some embodiments, the surface of the substrate is modified to contain one or more holes using techniques such as, but not limited to, embossing, microetching, or molding techniques. In some embodiments where the substrate includes one or more wells, the substrate may be concave or hollow slides. For example, the hole may be formed by one or more shallow depressions in the surface of the substrate. In some embodiments, when the substrate includes one or more holes, the holes can be formed by attaching a cartridge (eg, a cartridge containing one or more chambers) to the surface of the substrate structure.
In some embodiments, each substrate structure (eg, wells or features) can host a different capture probe. Different capture probes attached to each structure can be identified based on the location of the structure in or on the surface of the substrate. Examples of substrates include arrays in which individual structures are placed on a substrate, including, for example, those having openings for receiving features.
In some embodiments where the substrate is modified to contain one or more structures including, but not limited to, holes, protrusions, ridges, features, or markings, these structures may include physically altered sites. For example, substrates modified with various structures may include physical properties including but not limited to physical configuration, magnetic or compressive forces, chemical functionalization sites, chemical alteration sites, and/or electrostatic alteration sites. In some embodiments where the substrate is modified to contain various structures, including but not limited to holes, protrusions, ridges, features or markings, the structures are applied in a pattern. Alternatively, the structures may be randomly distributed.
A substrate (eg, a bead or array element) may include tens of thousands to hundreds of thousands or millions of individual oligonucleotide molecules (eg, at least about 10,000, 50,000, 100,000, 500,000, 1,000,000, 10,000,000, 100,000,000, 1,000,000,000, or 10,000,000,000 oligonucleotide molecules).
In some embodiments, the substrate includes one or more markers on the surface of the substrate, e.g., to provide guidance in associating spatial information with characterization of a target analyte. For example, the base may be marked with a grid of lines (eg to allow easy estimation of the size of objects seen under magnification and/or to provide a reference area for counting objects). In some embodiments, reference marks may be included on the substrate. Such indicia may be produced using techniques including, but not limited to, printing, sandblasting, and surface deposition.
In some embodiments, imaging may be performed using one or more reference markers, i.e., objects that are in the field of view of the imaging system and appear in the resulting image. Reference marks are often used as reference points or measuring rulers. Fiducial labels may include, but are not limited to, detectable labels such as fluorescent labels, radioactive labels, chemiluminescent labels, and colorimetric labels. For example, Carter et al. describe the use of fiducial markers to stabilize and orient biological samples,Applied optics46:421-427, 2007), the entire contents of which are incorporated herein by reference. In some embodiments, fiducial markers can be physical particles (eg, nanoparticles, microspheres, nanospheres, beads, pillars, or any other exemplary physical particles described herein or known in the art).
In some embodiments, reference marks may be present on the substrate to ensure orientation of the biological sample. In some embodiments, the microspheres can be attached to a substrate to help orient the biological sample. In some examples, the microspheres attached to the substrate can generate an optical signal (eg, fluorescence). In another example, the microspheres can be attached to a portion (eg, the corners) of the array in a particular pattern or design (eg, a hexagonal design) to help orient the biological sample over a series of features on the substrate. . In some embodiments, the quantum dots can be bound to a substrate to help orient the biological sample. In some examples, quantum dots attached to a substrate can generate optical signals.
In some embodiments, the fiducial marker can be an immobilized molecule with which a detectable signal molecule can interact to generate a signal. For example, a labeled nucleic acid can be attached or attached to a chemical moiety that can fluoresce when exposed to light of a specific wavelength (or range of wavelengths). Such marker nucleic acid molecules can be contacted with the array before, while or after the tissue sample is stained to visualize or represent a tissue section. Although not required, it may be useful to use a marker that can be detected using the same conditions (eg, imaging conditions) used to detect labeled cDNA.
In some embodiments, fiducial markers are included to facilitate orientation of the tissue sample or image thereof relative to immobilized capture probes on the substrate. Many methods can be used to label the arrays so that the labels can only be detected when tissue sections are imaged. For example, molecules, such as fluorescent signal-generating molecules, can be immobilized directly or indirectly on the substrate surface. Marks can be placed on the substrate in the form of a pattern (eg border, one or more lines, one or more lines, etc.).
In some embodiments, the reference markers may be placed randomly within the field of view. For example, oligonucleotides containing fluorophores can be randomly printed, imprinted, synthesized, or attached to random locations on a substrate such as glass. The tissue section may be contacted with a substrate such that the fluorophore-containing oligonucleotides are in contact with or in the vicinity of cells or cell components (eg, mRNA or DNA molecules) from the tissue section. Substrate and tissue cross-sectional images can be obtained, and the location of the fluorophore within the tissue cross-sectional image can be determined (eg, by viewing an optical image of a tissue cross-section overlaid with fluorophore detection). In some embodiments, the reference marks can be placed exactly in the field of view (eg, at known locations on the substrate). In this case, reference markers can be imprinted, attached or synthesized on the substrate and brought into contact with the biological sample. Typically, images of the sample and reference marks are taken, and the positions of the reference marks on the substrate can be confirmed by observing the images.
In some embodiments, fiducial markers can be immobilized molecules (eg, physical particles) attached to a substrate. For example, fiducial markers can be nanoparticles, such as nanorods, nanowires, nanocubes, nanopyramids, or spherical nanoparticles. In some examples, the nanoparticles may be made of heavy metals (eg, gold). In some embodiments, the nanoparticles may be made of diamond. In some embodiments, the reference marks may be visible to the naked eye.
As described herein, any of the fiducial markers described herein (eg, microspheres, beads, or any other physical particles described herein) may be located in a portion (eg, corner) of an array in a particular pattern or design (eg, corner). , hexagonal design) to aid in the placement of biological samples on a series of substrate elements. In some embodiments, reference markers located on a portion (eg, a corner) of an array (eg, an array on a substrate) may be shaped or designed with at least 1, at least 2, at least 3, or at least 4 unique patterns. In some examples, reference marks located at the corners of an array (eg, an array on a substrate) may have four unique patterns of reference marks.
In some examples, reference markers may surround the string. In some embodiments, fiducial markers enable the detection of mirror images, for example. In some embodiments, the reference markers may completely surround the array. In some embodiments, the reference markers may not completely surround the array. In some embodiments, the reference marks identify the corners of the array. In some embodiments, one or more reference markers identify the center of the array. In some embodiments, the reference marks include patterned dots, wherein one or more of the patterned reference marks are about 100 microns in diameter. The diameter of the reference marks can be any useful diameter including, but not limited to, 50 microns to 500 microns in diameter. The reference marks may be spaced such that the center of one reference mark is between 100 microns and 200 microns from the center of one or more other reference marks around the array. In some embodiments, the array with surrounding reference marks is approximately 8 mm x 8 mm. In some embodiments, the array without surrounding fiducial marks is less than 8 mm by 50 mm.
In some embodiments, the array may be enclosed within a frame. In other words, the scope of an array can have reference labels such that the array is surrounded or mostly surrounded. In some embodiments, the array perimeter can be a fiducial marker (eg, any of the fiducial markers described herein). In some embodiments, the extent of the array may be uniform. For example, fiducial markers may connect or substantially connect consecutive corners of the array in such a way that the non-corner portions of the perimeter of the array are the same on all sides (eg, four sides) of the array. In some embodiments, the fiducial markers attached to the non-corner portions of the perimeter may be shaped or designed to facilitate orientation of the biological sample on the array. In some embodiments, the particles attached to the non-corner portions of the perimeter may be shaped or formed into at least 1, at least 2, at least 3, or at least 4 patterns. In some embodiments, the pattern may have at least 2, at least 3, or at least 4 unique reference mark patterns on non-corner perimeter portions of the array.
In some embodiments, the array may include at least two reference markers (e.g., at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12. At least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100 reference marks or more (eg, hundreds, thousands or tens of thousands of reference marks)) at different locations on the surface of the substrate. Reference marks can be placed on the substrate in the form of a pattern (eg border, one or more lines, one or more lines, etc.).
A number of different substrates can be used for the above purposes. In general, the substrate can be any suitable support material. Examples of substrates include, but are not limited to, glass, modified and/or functionalized glass, hydrogels, films, membranes, plastics including, for example, acrylic, polystyrene, styrene copolymers and other materials, polypropylene, polyethylene, polybutylene, polyurethane, PTFE™ , cycloolefin, polyimide, etc.), nylon, ceramic, resin, Zeonor, silicon or silicon-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles and polymers such as polystyrene, cycloolefin copolymer (COC) , cycloolefin polymer (COP), polypropylene, polyethylene polycarbonate or their combinations.
Of the examples of substrate materials discussed above, polystyrene is a suitable hydrophobic material for binding negatively charged macromolecules because it generally contains few hydrophilic groups. For nucleic acids immobilized on glass slides, nucleic acid immobilization can be enhanced by increasing the hydrophobicity of the glass surface. This improvement may allow for a relatively denser formation (eg, providing improved specificity and resolution).
In another example, the substrate may be a flow cell. The flow cell may be formed from any of the foregoing materials and may include channels that allow reagents, solvents, elements, and analytes to pass through the flow cell. In some embodiments, the biological sample embedded in the hydrogel is assembled into a flow cell (eg, the flow cell is used to introduce the hydrogel into the biological sample). In some embodiments, the biological sample embedded in the hydrogel is not assembled into a flow cell. In some embodiments, the biological sample embedded in the hydrogel can then be prepared and/or isometrically expanded as described herein.
(ii) Conductive substrate
Conductive substrates (eg, electrophoretically compatible arrays) produced as described herein can be used for spatial detection of analytes. For example, electrophoretic fields can be applied to facilitate the migration of analytes into barcoded oligonucleotides (e.g., capture probes) on arrays (e.g., capture probes immobilized on paper, capture probes immobilized in hydrogel membranes) or capture probes immobilized on string) . slides with a conductive coating). In some versions, an electrophoretic assembly can be installed. For example, the anode and cathode can be positioned such that an array of capture probes (eg, capture probes immobilized on paper, in a hydrogel film, or on a glass slide with a conductive needle coating) and the biological sample are located between the anodes and cathodes. In such embodiments, the analytes in the biological sample actively migrate and are captured by the capture probes on the conductive substrate. Biological samples can be prepared (eg, permeabilized) according to any of the methods described herein. In some embodiments, after electrophoretic-assisted capture of the analyte, barcoded oligonucleotides (eg, capture probes) and the captured analyte.
In some embodiments, the conductive substrate may comprise glass (eg, a glass slide) that has been coated with a substance or otherwise modified to impart conductive properties to the glass. In some embodiments, the glass slides may be coated with a conductive coating. In some embodiments, the conductive coating includes tin oxide (TO) or indium tin oxide (ITO). In some embodiments, the conductive coating includes a transparent conductive oxide (TCO). In some embodiments, the conductive coating includes aluminum doped zinc oxide (AZO). In some embodiments, the conductive coating includes fluorine-doped tin oxide (FTO).
In some embodiments, oligonucleotides (e.g., capture probes, e.g., any of the various capture probes described herein) can be generated on a conductive substrate (e.g., any of the conductive substrates described herein).) in dot or printed arrays. For example, the arrays described herein may be compatible with active analyte capture methods (eg, any analyte capture method described herein, including but not limited to electrophoretic capture methods). In some embodiments, the conductive substrate is a porous medium. Non-limiting examples of porous media that can be used in the methods described herein that employ active analyte capture include nitrocellulose or nylon membranes. In some embodiments, porous media useful in the methods described herein that employ active analyte capture include paper. In some embodiments, the oligonucleotides can be printed on paper substrates. In some embodiments, printed oligonucleotides may interact with a substrate (eg, interact with paper fibers). In some embodiments, printed oligonucleotides may be covalently bound to a substrate (eg, bound to paper fibers). In some embodiments, the oligonucleotides in the molecular precursor solution can be printed onto a conductive substrate (eg, paper). In some embodiments, the molecular precursor solution can be polymerized to produce a gel pad on a conductive substrate (eg, paper). In some embodiments, the molecular precursor solution can be photopolymerized (eg, photocured). In some embodiments, gel beads (eg, any of the various gel beads described herein) containing oligonucleotides (eg, barcoded oligonucleotides such as capture probes) can be printed on a conductive substrate such as paper. In some embodiments, the printed oligonucleotides may be covalently linked to the gel matrix.
(iii) color
In some embodiments, the surface of the substrate may be coated with a cell-permissive coating to allow attachment of living cells. A "cell-permissive coating" is a coating that enables or assists cells to maintain cell viability (eg, remain viable) on a substrate. For example, a cell-permissive coating may enhance cell attachment, cell growth and/or cell differentiation, e.g. a cell-permissive coating can provide nourishment to living cells. Cell-permissive coatings may include biological and/or synthetic materials. Non-limiting examples of cell permissive coatings include coatings with one or more extracellular matrix (ECM) components (eg, proteoglycans and fibrin proteins such as collagen, elastin, fibronectin, and laminin), poly lysine, poly(L)-ornithine, and/or biocompatible silicones (eg CYTOSOFT®). For example, a cell-permissive coating that includes one or more extracellular matrix components may include collagen type I, collagen type II, collagen type IV, elastin, fibronectin, laminin, and/or vitronectin protein. In some embodiments, the cell permissive coating includes a solubilized basement membrane preparation (eg, MATRIGEL®) extracted from Engelbreth-Holm-Swarm (EHS) murine sarcoma. In some embodiments, the cell permissive coating includes collagen. Cell-permissive coatings can be used to grow adherent cells on spatial barcode arrays or to maintain cell viability in tissue samples or sections when in contact with spatial barcode arrays.
In some embodiments, the substrate is coated with a surface treatment, such as poly(L)-lysine. Additionally or alternatively, the substrate can be treated by silanization, for example, with epoxysilanes, aminosilanes and/or treatment with polyacrylamide.
In some embodiments, the substrate is treated to reduce or reduce non-specific hybridization of the analyte within or between features. For example, the treatment may include coating the substrate with a hydrogel, film and/or membrane, thereby creating a physical barrier to non-specific hybridization. Any suitable hydrogel can be used. For example, hydrogel matrices according to U.S. Pat. patents no. 6,391,937, 9,512,422 and 9,889,422 and U.S. patents no. Publications of applications no. US 2017/0253918 and US 2018/0052081. The entire contents of each of the above documents are incorporated herein by reference.
Treatment may involve the addition of a functional group that is reactive or can be activated to become reactive upon application of a stimulus (eg, a photoreactive functional group). Treatment may include treatment with a polymer that has one or more physical properties (eg, mechanical, electrical, magnetic, and/or thermal) that minimize non-specific binding (eg, activating the substrate at specific sites to allow the analyte to hybridize at those sites).
"Conditionally removable coating" is a coating that can be removed from the surface of the substrate after application of a release agent. In some embodiments, the conditionally removable coating comprises a hydrogel described herein, e.g., a hydrogel comprising a polypeptide-based material. Non-limiting examples of hydrogels containing peptide-based materials include synthetic peptide-based materials characterized by the incorporation of spider silk and transmembrane fragments of human muscle L-type calcium channels (eg, PEPGEL®), amphipathic16A peptide residue containing a repeating sequence of arginine-alanine-aspartate-alanine (RADARADARADARADA) (eg PURAMATRIX®), EAK16 (AEEAAKAKAEEAAKAK), KLD12 (KLDLKLDLKLDL) and PGMATRIX™.
In some embodiments, the hydrogel in the conditionally removable coating is a stimuli-responsive hydrogel. Stimuli-responsive hydrogels can undergo a gel-to-solution and/or gel-to-solid transition upon application of one or more external triggers (e.g., release agents). See, for example, Willner,cumulative chemistry. reservoir.50:657-658, 2017, which is incorporated herein by reference in its entirety. Non-limiting examples of stimuli-responsive hydrogels include heat-responsive hydrogels, pH-responsive hydrogels, light-responsive hydrogels, redox-responsive hydrogels, analyte-responsive hydrogels, or combinations thereof. In some embodiments, the stimuli-responsive hydrogel may be a multiple stimuli-responsive hydrogel.
A "release agent" or "external trigger" is a means that enables the release of the release coating from the substrate when the release agent is applied to the release coating. External triggers or release agents may include physical triggers such as thermal, magnetic, ultrasonic, electrochemical and/or optical stimuli as well as chemical triggers such as pH, redox reactions, supramolecular complexes and/or biocatalytically driven reactions. See, for example, Echeverria et al.,gel(2018), 4, 54; doi:10.3390/gels4020054, which is incorporated herein by reference in its entirety. The type of "releasing agent" or "external trigger" may depend on the type of conditionally removable coating. For example, conditionally removable coatings characterized by redox-responsive hydrogels can be removed after application of a release agent including a reducing agent such as dithiothreitol (DTT). As another example, pH-responsive hydrogels can be removed after application of a pH-altering release agent.
(iv) Gel substrate
In some embodiments, the hydrogel may form a substrate. The term "hydrogel" herein refers to a macromolecular polymer gel that contains a network. In a network, some polymer chains may optionally be cross-linked, although cross-linking does not always occur. In some embodiments, the substrate includes a hydrogel and one or more other materials. In some embodiments, the hydrogel is placed over one or more other materials. For example, a hydrogel can be preformed and then placed on top of, under, or in any other configuration one or more other materials. In some embodiments, hydrogel formation occurs upon contact with one or more other materials during substrate formation. Hydrogel formation can also occur within structures (eg pits, ridges, features, protrusions and/or markings) located on the substrate. When the substrate contains a gel (eg, hydrogel or gel matrix), the oligonucleotides within the gel can be attached to the substrate.
In some embodiments, the hydrogel may comprise hydrogel subunits. The hydrogel subunits may comprise any suitable hydrogel subunit such as, but not limited to, acrylamide, bisacrylamide, polyacrylamide and its derivatives, polyethylene glycol and its derivatives (eg, PEG-acrylate (PEG-DA), PEG-RGD) , gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polybutylene oxide, polyvinylpyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly( hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, sugars, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, etc., or combinations thereof.
In some embodiments, the hydrogel comprises a hybrid material, e.g. hydrogel material contains components of synthetic and natural polymers. Examples of suitable hydrogels are described, for example, in US Pat. Nos. 6,391,937, 9,512,422 and 9,889,422, and in US Pat. Nos. Application publication no. 2017/0253918, 2018/0052081 and 2010/0055733, the entire contents of which are incorporated herein by reference.
In some embodiments, cross-linking agents and/or initiators are added to the hydrogel subunits. Examples of cross-linking agents include, but are not limited to, bisacrylamide and diaziridines. Examples of initiators include, but are not limited to, azobisisobutyronitrile (AIBN), riboflavin, and L-arginine. The inclusion of cross-linkers and/or initiators can result in increased covalent bonding between biomacromolecules that interact in subsequent polymerization steps.
In some embodiments, the hydrogel may have a colloidal structure, such as agarose, or a polymeric network, such as gelatin. In some embodiments, the hydrogel is a homopolymeric hydrogel. In some embodiments, the hydrogel is a copolymer hydrogel. In some embodiments, the hydrogel is a polymer interpenetrating polymer hydrogel.
In some embodiments, some of the hydrogel subunits polymerize (eg, undergo "formation") and are covalently or physically cross-linked to form the hydrogel network. For example, hydrogel subunits can be polymerized by any method including, but not limited to, thermal crosslinking, chemical crosslinking, physical crosslinking, ionic crosslinking, photocrosslinking, free radical initiated crosslinking, addition reactions, condensation reactions, water soluble crosslinking reactions, radiation cross-linking (eg X-rays, electron beams) or their combinations. Techniques such as photolithographic photopolymerization can also be used to form hydrogels.
In some embodiments, gel beads containing oligonucleotides (eg, barcoded oligonucleotides, such as capture probes) can be deposited on a substrate (eg, glass slide). In some embodiments, the gel pad can be deposited on a substrate (eg, glass slide). In some embodiments, the gel pads or beads are deposited on the substrate in an array. In some embodiments where gel pads or gel beads are deposited on a substrate in an array, a hydrogel molecule precursor solution can be deposited on top of the array (eg, a gel pad or array of gel beads on a glass slide). In some embodiments, the hydrogel molecular precursor solution can be polymerized such that the deposited gel pad or gel beads are immobilized within the polymerized hydrogel. Any suitable polymerization method or (eg, any of the various methods described herein) may be used. In some embodiments, the polymer hydrogel comprising the gel pad or gel bead can be removed (eg, peeled off) from a substrate (eg, glass slide) such that the gel bead or gel bead is immobilized on the hydrogel in an adhesive. In some embodiments, a polymeric hydrogel comprising gel pads or beads is a conductive substrate (as described herein), which can be used in accordance with any of the various analyte capture methods described herein (eg, for electrophoretic migration of captured analytes).
Arrays can be prepared by depositing elements (eg, droplets, beads) onto a substrate surface to generate spatially labeled arrays. Methods of depositing (eg, droplet manipulation) features are known in the art (see U.S. Patent Application Publication No. 2008/0132429, Rubina, A.Y., et al.,BiotechnologyMay 2003, 34(5):1008-14, 1016-20, 1022 and Vasiliskov et al.Biotechnology1999 September;27(3):592-4, 596-8, p.600. Each is incorporated herein by reference in its entirety). Features can be printed or deposited at specific locations on the substrate (eg inkjet printing). In some embodiments, each feature may have a unique oligonucleotide that acts as a spatial barcode. In some embodiments, each feature may have capture probes for multiplexing (eg, capture of multiple analytes or analyte species, such as proteins and nucleic acids). In some embodiments, electric fields can be used to print or deposit features at specific locations. Features may include photocrosslinkable polymer precursors and oligonucleotides. In some embodiments, photo-crosslinkable polymer precursors can be deposited into structured features on the substrate (eg, wells).
"Photocrossable polymer precursor" refers to a compound that cross-links and/or polymerizes when exposed to light. In some embodiments, one or more photoinitiators may also be included to induce and/or promote polymerization and/or cross-linking. See e.g. Choi et al.Biotechnology2019 Jan;66(1):40-53, which is hereby incorporated by reference in its entirety.
Non-limiting examples of photocrosslinkable polymer precursors include poly(ethylene glycol) diacrylate (PEGDA), gelatin methacryloyl (GelMA), and methacrylic hyaluronic acid (MeHA). In some embodiments, the photocrosslinkable polymer precursors include poly(ethylene glycol) diacrylate (PEGDA), gelatin methacrylate (GelMA), methacrylated hyaluronic acid (MeHA), or a combination thereof. In some embodiments, the photo-crosslinkable polymer precursor (eg, PAZAM) can be covalently bound (eg, cross-linked) to the substrate. In some embodiments, the photocrosslinkable polymer precursor is not covalently bound to the surface of the substrate. For example, silane-free acrylamides can be used (see US Patent Application Publication No. 2011/0059865, which is incorporated herein by reference in its entirety). Photocrosslinkable polymer precursors in the elements (eg, droplets or beads) can be polymerized by any known method. Oligonucleotides can be polymerized (eg, copolymerized or copolymerized) in a cross-linked gel matrix. In some embodiments, features comprising photocrosslinkable polymer precursors deposited on the substrate surface may be exposed to ultraviolet light. UV light can induce polymerization of photocrosslinkable polymer precursors and cause features to become gel matrices (eg, gel pads) on substrate surfaces (eg, arrays).
The method of polymerization of the hydrogel subunits can be chosen to form hydrogels with different properties (eg, pore volume, swelling properties, biodegradability, electrical conductivity, transparency and/or permeability of the hydrogel). For example, the hydrogel may include pores of sufficient volume to allow macromolecules (eg, nucleic acids, proteins, chromatin, metabolites, gRNA, antibodies, carbohydrates, peptides, metabolites, and/or small molecules) to enter and exit the hydrogel. Samples (eg tissue sections). It is known that pore volume generally decreases with increasing hydrogel subunit concentration and generally increases with increasing hydrogel subunit to crosslinker ratio. Accordingly, hydrogel compositions can be prepared that include a concentration of hydrogel subunits that allow the passage of such biomacromolecules.
In some embodiments, hydrogel formation on the substrate occurs before, while, or after the features (eg, beads) are attached to the substrate. For example, when capture probes are attached (eg, directly or indirectly) to a substrate, hydrogel formation can be performed on a substrate that already contains capture probes.
(d) niz
In many of the methods described herein, features (as described further below) are located on a substrate. An "array" is a specific arrangement of a set of features, whether irregular or forming a regular pattern. Individual features in a sequence are distinguished from each other based on their relative position in space. Typically, at least two of the plurality of features in the array include different capture probes (eg, any of the example capture probes described herein).
The arrays can be used to measure a large number of analytes simultaneously. In some embodiments, oligonucleotides are used at least in part to generate the array. For example, one or more copies of a single oligonucleotide (eg, a capture probe) may correspond to or be associated directly or indirectly with a given feature in the array. In some embodiments, a given feature in an array includes two or more oligonucleotides (eg, capture probes). In some embodiments, two or more oligonucleotides (e.g., capture probes) linked directly or indirectly to a given feature on the array form a common (e.g., identical) spatial barcode.
(i) Analyte capture array
In some embodiments, the array may include capture probes attached directly or indirectly to the substrate. A capture probe can include a capture domain (eg, a nucleotide sequence) that can specifically bind (eg, hybridize) to an analyte of interest (eg, mRNA, DNA, or protein) in a sample. In some embodiments, binding (eg, hybridization) of a capture probe to a target can be detected and quantified by detecting a visual signal, eg, a fluorophore, heavy metal (eg, silver ion), or chemiluminescent label, that is incorporated into the target. In some embodiments, the intensity of the visual signal correlates with the relative abundance of each analyte in the biological sample. Because arrays can contain thousands or millions of capture probes (or more), arrays can probe many analytes in parallel.
In some embodiments, the substrate includes one or more capture probes designed to capture analytes from one or more organisms. In one non-limiting example, the substrate may comprise one or more capture probes designed to capture mRNA from one organism (eg, human) and one or more capture probes designed to capture mRNA from another organism. Capture probes that capture DNA from organisms (eg bacteria).
Various techniques can be used to attach capture probes to substrates or features. In some embodiments, capture probes are attached directly to features immobilized on the array. In some embodiments, the capture probes are immobilized on the substrate by chemical immobilization. For example, chemical immobilization can occur between a functional group on the substrate and the corresponding functional element on the capture probe. Examples of suitable functional elements in the capture probe may be intrinsic chemical groups of the capture probe, such as hydroxyl groups, or the functional element may be introduced onto the capture probe. An example of a functional group on a substrate is an amine group. In some embodiments, the capture probe to be immobilized includes or is chemically modified to include an amine functional group. Means and methods for such chemical modification are well known in the art.
In some embodiments, the capture probes are nucleic acids. In some embodiments, the capture probe is immobilized on a substrate or feature via its 5' end. In some embodiments, the capture probe is immobilized on a substrate or feature through its 5' end and includes from the 5' to the 3' end: one or more barcodes (eg, spatial barcodes and/or UMIs) and one or more covers the domain. In some embodiments, the capture probe is immobilized on a substrate or feature via its 5' end and comprises from the 5' to the 3' end: a barcode (eg, spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe is immobilized to a substrate or feature via its 5' end and includes from the 5' to the 3' end: a cleavage domain, a functional domain, one or more barcodes (eg, spatial barcode and/or UMI ) and grab the domain.
In some embodiments, the capture probe is immobilized to a substrate or feature via its 5' end and includes from the 5' to the 3' end: a cleavage domain, a functional domain, one or more barcodes (eg, spatial barcode and and/or UMI ), the second functional domain and the capture domain. In some embodiments, the capture probe is immobilized at its 5' end to a substrate or feature and includes from the 5' to the 3' end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain. In some embodiments, the capture probe is immobilized at its 5' end to the substrate or feature and does not include a spatial barcode. In some embodiments, the capture probe is immobilized at its 5' end to the substrate or feature and does not include a UMI. In some embodiments, the capture probe includes a sequence to initiate a sequencing reaction.
In some embodiments, the capture probe is immobilized on a substrate or feature via its 3' end. In some embodiments, the capture probe is immobilized on the substrate or feature through its 3' end and includes from the 3' to the 5' end: one or more barcodes (eg, spatial barcodes and/or UMIs) and one or more captures domain. In some embodiments, the capture probe is immobilized on a substrate or feature via its 3' end and comprises from the 3' to the 5' end: a barcode (eg, spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe is immobilized on a substrate or feature via its 3' end and includes, from the 3' to the 5' end: a cleavage domain, a functional domain, one or more barcodes (eg, a spatial barcode and/or UMI) and capture the domain. In some embodiments, the capture probe is immobilized by its 3' end to a substrate or feature and includes from the 3' to the 5' end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.
The positioning of functional groups within the capture probe to be immobilized can be used to control and shape the binding behavior and/or orientation of the capture probe, for example, functional groups can be placed at the 5' or 3' end of the capture. with a probe or inside a series of needles. In some embodiments, the capture probe may also include a substrate. Typical capture probe substrates to be immobilized include moieties capable of binding such capture probes, for example to amine-functionalized nucleic acids. Examples of such substrates are carboxylic, aldehyde or epoxy resin substrates.
In some embodiments, the substrate on which the capture probes can be immobilized can be chemically activated, e.g. by activating functional groups available on the substrate. The term "active substrate" refers to a material in which interactive or reactive chemical functional groups have been established or enabled by chemical modification procedures. For example, substrates containing carboxyl groups can be activated before use. In addition, some substrates contain functional groups that can react with specific moieties already present in the capture probe.
In some embodiments, a covalent bond is used to directly couple the capture probe to the substrate. In some embodiments, the capture probe is coupled to the substrate indirectly via a linker, eg, a chemical linker, that separates the "first" nucleotide of the capture probe from the substrate. In some embodiments, the capture probe does not directly bind the substrate, but acts indirectly, e.g., by binding a molecule that itself directly or indirectly binds the substrate. In some embodiments, the capture probes are indirectly attached to the substrate (eg, via a polymer-containing solution on the substrate).
In some embodiments where the capture probe is immobilized indirectly on the array property, for example, by hybridization to a surface probe capable of binding the capture probe, the capture probe may further include an upstream sequence (5' to the surface probe capable of hybridizing with the surface probe probe). The 5' end of the needle hybridizes to a nucleic acid, such as RNA from a tissue sample. Separately, the capture domain of the capture probe can be considered as a capture domain oligonucleotide, which can be implemented in the indirect immobilization of the capture probe on the array Example for Synthesis of Capture Probes.
In some embodiments, the substrate consists of an inert material or a substrate (eg, a slide) that has been functionalized, eg, by treating the substrate with a material containing reactive groups that can immobilize the capture probes. See, e.g., WO 2017/019456, the entire contents of which are incorporated herein by reference. Non-limiting examples include polyacrylamide hydrogels on inert substrates (eg, glass slides; see WO 2005/065814 and US Patent Application No. 2008/0280773, the entire contents of which are incorporated herein by reference).
In some embodiments, functionalized biomolecules (eg, capture probes) are immobilized on functionalized substrates using covalent methods. Covalent attachment methods include, for example, condensation of amines and activated carboxylates (eg, N-hydroxysuccinimide esters); condensation of amines and aldehydes under conditions of reductive amination; Diels-Alder[4+2] reaction, 1,3-dipolar cycloaddition reaction, [2+2] cycloaddition reaction and other cycloaddition reactions. Covalent attachment methods also include, for example, click chemistry reactions, including [3+2] cycloaddition reactions (eg, Huisgen 1,3-dipolar cycloaddition and copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC)); ene reactions; Diels-Alder reactions and inverse electron-demanding reactions Diels-Alder reactions; [4+1] cycloadditions of isocyanide and tetrazine; and nucleophilic opening of small carbon rings (eg opening of epoxides with amino oligonucleotides). Covalent attachment methods also include, for example, maleimides and thiols; and p-nitrophenyl ester functionalized oligonucleotides and polylysine functionalized substrates. Covalent bonding methods also include, for example, disulfide reactions; free radical reactions (see, e.g., US Patent No. 5,919,626, the entire content of which is incorporated herein by reference); functional groups directly or indirectly attached to the substrate) and aldehyde-functionalized oligonucleotides (see, for example,Eršovto wait. (1996)procedure. national team. college. know america93, 4913-4918, which is incorporated herein by reference in its entirety).
In some embodiments, functionalized biomolecules (eg, capture probes) are immobilized on functionalized substrates using photochemical covalent methods. Photochemical covalent attachment methods include, for example, immobilization of anthraquinone-conjugated oligonucleotides (see, e.g.,Kochto wait. (2000)bioconjugate chemistry11, 474-483, which is incorporated herein by reference in its entirety).
In some embodiments, functionalized biomolecules (eg, capture probes) are immobilized on a functionalized substrate using non-covalent methods. Non-covalent attachment methods include, for example, biotin-functionalized oligonucleotides and streptavidin-treated substrates (see, e.g.,Holmstromto wait. (1993)Analytical biochemistry209, 278-283 iGillesto wait. (1999)nature biotechnology17, 365-370, the entire contents of which are incorporated herein by reference).
In some embodiments, oligonucleotides (eg, capture probes) can be attached to substrates or features according to the methods set forth in US Pat. 5,888.88. 6,737,236, 7,259,258, 7,375,234, 7,427,678, 5,610,287, 5,807,522, 5,837,860 and 5,472,881; US Patent Application Publication No. 2008/0280773 and 201 1/0059865; Salon et al. (1996)genome research, 639-645;Rogersto wait. (1999)Analytical biochemistry266, 23-30;Stimpsonto wait. (1995)procedure. national team. college. scienceUSA 92, 6379-6383; Beatty et al. (1995)clinical. Chemical45, 700-706; Lamture et al. (1994)nucleic acid research22, 2121-2125; Bell et al. (1999)nucleic acid research27, 1970-1977; Joss et al. (1997)Analytical biochemistry247, 96-101; Nikiforov et al. (1995)Analytical biochemistry227, 201-209; Timofeev et al. (nineteen ninety six)nucleic acid research24, 3142-3148; Chris et al. (1996)nucleic acid research24, 3031-3039; Guo et al. (1994)nucleic acid research22. 5456-5465; Run and Urdia (1990.)Biotechnology8, 276-279; Fahy et al. (1993)nucleic acid research21, 1819-1826; Zhang et al. (1991) 19, 3929-3933 and Rogers et al. (1997)Gene therapy4, 1387-1392. The entire contents of each of the above documents are incorporated herein by reference.
(ii) Generation of capture probes in array format
Arrays can be prepared in several ways. In some embodiments, the arrays are prepared by synthesizing (eg, in situ synthesis) the oligonucleotides on the array, or by jet printing or photolithography. For example, light-guided synthesis of high-density DNA oligonucleotides can be achieved by photolithography or solid-phase DNA synthesis. To perform photolithographic synthesis, synthetic linkers modified with photochemical protecting groups can be attached to substrates, and photochemical protecting groups can be modified using a photolithographic mask (applied to specific regions of the substrate) and light, resulting in a photoreactive Protect. Many of these methods are known in the art and are described, for example, in "Basic Microarray Concepts and Potential Applications in Clinical Microbiology" by Miller et al.Clinical Microbiology Reviews22.4 (2009): 611-633; US201314111482A; US9593365B2; US2019203275; and WO2018091676, the entire contents of which are incorporated herein by reference.
locate or print
In some embodiments, oligonucleotides (eg, capture probes) can be "spotted" or "imprinted" on a substrate to form an array. Oligonucleotides can be applied by non-contact or contact printing. Contactless printers can eject small droplets of probe solution onto the substrate using the same methods as computer printers, such as bubble jet or inkjet. Dedicated inkjet printers can deliver nanoliter to picoliter volume droplets of oligonucleotide solutions (instead of ink) onto substrates. In contact printing, each printing needle applies an oligonucleotide solution directly to a specific spot on the surface. Oligonucleotides can be covalently linked by electrostatic interactions between negative charges of the phosphate backbone of DNA and positively charged coatings on the surface of the substrate or by UV cross-linking between thymidine bases in DNA and DNA on the surface of the substrate. Amino groups on the surface of the treated substrate. In some embodiments, the substrate is glass. In some embodiments, oligonucleotides (eg, capture probes) are bound to a substrate via covalent bonds to chemical substrates, eg, epoxysilane, aminosilane, lysine, polyacrylamide, and the like.
in situ synthesis
Arrays of capture probes can be prepared by in situ synthesis. In some embodiments, photolithography can be used to fabricate capture probe arrays. Photolithography typically relies on UV-masking and light-directed combinatorial chemical synthesis on substrates to selectively synthesize probes directly on the array surface, one nucleotide per spot, for multiple spots simultaneously. In some embodiments, the substrate comprises a covalent binding molecule with a photoremovable protecting group at the free end. UV light passes through the photolithographic mask to directly deprotect and activate selected sites with hydroxyl groups, which initiate coupling to incoming protected nucleotides attached to the activated sites. The mask is designed in such a way that exposure sites can be selected, specifying the coordinates at which each nucleotide on the array can bind. The process can be repeated, applying new masks to activate different sets of sites and joining different bases, allowing the construction of different oligonucleotides at each site. This procedure can be used to synthesize hundreds of thousands of different oligonucleotides. In some embodiments, unmasked field combining technology may be used. Programmable micromirrors can create digital masks that reflect desired patterns of UV light, thus removing the shielding.
In some embodiments, the inkjet blotting process can also be used for in situ synthesis of oligonucleotides. Various nucleotide precursors plus catalysts can be printed on the substrate, followed by coupling and deprotection steps. This method relies on printing picoliter volumes of nucleotides on the surface of the array in repeated base-by-base printing, extending the length of the oligonucleotide probes on the array.
electric field
Arrays can also be prepared by active electric field hybridization to control nucleic acid transport. Negatively charged nucleic acids can be delivered to specific sites or features when a positive current is applied to one or more test sites on the array. The surface of the array can contain binding molecules, such as streptavidin, which can form bonds (eg, streptavidin-biotin bonds) after electrically addressed biotinylated probes reach the target sites. The positive current is then removed from the active feature and new test sites can be activated by targeted application of positive current. This process is repeated until all places in the sequence are covered.
ligature
In some embodiments, arrays containing barcoded probes can be generated by ligation of multiple oligonucleotides. In some cases, one oligonucleotide of the plurality of oligonucleotides contains a portion of a barcode, and when the multiple oligonucleotides are linked, a complete barcode is generated. For example, a first oligonucleotide containing a first portion of a barcode can be attached to a substrate (e.g., using any of the methods described herein for attaching an oligonucleotide to a substrate), and an oligonucleotide containing a second portion of the barcode can be attached The second oligonucleotide is attached to the substrate . It is then ligated to the first oligo to create a complete barcode. Different combinations of the first, second and any additional part of the barcode can be used to increase the diversity of the barcode. In the event that the second oligonucleotide is also bound to the substrate prior to ligation, the first and/or second oligonucleotide may be linked to the substrate via a surface linker containing a cleavage site. After ligation, the ligated oligonucleotides can be linearized by cleavage at the cleavage site.
To increase barcode diversity, multiple second oligonucleotides containing two or more different barcode sequences can be ligated to multiple first oligonucleotides containing the same barcode sequence, resulting in two or more. There are many different types of barcodes. To achieve selective ligation, the first oligonucleotide bound to the substrate containing the first part of the barcode can be initially protected with a protecting group (eg, a cleavable protecting group), and the protecting group can be used between the first and Remove the second oligo before binding between of the first part. In cases where barcoded array probes are generated by ligation of two or more oligonucleotides, an oligonucleotide concentration gradient can be applied to the substrate so that different combinations of oligonucleotides are incorporated into the barcoded probe, depending on its position on the array matrix. .
Probes can be generated by directly attaching additional oligonucleotides to existing oligonucleotides via oligonucleotide splints. In some embodiments, the oligonucleotides on the existing array may include recognition sequences that hybridize to the probe oligonucleotides. The recognition sequence can be located at the free 5' or free 3' end of the oligonucleotides present on the array. Recognition sequences useful in the methods of the present disclosure may not contain restriction enzyme recognition sites or secondary structure (eg, hairpins), and may include high levels of guanine and cytosine nucleotides.
polymerase
Barcoded probes on arrays can also be generated by adding single nucleotides to existing oligonucleotides on the array, for example, using a polymerase that acts in a template-independent manner. Single nucleotides can be added to existing oligonucleotides in a concentration gradient to produce probes of different lengths, depending on the position of the probe on the array.
Modification of existing capture probes
Arrays can also be prepared by modifying existing arrays, for example, by modifying oligonucleotides already attached to the array. For example, capture probes can be generated on an array that already contains oligonucleotides fused to the array (or feature on the array) at the 3' end and have a free 5' end. In some cases, the array is any commercially available array (eg, any array that is commercially available as described herein). Oligonucleotides can be synthesized in situ using any of the in situ synthesis methods described herein. An oligonucleotide may include a barcode and one or more constant sequences. In some cases, the constant sequence is a cleavable sequence. Substrate-linked oligonucleotides (e.g., array) can be shorter than 100 nucleotides (e.g., less than 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15, and 10 nucleotides, respectively) . To generate probes using oligonucleotides, a primer complementary to a portion of the oligonucleotide (eg, a constant sequence shared by the oligonucleotides) can hybridize and extend the oligonucleotide (using the oligonucleotide acid as a template) to form a duplex and create a 3' overhang. 3' overhangs can be generated by template-independent ligases such as terminal deoxynucleotidyl transferase (TdT) or poly(A) polymerase. The 3' overhang allows additional nucleotides or oligonucleotides to be added to the duplex, for example, by enzymes. For example, capture probes can be generated by adding additional oligonucleotides to the 3' overhang (eg, via oligonucleotide-mediated ligation), wherein the additional oligonucleotides can include one or more sequences or portions of the capture domain sequence, or its complement.
Additional oligonucleotides (eg, the capture domain sequence or portion of the sequence) may include a degenerate sequence (eg, any degenerate sequence as described herein). Additional oligonucleotides (eg, the capture domain sequence or portion of the sequence) may include sequences compatible with hybridization or ligation of the analyte of interest in a biological sample. The analyte of interest can also be used as an oligonucleotide splint to attach additional oligonucleotides to the probe. When a scaffold oligonucleotide is used to facilitate ligation of additional oligonucleotides, the additional oligonucleotides may include a sequence complementary to that of the scaffold oligonucleotide. Ligation of oligonucleotides may involve the use of enzymes such as, but not limited to, ligase. Non-limiting examples of suitable ligases include Tth DNA ligase, Taq DNA ligase, Thermococcus sp. (strain 9oN) DNA ligase (9oNTM DNA Ligase, New England Biolabs), AmpligaseTM (available from Lucigen, Middleton, WI) and SplintR (available from New England Biolabs, Ipswich, MA). Arrays generated as described above can be used for spatial analysis of biological samples. For example, one or more capture domains can be used to hybridize to the polyA tail of an mRNA molecule. Reverse transcription can be performed using reverse transcriptase to generate cDNA complementary to the captured mRNA. The sequence and location of the captured mRNA can then be determined (eg, by sequencing captured probes containing barcodes and complementary cDNA).
Arrays for spatial analysis can be generated using the various methods described here. In some embodiments, the array has a plurality of capture probes that contain spatial barcodes. These spatial barcodes and their relationship to locations on the array can be determined. In some cases, this information is readily available because oligonucleotides have been spotted, printed, or synthesized on an array according to a predetermined pattern. In some cases, the spatial barcodes can be decoded by the methods described herein, such as in situ sequencing, different markers associated with the spatial barcodes, and the like. In some embodiments, a string can be used as a template for generating substrings. Therefore, spatial barcodes can be transferred into substrings with known patterns.
(iii) Features
A "signature" is an entity that acts as a support or repository for the various molecular entities used in sample analysis. In some embodiments, some or all of the features in the array are functionalized for analyte capture. In some embodiments, the functionalization features include one or more capture probes. Examples of features include, but are not limited to beads, dots of any two-dimensional or three-dimensional geometry (eg, inkjet dots, masked dots, grid squares), wells, and hydrogel cushions. In some embodiments, the features are directly or indirectly attached or fixed to the substrate. In some embodiments, the features are not directly or indirectly attached or fixed to the substrate, but are instead located, for example, within an enclosed or partially enclosed three-dimensional space (eg, a hole or recess).
In addition to those described above, a number of other features may be used to form the arrays described herein. For example, in some embodiments, features formed from polymers and/or biopolymers that are jet-printed, screen-printed, or electrostatically deposited onto a substrate can be used to form the array. For example, jet printing of biopolymers is described in PCT patent application publication no. WO 2014/085725. For example, de Gans et al. describe jet printing of polymers,advanced material16(3): 203-213 (2004). Methods of electrostatic deposition of polymers and biopolymers are described, for example, in Hoyer et al.,anus. Chemical68(21): 3840-3844 (1996). The entire contents of each of the above references are incorporated herein by reference.
As another example, in some embodiments, the features are formed from metal micro- or nanoparticles. Suitable methods for depositing such particles in the form of arrays are described, for example, in Lee et al.,Beilstein J. Nanotehnologija8: 1049-1055 (2017), which is hereby incorporated by reference in its entirety.
As a further example, in some embodiments, the features are formed from magnetic particles assembled on a substrate. Examples of such particles and array assembly methods are described by Ye et al.scientific report6:23145 (2016), which is incorporated herein by reference in its entirety.
As another example, in some embodiments, the feature corresponds to an area of the substrate where one or more optical indicia have been incorporated and/or have been altered by a process such as permanent photobleaching. Suitable substrates for implementing features in this way include, for example, various polymers. Methods for forming such features are described, for example, in Moshrefzadeh et al.,Apply for physics. Wright62:16 (1993), which is incorporated herein by reference in its entirety.
As another example, in some embodiments, the feature may correspond to colloidal particles that assemble (eg, by self-assembly) to form an array. Suitable colloidal particles are described, for example, in Sharma,resonance23(3): 263-275 (2018), which is hereby incorporated by reference in its entirety.
As a further example, in some embodiments, features can be formed by lattice photopolymerization of monomer solutions on a substrate. In particular, two-photon and three-photon polymerization can be used to fabricate features of relatively small (eg, submicron) size. Suitable methods for making features on substrates in this way are described, for example, in Nguyen et al.,today's material20(6): 314-322 (2017), the entire contents of which are incorporated herein by reference.
In some embodiments, the features are directly or indirectly attached or fixed to the fluid permeable substrate. In some embodiments, the features are directly or indirectly attached or fixed to a biocompatible substrate. In some embodiments, the features are directly or indirectly attached or fixed to the substrate as a hydrogel.
perla
"Beads" can be particles. Balls can be porous, non-porous, solid, semi-solid and/or combinations thereof. In some embodiments, the beads may be soluble, cleavable, and/or degradable, while in some embodiments, the beads are nondegradable. The semi-solid beads can be liposomal beads. The solid beads may include metals including, but not limited to, iron oxide, gold, and silver. In some embodiments, the beads may be silica beads. In some embodiments, the beads may be rigid. In some embodiments, the beads may be flexible and/or compressible.
The beads can be large molecules. Beads can be formed from nucleic acid molecules linked together. Beads can be formed by covalent or non-covalent assembly of molecules (eg macromolecules), such as monomers or polymers. Polymers or monomers can be natural or synthetic. A polymer or monomer may be or include, for example, a nucleic acid molecule (eg, DNA or RNA).
Balls can be rigid, flexible and/or compressible. The beads may include a coating consisting of one or more polymers. Such coatings can be degradable or soluble. In some embodiments, the bead includes a spectral or optical label (eg, dye) attached to the bead either directly or indirectly (eg, via a linker). For example, beads can be prepared as colored formulations (eg, beads that exhibit different colors within the visible spectrum) that change color upon application of desired stimuli (eg, heat and/or chemicals) (eg, colorimetric bead reactions) to form beads of different colors (eg opaque and/or transparent beads).
Balls may contain natural and/or synthetic materials. For example, the beads may contain natural polymers, synthetic polymers, or both natural and synthetic polymers. Examples of natural polymers include, but are not limited to, proteins, sugars such as deoxyribonucleic acid, rubber, cellulose, starch (eg, amylose, pullulan), enzymes, polysaccharides, silk, polyhydroxyalkanoates, chitosan, sugar, dextran, collagen, carrageenan, ispaghula, gum arabic, agar, gelatin, shellac, fatty marine gum, xanthan gum, corn sugar gum, guar gum, karaya gum, agarose, seaweed acid, alginate or its natural polymers. Examples of synthetic polymers include, but are not limited to, acrylic, nylon, silicone, spandex, viscose rayon, polycarboxylate, polyvinyl acetate, polyacrylamide, polyacrylate, polyethylene glycol, polyurethane, polylactic acid, silicon, polystyrene, polyacrylonitrile, polybutadiene, polycarbonate, polyethylene, polyethylene terephthalate, poly(chlorotrifluoroethylene), poly(ethylene oxide), poly(ethylene terephthalate), polyethylene, polyisobutylene, poly(methyl methacrylate), poly(oxymethylene), polyoxymethylene, polypropylene, polystyrene, poly(tetrafluoroethylene), poly( vinyl acetate ), poly(vinyl alcohol), poly(vinyl chloride), poly(vinylidene chloride), poly(vinylidene fluoride), poly(vinyl fluoride) and/or combinations thereof (eg copolymer materials). The beads can also be formed from materials other than polymers, including, for example, lipids, micelles, ceramics, glass ceramics, composites of materials, metals, and/or other inorganic materials.
In some embodiments, the beads are degradable beads. Degradable beads may include one or more substances with labile bonds (eg, disulfide bonds, primers, other oligonucleotides, etc.) so that when the bead/substance is exposed to an appropriate stimulus, the labile bonds are destroyed. breakage and balls are broken down. A labile bond can be a chemical bond (eg covalent bond, ionic bond) or it can be another type of physical interaction (eg van der Waals interaction, dipole-dipole interaction, etc.). In some embodiments, the cross-linking agents used to form the beads may include labile linkages. When exposed to the right conditions, the unstable bonds break and the grains degrade. For example, when polyacrylamide gel beads containing a cystamine crosslinker are exposed to a reducing agent, the cystamine disulfide bonds are broken and the beads are degraded.
Degradation can refer to the dissociation of bound or incorporated species (eg, disulfide bonds, primers, other oligonucleotides, etc.) from the beads, regardless of whether the physical bead itself is structurally degraded. For example, trapped substances can be released from the beads due to differences in osmotic pressure, for example, due to changes in the chemical environment. For example, changes in bead pore volume due to differences in osmotic pressure can often occur without structural degradation of the bead itself. In some embodiments, the increase in pore volume due to osmotic swelling of the beads may allow release of substances trapped within the beads. In some embodiments, osmotic shrinkage of the beads may result in the beads better retaining trapped species due to shrinkage of pore volume.
Any suitable reagent capable of breaking down the beads can be used. In some embodiments, changes in temperature or pH can be used to degrade heat- or pH-sensitive bonds within the beads. In some embodiments, chemical degradation agents can be used to degrade the chemical bonds within the beads through oxidation, reduction, or other chemical changes. For example, the chemical degrading agent can be a reducing agent such as DTT, where DTT can break down the disulfide bonds formed between the crosslinker and the gel precursor, thereby breaking down the beads. In some embodiments, a reducing agent may be added to degrade the beads, which may cause release of the bead contents. Examples of reducing agents may include, but are not limited to, dithiothreitol (DTT), β-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris (2-carboxyethyl)phosphine (TCEP), or their combinations.
Any of a number of chemical reagents can be used to initiate degradation of the beads. Examples of chemical agents include, but are not limited to, pH-mediated changes in the integrity of the components within the beads, degradation of the bead components by cleavage of crosslinks, and deaggregation of the bead components.
In some embodiments, the beads may be formed from a material that includes a degradable chemical cross-linking agent, such as N,N'-bis-(acryloyl)cystamine (BAC) or cystamine. Degradation of such degradable cross-linkers can be achieved by any number of different mechanisms. In some examples, the beads may come into contact with chemical degradants that may cause oxidation, reduction, or other chemical changes. For example, the chemical degradation agent may be a reducing agent such as dithiothreitol (DTT). Other examples of reducing agents may include beta-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof.
In some embodiments, exposure to an aqueous solution, such as water, can initiate hydrolytic degradation and thereby degradation of the beads. The beads can also be induced to release their contents when a heat stimulus is applied. Changes in temperature cause different changes to the balls. For example, heat can cause solid beads to liquefy. The change in heat causes the beads to melt, which degrades part of the bead. In some embodiments, the heat can increase the internal pressure of the bead components, causing the beads to crack or explode. Heat can also be applied to heat-sensitive polymers used as bead building materials.
When degradable beads are used, it may be beneficial to avoid exposing such beads to stimuli or stimuli that lead to such degradation before a certain time, in order to avoid, for example, premature degradation of the beads and problems caused by such degradation. These include, for example, poor flow and aggregation characteristics. For example, when the beads include reducing cross-linking groups such as disulfide groups, it is desirable to avoid contacting such beads with reducing agents such as DTT or other disulfide-cleaving reagents. In such embodiments, in some embodiments, the bead processing described herein will be provided in the absence of reducing agents such as DTT. Since reducing agents are often found in commercial enzyme preparations, it may be desirable to provide enzyme preparations without a reducing agent (or without DTT) when processing the grains described herein. Examples of such enzymes include, for example, polymerase compositions, reverse transcriptase compositions, ligase compositions, and many other enzyme compositions that can be used to process the beads described herein. The term "reducing agent-free" or "DTT-free" formulation refers to the formulation used to break down the beads. For example, for DTT, a formulation without a reducing agent may have less than about 0.01 millimolar (mM), 0.005 mM, 0.001 mM DTT, 0.0005 mM DTT, or less than about 0.0001 mM DTT. In some embodiments, the amount of DTT may not be detectable.
When an appropriate stimulus is applied to the bead, degradable beads can be used to release attached capture probes (eg, nucleic acid molecules, spatial barcode sequences, and/or primers) from the bead faster than non-degradable beads. For example, for a substance bound to the inner surface of a porous bead or in the case of an encapsulated substance, the substance may have greater mobility and accessibility to other substances in solution as the bead degrades. In some embodiments, the substances can also be attached to the degradable beads via degradable linkages (eg, disulfide linkages). The degradable linker may respond to the same stimulus as the degradable seed, or the two degradable substances may respond to different stimuli. For example, capture probes with one or more spatial barcodes can be disulfide-linked to cystamine-containing polyacrylamide beads. Upon exposure of the spatially barcoded beads to a reducing agent, the beads are degraded and capture probes having one or more spatially barcoded sequences have disulfide bonds between the capture probe and the bead and disulfide bonds between the capture probe and the bead. It is released when the connection is broken. Cystamine in balls.
Adding multiple types of labile bonds to the beads can lead to beads that respond to different stimuli. Each type of labile bond can be sensitive to relevant stimuli (eg chemical stimuli, light, temperature, pH, enzymes, etc.) and therefore can be controlled by applying appropriate stimuli. Some non-limiting examples of labile bonds that can be attached to precursors or beads include ester bonds (e.g., cleavable by acids, bases, or hydroxylamine), vicinal diol bonds (e.g., cleavable by sodium periodate), Diels-Alder bonds (e.g., cleavable by heat) , sulfonic bonds (e.g., cleaved by bases), silyl ether bonds (e.g., cleaved by acids), glycosidic bonds (e.g., cleaved by amylases), peptide bonds (e.g., cleaved by proteases) or phosphodiester bonds (e.g. .which can be cleaved by nucleases (eg DNase)). The bonds can be cleaved by other enzymes that target nucleic acid molecules, such as restriction enzymes (eg, restriction endonucleases). This functionality can be used for controlled release of reagents from beads. In some embodiments, a second reagent comprising a labile bond can be attached to the beads after gel bead formation via, for example, an activating functional group of the bead, as described above. In some embodiments, gel beads containing labile linkages are reversible. In some embodiments, gel beads with reversibly labile bonds are used to capture one or more regions of interest in a biological sample. For example and without limitation, beads containing thermolabile bonds can be heated by a light source (eg, a laser), which causes a change in the gel beads, facilitating the capture of biological samples in contact with the gel beads. Capture probes having one or more spatial barcodes that can be detached, grafted, or reversibly attached to the beads described herein include capture probes that are releasable or releasable by cleaving the bond between the capture probe and the bead. Release, or release of the core sama bead by degradation, provides access to a capture probe with one or more spatial barcodes that can be accessed or made available by other reagents, or both.
Balls can have different physical properties. The physical properties of the beads can be used to characterize the beads. Non-limiting examples of physical properties of spheres that may vary include volume, shape, roundness, density, symmetry, and stiffness. For example, beads can have different volumes. Beads of different diameters can be obtained using a network of microfluidic channels configured to provide a specific volume of beads (eg, based on channel size, flow rate, etc.). In some embodiments, the beads have different hardness values, which can be obtained by varying the concentration of the polymer used to make the beads. In some embodiments, the physical properties of the capture probes can be used to make the spatial barcode attached to the bead optically detectable. For example, nucleic acid origami, such as deoxyribonucleic acid (DNA) origami, can be used to generate optically detectable spatial barcodes. To this end, a nucleic acid molecule or multiple nucleic acid molecules can be folded to produce two-dimensional and/or three-dimensional geometries. Different geometries can be detected optically.
In some embodiments, the beads can be made physically distinguishable by using specific types of nanoparticles that have more than one distinct physical property. For example, Janus particles with hydrophilic and hydrophobic surfaces can be used to provide unique physical properties.
The beads can generally be of any suitable shape. Examples of bead shapes include, but are not limited to, spherical, non-spherical, elliptical, oval, amorphous, circular, cylindrical, cubic, hexagonal, and variations thereof. In some embodiments, non-spherical (eg, hexagonal, cubic, shaped beads) can be packed more tightly (eg, tighter) than spherical beads. In some embodiments, the beads may self-assemble into monolayers. The cross-section (eg, the first cross-section) may correspond to the diameter or the largest cross-sectional dimension of the grain. In some embodiments, the beads may be approximately spherical. In such embodiments, the first cross-section may correspond to the diameter of the ball. In some embodiments, the balls may be approximately cylindrical. In such embodiments, the first cross-section may correspond to the diameter, length, or width along the approximately cylindrical grain.
Balls can be uniform or heterogeneous in size. "Polydispersity" generally refers to heterogeneity in the size of molecules or particles. The polydispersity index (PDI) of the beads can be calculated using the equation PDI = Mw/Mn, where Mw is the average molar mass and Mn is the average molar mass. In certain embodiments, the beads may be provided as a population or plurality of beads having a relatively monodisperse size distribution. In situations where there may be a need to provide relatively consistent amounts of reagent, maintaining relatively consistent bead characteristics, such as size, can aid in overall consistency.
In some embodiments, the balls provided herein may have a size distribution with a coefficient of variation of cross-sectional dimensions of less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 5 % % or lower. In some embodiments, the plurality of beads provided herein has less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15% , less than 10%, less than 5% or less.
In some embodiments, the diameter or largest dimension of the beads may not exceed 100 µm (eg, no greater than 95 µm, 90 µm, 85 µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm µm, 45 µm, 40 µm, 35 µm, 30 µm, 25 µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6 µm , 5 µm, 4 µm, 3 µm, 2 µm or 1 µm.)
In some embodiments, the plurality of beads have an average diameter of no greater than 100 µm. In some embodiments, the plurality of beads have an average diameter or maximum dimension of no greater than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm. , 35 µm, 30 µm, 25 µm µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm or 1 µm.
In some embodiments, the beads may have a volume of at least about 1 µm3, for example, at least 1 µm3, 2 microns3, 3 microns3, 4 microns3, 5 microns3, 6 microns3, 7 microns3, 8 microns3, 9 microns3, 10 microns3, 12 microns3, 14 microns3, 16 microns3, 18 microns3, 20 microns3, 25 microns3, 30 microns3, 35 microns340 microns3, 45 microns3, 50 microns355 microns3, 60 microns3, 65 microns3, 70 microns3, 75 microns3, 80 microns3, 85 microns3, 90 microns3, 95 microns3, 100 microns3, 125 microns3, 150 microns3, 175 microns3, 200 microns3, 250 microns3, 300 microns3, 350 microns3, 400 microns3, 450 microns3, 500 microns3, 550 microns3, 600 microns3, 650 microns3, 700 microns3, 750 microns3, 800 microns3, 850 microns3, 900 microns3, 950 microns3, 1000 microns3, 1200 microns3, 1400 microns3, 1600 microns3, 1800 microns3, 2000 μm3, 2200 microns3, 2400 microns3, 2600 microns3, 2800 microns3, 3000 microns3, or greater.
In some embodiments, the volume of the beads can be between about 1 µm3and 100 microns3, for example between about 1 µm3and 10 microns3, between about 10 µm3and 50 microns3or between about 50 µm3and 100 microns3In some embodiments, the beads can have a volume between about 100 µm3and 1000 microns3, for example between about 100 µm3and 500 microns3or between approximately 500 µm3and 1000 microns3In some embodiments, the beads can have a volume between about 1000 µm3and 3000 microns3, for example between about 1000 µm3and 2000 microns3or between about 2000 µm3and 3000 microns3In some embodiments, the beads may have a volume between about 1 µm3and 3000 microns3, for example between about 1 µm3and 2000 microns3, between about 1 µm3and 1000 microns3, between about 1 µm3and 500 microns3or between about 1 µm3and 250 microns3.
Beads may contain one or more identical or different sections. In some embodiments, the balls may have a first cross-section different from the second cross-section. The beads may have a first cross-section of at least about 0.0001 micron, 0.001 micron, 0.01 micron, 0.1 micron, or 1 micron. In some embodiments, the beads may include a cross-section (e.g., first cross-section) of at least about 1 micrometer (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm , 10 μm, 11 μm micron, 12 micron, 13 micron, 14 micron, 15 micron, 16 micron, 17 micron, 18 micron, 19 micron, 20 micron, 25 micron, 30 micron, 35 micron, 40 micron , 45 µm, 50 µm, 55 µm, 60 µm, 65 µm, 70 µm, 75 µm, 80 µm, 85 µm, 90 µm, 100 µm, 120 µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300 µm , 350 µm, 400 µm, 450 µm, 500 µm, 550 µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900 µm, 950 µm, 1 millimeter (mm), or larger. In some embodiments, the beads may comprise a cross section between about 1 μm and 500 μm, such as between about 1 μm and 100 μm, between about 100 μm and 200 μm, between about 1 μm and about 200 μm (For example, the first cross section ). 200 µm and 300 µm, approximately between 300 µm and 400 µm, or approximately between 400 µm and 500 µm. For example, the bead may have a cross-section (eg, first cross-section) between about 1 µm and 100 µm. In some embodiments, the beads may have a second cross-section of at least about 1 µm. For example, the beads may comprise at least about 1 micron (μm), 2 μm, 3 μm, 4 μm, 5 μm, 6 μm, 7 μm, 8 μm, 9 μm, 10 μm, 11 micron, 12 micron, 13 micron, 14 micron, 15 micron, 16 micron, 17 micron, 18 micron, 19 micron, 20 micron, 25 micron, 30 micron, 35 micron, 40 micron, 45 micron, 50 micron, 55 micron, 60 micron, 65 micron, 70 µm , 75 µm, 80 µm, 85 µm, 90 µm, 100 µm, 120 µm, 140 µm, 160 µm, 180 µm, 200 µm, 250 µm, 300 µm, 350 µm, 400 µm, 450 µm, 500 µm, 550 µm µm, 600 µm, 650 µm, 700 µm, 750 µm, 800 µm, 850 µm, 900 µm, 950 µm, 1 millimeter (mm) or larger. In some embodiments, the beads may comprise a second cross section between about 1 µm and 500 µm, for example between about 1 µm and 100 µm, between about 100 µm and 200 µm, between about 200 µm and 300 µm, between about 300 µm and 400 µm. , or between about 400 µm and 500 µm. For example, the beads may include a second cross-section between about 1 µm and 100 µm.
In some embodiments, the beads may be nanoscale (e.g., the beads may have a diameter or largest cross-sectional dimension (e.g., 850 nm or less, 800 nm or less, 750 nm or less, 650 nm or less, 550 nm or less, 450 nm or less, 400 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nanometers or less).The plurality of beads may have an average diameter or average largest cross-sectional dimension of about 100 nanometers (nm) to about 900 nanometers (nm) (eg, 850 nm or less, 800 nm or less, 750 nm or less, 700 nm or less, 650 nm or less, 600 nm or less, 550 nm or less, 500 nm or less, 450 nm or less, 400 nm or less, 350 nm or less, 300 nm or less, 250 nm or less, 200 nm or less, 150 nm or less. In some embodiments, the diameter or volume of the beads is approximately the diameter of one cell (eg .one cell being evaluated).
In some embodiments, the beads are capable of recognizing multiple analytes (eg, nucleic acids, proteins, chromatin, metabolites, drugs, gRNA, and lipids) from a single cell. In some embodiments, the beads are capable of recognizing a single analyte (eg, mRNA) from a single cell.
The beads can have adjustable pore volumes. The pore volume can be selected, for example, to retain denatured nucleic acids. The pore volume can be selected to maintain diffusional permeability to exogenous chemicals such as sodium hydroxide (NaOH) and/or endogenous chemicals such as inhibitors. The beads can be formed from biocompatible and/or biochemically compatible materials and/or materials that maintain or increase cell viability. The beads can be formed from materials that can be depolymerized thermally, chemically, enzymatically, and/or optically.
In some embodiments, the beads may be non-covalently loaded with one or more reagents. The beads can be filled non-covalently, eg, by subjecting the beads to conditions sufficient to swell the beads, allowing sufficient time for the reagents to diffuse into the interior of the beads, and subjecting the beads to conditions sufficient to deflate the beads. The beads can be swollen, for example, by placing the beads in a thermodynamically favorable solvent, exposing the beads to higher or lower temperatures, exposing the beads to higher or lower ion concentrations, and/or exposing the beads to an electric field.
The swelling of the balls can be achieved by different swelling methods. In some embodiments, the swelling is reversible (eg, by subjecting the beads to conditions that promote de-swelling). In some embodiments, for example, by transferring the beads to a thermodynamically unfavorable solvent, exposing the beads to lower or higher temperatures, exposing the beads to lower or higher ion concentrations, and/or adding or removing an electric field. De-swelling of balls can be achieved by different methods of de-swelling. In some embodiments, de-swelling is reversible (eg, subjecting the beads to conditions that promote swelling). In some embodiments, de-swelling the beads may include moving the beads to cause the pores in the beads to shrink. The contraction then prevents the reagents inside the bead from diffusing outside the interior of the bead. The resulting barrier may be due to spatial interactions between the reagent and the interior of the bead. Transfer can be achieved microfluidically. For example, transfer can be achieved by moving beads from one DC solvent stream to another DC solvent stream. The swelling and/or pore volume of the beads can be adjusted by changing the polymer composition of the beads.
The beads may contain temperature-sensitive polymers so that when the beads are heated or cooled, the properties or dimensions of the beads may change. For example, the polymer may include poly(N-isopropylacrylamide). The gel beads may contain poly(N-isopropylacrylamide) and the gel beads may shrink in one or more dimensions (eg, cross-sectional diameter, multiple cross-sectional diameters) when heated. A temperature sufficient to alter one or more properties of the gel beads may be, for example, at least about 0°C (°C), 1°C, 2°C, 3°C, 4°C. , 5° C, 10° C or more. For example, the temperature can be around 4°C. In some embodiments, the temperature sufficient to alter one or more properties of the gel beads may be, for example, at least about 25°C, 30°C, 35°C. C., 37°C, 40°C, 45°C, 50°C or more. For example, the temperature can be around 37°C.
Functionalization of beads for attachment of capture probes can be achieved by a number of different methods including, but not limited to, activation of chemical groups within the polymer, incorporation of reactive or activating functional groups into the polymer structure, or the bond is attached. Polymer or monomer stage in the production of balls. The beads can be functionalized to bind analytes of interest, such as nucleic acids, proteins, carbohydrates, lipids, metabolites, peptides, or other analytes.
In some embodiments, the beads can contain molecular precursors (eg, monomers or polymers) that can form polymer networks via polymerization of the molecular precursors. In some embodiments, the precursor may be an already polymerized species capable of further polymerization, e.g., by chemical cross-linking. In some embodiments, the precursor may include one or more monomers, oligomers, or polymers of acrylamide or methacrylamide. In some embodiments, the beads may include prepolymers, which are oligomers capable of further polymerization. For example, prepolymers can be used to make polyurethane beads. In some embodiments, the beads may contain individual polymers that can further be polymerized together (eg, to form a copolymer). In some embodiments, the beads can be produced by polymerizing different precursors to include hybrid polymers, copolymers, and/or block copolymers. In some embodiments, beads may include covalent bonds or ions between polymeric precursors (eg, monomers, oligomers, and linear polymers), nucleic acid molecules (eg, oligonucleotides), primers, and other entities. In some embodiments, the covalent bond may be a carbon-carbon bond or a thioether bond.
Crosslinking of the polymer can be permanent or reversible, depending on the crosslinking agent used. Reversible cross-linking allows polymers to be linearized or dissociated under appropriate conditions. In some embodiments, reversible cross-linking may also allow for reversible attachment of materials bound to the surface of the bead. In some embodiments, the cross-linking agent can form disulfide bonds. In some embodiments, the disulfide bond that forms the chemical cross-linker can be cystamine or modified cystamine.
For example, where the polymeric precursor material includes a linear polymeric material such as linear polyacrylamide, PEG, or other linear polymeric material, the activator may include a cross-linking agent or a chemical form that internally activates the droplet cross-linking agent. Similarly, for polymer precursors that include polymerizable monomers, the activator may include a polymerization initiator. For example, in certain embodiments, when the polymer precursor includes a mixture of acrylamide monomer and N,N'-bis-(acryloyl)cystamine (BAC) comonomer, such as tetraethylamethylene methyldiamine (TEMED) is a reagent that can initiate copolymerization of acrylamide and BAC for cross-linking polymer network, or other conditions sufficient for polymerization or gelation of the precursor. Conditions sufficient to polymerize or gel the precursor may include exposure to heat, cooling, electromagnetic radiation, and/or light.
After polymerization or gelation, a polymer or gel can be formed. Polymers or gels can diffuse and permeate chemical or biochemical agents. The polymer or gel may be diffusion impermeable to the macromolecular components. Polymers or gels may include disulfide cross-linked polyacrylamide, agarose, alginate, polyvinyl alcohol, polyethylene glycol (PEG)-diacrylate, PEG-acrylate, PEG-thiol, one or more PEG-azides, PEG-alkynes, other acrylates, chitosan, hyaluronic acid, collagen, fibrin, gelatin or elastin. A polymer or gel may include any other polymer or gel.
In some embodiments, disulfide bonds may be preceded by molecular precursor units (eg, monomers, oligomers, or linear polymers) or may be incorporated into beads and nucleic acid molecules (eg, oligonucleotides, capture probes) formed between bodies. For example, cystamine (including modified cystamine), an organic reagent containing disulfide bonds, can be used as a crosslinker between individual monomeric or polymeric bead precursors. Polyacrylamide can be polymerized in the presence of cystamine or cystamine-containing substances (eg, modified cystamine) to produce polyacrylamide gel beads containing disulfide bonds (eg, chemically degradable beads containing chemically reducing crosslinkers). Disulfide bonds allow the particles to break down (or dissolve) when exposed to reducing agents.
In some embodiments, chitosan, a linear polysaccharide polymer, can be cross-linked with glutaraldehyde through hydrophilic chains to form beads. Cross-linking of chitosan polymers can be achieved by chemical reactions initiated by heat, pressure, pH changes and/or radiation.
In some embodiments, the bead may comprise an acrylic portion, which may be used in certain aspects to attach one or more capture probes to the bead. In some embodiments, an acridite moiety may refer to an acridite analog resulting from the reaction of acridite with one or more species (e.g., disulfide linkers, primers, other oligonucleotides, etc.), such as, but not limited to, acridite with a polymer Other monomers and crosslinkers during the reaction. The acridite moiety can be modified to form a chemical bond with the species to be attached, such as a capture probe. The acridite moiety can be modified with a thiol group that can form a disulfide bond or it can be modified with a group that already contains a disulfide bond. A thiol or disulfide (disulfide exchange) can be used as an anchor for the species being attached, or another part of the acrylate moiety can be used for attachment. In some embodiments, the bond may be reversible such that when the disulfide bond is broken (eg, in the presence of a reducing agent), the bound species is released from the bead. In some embodiments, the acridite moiety may contain reactive hydroxyl groups that can be used to bind substances.
In some embodiments, the precursors (eg, monomers or crosslinkers) that are polymerized to form beads may include acrylate moieties such that when the beads are produced, the beads also include acrylate moieties. The acridite moiety may be linked to a nucleic acid molecule (e.g., an oligonucleotide), which may include a primer (e.g., a target nucleic acid amplification primer, a random primer, a messenger RNA primer) and/or one or more probes. for catching. One or more capture probes may contain the same sequence for all capture probes coupled to a given bead and/or a sequence that differs among all capture probes coupled to a given bead. Capture probes can be embedded in balls. In some embodiments, the capture probes can be incorporated or attached to the beads such that the capture probes retain free 3' ends. In some embodiments, the capture probe can be incorporated or attached to the bead such that the capture probe retains the free 5' end. In some embodiments, the beads can be functionalized such that each bead contains multiple different capture probes. For example, a bead may include multiple capture probes, such as capture probe 1, capture probe 2, and capture probe 3, each of capture probe 1, capture probe 2, and capture probe 3 comprising another capture probe. Domains (for example, the capture domain of Capture Probe 1 includes a poly(dT) capture domain, the capture domain of Capture Probe 2 includes a gene-specific capture domain, and the capture domain of Capture Probe 3 includes a CRISPR-specific capture domain). The level of multiplexability for analyte detection can be increased by functionalizing the beads so that each bead contains multiple different capture domains.
In some embodiments, the precursor (e.g., monomer or cross-linking agent) that is polymerized into beads may include reactive or activatable functional groups such that when it becomes reactive, it may polymerize with other precursors to produce beads that include activated or activating functional groups. Functional groups can then be used to attach other substances (eg, disulfide bonds, primers, other oligonucleotides, etc.) to the beads. For example, some precursors that include carboxylic acid (COOH) groups can be copolymerized with other bead precursors that also include COOH functional groups. In some examples, acrylic acid (a species containing free COOH groups), acrylamide, and bis(acrylic)cystamine can be copolymerized together to produce beads containing free COOH groups. The COOH groups of the beads can be activated (for example, by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or 4-(4,6-dimethoxy-1,3,5-triazine -2-yl)-4-methylmorpholine chloride (DMMTMM)), making them reactive (for example, reactive towards amine functions when activated with EDC/NHS or DMTMM). The activated COOH group can then react with a suitable species (eg, a species containing an amine function where the carboxylic acid group is activated to react with the amine function) as a functional group on the bead-binding moiety.
Beads containing disulfide bonds in their polymer network can be functionalized with other species (eg, disulfide bonds, primers, other oligonucleotides, etc.) by reducing some of the disulfide bonds to free thiols. Disulfide bonds can be reduced using, for example, reducing agents (eg, DTT, TCEP, etc.) to generate free thiol groups without dissolving the beads. The free thiol of the bead can then react with the free thiol of a species or species containing another disulfide bond (eg, by thiol-disulfide exchange), so that the species can bind to the bead (eg, by generated disulfide bonds). In some embodiments, the free thiols of the beads can be reacted with any other suitable group. For example, the free thiols of the beads can react with species containing acrylate moieties. The free thiol groups of the beads can react with acridite via Michael addition chemistry, allowing acridite-containing species to bind to the beads. In some embodiments, uncontrolled reactions can be prevented by including a thiol capping agent such as N-ethylmaleamide or iodoacetate.
The activation of disulfide bonds within the beads can be controlled so that only a small number of disulfide bonds are activated. Control can be performed, for example, by controlling the concentration of the reducing agent used to generate free thiol groups and/or the concentration of the reagent used to form disulfide bonds in the polymerization of the beads. In some embodiments, a low concentration of reducing agent (e.g., ratio of reducing agent molecules:gel beads) less than or equal to about 1:100,000,000,000, less than or equal to about 1:10,000,000,000, less than or equal to about 1:1,000,000,000, less than or equal to about 1 :100,000,000, less than or equal to about 1:10,000,000, less than or equal to about 1:1,000,000, less than or equal to about 1:100,000, or less than or equal to about 1:10,000) can be used to reduce. Controlling the number of disulfide bonds that are reduced to free thiols can be used to ensure the structural integrity of the beads during functionalization. In some embodiments, optically active agents, such as fluorescent dyes, can be coupled to the beads via their free thiol groups and used to quantify the amount of free thiols present in the beads and/or track the beads.
In some embodiments, it may be useful to add residues to the beads after the beads are formed. For example, adding capture probes after bead formation can avoid loss of species (eg, disulfide adapters, primers, other oligonucleotides, etc.) during strand transfer termination that can occur during polymerization. In some embodiments, smaller precursors (eg, monomers or cross-linkers that do not include pendant groups and linkers) can be used for polymerization and can minimally inhibit chain end addition due to stickiness effects. In some embodiments, post-synthesis functionalization of the beads can minimize exposure to substances (eg, oligonucleotides) to be loaded with potentially damaging agents (eg, free radicals) and/or the chemical environment. In some embodiments, the resulting hydrogel may have an upper critical solution temperature (UCST), which may allow temperature-induced swelling and collapse of the grains. Such functionality facilitates the infiltration of oligonucleotides (eg primers) into the beads during subsequent functionalization of the beads with oligonucleotides. Post-production functionalization can also be used to control the loading ratio of substances in beads, for example, so that loading ratio variability can be minimized. Substance loading can also be performed in a batch process so that multiple beads can be functionalized with the substance in one batch.
During bead formation (eg during precursor polymerization), reagents can be encapsulated within the beads. These reagents may or may not be included in the polymerization. Such reagents can be added to the polymerization reaction mixture so that the resulting beads incorporate the reagents at the time of bead formation. In some embodiments, such reagents may be added to the beads after formation. Such reagents may include, for example, capture probes (e.g., oligonucleotides), reagents for nucleic acid amplification reactions (e.g., primers, polymerases, dNTPs, cofactors (e.g., ionic cofactors), buffers), including those herein described, reagents for enzymatic reactions (e.g., enzymes, cofactors, chemical substrates, buffers), reagents for nucleic acid modification reactions (e.g., polymerization, ligation, or digestion) and/or for one or more reagents for template preparation (e.g., labeling) Multiple sequencing platforms (eg Nextera® for Illumina®). Such reagents may include one or more of the enzymes described herein, including but not limited to polymerases, reverse transcriptases, restriction enzymes (eg, endonucleases), transposases, ligases, proteinase K, deoxyribonuclease enzymes, etc. Such reagents may also or alternatively include one or more reagents such as lytic agents, inhibitors, inactivators, chelators, stimulators, and the like. Entrapment of such agents can be controlled by the density of the polymer network created during polymerization of the precursor, by controlling the ionic charge within the bead (eg, by ionic species bound to the polymer species), or by the release of another species. Encapsulated reagents can be released from the beads upon degradation of the beads and/or by application of a stimulus that can release the reagents from the beads. In some embodiments, the beads or arrays of beads can be incubated in the permeabilization reagents described herein.
In some embodiments, the bead may also include (eg, encapsulate or attach to) a plurality of capture probes comprising a spatial barcode, and the optical properties of the spatial barcode may be used to optically detect the bead. For example, light absorption using spatial barcodes can be used to distinguish beads. In some embodiments, a detectable label can be attached directly or indirectly to the spatial barcode and enable optical detection of the bead. In some embodiments, each bead in the pool of one or more beads has a unique detectable label, and detection of the unique detectable label determines the location of the spatial barcode sequence associated with the bead.
The optical properties that cause the optical detection of the pellet may be due to the optical properties of the surface of the pellet (e.g., a detectable label attached to the pellet) or the optical properties of a larger area of the pellet (e.g., a detectable label incorporated during the pelleting process ) its own formation or optical properties). In some embodiments, the detectable tag may be associated with the bead or one or more units associated with the bead.
In some embodiments, the bead includes a plurality of detectable indicia. For example, fluorescent dyes can be attached to the surface of the beads and/or can be incorporated into the beads. Different intensities of different fluorochromes can be used to increase the number of optical combinations that can be used to distinguish beads. For example, if N is the number of fluorochromes (eg, 2 to 10 fluorochromes, such as 4 fluorochromes) and M is the possible color intensity (eg, 2 to 50 intensities, such as 20 intensities), then MYesVarious optical combinations are possible. In one example, 4 fluorescent colors with 20 possible intensities can be used to create 160,000 different optical combinations.
One or more optical properties of the beads or biological content (eg, cells or nuclei) can be used to distinguish individual beads or biological content from other beads or biological content. In some embodiments, the beads can be optically detected by including a detectable label with optical properties to distinguish the beads from each other.
In some embodiments, the optical properties of the beads can be used for optical detection of the beads. For example, without limitation, optical properties may include absorbance, birefringence, color, fluorescence, luminosity, photosensitivity, reflectance, refractive index, scattering, or transmittance. For example, the beads may have different birefringence values based on the degree of polymerization, chain length, or monomer chemistry.
In some embodiments, nanospheres, such as quantum dots or Janus spheres, can be used as optical labels or components thereof. For example, quantum dots can be attached to beads with spatial barcodes.
Optical bead labeling can provide improved spectral resolution for distinguishing (eg, identifying) beads with unique spatial barcodes (eg, beads containing unique spatial barcode sequences). That is, the balls are manufactured in such a way that the optical markings and barcodes (eg spatial barcodes) on the balls are correlated. In some aspects, the beads may be introduced into a flow cell such that the beads are arranged in a compact fashion (eg, single cell resolution). Imaging can be performed and the spatial location of the barcode can be determined (for example, based on information from a look-up table (LUT)). Optical tags for spatial analysis enable fast deconvolution identification of ball barcodes (eg spatial barcodes).
In some examples, a look-up table (LUT) can be used to correlate bead properties (eg, optical markers, such as color and/or intensity) with barcode sequences. This property can originate from particles (eg beads) or from optical labels associated with the beads. The beads can be imaged to obtain optical information about the beads, including, for example, properties of the beads (eg, color and/or intensity) or optical markers associated with the beads, as well as optical information about the biological sample. For example, an image may contain optical information in the visible spectrum, the invisible spectrum, or both. In some embodiments, multiple images can be acquired over different optical frequencies.
In some embodiments, the first grain includes a first optical tag and spatial barcodes, each spatial barcode having a first spatial barcode. The second grain includes a second optical tag and spatial barcodes, each spatial barcode having a second spatial barcode. The first optical label and the second optical label can be different (eg they are provided by two different fluorescent colors or two different intensities of the same fluorescent color). The first and second spatial barcode sequences may be different nucleic acid sequences. In some embodiments, the beads may be imaged to identify the first and second optical indicia, which may then be used to correlate the first and second optical indicia with the first and second spatial barcode arrays, or pair. In some embodiments, the nucleic acid comprising the spatial barcode may also have an analyte capture domain (eg, mRNA). In some embodiments, nucleic acids (eg, nucleic acids containing spatial barcodes) may have unique molecular identifiers, cleavage domains, functional domains, or combinations thereof.
In some embodiments, the optical tag has a characteristic electromagnetic spectrum. As used herein, "electromagnetic spectrum" refers to the frequency range of electromagnetic radiation. In some embodiments, the optical label has a characteristic absorption spectrum. As used herein, "absorption spectrum" refers to the frequency range of electromagnetic radiation that is absorbed. "Electromagnetic spectrum" or "absorption spectrum" can lead to different characteristic spectra. In some embodiments, peak emission or peak absorption occurs at 380-450 nm (violet), 450-485 nm (blue), 485-500 nm (cyan), 500-565 nm (green), 565-590 nm (yellow ), 590 nm -625 nm (orange) or 625-740 nm (red). In some embodiments, peak emission or peak absorption occurs around 400 nm, 460 nm, or 520 nm.
An optical tag contained on the grain can identify an associated spatial barcode on the grain. Since the variety of optical labels is relatively limited, it can be useful to limit the size of the spatial array used for deconvolution. For example, the substrate can be divided into two or more partitions (eg containers). In some embodiments, the substrate can be divided into three or more partitions. In some embodiments, the substrate can be divided into four or more compartments (eg, containers). In some embodiments, a set of balls is placed on the baffle. In each pool of beads, one or more beads (for example, equal to or greater than 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, or 5000 beads) may have unique optical indicia.
In some cases, balls within the same partition may have different coordinates on the substrate. These coordinates can be determined, for example, by different recording techniques, for example by microscopy under appropriate conditions. In some embodiments, beads within the same partition may share the same spatial barcode. In some embodiments, the beads (eg, beads that have capture probes with barcodes (eg, spatial barcodes or UMIs)) are different from each other for different bulk containers. In some embodiments, capture probe beads with barcodes (eg, spatial barcodes or UMIs) may have different barcodes. For example, in some cases, which beads are associated with capture probes in each set of beads, the capture probes on each bead may have a unique barcode. In some cases, a single bead may have a unique capture probe with a barcode in all beads (eg, in two or more sets of beads).
In some aspects, this disclosure provides a substrate. Substrates can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 , 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more than 1000 partitions (for example, containers or predefined areas). Partitions can have the same shape or different shapes. In some embodiments, the substrate has only one partition (eg, a container or a predefined area).
In some embodiments, a first partition (eg, a first predefined area, or a single vessel on a substrate) may have a first set of beads. In some embodiments, at least one bead of the first set of beads comprises an optical label and a capture probe (eg, an oligonucleotide capture probe) comprising a barcode and a capture domain. In the first array of beads, at least one bead may have a unique optical label. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% % , 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the beads in the first set of beads had unique optical markers. In some embodiments, each bead in the first set of beads has a unique optical label.
In some embodiments, the substrate may have another partition (eg, another predefined area or another box). The second compartment may have a second set of beads. In some embodiments, at least one bead of the second set of beads comprises an optical label and a capture probe (eg, an oligonucleotide capture probe) comprising a barcode and a capture domain. In the second array of beads, at least one bead may have a unique optical indicia. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% % , 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the beads in the second set of beads had unique optical markers. In some embodiments, each bead in the second set of beads has a unique optical marker.
In some embodiments, the substrate may have a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth partition or the like. In some embodiments, the substrate may have multiple partitions. In some cases, each of these partitions has properties similar to the first or second partition described herein. For example, at least one bead in each set of beads contains an optical label and a capture probe (eg, an oligonucleotide capture probe) that contains a barcode and a capture domain. At least one bead may have a unique optical marking within each bead set. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96% % , 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% The beads in each set of beads have unique optical markings. In some embodiments, each bead in each set of beads has a unique optical marker.
In some embodiments, the beads are deposited on a substrate. In some embodiments, the beads can be deposited directly onto or into a biological sample. Therefore, in some cases the biological sample can be immobilized or attached to the support before the beads are placed on the support.
In some embodiments, the beads are deposited only on areas of interest (eg, specific locations on the substrate, specific cell types, and specific tissue structures). Therefore, the deposited granules do not necessarily cover the entire biological sample. In some embodiments, one or more regions of the substrate may be masked or modified (eg, closed capture domains) such that the masked regions do not interact with corresponding regions of the biological sample.
In some embodiments, two or more sets of balls (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 sets) are laid out in two or more partitions (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10 partitions). These partitions do not have to be next to each other. The identity of the ball can be determined from the optical marker by simply recording the position of the baffle on the substrate.
In some embodiments, the pool of beads may have equal to or more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000 balls. In some embodiments, a set of 25 balls may have less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000 or 5000 balls.
Optical markers can be turned on when generating beads. For example, optical labels can be incorporated into the polymer structure of gel beads, or attached in the prepolymer or monomer phase of bead production. In some embodiments, the bead includes a portion (eg, on the surface of the bead and/or within the bead) attached to one or more optical markers. In some embodiments, optical labels may be beaded with one or more reagents. For example, reagents and optical labels can be loaded into the beads by diffusion of reagents (eg, solutions of reagents including optical labels). In some embodiments, an optical tag may be included in the preparation of the spatial barcode. For example, spatial barcodes can be prepared by synthesizing molecules that incorporate the barcode sequence (eg, using split assembly or combinatorial approaches). The optical label can be attached to the spatial barcode before attaching the spatial barcode to the bead. In some embodiments, an optical label may be included after attaching the spatial barcode to the bead. For example, optical tags can be attached to spatial barcodes associated with beads. In some embodiments, the spatial barcode or sequence thereof may be separated or split into grains. Optical markers can be detached or integrally attached to the balls. In some embodiments, a first bead (eg, a bead containing a plurality of spatial barcodes) can be connected to a second bead containing one or more optical indicia. For example, the first bead may be covalently linked to the second bead via a chemical bond. In some embodiments, the first bead may be non-covalently attached to the second bead.
The first and/or second grain may contain multiple spatial barcodes. Multiple spatial barcodes attached to a particular grain may include the same barcode sequence. Where both the first bead and the second bead comprise a spatial barcode, the first bead and the second bead may comprise a spatial barcode, the spatial barcode comprises the same barcode sequence or different barcode sequences.
Arrays of beads containing captured analytes can be processed in batches or separated into droplet emulsions to prepare libraries for sequencing. In some embodiments, next generation sequencing reads are clustered and associated with the spatial location of the spatial barcode on the bead array. For example, information can be computer-superimposed on high-resolution images of tissue sections to identify where analytes are detected.
In some embodiments, decrosslinking can be performed to account for decrosslinking chemistries that may be incompatible with certain barcode/library biochemical preparations (eg, presence of proteases). For example, a two-step process is possible. In a first step, the beads can be delivered in droplets so that the DNA binds to the beads after performing conventional cross-linking chemistry. In the second step, the emulsion is broken and the beads are collected, then re-encapsulated after washing for further processing.
In some embodiments, the beads can be immobilized or attached to a substrate using photochemical methods. For example, beads can be functionalized with perfluorophenylazidosilane (PFPA silane), contacted with a substrate, and then exposed to radiation (see, e.g., Liu et al. (2006)Journal of the American Chemical Society128, 14067-14072). For example, immobilization of anthraquinone-functionalized substrates (see, e.g., Koch et al. (2000)bioconjugate chemistry11, 474-483, which is incorporated herein by reference in its entirety).
Arrays can also be prepared by self-assembling beads. Each bead can cover hundreds of thousands of copies of a particular oligonucleotide. In some embodiments, each bead can be coated with about 1,000 to about 1,000,000 oligonucleotides. In some embodiments, each bead can be coated with about 1,000,000 to about 10,000,000 oligonucleotides. In some embodiments, each bead may be coated with about 2,000,000 to about 3,000,000, about 3,000,000 to about 4,000,000, about 4,000,000 to about 5,000,000, about 5,000,000 to about 6,000,000, about 6,000,000 to about 7,000,000 about 7,000,000 to about 8,000,000 about 8,000,000 to about 9,000,000, or about 9,000,000 to about 10,000,000 oligonucleotides. In some embodiments, each bead can be coated with about 10,000,000 to about 100,000,000 oligonucleotides. In some embodiments, each bead can be coated with about 100,000,000 to about 1,000,000,000 oligonucleotides. In some embodiments, each bead can be coated with about 1,000,000,000 to about 10,000,000,000 oligonucleotides. Beads can be irregularly distributed across the etched substrate during array production. During this process, the beads can self-assemble into arrays (for example, on fiber optic bundle substrates or silica substrates). In some embodiments, the balls irregularly arrive at their final position on the array. Therefore, bead positions may need to be mapped, or oligonucleotides may need to be synthesized according to a predetermined pattern.
Balls can be fixed or attached to the substrate covalently, non-covalently, with glue or a combination thereof. The attached beads may be layered, for example, in single layers, double layers, triple layers or clusters. As defined herein, a "monolayer" generally refers to an array of probes, beads, spots, dots, features, microsites, or islands immobilized or attached to a substrate such that the beads are arranged in a single layer of beads. In some embodiments, the beads are tightly packed.
As defined herein, the phrase "substantially monolayer" or "substantially forming a monolayer" generally refers to (forming) an array of arrays of probes, beads, microspheres, spots, dots, features, microsites, or islands immobilized or attached to a substrate as if about 50% to about 99% (eg, about 50% to about 98%) of the beads arranged in a single layer of beads. This alignment can be determined by various methods, including microscopic imaging.
In some embodiments, one layer of beads is located in a predetermined area on the substrate. For example, predefined areas can be demarcated by physical barriers, photomasks, depressions in the substrate, or holes in the substrate.
As used herein, the term "reactive element" generally refers to a molecule or part of a molecule that can react with another molecule or part of a molecule to form a covalent bond. Reactive elements include, for example, amines, aldehydes, alkynes, azides, thiols, haloacetyls, pyridyl disulfides, hydrazides, carboxylic acids, alkoxyamines, mercaptoses, maleimides, Michael acceptors, hydroxyl groups, and active esters. Some reactive elements, such as carboxylic acids, can be treated with one or more activators (eg, acylating agents, isourea formers) to increase the susceptibility of the reactive element to nucleophilic attack. Non-limiting examples of activators include N-hydroxysuccinimide, N-hydroxysulfosuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, bicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-hydroxybenzotriazole, (benzotriazol-1-yloxy)tripyrrolidinium phosphonium hexafluorophosphate, (benzotriazol-1-yloxy) yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 4-(N,N-dimethylamino)pyridine and carbonyldiimidazole.
In some embodiments, the reactive elements are attached directly to the beads. For example, hydrogel beads can be treated with acrylic acid monomers to form acrylic functionalized hydrogel beads. In some cases, the reactive element is indirectly attached to the bead via one or more linkers. As used herein, "linker" generally refers to a multifunctional (eg, bifunctional, trifunctional) reagent for conjugating two or more chemical moieties. The linker can be a cleavable linker that can undergo induced dissociation. For example, dissociation can be induced by solvents (eg, hydrolysis and solvolysis); radiation (eg photolysis); enzymes (eg enzymolysis); or using specific pH values (eg pH 4, 5, 6). , 7 or 8) for processing.
In some embodiments, the reactive element is directly bound to the substrate. For example, subject glasses may be coated with (3-aminopropyl)triethoxysilane. In some embodiments, the reactive element is indirectly attached to the substrate via one or more linkers.
Zrnca gel/hydrogel
In some embodiments, the beads may be gel beads. "Gel" is a semi-hard material that is permeable to liquids and gases. Examples of gels include, but are not limited to, those with colloidal structures, such as agarose; polymeric networks, such as gelatin; hydrogels; and cross-linked polymer structures, such as polyacrylamide, SFA (see, e.g., US Patent Application Publication 2011/0059865, which is incorporated herein by reference in its entirety), and PAZAM (see, e.g., US Patent No. 2011/ 0059865). Application publication no. 2014/0079923, the entire contents of which are incorporated herein by reference).
Gels can be made in different shapes and sizes depending on the purpose. In some embodiments, the gels are prepared and formulated as gel beads (eg, gel beads containing capture probes attached or linked to the gel beads). The gel beads can be hydrogel beads. Hydrogel beads can be formed from molecular precursors, such as polymeric or monomeric species.
In some cases, the beads contain polymers or hydrogels. The polymer or hydrogel may determine one or more properties of the hydrogel bead, such as volume, fluidity, porosity, stiffness, organization, or one or more other characteristics of the hydrogel bead. In some embodiments, the hydrogel beads may include a polymeric matrix (eg, a matrix formed by polymerization or cross-linking). The polymer matrix may include one or more polymers (eg, polymers with different functional groups or repeating units). Cross-linking can occur through covalent, ionic and/or inductive interactions and/or physical entanglement.
A polymer or hydrogel can be formed, for example, when one or more crosslinkable molecules are crosslinked within a hydrogel bead. For example, hydrogels can be formed by cross-linking one or more molecules within hydrogel beads. Hydrogels can be formed by polymerizing multiple monomers within hydrogel beads. Hydrogels can be formed by polymerizing different polymers inside hydrogel beads. A polymer or hydrogel precursor can be added to the hydrogel beads, and the polymer or hydrogel cannot be formed without the application of a stimulus (eg, as described herein). In some cases, hydrogel beads can be encapsulated within a polymer or hydrogel. The formation of hydrogel beads can occur after one or more other changes in the cell that can be caused by one or more other conditions.
The methods described here can be applied to a single hydrogel bead or to multiple hydrogel beads. The method of processing the plurality of hydrogel beads may include providing the plurality of hydrogel beads within a container and subjecting the plurality of hydrogel beads to conditions sufficient to alter one or more properties of the hydrogel beads. For example, a plurality of hydrogel beads may be subjected to a first condition or set of conditions comprising a chemical species, and the cross-section of the hydrogel beads in the plurality of hydrogel beads may be changed from a first cross-section to a second cross-section, the second cross-section being smaller than the first. cross section. Chemicals may include, for example, organic solvents such as ethanol, methanol or acetone. The plurality of hydrogel beads may then be subjected to another condition or set of conditions involving a chemical species, and cross-links may form within each hydrogel bead. Chemicals may include, for example, cross-linking agents. A plurality of processed hydrogel beads can be provided in an aqueous liquid. In some cases, the second cross-section of the plurality of hydrogel beads remains largely in the aqueous liquid. Multi-processed hydrogel beads can be distributed among multiple partitions. The barriers can be, for example, water droplets contained in a water-in-oil emulsion. The manifold can be, for example, multiple holes. Multiple immobilized hydrogel beads can be co-distributed with one or more reagents. In some cases, multiple immobilized hydrogel beads may be co-distributed with one or more beads, each bead containing multiple nucleic acid barcode molecules bound to it. Nucleic acid barcode molecules attached to a given bead may contain a common barcode sequence, and nucleic acid barcode molecules attached to each different bead may contain a sequence containing a different common barcode sequence. The nucleic acid barcode molecule or portion thereof can then be used in a reaction with a target molecule associated with a hydrogel bead of a plurality of hydrogel beads.
Core/shell beads
In some embodiments, the beads are core/shell consisting of an inner core (eg, nanospheres or microspheres) and an outer shell (eg, a hydrogel coating the nanospheres or microspheres). In some embodiments, the inner core may be a solid nanoparticle or a solid microparticle. In some embodiments, the inner core may be a silica inner core (eg, silicon nanoparticles or silicon microparticles). In some embodiments, the inner core of the core/shell grain may have an average diameter of about 1 micron. In some embodiments, the inner core may have an average diameter of about 2 microns. In some embodiments, the inner core may have an average diameter of about 3 microns. In some embodiments, the inner core may have an average diameter of about 4 microns. In some embodiments, the inner core may have an average diameter of about 5 microns. In some embodiments, the inner core may have an average diameter of about 6 microns. In some embodiments, the inner core may have an average diameter of about 7 microns. In some embodiments, the inner core may have an average diameter of about 8 microns. In some embodiments, the inner core may have an average diameter of about 9 microns. In some embodiments, the inner core may have an average diameter of about 10 microns. In some embodiments, the inner core may have an average diameter of about 100 nanometers to about 10 microns.
In some embodiments, the core/shell pellet may have a reduced shell volume by removing solvent, salt, or water from the shell (eg, dehydrating, drying, drying, dehydrating) to form a shrunken core/shell pellet. In another example, core/shell beads can be reduced in shell volume by adjusting temperature or pH, as described above. In some embodiments, the core/shell bead can increase its shell volume, eg, by adding a solvent, salt, or water (eg, rehydration) to form an expanded core/shell bead. In some embodiments, the coating (eg, coated core) may have an average thickness of about 1 micron. In some embodiments, the shell may have an average thickness of about 2 microns. In some embodiments, the shell may have an average thickness of about 3 microns. In some embodiments, the shell may have an average thickness of about 4 microns. In some embodiments, the shell may have an average thickness of about 5 microns.
In some embodiments, the core/shell beads may have an average diameter of about 1 micron to about 10 microns. In some embodiments, the core/shell beads may have an average diameter of about 1 micron. In some embodiments, the core/shell beads may have an average diameter of about 2 microns. In some embodiments, the core/shell beads may have an average diameter of about 3 microns. In some embodiments, the core/shell beads may have an average diameter of about 4 microns. In some embodiments, the core/shell beads may have an average diameter of about 5 microns. In some embodiments, the core/shell beads may have an average diameter of about 6 microns. In some embodiments, the core/shell beads may have an average diameter of about 7 microns. In some embodiments, the core/shell beads may have an average diameter of about 8 microns. In some embodiments, the core/shell beads may have an average diameter of about 9 microns. In some embodiments, the core/shell beads may have an average diameter of about 10 microns.
Methods of covalent binding of properties to substrates
Methods for covalently attaching features (eg, optically labeled beads, hydrogel beads, microspherical beads) to substrates are provided here.
In some embodiments, the features (eg, beads) are attached to the substrate via a covalent bond between the first reactive element and the second reactive element. In some embodiments, the covalently bound beads form a substantially monolayer feature on the substrate (eg, hydrogel beads, microspherical beads).
In some embodiments, the feature (eg, bead) is functionalized with a first reactive element that binds directly to the feature. In some embodiments, the feature is functionalized with a first reactive element, which is indirectly attached to the bead via a linker. In some embodiments, the linker is benzophenone. In some embodiments, the linker is aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the link is a cleavable link.
In some embodiments, the substrate is functionalized with another reactive element attached directly to the substrate. In some embodiments, the substrate is functionalized with another reactive element, which is indirectly attached to the bead via a linker. In some embodiments, the linker is benzophenone. For example, the linker may be benzophenone. In some embodiments, the linker is aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the link is a cleavable link.
In some embodiments, the substrate is glass. In some embodiments, the substrate is pre-functionalized glass.
In some embodiments, about 99% of the covalently bound beads form a monolayer of beads on the substrate. In some embodiments, from about 50% to about 98% constitutes a monolayer of beads on the substrate. For example, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70% About 50% to about 65%, about 50% to about 60%, or about 50% to about 55% of the covalently bonded beads form a monolayer of beads on the substrate. In some embodiments, about 55% to about 98%, about 60% to about 98%, about 65% to about 98%, about 70% to about 98%, about 75% to about 98%, about 80% to about 98 %, about 85% to about 98%, about 90% to about 95%, or about 95% to about 98% of the covalently bound beads form a monolayer of beads on the substrate. In some embodiments, about 55% to about 95%, about 60% to about 90%, about 65% to about 95%, about 70% to about 95%, about 75% to about 90%, about 75% to about 95 %, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, or about 85% to about 95% of the covalently bound beads is used in a monolayer on the substrate beads.
In some embodiments, at least one of the first reactive element and the second reactive element is selected from the group consisting of:
u
- R1from H, C1-C6alkyl or -SO3;
- R2is C1-C6alkyl; i
- X is the halo part.
In some embodiments, at least one of the first reactive element or the second reactive element includes
or
one of them
Indicates the point of attachment of the first reactive element or the second reactive element to the bead (eg, hydrogel bead or microsphere bead) or substrate.
In some embodiments, at least one of the first reactive element or the second reactive element is selected from the group consisting of:
i
u
- R1from H, C1-C6alkyl or -SO3;
- R2is C1-C6alkyl; i
- X is the halo part.
In some embodiments, at least one of the first reactive element or the second reactive element includes
where R1from H, C1-C6alkyl or -SO3In some embodiments, R1It is H. In some embodiments, R1is C1-C6alkyl. In some embodiments, R1Yes bye3.
In some embodiments, at least one of the first reactive element or the second reactive element includes
where R2is C1-C6alkyl. In some embodiments, R2is methyl.
In some embodiments, at least one of the first reactive element or the second reactive element includes
In some embodiments,
It can react with activators to form active esters. In some embodiments, the active ester is
In some embodiments, the activator is an acylating agent (eg, N-hydroxysuccinimide and N-hydroxysulfosuccinimide). In some embodiments, the activator is an O-acylisourea generator (eg, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), dicyclohexylcarbodiimide amines, and diisopropylcarbodiimide). In some embodiments, the activator is at least one acylating agent and at least one O-isourea generator (e.g., N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-di(methylaminopropyl) carbodiimide (EDC), N-hydroxysulfosuccinimide ( sulfo-NHS) and their combinations).
In some embodiments, at least one of the first reactive element or the second reactive element includes
In some embodiments, at least one of the first reactive element or the second reactive element includes
where X is a halogen residue. For example, X is chlorine, bromine or iodine.
In some embodiments, at least one of the first reactive element or the second reactive element includes
In some embodiments, at least one of the first reactive element or the second reactive element includes
In some embodiments, at least one of the first reactive element or the second reactive element includes
In some embodiments, at least one of the first reactive element or the second reactive element is selected from the group consisting of:
i
u
- R3Is it H or C1-C6alkyl; i
- R4R 2 is H or trimethylsilyl.
In some embodiments, at least one of the first reactive element or the second reactive element comprises H.
where R4R 2 is H or trimethylsilyl. In some embodiments, R4And
In some embodiments, at least one of the first reactive element or the second reactive element is selected from the group consisting of:
i
where R3Is it H or C1-C6alkyl. In some embodiments, R3It is H. In some embodiments, R3is C1-C6alkyl.
In some embodiments, at least one of the first reactive element or the second reactive element includes
where R3Is it H or C1-C6alkyl. In some embodiments, R3It is H. In some embodiments, R3is C1-C6alkyl.
In some embodiments, at least one of the first reactive element or the second reactive element comprises
In some embodiments, at least one of the first reactive element or the second reactive element comprises
In some embodiments, one of the first reactive element or the second reactive element is selected from the group consisting of:
- u
- R1from H, C1-C6alkyl or -SO3;
- R2is C1-C6alkyl;
- X is a halogen residue;
- The second of the first reactive element or the second reactive element is selected from the group consisting of:
-
- u
- R3Is it H or C1-C6alkyl; i
- R4R 2 is H or trimethylsilyl.
In some embodiments, one of the first reactive element or the second reactive element is selected from the group consisting of:
i
where R3Is it H or C1-C6alkyl;
The second of the first reactive element or the second reactive element is
where R4R 2 is H or trimethylsilyl. In some embodiments, R3It is H. In some embodiments, R3is C1-C6alkyl. In some embodiments, R4It is H. In some embodiments, R4is a trimethylsilyl group.
In some embodiments, one of the first reactive element or the second reactive element is selected from the group consisting of:
- u
- R1from H, C1-C6alkyl or -SO3;
- R2is C1-C6alkyl;
- X is a halogen residue;
- The second of the first reactive element or the second reactive element is selected from the group consisting of:
-
- where R3Is it H or C1-C6alkyl. In some embodiments, R1It is H. In some embodiments, R1is C1-C6alkyl. In some embodiments, R1Yes bye3In some embodiments, R2is methyl. In some embodiments, X is iodine. In some embodiments, R3It is H. In some embodiments, R3is C1-C6alkyl.
In some embodiments, one of the first reactive element or the second reactive element is selected from the group consisting of:
u
- R1from H, C1-C6alkyl or -SO3;
- R2is C1-C6alkyl;
The second of the first reactive element or the second reactive element includes
where R3Is it H or C1-C6alkyl. In some embodiments, R1It is H. In some embodiments, R1is C1-C6alkyl. In some embodiments, R1Yes bye3In some embodiments, R2is methyl. In some embodiments, R3It is H. In some embodiments, R3is C1-C6alkyl.
In some embodiments, one of the first reactive element or the second reactive element is selected from the group consisting of:
- where X is a halogen residue;
- The second of the first reactive element or the second reactive element includes
-
- In some embodiments, X is bromine. In some embodiments, X is iodine.
In some embodiments, one of the first reactive element or the second reactive element is selected from the group consisting of:
i
The second of the first reactive element or the second reactive element includes
The term "halogen" refers to fluorine (F), chlorine (Cl), bromine (Br) or iodine (I).
The term "alkyl" refers to a hydrocarbon chain that can be straight or branched, contain the indicated number of carbon atoms. For example, C1-10It means that there can be from 1 to 10 (inclusive) carbon atoms in the group. Non-limiting examples include methyl, ethyl, isopropyl, t-butyl, n-hexyl.
The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms have been replaced by an independently selected halogen.
"Alkoxy" refers to -O-alkyl (eg -OCH3).
The term "alkylene" refers to a divalent alkyl group (eg, -CH2—).
The term "alkenyl" refers to a hydrocarbon chain that may be straight or branched and have one or more carbon-carbon double bonds. Alkenyl moieties contain the indicated number of carbon atoms. For example, C2-6Indicates that there may be 2 to 6 (inclusive) carbon atoms in the group.
The term "alkynyl" refers to a hydrocarbon chain which may be straight or branched and have one or more carbon-carbon triple bonds. The alkynyl part contains the indicated number of carbon atoms. For example, C2-6Indicates that there may be 2 to 6 (inclusive) carbon atoms in the group.
The term "aryl" refers to a monocyclic, bicyclic, tricyclic or polycyclic group of 6-20 carbons, where at least one ring in the system is aromatic (e.g. monocyclic with 6 carbons, bicyclic with 10 carbons or tricyclic aromatic with 14 carbons ring system ); wherein 0, 1, 2, 3 or 4 atoms of each ring may be substituted by a substituent. Examples of aryl groups include phenyl, naphthyl, tetrahydronaphthyl and the like.
The methods provided herein for non-covalently attaching features to substrates are methods for non-covalently attaching features (eg, optically labeled beads, hydrogel beads, or microsphere beads) to substrates.
In some embodiments, the features (eg, beads) are attached to the substrate via a non-covalent bond between the first affinity group and the second affinity group. In some embodiments, the non-covalently bound features (eg, beads) form a substantially monolayer of beads (eg, hydrogel beads, microspherical beads) on a substrate.
In some embodiments, the feature is functionalized with a first affinity group that binds directly to the feature. In some embodiments, the feature is functionalized with a first affinity group, which is indirectly attached to the bead via a linker. In some embodiments, the linker is benzophenone. In some embodiments, the linker is aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the link is a cleavable link.
In some embodiments, the substrate is functionalized with another affinity group attached directly to the substrate. In some embodiments, the substrate is functionalized with another affinity group, which binds indirectly to the bead via a linker. In some embodiments, the linker is benzophenone. In some embodiments, the linker is aminomethacrylamide. For example, the linker can be 3-aminopropylmethacrylamide. In some embodiments, the linker is a PEG linker. In some embodiments, the link is a cleavable link.
In some embodiments, the first affinity group or the second affinity group is biotin and the second of the first affinity group or the second affinity group is streptavidin.
In some embodiments, about 99% of the non-covalently bound beads form a monolayer of beads on the substrate. In some embodiments, from about 50% to about 98% constitutes a monolayer of beads on the substrate. For example, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70% about 50% to about 65%, about 50% to about 60%, or about 50% to about 55% of the non-covalently bound beads form a monolayer of beads on the substrate. In some embodiments, about 55% to about 98%, about 60% to about 98%, about 65% to about 98%, about 70% to about 98%, about 75% to about 98%, about 80% to about 98 %, about 85% to about 98%, about 90% to about 95%, or about 95% to about 98% of the non-covalently bound beads form a monolayer of beads on the substrate. In some embodiments, about 55% to about 95%, about 60% to about 90%, about 65% to about 95%, about 70% to about 95%, about 75% to about 90%, about 75% to about 95 %, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, or about 85% to about 95% of non-covalently bound beads is used for a single layer of beads on a substrate.
In some embodiments, a single layer of beads is formed in predetermined areas on the substrate. In some embodiments, the predefined area is divided by physical barriers. For example, pits or wells in the substrate. In some embodiments, predefined regions are delineated using a photomask. For example, the substrate is coated with a photoactivating solution, dried and then irradiated under a photomask. In some embodiments, the photoactivated solution is UV-activated.
As used herein, "adhesive" generally refers to a substance used to join objects or materials together. Adhesives include, for example, adhesives, pastes, liquid tapes, epoxies, bioadhesives, gels and slimes. In some embodiments, the adhesive is a liquid tape. In some embodiments, the adhesive is an adhesive.
In some embodiments, the beads are adhered to the substrate using an adhesive (eg, liquid tape, glue, paste). In some embodiments, the bonded beads form substantially monolayer beads on a substrate (eg, glass). In some embodiments, the beads are hydrogel beads. In some embodiments, the beads are microspherical beads. In some embodiments, the beads are coated with an adhesive, and the beads are then contacted with a substrate. In some embodiments, the substrate is coated with an adhesive, and the substrate is then contacted with the beads. In some embodiments, both the substrates are coated with the adhesive and the beads are both coated with the adhesive, and then the beads and the substrate are in contact with each other.
In some embodiments, about 99% of the bonded beads form a monolayer of beads on the substrate. In some embodiments, from about 50% to about 98% constitutes a monolayer of beads on the substrate. For example, about 50% to about 95%, about 50% to about 90%, about 50% to about 85%, about 50% to about 80%, about 50% to about 75%, about 50% to about 70% about 50% to about 65%, about 50% to about 60%, or about 50% to about 55% of the adhered beads form a monolayer of beads on the substrate. In some embodiments, about 55% to about 98%, about 60% to about 98%, about 65% to about 98%, about 70% to about 98%, about 75% to about 98%, about 80% to about 98 %, about 85% to about 98%, about 90% to about 95%, or about 95% to about 98% of the adhered beads form a monolayer of beads on the substrate. In some embodiments, about 55% to about 95%, about 60% to about 90%, about 65% to about 95%, about 70% to about 95%, about 75% to about 90%, about 75% to about 95 %, about 80% to about 90%, about 80% to about 95%, about 85% to about 90%, or about 85% to about 95% of the bonded beads is used for one layer of beads on the grain of the substrate.
In some embodiments, the beads may be deposited on the biological sample such that the deposited beads form a monolayer of beads on the biological sample (eg, above or below the biological sample). In some embodiments, beads deposited on a substrate may self-assemble into a bead monolayer that saturates the expected surface area of the biological sample being studied. In this approach, bead arrays can be designed, formulated and prepared to assess multiple analytes from biological samples of any size or dimension. In some embodiments, the concentration or density of beads (eg, gel beads) applied to the biological sample is such that the entire region or one or more regions of interest in the biological sample is saturated with monolayer beads. In some embodiments, the beads are contacted with the biological sample by pouring, pipetting, spraying, etc. onto the biological sample. Any suitable form of bead deposition may be used.
In some embodiments, biological samples may be restricted to specific regions or areas of the array. For example, a biological sample can be attached to a slide and a chamber, seal, or cage placed above the biological sample to serve as a holding space or frame within which the beads are deposited. It will be apparent that one of ordinary skill in the art can readily determine the density or concentration of beads required to saturate an area or biological sample (eg, by microscopic observation of the beads in a biological sample). In some embodiments, the bead array contains microfluidic channels for directing reagents to the dots or beads of the array.
feature geometry
Features on an array can be of multiple sizes. In some embodiments, the array features may have an average diameter or greatest dimension between 500 nm µm and 100 µm. For example, 500 nm to 2 µm, 1 µm to 3 µm, 1 µm to 5 µm, 1 µm to 10 µm, 1 µm to 20 µm, 1 µm to 30 µm, 1 µm to 40 µm, 1 µm to 50 µm. , 1 µm to 60 µm, 1 µm to 70 µm, 1 µm to 80 µm, 1 µm to 90 µm, 90 µm to 100 µm, 80 µm to 100 µm, 70 µm to 100 µm, 60 µm to 100 µm, 50 µm to 100 µm, 40 µm to 100 µm, 30 µm to 100 µm, 20 µm to 100 µm, 10 µm to 100 µm, about 40 µm to about 70 µm or about 50 µm to about 60 µm. In some embodiments, the features have an average diameter or maximum dimension between 30 µm to 100 µm, 40 µm to 90 µm, 50 µm to 80 µm, 60 µm to 70 µm, or any range within the disclosed subrange. In some embodiments, the features have an average diameter or greatest dimension of no greater than 95 µm, 90 µm, 85 µm, 80 µm, 75 µm, 70 µm, 65 µm, 60 µm, 55 µm, 50 µm, 45 µm, 40 µm, 35 μm, 30 μm, 25 μm, 20 micron, 15 micron, 14 micron, 13 micron, 12 micron, 11 micron, 10 micron, 9 micron, 8 micron, 7 micron, 6 micron, 5 micron, 4 micron, 3 micron , 2 microns or 1 micron. In some embodiments, the features have an average diameter or greatest dimension of about 65 μm. In some embodiments, the features have an average diameter or greatest distance of about 55 μm.
In some embodiments, the plurality of array features are approximately uniform in size and/or shape. In some embodiments, the multiple array features are not uniform in size and/or shape. For example, in some embodiments, features in an array may have an average cross-sectional dimension, and the distribution of cross-sectional dimensions among features may have a full width and half maximum value of 0% or greater (e.g., 5% or greater, 10% or greater, 20% or more, 30% or more, 40% or more, 50% or more, 70% or more, or 100% or more) of the average cross-sectional size distribution.
In certain embodiments, the features in the array may have an average cross-sectional dimension between about 1 µm and about 10 µm. This range of average characteristic cross-sectional dimensions corresponds to the approximate diameter of a single mammalian cell. Accordingly, an array of such features can be used to detect analytes at or below the resolution of a single mammalian cell.
In some embodiments, the plurality of features have an average diameter or average largest dimension of about 0.1 μm to about 100 μm (e.g., about 0.1 μm to about 5 μm, about 1 μm to about 10 μm, about 1 μm to about 20 μm ), about 1 μm to about 30 μm, about 1 μm to about 40 μm, about 1 μm to about 50 μm, about 1 μm to about 60 μm, about 1 μm to about 70 μm, about 1 μm to about 80 μm, about 1 μm to about 90 μm, about 90 μm to about 100 μm, about 80 μm to about 100 μm, about 70 μm to about 100 μm, about 60 μm to about 100 μm, about 50 μm to about 100 μm, about 40 μm to about 100 μm, about 30 μm to about 100 μm, about 20 μm to about 100 μm, or about 10 μm to about 100 μm). In some embodiments, the plurality of features have an average diameter or average largest dimension between 30 µm to 100 µm, 40 µm to 90 µm, 50 µm to 80 µm, 60 µm to 70 µm, or detected subranges Any range within. In some embodiments, the plurality of features has a range of no greater than 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm. 30 µm, 25 µm µm, 20 µm, 15 µm, 14 µm, 13 µm, 12 µm, 11 µm, 10 µm, 9 µm, 8 µm, 7 µm, 6 µm, 5 µm, 4 µm, 3 µm, 2 µm µm or 1 µm. In some embodiments, the plurality of features has an average diameter or average largest dimension of about 65 μm, about 60 μm, about 55 μm, about 50 μm, about 45 μm, about 40 μm, about 35 μm, about 30 μm, about 25 μm, about 20 µm, about 15 µm, about 10 µm, about 5 µm, about 4 µm, about 3 µm, about 2 µm or about 1 µm.
(iv) Array geometry attributes
In some embodiments, the array includes a plurality of characteristics. For example, an array containing 4,000 to 50,000 features, or any range between 4,000 and 40,000 features. For example, an array with 4,000 to 35,000 features, 4,000 to 30,000 features, 4,000 to 25,000 features, 4,000 to 20,000 features, 4,000 to 15,000 features, 4,000 to 10,000 features, 4,000 to 6,000 features. or 4, 400 to 6000 features. In some embodiments, the array includes 4100 to 5900 features, 4200 to 5800 features, 4300 to 5700 features, 4400 to 5600 features, 4500 to 5500 features, 4600 to 5400 features, 4700 to 5300 features, 4700 to 5300 features, 4800 to 5300 features. 0 and 5200 features, 4900 to 5100 features or any range within the detected subrange. For example, the array may include about 4,000 features, about 4,200 features, about 4,400 features, about 4,800 features, about 5,000 features, about 5,200 features, about 5,400 features, about 5,600 features, or about 6,000 features, about 10,000 features, about 20,000 features, around 30,000 features, around 40,000 features or around 50,000 features. In some embodiments, the array includes at least 4000 features. In some embodiments, the array includes approximately 5,000 features.
In some embodiments, the features within the array have an irregular arrangement or relationship to each other such that there is no apparent pattern or regularity in the geometric spacing relationship between the features. For example, features in an array may be randomly placed relative to each other. Alternatively, the features within the array may be placed irregularly, but the spacing may be chosen deterministically to ensure that the resulting arrangement of features is irregular.
In some embodiments, the features within the array are properly positioned relative to one another to form a pattern. Various feature modes can be implemented in arrays. Examples of such patterns include, but are not limited to, arrays of square features, arrays of rectangular features, arrays of hexagonal features (including hexagonal close-packed arrays), arrays of radial features, arrays of spiral features, arrays of triangular features, and generally, any array in which adjacent features in series they reach each other by regular increase of linear and/or angular coordinate dimensions.
In some embodiments, the features within the array are placed with a degree of regularity relative to each other such that the array of features is neither fully regular nor fully irregular (ie, the array is "partially regular"). For example, in some embodiments, adjacent features in an array may be separated by an offset in one or more linear and/or angular coordinate dimensions of 10% or more (e.g., 20% or more, 30% or more, 40% or more, 50 % or more, 60% or more, 70% or more, 80% or more, 90% or more, 100% or more, 110% or more, 120% or more, 130% or more, 140% or more, 150 % or more, 160% or more, 170% or more, 180% or more, 190% or more, 200% or more) the average or nominal offset between adjacent features in a row. In some embodiments, the displacement distribution (linear and/or angular) between adjacent features in the array has half the range between 0% and 200% (eg, between 0% and 100%, between 0%) Maximum (75% , 0% and 50 %, 0% and 25%, 0% and 15%, 0% and 10%) of the average or nominal offset between adjacent features in a full-width array).
In some embodiments, the array of features may have a variable geometry. For example, a first subset of features in the array may be arranged according to a first geometric pattern, while a second subset of features in the array may be arranged according to a second geometric pattern different from the first pattern. For example, any of the patterns described above may correspond to the first and/or second geometric patterns.
In general, arrays of different feature densities can be prepared by adjusting the spacing between adjacent features in the array. In some embodiments, the geometric center-to-center spacing (e.g., pitch) between adjacent features in the array is between 100 nm to 10 μm, 500 nm to 2 μm, 1 μm to 5 μm, and 20 μm to 200 μm. For example, the center-to-center spacing can be between 100 nm to 10 μm, 500 nm to 2 μm, 1 μm to 5 μm, 20 μm to 40 μm, 20 μm to 60 μm, 20 μm to 80 μm, 80 μm to 100 µm, 100 µm to 120 µm, 120 µm to 140 µm, 140 µm to 160 µm, 160 µm to 180 µm, 180 µm to 200 µm, 60 µm to 100 µm or 40 µm to 100 µm, 50 µm to 150 µm. µm µm, 80 µm to 120 µm or 90 µm to 110 µm. In some embodiments, the spacing between adjacent array features is between 30 µm and 100 µm, 40 µm and 90 µm, 50 µm and 80 µm, 60 µm and 70 µm, 80 µm and 120 µm, or all public subranges. In some embodiments, the spacing between adjacent array elements is about 65 μm, about 60 μm, about 55 μm, about 50 μm, about 45 μm, about 40 μm, about 35 μm, about 30 μm, about 25 μm, about 20 μm, about 15 µm µm, about 10 µm, about 5 µm, about 4 µm, about 3 µm, about 2 µm or about 1 µm. In some embodiments, the spacing between adjacent array features is less than 100 μm.
The feature set can have any suitable resolution. In some embodiments, the array of features may have a spatially constant (eg, within error) resolution. In general, an array of spatially uniform resolution is one in which the spacing between adjacent features in the array is constant (eg, within error bounds). Such arrays can be used in various applications. In some embodiments, the array of features may have a spatially varying resolution. In general, an array with spatially variable resolution is one in which the center-to-center spacing (eg, pitch) between adjacent features in the array varies (along linear, angular, or both linear and angular coordinate dimensions). Such arrays can be used in various applications. For example, in some embodiments, depending on the spatial resolution of the sample under study, the sample may be selectively associated with a portion of the array that approximately corresponds to the desired spatial resolution of the measurement.
In some embodiments, it may be useful to describe the resolution of a set of features in terms of functional aspects, e.g., the number of reads each feature can produce (which may represent sequencing saturation), the number of transcripts each feature can detect, the number of genes detected, or the number of genes detectable for every feature. For example, in some embodiments, between 50,000 and 1,000,000 reads may be performed per signature. For example, the number of reads that can be performed per feature can be between 50,000 and 100,000, 50,000 and 150,000, 50,000 and 200,000, 50,000 and 250,000, 50,000 and 300,000, 50,000 and 350,000, 50,000 and 400,000, 50,000 and 500,000, 50,000 and 550,000, 50,000 and 600,000, 50,000 and 650,000, 50,000 and 700,000, 50,000 and 750,000, 50,000 and 800,000, 50,000 and 850,000, 50,000 and 900,000, 50,000 to 950,000, 50,000 up to 1 million, 100,000 to 500,000, 150,000 to 500,000, 200,000 to 500,000, 250,000 to 500,000, 300,000 and 500,000, 350,000 and 500,000, 400,000 and 500,000, 450,000 and 500,000, 150,000 to 250,000, or 300,000 to 400,000. In some embodiments, the number of reads that can be performed per signature is approximately 70,000. In some embodiments, the number of reads that can be performed per signature is approximately 170,000. In some embodiments, the number of reads that can be performed per signature is approximately 330,000. In some embodiments, the number of reads that can be performed per signature is approximately 500,000. In some embodiments, the number of reads that can be performed per signature is approximately 800,000.
In some embodiments, the number of detectable transcripts per signature is between 20,000 and 200,000. For example, in some embodiments, the number of detectable transcripts per feature can be between 20,000 and 30,000, 20,000 and 40,000, 20,000 and 50,000, 30,000 and 60,000, 40,000 and 60,000, 50,000 and 60,000, 20,000. 000 and 100,000, 30,000 and 100,000, 40,000 and 200,000, 50,000 and 200,000, respectively 30,000 and 200,000. In some embodiments, the number of detectable transcripts per signature is approximately 40,000. In some embodiments, the number of detectable transcripts per signature is approximately 60,000. In some embodiments, the number of detectable transcripts per signature is approximately 80,000. In some embodiments, the number of detectable transcripts per signature is approximately 100,000.
In some embodiments, the number of detectable genes per signature is between 1,000 and 5,000. For example, the number of detectable genes per trait may be between 1000 and 1500, 1000 and 2000, 1000 and 2500, 1000 and 3000, 1000 and 3500, 1000 and 4000, 1000 and 4500, 1500 and 5000, 2000 and 5000. 000, 2500 and 5000, 3000 and 5000, 3500 and 5000, 4000 and 5000, 4500 and 5000, 1500 and 2500, 2500 and 3500 or 3500 and 4000. In some embodiments, the number of detectable genes per signature is approximately 2,000. In some embodiments, the number of genes detectable per signature is approximately 3,000. In some embodiments, the number of detectable genes per signature is approximately 4,000.
In some embodiments, it may be useful to describe the resolution of a set of features in terms of functionality, eg, the number of UMI counters per feature. For example, in some embodiments, the number of UMI counts that each feature can perform is between 1,000 and 50,000. In some embodiments, the number of UMI counts may be averaged to obtain an average UMI for each feature. In some embodiments, the number of UMI counts may be averaged to obtain a mean UMI count for each feature. For example, the median UMI number for each characteristic can be between 1,000 and 50,000, 1,000 and 40,000, 1,000 and 30,000, 1,000 and 20,000, 1,000 and 10,000, 1,000 and 5,000. In some embodiments, the mean UMI number per signature is approximately 5000. In some embodiments, the mean UMI number per signature is approximately 10,000.
These components can be used to determine the saturation of array sequencing. Sequencing saturation can be a measure of library complexity and sequencing depth. For example, different cell types will have different amounts of RNA and therefore produce different numbers of transcripts, affecting the complexity of the library. Furthermore, sequencing depth is related to the number of sequencing reads. In some embodiments, the inverse sequencing saturation is the number of additional reads required to detect new transcripts. One way to measure the saturation of sequencing arrays is to determine the number of reads that reveal new UMIs. For example, if a new UMI is detected every 2 signature reads, sequencing saturation will be 50%. As another example, if a new UMI is detected for every 10 signatures read, sequencing saturation will be 90%.
Arrays of spatially different resolutions can be realized in several ways. In some embodiments, for example, the spacing between adjacent features in an array varies continuously along one or more linear and/or angular coordinate directions. Therefore, for a rectangular array, the spacing between consecutive rows of features, between consecutive columns of features, or between consecutive rows and columns of features can vary continuously.
In some embodiments, the array of spatially varying resolutions may include discrete domains that have a population of features. Within each domain, adjacent features may have regular spacing. Thus, for example, the array may include a first domain and a second domain in which adjacent features are spaced from each other along a linear and/or angular coordinate dimension by a first set of uniform coordinate offsets, and a second domain in which adjacent features are spaced from each other by others along other Linear and/or angular coordinate dimensions are offset by another set of uniform coordinates. The first and second sets of offsets differ by at least one coordinate offset such that the spacing of adjacent features differs in the two domains, and the resolution of the array in the first domain is therefore different from the resolution of the array in the second domain.
In some embodiments, the pitch of the array features may be small enough to effectively position the array features continuously or nearly continuously along one or more dimensions of the array with little or no displacement between array features along those dimensions. For example, in a feature array where the features correspond to regions of the substrate (ie, the oligonucleotides are directly bound to the substrate), the offset between adjacent oligonucleotides can be very small—in fact, one molecular width of the oligonucleotide. In such embodiments, each oligonucleotide may include a different spatial barcode such that the spatial location of each oligonucleotide in the array can be determined during sample analysis. Arrays of this type can have very high spatial resolution, but may contain only one oligonucleotide corresponding to each separate spatial location in the sample.
In general, array size (corresponding to the largest dimension along the smallest boundary encompassing all features in the array in one coordinate direction) can be chosen arbitrarily based on criteria such as sample size, feature diameter, and density of recorded probes in each feature. For example, in some embodiments, the array may be a rectangular or square array, where the largest dimension of the array along each coordinate direction is 10 mm or less (eg, 9 mm or less, 8 mm or less, 7 mm or less, 6 mm or less , 5 mm or less, 4 mm or less, 3 mm or less). Thus, for example, a square array of features may have dimensions of 8 mm x 8 mm, 7 mm x 7 mm, 5 mm x 5 mm, or less than 5 mm x 5 mm.
(v) Row of balls
As used herein, the term "bead array" refers to an array that contains a plurality of beads as an array feature. In some embodiments, two or more beads are sputtered onto a substrate to form an array, where each bead is a feature on the array. In some embodiments, the balls are attached to the substrate. For example, the beads can optionally be attached to a substrate such as a subject microscope slide and brought into proximity to a biological sample (eg, a tissue section including cells). Beads can also be suspended in solution and deposited on surfaces (eg membranes, tissue sections or matrices (eg microscope slides)). The beads can optionally be dispersed into wells on the substrate, for example, such that each well contains only one bead.
Examples of bead arrays on or within a substrate include beads-in-wells such as BeadChip microarray genotyping arrays (available from Illumina Inc., San Diego, CA), and arrays for sequencing platforms, from Ion Torrent (a subsidiary of Life Technologies, Carlsbad, CA ). Examples of ball arrays are described, for example, in US Pat. Nos. 6,266,459; 6,355,431; 6,770,441; 6,859,570; 6,210,891; 6,258,568; 2010/0137143; 2019/0177777; and 20 10/0282617 and PCT patents. Application publications no. WO 00/063437 and WO 2016/162309, the entire contents of each are incorporated herein by reference.
In some embodiments, the bead array includes a plurality of beads. For example, an array of beads may contain at least 10,000 beads (eg, at least 100,000 beads, at least 1,000,000 beads, at least 5,000,000 beads, at least 10,000,000 beads). In some embodiments, the plurality of grains comprises a single type of grain (eg, substantially uniform in volume, shape, and other physical properties (eg, transparency)). In some embodiments, the plurality of beads includes two or more types of different beads.
Arrays of beads can be generated by attaching beads (eg barcoded beads) to a substrate in a regular pattern or an irregular arrangement. In some embodiments, the barcode sequences are known prior to their attachment to the substrate. In some embodiments, the barcode sequences are not known prior to their attachment to the substrate. The beads can be attached to selective regions on the substrate, for example, by selectively activating regions on the substrate to allow attachment of the beads. Activating a selective region on a substrate may include activating or degrading a coating (eg, a conditionally removable coating as described herein) on the selective region where the coating is applied to the substrate such that the selective region is compatible with the selected region. area outside the area. Areas that are more allowable for ball attachment can be configured to fit only one ball or multiple balls (eg, limited by hole size or surface pattern, eg, manufacturing technique). Beads bound to selected areas can form two-dimensional arrays on a substrate. The substrate can be evenly or unevenly coated. The beads may be any suitable beads described herein, including beads attached to one or more spatial barcodes. The beads can be attached to the selected regions by any method suitable for attaching the beads to the substrates described herein, such as covalent bonds, non-covalent bonds, or chemical linkers.
The beads can be attached to the surface of the substrate using any of a variety of suitable patterning techniques. In some embodiments, without limitation, physical techniques such as inkjet printing, optical and photoelectric cell trapping, laser patterning, acoustic patterning, dielectrophoretic or magnetic techniques may be used to pattern the substrate. Alternatively, chemical and/or physicochemical techniques may be used, such as, but not limited to, surface chemical methods, microcontact printing, micropores and filtration, DUV sampling, or sampling in microfluidic devices combined with microcontact printing (see, e.g., Martinez Martinez -Rivas, A., Micropatterning and Cell Manipulation Methods for Biomedical Applications, Micromachines (Basel) 8, (2017), incorporated herein by reference).
The coating may be photoreactive, and selective activation or degradation of the coating involves exposing selected areas of the coating to light or radiation. Selectivity can be achieved by applying a photomask. Areas of the coating exposed to light may become more receptive to bead attachment (eg, more sticky) than areas not exposed to light (eg, areas protected from light by a photomask). When applied to a substrate, the beads are therefore preferentially attached to looser areas of the substrate, and unattached beads can be optionally removed from the substrate. The light source and/or photomask can be adjusted to make more spots on the substrate more acceptable for bead attachment, thus allowing for additional beads at those spots. Alternatively, a different light source or a different photomask can be applied. Thus, the photopatterning process allows beads to be attached at predetermined locations on the substrate, thereby generating arrays of beads.
Beads may be bonded repeatedly, for example, one subset of beads may be bonded at a time, and the process may be repeated to bond one or more additional subsets of beads. In some embodiments, the size of the actuated spot (eg, spot on the substrate) is smaller than the size of the bead. For example, balls can be attached to an actuated base (eg, a dot) such that only one ball is attached to the actuated base. In some embodiments, the substrate can be washed to remove unbound beads. In some embodiments, the substrate can be activated at a different location and the second bead can be attached to the activated surface of the substrate. This process can be repeated to attach the beads to all or part of the substrate. Alternatively, the balls can be attached to the substrate in one step. Furthermore, methods of attaching beads to substrates are known in the art. Any suitable method may be used, including but not limited to specific chemical bonds, non-specific chemical bonds, linkers, physical capture beads (eg, polymers, hydrogels), or any of the methods described herein.
An exemplary workflow for generating bead arrays may include selectively patterning a first set of one or more selected regions on a coated substrate that is more permissive for bead attachment than regions outside of the selected regions, placing a plurality of beads applied to the array allows the beads to attach to the first set selected areas, optionally removing unattached beads so that another set of one or more selected areas is more bead-allowable than areas outside the second set of selected areas Pinning, applying more beads and allowing beads to attach to another set of selected areas and optionally removing beads that are not attached. This iterative process can be performed an unlimited number of times to generate arrays of patterned beads.
Another exemplary process includes activating a first region on a coated substrate and exposing the activated first region to a plurality of barcode beads such that a first set of one or more beads bind to the first region; [0032] Activation coating the second region on the substrate and exposing the activated second region to a plurality of barcode beads such that the second set of one or more beads binds to the second region, wherein the first set of one or more beads comprises the first same first oligonucleotide sequence specific for region, and the second set of one or more beads contains the same second oligonucleotide sequence specific for the second region of the substrate surface, and wherein the first and second oligonucleotide sequences are different. Other areas on the coated substrate can be activated and exposed to additional barcode beads. Each set of barcoded beads may contain an oligonucleotide sequence that is different from all other sets of barcoded beads and that is unique to the location of the activation region. In some cases, a set of barcoded beads includes a two-part barcode, where the first part is associated with the position at which the row of beads is attached and the second part is associated with the position of the bead relative to the bead in the set. For example, the position of a bead in a set of substrate-bound beads can be identified based on the sequence information of the first part of the barcode and the second part of the barcode. The first part of the barcode can be the same among beads within the same set and can be attached to the beads before or after they are adhered to the substrate. The second part of the barcode can be distinguished between the two beads within the set and can be attached to the bead (eg attached to the first part of the barcode) before or after the bead is attached to the substrate. In some cases, a second part of the barcode is attached to the bead after the bead is bonded to the substrate.
Additionally, the first set of one or more balls and the second set of one or more balls may differ. In other words, the first set of one or more beads and the second set of one or more beads may have different surface chemistries, different compositions (eg, solid beads, gel beads, silicate beads) (eg, nanoparticles and microparticles), and/or physical volumes. . In some embodiments, the third set of one or more beads, the fourth set of one or more beads, the fifth set of one or more beads or more may have different surface chemistries, different compositions (eg, solid beads, gel beads, silica beads) (eg nanoparticles and microparticles), and/or physical volumes can be attached to the surface of the substrate. The method may include removing beads that do not bind the first, second and/or any additional regions. In some embodiments, removing the beads includes washing the beads from the surface of the substrate. Removal can be performed after each round or several rounds of activating an area (eg, a first, second or additional area on a substrate surface) and binding beads to the activated area. In some cases, each ball is attached to the substrate in one place. The beads bound to the first, second and additional regions can form a two-dimensional array of beads on the substrate.
A photoreactive coating may contain a plurality of photoreactive molecules that can undergo a chemical reaction (eg, hydrolysis, oxidation, photolysis) upon exposure to light of a specific wavelength or range of wavelengths. Photoreactive molecules can become reactive when exposed to light and can react and form chemical bonds with other molecules.
The coating may contain a polymer, and activating selected areas on the substrate involves modifying the polymer at the appropriate areas. Modifying the polymer includes, for example, photochemically modifying the polymer by exposing the polymer to radiation or light. Alternatively or additionally, modifying the polymer may include chemically modifying the polymer by contacting the polymer with one or more chemical agents. In some cases, the coating is a hydrogel. In some cases, the coating includes a photoreactive polymer. Examples of photoresponsive polymers include poly(ethylene glycol) (PEG)-based polymers, poly(L-lysine) (PLL)-based polymers, functionalized or non-functionalized polymers containing PEG and PLL unit copolymers (for example, poly-L-polyethylene glycol with grafted lysine (PLL-g-PEG)) and gelatin methacrylate polymers (GelMA).
The beads can also be attached to selected areas on the substrate by selectively cross-linking the beads to a coating already applied to the substrate. For example, multiple beads can be deposited on a substrate with a photocrosslinked coating, and after a subset of the beads are crosslinked with the coating, the uncrosslinked beads can be removed, leaving only the crosslinked beads in the substrate. This procedure can be repeated several times. The coating may contain a photocrosslinkable polymer. Examples of photocrosslinkable polymers are described, for example, in Shirai,Polymer Journal46:859-865 (2014), orgy,photocrosslinked polymer, Light-dependent reactions of synthetic polymers. Springer, New York, NY (2006) and Ferreira et al.Photocrosslinked polymers for biomedical applications, biomedical engineering—limits and challenges, prof. Reza Fazel (ed.), ISBN: 978-953-307-309-5 (2011), each of which is incorporated herein by reference in its entirety.
Suitable light sources for activating, degrading, or cross-linking coatings as described herein include, but are not limited to, ultraviolet (UV) light (eg, 250-350 nm or 350-460 nm UV light) and visible light (eg, broad-spectrum visible light). A digital micromirror device (DMD) can also be used as a light source.
The distance between the first pair of adjacent selected regions according to the methods described herein may be the same or different from the second pair of adjacent selected regions.
Barcode beads or beads containing multiple barcode probes can be prepared by first preparing multiple barcode probes on a substrate, placing the multiple beads on the substrate, and using the probes on the substrate to generate probes attached to the template generation beads.
Large-scale commercial production methods allow millions of oligonucleotides to be attached to an array. Commercially available arrays include those from Affymetrix (ThermoFisher Scientific).
In some embodiments, the arrays may be in accordance with WO 2012/140224, WO 2014/060483, WO 2016/162309, WO 2017/019456, WO 2018/091676 and WO 2012/140224 and US Pat. Application no. 2018/ 0245142. The entire contents of each of the above documents are incorporated herein by reference.
In some embodiments, an array of beads is formed when the beads are embedded in a layer of hydrogel, wherein the hydrogel polymerizes and fixes the associated positions of the beads. Bead arrays can be pre-equilibrated and combined with reaction buffers and enzymes (eg, reverse transcription mix). In some embodiments, bead arrays can be stored (eg, frozen) for an extended period of time (eg, days) prior to use.
(vi) Flexibilni niz
A "flexible array" includes multiple spatial barcode features attached or embedded in a flexible substrate (eg, membrane, hydrogel, or tape) placed on or near a biological sample. In some embodiments, the flexible array includes a plurality of spatially labeled features embedded within the hydrogel.
Flexible arrays can be very modular. In some embodiments, spatially barcoded features (eg, beads) can be loaded onto substrates (eg, glass slides) to create high-density self-assembled arrays. In some embodiments, features (eg, beads) can be loaded onto a substrate using a flow cell. In some embodiments, the features (eg, beads) are embedded in a hydrogel (eg, a hydrogel pad or layer placed on top of the self-assembled features). In some embodiments, the hydrogel can be polymerized, thereby immobilizing the characteristics in the hydrogel. In some embodiments, the hydrogel containing the features can be removed from the substrate and used as a flexible array. In some embodiments, the flexible arrays can be deconvoluted by optical sequencing or any other method described herein. In some embodiments, the features (eg, beads) may be about 1 µm to about 25 µm in diameter. In some embodiments, about 25 μm diameter features in a flexible array can provide about 1000 DPI and a resolution of about 1 million pixels. In some embodiments, the features (eg, beads) may be about 13.2 μm in diameter. In some embodiments, about 13.2 µm balls in a flexible array can provide a resolution of about 1920 x 1080.
Flexible arrays (eg, beads embedded in hydrogels) created by any of the methods described herein can contain thermolabile polymers. In some embodiments, the flexible array with heat-labile beads may be in contact with the biological sample. In some embodiments, a region of interest in a biological sample can be identified such that an infrared laser can be used to select the region of interest. In some embodiments, infrared laser light can cause deformation and jamming of flexible arrays (eg, heat-resistant beads). In some embodiments, the adhesive portion of the flexible array may adhere (e.g., bind) to the region of interest (e.g., cell) directly above or directly below. The process of identifying an area of interest, applying infrared laser light to the area of interest, and adhering the underlying biological sample (such as cells) to the flexible array can be repeated. In some embodiments, the flexible array can be removed so that only adhered biological samples (eg, cells) from one or more regions of interest can also be removed with the flexible array. In some embodiments, the flexible array and the attached biological sample can be further processed (eg, amplified, quantified and/or sequenced) according to any of the methods described herein.
Flexible arrays can be pre-equilibrated with working concentrations of reaction buffer and enzyme (eg reverse transcription mix). In some embodiments, the flexible arrays can be stored for extended periods of time (eg, several days) or frozen until ready for use. In some embodiments, permeabilization of biological samples (eg, tissue sections) can be performed by adding enzymes/determinants prior to contact with the flexible array. In some embodiments, the flexible array may be placed directly on the sample or placed in indirect contact with the sample (eg, with an interlayer or substance between the biological sample and the flexible bead array). In some embodiments, the flexible array can be mechanically applied (eg, pressed or compressed between two surfaces) to a biological sample to enhance analyte capture. In some embodiments, flexible arrays can be applied to the sides of the biological sample. For example, biological samples can be cut (eg, cut) in any direction, and flexible arrays can be applied to exposed analytes. In some embodiments, the flexible array can be dissolved (eg, by thermal, chemical, or enzymatic degradation). In some embodiments, after the flexible array is applied to the sample, the microspheres or the first volume of beads and the second volume of beads or any of the beads described herein can be subjected to reverse transcription and targeted analyte capture. In some embodiments, after the flexible array is applied to the biological sample and analyte capture is enabled, the flexible array can be removed (e.g., stripped) from the biological sample for further processing (e.g., amplification, quantification, and/or sequencing).), according to any of the methods described here.
Flexible arrays can also be used with any of the methods described herein (eg, active capture methods such as electrophoresis). For example, the flexible array may be in contact with a biological sample on a conductive substrate (eg, indium tin oxide coated glass), such that an electric field may be applied to the conductive substrate to facilitate analyte migration through, across, within, or within the flexible array. array orientations. Additionally and alternatively, the flexible array may be in contact with the biological sample in an electrophoretic assembly (eg, electrophoretic chamber) such that an electric field may be applied to migrate the analyte toward, or through, through, or within the flexible array. A flexible array.
In some embodiments, flexible arrays can be generated using a substrate holder (eg, any array alignment device). For example, an array of spatially labeled beads can be placed in one substrate holder site holder, and another substrate (eg, glass slide) can be placed in another substrate holder site holder. In some embodiments, the array is optionally optically decoded, and a gel prepolymer solution is introduced between the array of spatially labeled beads and another substrate. In some embodiments, the substrate holder is closed so that the second substrate is on top of (eg, above, parallel to) the row of spatial barcode beads. Gel prepolymer solutions can be polymerized by any of the methods described herein and result in cross-linked spatially barcoded features in the hydrogel, resulting in flexible arrays. In some embodiments, the substrate holder can be opened and a second substrate having hydrogel characteristics and a crosslinking spatial barcode can be removed from the substrate holder (optionally the flexible array can be removed from the second substrate) for spatial analysis using any of the methods described herein.
(vii) Shrinkable hydrogel features/arrays
As used herein, "shrinkage" or "shrinkage" of a hydrogel refers to any process that results in physical shrinkage of the hydrogel and/or reduction in size of the hydrogel in volume. For example, gel scaffolds can collapse or “implode” upon removal of solvent (see, e.g., Long and Williams.science2018;362(6420):1244-1245, and Oran et al.science2018;362(6420):1281-1285; each of which is hereby incorporated by reference in its entirety). As another example, the process of shrinking or reducing the volume of the hydrogel may be the process of removing water from the hydrogel (ie, the dehydration process). There are many methods known to those skilled in the art for shrinking or reducing the volume of hydrogels. Non-limiting examples of methods of shrinking or reducing the volume of a hydrogel include exposing the hydrogel to one or more of the following: dehydrating solvents, salt, heat, vacuum, lyophilization, drying, filtration, air drying, or combinations thereof.
In some embodiments, the hydrogel beads may be reduced in volume (eg, shrunken hydrogel beads) prior to attachment or incorporation into the hydrogel. In some embodiments, the hydrogel beads may be reduced in volume (eg, shrunken hydrogel beads) after being attached or incorporated into the hydrogel. In some embodiments, one or more hydrogel beads may be attached or embedded in the hydrogel. In some embodiments, one or more hydrogel beads may be reduced in volume prior to attachment or incorporation into the hydrogel (eg, one or more shrunken hydrogel beads). In some embodiments, one or more hydrogel beads may decrease in volume after being attached or incorporated into the hydrogel (eg, one or more hydrogel beads that shrink). In some embodiments, the volume of one or more hydrogel beads attached or embedded in the hydrogel may be reduced. For example, the volume of one or more hydrogel beads and the hydrogel to which the hydrogel beads are attached or embedded simultaneously decreases (eg, a hydrogel containing shrinking hydrogel beads). In some embodiments, the volume of one or more hydrogel beads attached or embedded in the hydrogel may be reduced equidistantly.
In some embodiments, the volume of one or more hydrogel beads attached or embedded in the hydrogel may be reduced by about 3-fold to about 4-fold. For example, the volume of one or more hydrogel beads attached or embedded in the hydrogel can be reduced by removing or changing the solvent, salt, or water (eg, dehydration). In another example, the volume of one or more hydrogel beads attached or embedded in the hydrogel can be reduced by controlling temperature or pH. See eg Ahmed, E.M.Journal of Advanced Research2015, March;6(2):105-121, which is hereby incorporated by reference in its entirety. In some embodiments, the volume of one or more hydrogel beads attached or embedded in the hydrogel can be reduced by removing water.
In some embodiments, reducing the volume of one or more hydrogel beads attached or embedded in the hydrogel can increase the spatial resolution of subsequent sample analysis. Increased resolution in spatial analysis can be achieved by comparing the sample space using one or more shrinking hydrogel beads attached or embedded in the hydrogel versus one or more non-shrinking hydrogel beads attached or embedded in the hydrogel analysis to determine.
In some embodiments, the volume of the hydrogel beads is not reduced. In some embodiments, the hydrogel beads may be reduced in volume (eg, shrunken hydrogel beads). In some embodiments, the aggregated hydrogel gel beads are stable. For example, the volume of the hydrogel beads can be reduced by removing solvent, salt, or water from the hydrogel beads (eg, dehydration, drying, drying, drying) to form shrunken hydrogel beads. In another example, the volume of hydrogel beads can be reduced by controlling temperature or pH. See eg Ahmed, E.M.Journal of Advanced Research2015, March;6(2):105-121, which is hereby incorporated by reference in its entirety. Non-limiting examples of solvents that can be used to form shrinkable hydrogel beads or arrays of shrinkable hydrogel beads include ketones such as methyl ethyl ketone (MEK), isopropanol (IPA), acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide (DMSO), glycerin, propylene glycol, ethylene glycol, ethanol, (k)1,4-dioxane, propylene carbonate, furfuryl alcohol, N,N-dimethylformamide (DMF), acetonitrile, aldehydes such as formaldehyde or glutaraldehyde or any what combination of that
In some embodiments, the hydrogel bead or array of hydrogel beads is collected or stabilized by a cross-linking agent. For example, cross-linking agents may include disuccinimidyl suberate (DSS), dimethyl suberimide (DMS), formalin and dimethyl adipimide (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartarate (DST), and ethylene glycol bis (succinimidyl succinate) (EGS).
In some embodiments, the hydrogel beads or array of hydrogel beads are treated with salt to form the aggregated hydrogel beads or array of aggregated hydrogel beads. Non-limiting examples of salts that can be used to form shrinkable hydrogel beads or arrays of shrinkable hydrogel grains are inorganic salts including aluminum, ammonium, barium, beryllium, calcium, cesium, lithium, magnesium salts, potassium, rubidium, sodium, and strontium Others non-limiting examples of inorganic salts include sodium chloride, potassium chloride, lithium chloride, cesium chloride, sodium fluoride, sodium bromide, sodium iodide, sodium nitrite, potassium sulfate, potassium nitrate, potassium carbonate, potassium bicarbonate, sodium sulfate, sodium nitrate, sodium carbonate, sodium bicarbonate, calcium sulfate, copper oxychloride, calcium chloride, calcium carbonate, calcium bicarbonate, magnesium sulfate, magnesium nitrate, magnesium chloride, magnesium carbonate, magnesium bicarbonate, ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium carbonate, ammonium sodium bicarbonate, trisodium phosphate, tripotassium phosphate, calcium phosphate, copper(II) sulfate, sodium sulfide, potassium sulfide, calcium sulfide, potassium permanganate, iron(II) chloride, iron(III) chloride, iron(2+) sulfate, iron(III) sulfate , iron(II) nitrate, iron(III) nitrate ), manganese(II) chloride, manganese(III) chloride, manganese(II) sulfate, manganese(II) nitrate, zinc chloride, zinc nitrate, zinc sulfate, ammonium orthomolybdate, potassium dihydrogen phosphate, sulfuric acid nickel(II), nickel(II) nitrate, sodium metavanadate, sodium paravanadate, potassium dichromate, ammonium dichromate, diazepam, ammonium nitrite, potassium fluoride, sodium fluoride, ammonium fluoride, calcium fluoride, chromium alum, potassium alum, potassium iodide, sodium hypochlorite, tin(II) sulfate, tin(II) nitrate, gold selenite, dicesium chromate, potassium perchlorate, calcium perchlorate, aluminum sulfate, hydrogen sulfate Lead(II), barium phosphate, barium hydrogen orthophosphate, barium Dihydrogen Phosphate, Silver Dichromate, Potassium Bromate, Sodium Bromate, Sodium Iodate, Sodium Silicate, Diammonium Phosphate, Ammonium Phosphate, Ammonium Dihydrogen Phosphate, Chromium Orthophosphate, Copper Chloride, Copper Chloride, Sodium Tetrametaphosphate, Potassium Heptafluoroniobate, Z inc Phosphate, Sodium Sulfite, copper(I) nitrate, copper(II) nitrate, potassium silicate, basic copper carbonate (II), copper(II) carbonate salts of acrylic acid and sulfopropyl acrylic acid.
In some embodiments, the water removal includes an acid. Non-limiting examples of acids include: HCl, HI, HBr, HClO4, HClO3, HNO3, H2SO4, phosphoric acid, phosphoric acid, acetic acid, oxalic acid, ascorbic acid, carbonic acid, sulfuric acid, tartaric acid, citric acid, malonic acid, phthalic acid acid formic acid, barbituric acid, cinnamic acid, glutaric acid, caproic acid, malic acid, folic acid, propionic acid, stearic acid, trifluoroacetic acid, acetylsalicylic acid, glutamic acid, azelaic acid, benzoic acid, malic acid, gluconic acid, lactic acid, oleic acid, propyl acid, pink acid, tannic acid, uric acid, gallic acid and combinations of two or more of them. In some embodiments, the hydrogel is exposed to different pH environments. For example, the hydrogel can be exposed to acidic pH or basic pH. In some embodiments, the hydrogel is exposed to a pH of less than about 6.5, such as a pH of about 6, about 5.5, about 5, about 4.5, about 4, about 3.5, about 3, about 2 .5, about 2, about 3. 1.5 or about 1. In some embodiments, the hydrogel is exposed to a pH greater than about 7.5, such as about 8, about 8.5, about 9, about 9.5 , about 10, about 10.5, about 11, about 11.5, about 12, about 12.5, about 13 , about 13.5 or about 14.
In some embodiments, water removal includes dehydration processes such as heat, vacuum, freeze drying, drying, filtration, and air drying. In some embodiments, the hydrogel beads or array of hydrogel beads are subjected to a pH change to form the aggregated hydrogel beads or array of aggregated hydrogel beads (e.g., from about pH 7 to about pH 5, from about pH 7 to about pH 5.5, from about pH 7 to about pH 6, from about pH 7 to about pH 6.5, from about pH 6.5 to about pH 5, from about pH 6 to about pH 5, from about pH 6 to about pH 5.5, or include any pH changes within these ranges).
In some embodiments, the hydrogel beads or arrays of hydrogel beads are subject to changes in temperature (eg, changes from about 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C , 45°C, 46°C, 47°C, 48°C, 49°C to about 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57 °C, 58 °C, 59 °C 60 °C, 61 °C, 62 °C, 63 °C, 64 °C, 65 °C, 66 °C, 67 °C, 68 °C, 69 ° C, 70°C or higher, or any temperature change within these ranges) to form shrinkable hydrogel beads or arrays of shrinkable hydrogel beads.
In some embodiments, the linear dimension of the hydrogel beads may be reduced by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or any other interval. In some embodiments, the volume of the hydrogel beads can be reduced by about 1-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold , about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, or any interval therein. In some embodiments, the hydrogel grain size can be reduced such that the hydrogel grains have an average diameter of about 1 µm to about 15 µm.
In some embodiments, the linear dimension of the plurality of hydrogel beads may be reduced by about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, or any interval. between. In some embodiments, the plurality of hydrogel grains may be reduced in size such that the average diameter of the hydrogel grains is from about 1 µm to about 15 µm. In some embodiments, the volume of the plurality of hydrogel beads may be reduced by about 1-fold, about 5-fold, about 10-fold, about 15-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold times, about 50 times, about 55 times, about 60 times, about 65 times, about 70 times, about 75 times, about 80 times, or any interval therein.
在一些实施例中，多个水凝胶珠的体积可以减小，使得水凝胶珠具有约1μm至约15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约4μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约3μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约2μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约1μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约14-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约13-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约12-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约11-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约10-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-15μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约1-10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约1-5μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约1-3μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约13-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约12-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约11-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约10-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-14μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约12-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约11-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约10-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-13μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约11-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约10-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-12μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约10-11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9-11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-11μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约9-10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-10μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约8-9μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-9μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-9μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-9μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约7-8μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-8μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-8μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约6-7μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-7μm的平均直径。在一些实施例中，多个收缩的水凝胶珠粒具有约5-6μm的平均直径。
In some embodiments, the volume of one or more hydrogel beads can be reduced simultaneously. In some embodiments, the volume of one or more hydrogel beads may be reduced at different times. In some embodiments, one or more hydrogel beads may be assembled into an array prior to reducing the volume of the one or more hydrogel beads. In some embodiments, one or more hydrogel beads may be assembled into an array after reducing the volume of one or more hydrogel beads. In some embodiments, one or more aggregated hydrogel beads can be reversibly attached to the substrate. In some embodiments, one or more aggregated hydrogel beads may be irreversibly attached to the substrate. In some embodiments, one or more of the aggregated hydrogel beads may be re-swelled. In some embodiments, one or more of the shrunk hydrogel beads can be isometrically re-swelled. In some embodiments, one or more of the collapsed hydrogel beads can be re-expanded primarily in the z-dimension. In some embodiments, one or more of the shrunk hydrogel beads attached to the substrate (eg, reversibly or irreversibly) may re-swell primarily in the z-dimension. In some embodiments, the one or more aggregated hydrogel beads attached to the substrate (eg, reversibly or irreversibly) can re-swell isometrically primarily in the z-dimension.
In some embodiments, reducing the volume of the hydrogel beads (eg, shrinking the hydrogel beads) can increase the spatial resolution of subsequent sample analysis. The improvement in resolution in spatial analysis can be determined by comparing the spatial analysis of samples using shrinkable hydrogel beads versus non-shrinkable hydrogel beads. For example, in some embodiments, subsequent analysis of the sample may include any of the array-based spatial analysis methods disclosed herein.
In some embodiments, one or more physical parameters or dimensions and/or one or more other characteristics of the hydrogel beads may be altered. For example, the cross-section of the hydrogel beads can be changed from a first cross-section to a second cross-section. The first section can be smaller or larger than the second section. Alternatively or additionally, one or more other properties of the hydrogel beads may be altered. For example, the fluidity, density, stiffness, porosity, refractive index, polarity, and/or other properties of the hydrogel bead or one or more of its components may be altered. In a non-limiting example, the hydrogel beads comprise hydrogels. In another example, hydrogel beads. The hydrogel can form crosslinks within the grains. The same or different conditions can be used to change or influence different properties of the hydrogel beads at the same or different times. In some cases, a first condition or set of conditions can be used to change a first characteristic or set of characteristics (eg, cross-section) of a hydrogel bead, and a second condition or set of conditions can be used to change a second characteristic or set of properties of the hydrogel bead. The first condition or set of conditions may apply at the same time or at a different time than the second condition or set of conditions. For example, a first property or set of properties may change under a first condition or set of conditions, after which a second property or set of properties may change under a second condition or set of conditions.
A property or set of properties of a hydrogel bead can be changed under one or more conditions. Conditions suitable for changing a property or set of properties of the hydrogel beads may be, for example, temperature, pH, ion or salt concentration, pressure, chemical species, any combination thereof, or other conditions. For example, the hydrogel beads may be exposed to chemicals that may cause one or more properties of the hydrogel beads to change. In some cases, the stimulus can be used to change one or more properties of the hydrogel bead. For example, one or more properties of the hydrogel bead may change upon application of a stimulus. The stimuli may be, for example, thermal, optical, chemical or other stimuli. A temperature sufficient to alter one or more properties of the hydrogel beads may be, for example, at least about 0°C (°C), 1°C, 2°C, 3°C, 4°C. , 5° C, 10° C or more. For example, the temperature can be around 4°C. In other cases, the temperature sufficient to change one or more properties of the hydrogel bead may be, for example, at least about 25°C, 30°C, 35°C. C., 37°C, 40°C, 45°C, 50°C or more. For example, the temperature can be around 37°C. A pH sufficient to alter one or more properties of the hydrogel bead may be, for example, about 5 to 8, such as about 6 to 7.
In some cases, chemicals or chemical stimuli can be used to alter one or more properties of the hydrogel beads. For example, chemicals or chemical stimuli can be used to change the dimensions (eg, cross-section, diameter, or volume) of the hydrogel grains. In some cases, chemicals or chemical stimuli can be used to change the size of the hydrogel beads (eg, cross-sectional diameter) from a first dimension to a second dimension (eg, second cross-sectional diameter), which is comparable to the first dimension. Then the second dimension is reduced . Chemicals may include organic solvents such as alcohols, ketones or aldehydes. For example, chemicals may include acetone, methanol, ethanol, formaldehyde, or glutaraldehyde. Chemical substances may contain crosslinkers. For example, cross-linking agents may include disuccinimidyl suberate (DSS), dimethyl suberimide (DMS), formalin and dimethyl adipimide (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartarate (DST), and ethylene glycol bis (succinimidyl succinate) (EGS) and any combination. In some cases, the crosslinking agent may be a photocleavable crosslinking agent. In some cases, a chemical stimulus can be used to alter one or more properties of the hydrogel bead (eg, hydrogel bead size), where the chemical stimulus includes one or more chemicals. For example, the chemical stimulus may include a first chemical substance and a second chemical substance, where the first chemical substance is an organic solvent and the second chemical substance is a cross-linking agent. In some cases, the chemical stimuli may include chemicals that act as fixatives that can fix or preserve the hydrogel beads. For example, organic solvents such as alcohols (eg, ethanol or methanol), ketones (eg, acetone), or aldehydes (eg, formaldehyde or glutaraldehyde), or any combination thereof, can be used as fixatives. Alternatively, or additionally, the cross-linking agent may act as a fixative. In some cases, fixatives may include disuccinimidyl suberate (DSS), dimethyl suberimide (DMS), formalin and dimethyl adipimide (DMA), dithio-bis(-succinimidyl propionate) (DSP), disuccinimidyl tartarate (DST), and/or ethylene glycol. bis(succinimidyl succinate) (EGS) and any combination thereof. In some cases, a first chemical and/or immobilizing agent may be placed in or contacted with the hydrogel beads to cause a change in the first characteristic or set of characteristics of the hydrogel beads, and a second chemical and/or may immobilize the beads. The agent or fixative is administered or comes into contact with the hydrogel beads to cause a change in another property or set of properties of the hydrogel beads. For example, a first chemical and/or fixative may be placed in or contacted with the hydrogel bead to cause a dimensional change (eg, decrease in cross-sectional diameter) of the hydrogel bead, and the second chemical may be two chemical substances and/or fixing agents or bring them into contact to cause a change in another property or set of properties of the hydrogel beads (eg, to form cross-links in and/or around the hydrogel beads). The first and second chemicals and/or fixatives may be introduced or contacted with the hydrogel beads at the same time or at different times.
In some embodiments, immobilization may affect one or more parameters or properties of the hydrogel bead. For example, immobilization can result in shrinkage or volume reduction of hydrogel beads. Fixation may involve dehydration of the hydrogel beads. Adding a fixative to the hydrogel beads results in a change in the size of the hydrogel beads. For example, the addition of a fixing agent to the hydrogel beads may result in a change in the volume or diameter of the hydrogel beads. Addition of a fixative to the hydrogel beads can result in a change in the cross-section (eg, cross-sectional diameter) of the hydrogel beads. For example, the first cross-section of the hydrogel beads before immobilization may be different (eg, larger) than the second cross-section of the hydrogel beads after immobilization. In one example, the approximately spherical hydrogel bead may include a first cross-section (eg, cross-sectional diameter) prior to immobilization that is reduced in size to a second cross-section after immobilization. Administering the fixative to the hydrogel beads can result in a reduction of the second cross-section by at least about 5% compared to the first cross-section. In some cases, the second slice is relative to the first slice. For example, the second cross-section may be reduced by at least about 10%, 15%, 25%, or 50% relative to the first cross-section. Fixation can also affect other characteristics of hydrogel beads. For example, immobilization can result in a change in the porosity of the hydrogel bead membrane or wall, a reorganization of the components of the hydrogel bead, a change in the fluidity or stiffness of the hydrogel bead, or other changes. In one example, a first fixative is provided to the hydrogel beads as an organic solvent to alter a first property (eg, volume of the hydrogel beads) and a second fixative is provided to the hydrogel beads as a cross-linking agent to alter a second property (eg, fluidity or stiffness of the hydrogel beads). The first fixative can be placed on the hydrogel beads before the second fixative.
In some cases, the approximately spherical hydrogel beads, prior to immobilization (eg, with an organic solvent), may include a first diameter whose volume is reduced compared to a second diameter after immobilization when maintained in a non-aqueous environment. After immobilization and volume reduction to a second diameter, the hydrogel beads can increase in volume to have a diameter substantially similar to the first diameter when maintained in an aqueous environment. In some cases, the approximately spherical hydrogel beads, prior to immobilization (eg, with an organic solvent), may include a first diameter whose volume is reduced compared to a second diameter after immobilization. After fixation and volume reduction to the second diameter, the hydrogel beads can be cross-linked with another fixative, whereby the second diameter remains mostly in an aqueous environment after cross-linking with the second fixative.
The change of one or a number of hydrogel bead properties can be reversible or irreversible. In some cases, a change in a property or set of properties of a hydrogel bead may be irreversible such that the change cannot be easily reversed. For example, the volume, morphology, or other characteristics of hydrogel beads can be altered in ways that cannot be easily reversed. In the example, the change from the first hydrogel bead cross-section to the second hydrogel bead cross-section is irreversible. In some cases, irreversible changes can be at least partially reversed by applying appropriate conditions and/or over time. In other cases, a change in one or a number of hydrogel bead properties may be reversible. For example, the volume of the hydrogel bead may decrease when subjected to the first condition or set of conditions, while the volume of the hydrogel bead may increase to approximately its original volume when subjected to the second condition or set of conditions. So, from water The change from the first cross-section to the second cross-section of the gel beads can be reversible. Reversible changes (eg, reversible volume reduction) can be used, for example, to deliver a specific volume of hydrogel beads to a specific location, such as a septum. In some cases, a change in one or a number of hydrogel bead properties may be only partially reversible. For example, the volume of hydrogel grains can be reduced (e.g., by dehydration), and the reduction in hydrogel grain volume can be accompanied by reorganization of components within the hydrogel grains. After a change in the volume of the hydrogel beads (e.g., by rehydration), the alignment of one or more components may not return to the original alignment of the hydrogel beads prior to volume reduction. A change in a characteristic or set of characteristics of a hydrogel bead, such as the cross-section of a hydrogel bead, may be reversible upon application of a stimulus. The stimuli can be, for example, thermal, optical or chemical stimuli. In some cases, stimulation may involve changes in pH and/or the use of reducing agents such as dithiothreitol. The application of the stimulus can completely or partially reverse, for example, the change from the first cross-section to the second cross-section.
In some embodiments, the plurality of hydrogel beads may be shrinkable hydrogel beads produced by removing water from the first plurality of hydrogel beads. In some embodiments, the plurality of aggregated hydrogel beads have an average diameter of no greater than about 15 microns. For example, the plurality of hydrogel beads that are collected have an average diameter of no greater than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron. In some embodiments, each member of the plurality of aggregated hydrogel beads has a diameter of no greater than about 15 microns. For example, each member of the plurality of collapsible hydrogel beads may have a diameter of no greater than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 micron. In some embodiments, the average diameter of the plurality of aggregated hydrogel beads is no greater than 10 microns. In some embodiments, each member of the plurality of aggregated hydrogel beads has a diameter of no greater than 10 microns. In some embodiments, the plurality of aggregated hydrogel beads have an average diameter of no greater than 5 microns. In some embodiments, each member of the plurality of contracted hydrogel beads has a diameter no greater than 5 microns. In some embodiments, the average diameter of the plurality of aggregated hydrogel beads is no greater than 1 micron. In some embodiments, each member of the plurality of tapered hydrogel beads has a diameter of no greater than 1 micron. In some embodiments, the average diameter of the plurality of hydrogel beads to be aggregated is no greater than the diameter of a cell (eg, a mammalian cell, a plant cell, or a fungal cell). In some embodiments, the diameter of each member of the plurality of tapered hydrogel beads is no greater than the diameter of a cell (eg, a mammalian cell, a plant cell, or a fungal cell).
In some embodiments, the plurality of aggregated hydrogel beads have a polydispersity index of less than about 25%. For example, a plurality of aggregated hydrogel beads may have a polydispersity index of less than about 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the plurality of aggregated hydrogel beads have a polydispersity index of less than 15%. In some embodiments, the plurality of aggregated hydrogel beads have an average diameter of about 8 to about 13 microns. In some embodiments, the plurality of aggregated hydrogel beads have an average diameter of about 10 to about 12 microns. In some embodiments, the average diameter of the plurality of hydrogel beads to be aggregated is approximately the diameter of a cell (eg, a mammalian cell, a plant cell, or a fungal cell). In some embodiments, the average diameter of the plurality of hydrogel beads being collected is smaller than the diameter of a cell (eg, a mammalian cell, a plant cell, or a fungal cell). In some embodiments, multiple capture probes on multiple gel beads bind cellular analytes at single cell resolution. In some embodiments, a plurality of capture probes on a plurality of tapered gel beads binds a cellular analyte at a higher resolution than a single cell (eg, at a higher density than the cell diameter).
In some embodiments, bead arrays having a plurality of hydrogel beads distributed on a substrate are produced by patterning or self-assembling larger gel beads, followed by assembling the array of larger gel beads (e.g., by any of the methods provided herein). In some embodiments, the larger gel beads are not small enough to resolve a single cell, while the shrinking gel beads are small enough to resolve a single cell. In some embodiments, bead arrays with multiple hydrogel beads distributed on a substrate are produced by patterning or self-assembling aggregated gel beads previously made by assembly of larger gel beads. Production of beads (eg, by any of a number of methods provided, the beads may be sterically constrained by any of a variety of methods, including, but not limited to, reversible or irreversible cross-linking.
In some embodiments, the bead array contains spatially confined gel beads that have a high aspect ratio (eg, a columnar array). For example, bead arrays with a plurality of hydrogel beads distributed on a substrate can be generated by any of a number of methods described herein (eg, patterning or self-assembly of shrinkable gel beads, or patterning of larger gels or self-assembled beads followed by shrinkage), after whereupon the arrays of high-density beads expand (or re-expand). When spreading, spatial constraints direct the beads to spread primarily in the Z dimension (away from the substrate), forming a columnar array. In some embodiments, the gel beads of the column array have a high aspect ratio. In some embodiments, the extended array of spatially confined shrinkable hydrogel beads has an aspect ratio of at least 2. In other embodiments, the extended array of spatially confined hydrogel shrinkable beads has an aspect ratio of at least 3. In some embodiments, the plurality of spatially confined hydrogel beads that are shrinking has an average aspect ratio of at least 4, 5, 6, 7, 8 or greater.
In some embodiments, the method of removing water from the hydrogel is the same for each hydrogel (eg, first hydrogel, second hydrogel, or third hydrogel). In some embodiments, water is removed from one hydrogel (eg, a first hydrogel) differently than from at least one other hydrogel (eg, a second hydrogel, a third hydrogel, or a method for removing water in a second hydrogel). fourth hydrogel). For example, the method of removing water from one hydrogel may differ from the method of removing water from other hydrogels (eg, a second hydrogel, a third hydrogel, or a fourth hydrogel). In some embodiments, the method for removing water is different for each hydrogel (eg, first hydrogel, second hydrogel, third hydrogel, and fourth hydrogel).
In some embodiments, the contracted hydrogel is at least about 2 times smaller in linear dimension (eg, along one axis) than the previously contracted hydrogel. For example, at least about 2.5, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more times smaller linear dimensions than the pre-shrunk hydrogel.
In some embodiments, the size of the hydrogel is reduced along more than one axis, such as along 2 or 3 axes. In some embodiments, each axis intersects each other's axes at 90 degrees. In some embodiments, the dimension of the hydrogel along the first axis is about 2 times to about 10 times or more smaller than the pre-shrunk hydrogel, such as about 2, about 3, about 4, about 5, about 6, about 7. 8 times, about 9 times, about 10 times or more smaller than the pre-shrunk hydrogel. In some embodiments, the dimension of the hydrogel along the second axis is about 2 times to about 10 times or more smaller than the pre-shrunk hydrogel, such as about 2, about 3, about 4, about 5, about 6, about 7. 8 times, about 9 times, about 10 times or more smaller than the pre-shrunk hydrogel. In some embodiments, the dimension of the hydrogel along the third axis is about 2 times to about 10 times or more smaller than the pre-shrunk hydrogel, such as about 2, about 3, about 4, about 5, about 6, about 7. 8 times, about 9 times, about 10 times or more smaller than the pre-shrunk hydrogel. In some embodiments, the reduction in hydrogel volume is equidistant.
In some embodiments, each hydrogel (eg, first hydrogel, second hydrogel, third hydrogel, or fourth hydrogel) has the same volume. In some embodiments, the at least one hydrogel varies in volume. For example, in some embodiments, one hydrogel is volumetrically different from other hydrogels (eg, a second hydrogel, a third hydrogel, or a fourth hydrogel). In some embodiments, each hydrogel differs in volume from every other hydrogel.
In some embodiments, members of the array of features are cross-linked with the hydrogel (eg, a first hydrogel, a second hydrogel, a third hydrogel, or a fourth hydrogel).
In one embodiment, the features of the array can be replicated in the hydrogel and the volume of the hydrogel reduced by removing water. These steps can be performed multiple times. For example, a method of making a flexible high-density spatial barcode array may include replicating multiple spatial barcode features from the array into a first hydrogel, wherein the first hydrogel is in contact with the array; hydrogel volume, formation of the first contracted hydrogel incorporating replicated features; replicating the features in the first contracted hydrogel into the second hydrogel, wherein the second hydrogel is identical to the first. Two hydrogels are in contact; the volume of the second hydrogel containing the replicated features is reduced by removing water to form a second contracted hydrogel containing the replicated features, thereby creating a high-density array of spatial barcodes. In some cases, the array includes one or more first oligonucleotides and the first hydrogel includes one or more second oligonucleotides. After contacting the hydrogel with the array, one or more members of the first plurality of oligonucleotides may be linked to one or more members of the second plurality of oligonucleotides. The array may include a greater number of first oligonucleotide species than second oligonucleotide species in the hydrogel so that a first oligonucleotide containing the same sequence can be separated from a second oligonucleotide containing a different sequence. splicing of nucleotides. This can increase the diversity of oligonucleotides (eg spatial barcodes) in the first hydrogel. Copying the spatial barcode features from the array into the first hydrogel, removing water from the first hydrogel to form the first constricted hydrogel, and copying the spatial barcode features from the first constricted hydrogel to one or more. The procedure of subsequent hydrogelation can be performed several times (eg 2, 3, 4, 5, 6, 7, 8, 9 or 10 times). The result is a flexible, high-density array containing spatially barcoded features.
In some embodiments, replicating the feature array members from the array comprises replicating by PCR. In some embodiments, the hydrogel (eg, the first hydrogel, the second hydrogel, the third hydrogel, and/or the fourth hydrogel) contains the PCR reagents described herein. In some embodiments, the members of the multiple features are replicated using replica cloaking techniques (see, e.g., Mitra and Church,nucleic acid research1999 Dec 15;27(24):e34, which is incorporated herein by reference in its entirety). In some embodiments, after replicating the array of features from the array into the first hydrogel, the array features are amplified (eg, clonally expanded) in the first hydrogel. In some embodiments, members of the plurality of features are replicated in the first hydrogel such that the pattern of the plurality of features of the first hydrogel is identical or substantially similar (eg, at least 80%) to the pattern of the plurality of features of the array.
In some embodiments, one or more array features are split. For example, each partition may include a multitude of characteristics that differ from the characteristics of other partitions. For example, partitioning the members of multiple features is similar to partitioning the multiple features of an array. In some embodiments, the array features are replicated into the first hydrogel such that the volume or diameter of the preassembled features of the first hydrogel is similar to the volume or diameter of the array features.
In some embodiments, the volume of the hydrogel containing the replicated features is reduced, thereby increasing the density of the replicated features. In some embodiments, the replicated features within the hydrogel further increase in density with each subsequent replication and contraction of the hydrogel. For example, the replicated features of the second shrinking hydrogel have a higher density than the replicated features of the first shrinking hydrogel. Similarly, the density of replicated features of the third shrinkable hydrogel is greater than the density of replicated features of the second shrinkable hydrogel. Similarly, the density of replicated features of the fourth shrinkable hydrogel is greater than the density of replicated features of the third shrinkable hydrogel. In some embodiments, as the volume of the hydrogel decreases, the volume of the partition members of the feature array in the hydrogel decreases.
In some embodiments of the methods described herein, the array contains aggregated gel features (eg, beads). In some embodiments, the methods described herein produce collapsible arrays of gel beads. In some embodiments, the collapsible gel beads are collapsible hydrogel beads.
A "reduced array" includes a plurality of spatially marked features that are affixed or embedded in a substrate that is reduced in volume (eg, reduced in diameter or volume). The biological sample can be contacted with the reduced array and further contacted with a solution that can rehydrate the reduced array. In some embodiments, analyte transport and capture is driven by molecular diffusion. The process of rehydrating the pooled array by supplying the sample with a permeabilization solution or staining the tissue can enhance the orientation of analytes (eg, transcripts) present in biological samples to spatially tagged features, thereby increasing the efficiency of analyte capture. See, eg, J. Vlassakis, A.E. Herr. "Effect of Polymer Hydration State on In-Gel Immunoassays."anus. Chemical2015, 87(21):11030-8, which is hereby incorporated by reference in its entirety.
Reduced arrays can be generated using features (eg, balls) that contain spatial barcodes from existing arrays. For example, an array (e.g., an array of hydrogel beads) described and prepared by any of the methods herein can be contacted with an agent capable of dehydrating (e.g., removing water) the feature (e.g., beads) to produce a reduced array (e.g. .reduced array of balls). Methods of dehydrating properties such as beads are known in the art. Any suitable method of dehydration (eg, removal of water) may be used. For example, without limitation, features such as beads can be dehydrated with ketones such as methyl ethyl ketone (MEK), isopropanol (IPA), acetone, 1-butanol, methanol (MeOH), dimethyl sulfoxide (DMSO), glycerol, propylene glycol, ethylene glycol, ethanol, (k) 1,4-dioxane, propylene carbonate, furfuryl alcohol, N,N-dimethylformamide (DMF), acetonitrile, aldehydes such as formaldehyde or glutaraldehyde, or any combination thereof. Additional dehydrating agents include a variety of salts, including inorganic salts (see, e.g., Ahmed, E.M., Hydrogel: Preparation, characterization, and applications: A review,Journal of Advanced Research, 6 (2) 105-121 (2015), which is incorporated herein by reference).
In some embodiments, the dehydrating features (e.g., beads) may result in shrinkable arrays (e.g., shrinkable bead arrays or shrinkable hydrogel arrays), wherein the average diameter of the dehydrated features (e.g., bead) may be smaller than the features before dehydration. In some embodiments, the average diameter of the dehydrating features (eg, the bead) may be at least two times smaller than the average diameter of the features prior to dehydration. In some embodiments, the average diameter of the dehydrating features (eg, the bead) may be at least three times smaller than the average diameter of the dehydrating features prior to dehydration. In some embodiments, the average diameter of the dehydration features (e.g., bead) may be at least four times or less than the average diameter of the pre-dehydration features (e.g., bead).
After the contraction array is created, a biological sample (eg, a tissue sample) can be contacted with the contraction array (eg, a contraction bead array). The rehydration solution can be introduced into the biological sample and collected array by any convenient method (eg, pipetting). The rehydration solution may contain reagents that rehydrate (eg, water or buffer) the contractile array features (eg, beads). In some embodiments, the rehydration solution can be applied to the entire biological sample. In some embodiments, the rehydration solution can be selectively applied (eg, to an area of interest). In some embodiments, absorption of water from the rehydration solution can increase the diameter of at least one feature (e.g., beads) in the tapered array. In some embodiments, the rehydration solution can increase the diameter of at least one feature (e.g., beads) by at least two-fold. In some embodiments, the rehydration solution can increase the diameter of at least one feature (e.g., beads) by at least three times. In some embodiments, the rehydration solution can increase the diameter of at least one feature (e.g., beads) by at least four times. In some embodiments, the rehydration solution can increase the diameter of at least one feature (e.g., beads) by at least five times or more.
In some embodiments, the rehydration solution may include a permeabilizing agent. Biological samples can be permeabilized using permeabilization reagents and techniques known in the art or otherwise described herein. Biological samples from different sources (eg brain, liver, ovary, kidney, breast, colon, etc.) may require different permeabilization treatments. For example, permeabilizing biological samples (eg, with proteases) can facilitate the migration of analytes to substrate surfaces (eg, spatially marked features). In some embodiments, the permeability-enhancing agent can be a detergent (eg, saponin, Triton X-100™, Tween-20™). In some embodiments, organic solvents (eg, methanol, acetone) can permeabilize the cells of the biological sample. In some embodiments, an enzyme (eg, trypsin) can permeabilize the biological sample. In another embodiment, an enzyme (eg, collagenase) can permeabilize the biological sample.
In some embodiments, the solution may permeabilize the biological sample and rehydrate the features (eg, beads) of the shrinkable array (eg, shrinkable hydrogel). In some embodiments, the rehydration solution can stain the biological sample and rehydrate the features (eg, beads) of the pooled array.
In some embodiments, the rehydration solution (eg, permeabilization solution or staining solution) may diffuse through the biological sample. In some embodiments, the rehydration solution can reduce the diffusion of the analyte from the substrate. In some embodiments, while diffusing through the biological sample, the rehydration solution may migrate the analyte to the surface of the substrate and increase analyte capture efficiency.
(viii) Microcapillary array
A "microcapillary array" is characterized by a series of arrangements separated by microcapillaries. "Microcapillary channels" are individual compartments that create microcapillaries. For example, microcapillary channels may be fluidly isolated from other microcapillary channels such that the fluid or other contents in one microcapillary channel in the array are separated from the fluid or other contents in adjacent microcapillary channels in the array. The density and order of the microcapillary channels can be any suitable density or order of discrete sites.
In some embodiments, the microcapillary arrays are treated to create conditions that facilitate loading. An example is the use of a corona rod (BD-20AC, Electro Technic Products) to create a hydrophilic surface. In some embodiments, features (eg, beads with attached capture probes) are loaded onto the microcapillary array such that the exact location of the features within the array is known. For example, a capture probe containing a spatial barcode can be placed in a microcapillary channel such that the spatial barcode identifies the site of origin of the barcoded nucleic acid molecule. In some embodiments, the features are introduced into the microcapillary array by flowing the features through the microcapillary channels. In some embodiments, the microcapillary channels may reduce the cross-sectional area of the feature.
In some embodiments, when a random distribution is used to distribute the characteristics, empirical testing can be performed to generate loading/distribution conditions that promote individual characteristics per microcapillary. In some embodiments, it may be desirable to achieve distribution conditions that promote only one feature (eg, bead) per microcapillary channel. In some embodiments, it may be desirable to achieve distribution conditions that promote more than one feature (eg, beads) per microcapillary channel by flowing the features through the microcapillary channel.
In some embodiments, the microcapillary array is placed in contact with the sample (eg, on top or below), such that the microcapillaries containing features (eg, beads, which may include capture probes) are in contact with the biological sample. In some embodiments, placing the biological sample on the exposed side of the microcapillary array and applying mechanical compression moves the biological sample into the microcapillary channel to create a liquid-isolated reaction chamber containing the biological sample.
In some embodiments, the biological sample is dispersed by contacting the microcapillary array with the biological sample, thereby creating a microcapillary channel that includes the beads and a portion of the biological sample. In some embodiments, the portion of the biological sample contained in the microcapillary channel is one or more cells. In some embodiments, the portion of the biological sample located within the microcapillary channel is a cell. In some embodiments, features are introduced into the microcapillary array by flow after one or more cells are added to the microcapillary channel.
In some embodiments, reagents are added to the microcapillary array. Reagents may include enzymatic reagents or reagent mixtures for performing nucleic acid amplification. In some embodiments, the reagents include a reverse transcriptase, a ligase, one or more nucleotides, or any combination thereof. After adding the reagent to the microcapillary channels, one or more microcapillary channels can be closed, for example, using silicone oil, mineral oil, a non-porous material, or a plug. In some embodiments, the microcapillary array is incubated in a humidified chamber. In some embodiments, the microcapillary array is incubated at a temperature for a period of time suitable to allow nucleic acid amplification.
In some embodiments, the reagent solution is removed from each microcapillary channel after incubation at a temperature or temperature range for a specified time, eg, after a hybridization or amplification reaction. Reagent solutions can be processed separately for sequencing or combined for sequencing analysis. In some embodiments, the sequencing information from the pooled reaction solutions is spatial information for one or more biological analytes.
(ix) Hydrogel array/well
In some embodiments, there are methods of creating arrays of patterned hydrogels using pores (eg, arrays of nanopores or microwells). In some embodiments, the pores are three-dimensional structures. In some embodiments, the top view of the well is any suitable two-dimensional shape that, when extended along the z-axis, produces a three-dimensional structure that may contain one or more features (eg, beads) and/or reagents. Non-limiting examples of wells that can be arrayed include triangular, square or rectangular, pentagonal, hexagonal, heptagonal, octagonal, n-sided, or cylindrical arrays (eg, "microcapillary arrays"). In some embodiments, the wells of a well array share at least one well wall (or part of a well wall, in the case of a microcapillary array) with adjacent wells. In some embodiments, the well does not share any wall or part of the wall with another well of the string. In some embodiments, the array of wells is attached to the substrate such that the wells of the array of wells are fluidly isolated from each other. In some embodiments, one end of the array of wells is open (eg, exposed), wherein the open end can be used to dispense properties or reagents into the wells.
In some embodiments, the method includes providing aggregated (e.g., dehydrated) hydrogel features (e.g., beads) to a series of wells. The hydrogel component can be dehydrated (eg, remove water) by any of a number of methods described herein. The features described elsewhere herein may be provided such that the number of features is less than the number of holes in the array, the features may be provided such that the number of features is equal to the number of holes in the array, or the features may provide more than the number of holes in the array. In some embodiments, the array of wells is manipulated so that one or more shrinking characteristics of the hydrogel are moved down from the top surface of the array into the wells. For example, a series of holes can be placed on the shake table for a period of time to arrange the features into the holes. Other non-limiting examples of manipulations that can cause the shrinking hydrogel features to enter the wells include physically shaking, tilting, or rolling the array of wells, or a combination thereof; using forced air to blow the features into the wells, using a magnet to pull the hydrogel features, using a microfluidic system to distribute the features into the wells, using a printer to deposit the features into the wells, or any other method to distribute the features into the wells. In some embodiments, when a well contains one feature, the well may not accept or retain another feature. In other embodiments, the hole may contain more than one feature.
In some embodiments, the method includes rehydrating (eg, adding water) the pooled hydrogel features, wherein the pooled hydrogel features are located in the pores. The rehydration shrinkage characteristic of the hydrogel can be achieved by any of the methods described herein. Rehydration of the pooled hydrogel characteristics in the pores can cause expansion of the pooled hydrogel characteristics. In some embodiments, the pooled characteristics of the hydrogel expand to fill the wells. In some embodiments, the contracted hydrogel feature extends in the z-direction such that the feature extends beyond the unclosed (ie, open) end of the pore. Rehydration of exposed feature areas can create arrays of patterned hydrogels (eg arrays of wells). The rehydration features contained in the well may be stable. In some embodiments, the rehydration features (eg, hydrogel features reduced by rehydration) are immobilized within the wells such that typical use of the array does not separate the rehydration features from the wells. Arrays of patterned hydrogels can be used to capture analytes according to the methods described herein.
(x) perle
"Bead tether" can refer to an arrangement of beads, where the arrangement may or may not form a string. The bound beads can be contacted with the sample and processed according to the methods described here. Furthermore, contacting biological samples with individual beads or beads bound together in different arrangements can enable more precise spatial detection of analytes, such as regions of interest. Methods of interconnecting the balls are known in the art. Some suitable, non-limiting methods of linking the beads together may be, for example, chemical linkers, proximity ligation, or any of the other methods described herein. In some embodiments, the beads may be bound together independently of the base. In some embodiments, the beads can be bonded to the existing substrate in various arrangements. In some embodiments, a substrate (eg, a hydrogel) can be formed around an existing binder bead. In some embodiments, the bead or array of beads may contact a portion of the biological sample. In some embodiments, a bead or array of beads may contact the region of interest. In some embodiments, the bead or array of beads may contact the entire biological sample. In some embodiments, the beads or array of beads are in contact with random locations on the biological sample. In some embodiments, the beads are contacted according to a specific pattern on the biological sample.
The beads can be tied in different arrangements. In some embodiments, one (eg, one) bead may contact the biological sample. In some embodiments, two or more beads may be bonded (eg, connected to each other) in different arrangements. For example, in a non-limiting manner, the beads may be linked together in clusters, rows, or arranged in a mesh (eg, net). In some embodiments, at least three beads may be linked together in a two-dimensional (2D) array (eg, a cluster). In some embodiments, at least two beads may be linked together in a one-dimensional (1D) array (eg, a row). In such embodiments, the balls are arranged in such a way that they can come into direct contact with each other. In some embodiments, at least two balls may be linked together in a rope-like arrangement. In such embodiments, the beads are arranged in such a way that the beads can indirectly contact each other (eg, the beads are connected by a binder). In some embodiments, the at least two beads may be linked together in a mesh-like arrangement (eg, a mesh). In some embodiments, beads bound together in 2D arrays, 1D arrays, beads on an array, and beads on a grid can be used on biological samples in any combination with each other.
In some embodiments, at least about 2 to about 10 beads may be connected together in various arrangements. In some embodiments, at least about 3, about 4, about 5, about 6, about 7, about 8, about 9 or more beads may be connected together. In some embodiments, from about 10 to about 100 beads may be connected together in various arrangements. In some embodiments, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90 or more beads may be in different arrangements to be connected together. In some embodiments, from about 100 to about 1000 beads can be linked together in various arrangements. In some embodiments, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, or about 900 or more beads may be in different arrangements. In some embodiments, from about 1,000 to about 10,000 beads may be linked together in various arrangements. In some embodiments, about 1000, about 2000, about 3000, about 4000, about 5000, about 6000, about 7000, about 8000, about 9000, about 10000 or more beads can be connected together.
In some embodiments, the bound beads may have capture probes comprising spatial barcodes, functional domains, unique molecular identifiers, cleavage domains, and capture domains, or combinations thereof. In some embodiments, each grain may be associated with a unique spatial barcode. In some embodiments, the spatial barcode is known prior to contacting the bead or array of beads with the biological sample. In some embodiments, the spatial barcode is unknown prior to contacting the bead or array of beads with the biological sample. The identity of each bead (eg, spatial barcode) in the array can be deconvoluted, eg, by direct optical sequencing, as discussed herein.
(xi) Printing arrays in liquid
In some embodiments, the arrays can be printed in liquid. Due to the proliferation of printing solutions, the resolution of traditional printed arrays can be limited. Printing arrays in highly viscous liquids can improve resolution by preventing the spreading of the printing solution. Accordingly, various methods and materials are disclosed herein for attaching and/or introducing capture probes (e.g., nucleic acid capture probes) having barcodes (e.g., spatial barcodes) to substrates (e.g., slides), while to which the connection is made (eg printing). in liquid.
In some aspects, the capture probes are printed on a substrate (eg, glass slide or bead). In some aspects, the substrate is a glass slide. In some aspects, the substrate is a 96-well microtiter plate. In some aspects, the methods provided herein are also applicable to other substrates commonly used in nucleic acid analysis, including but not limited to beads, particles, membranes, filters, dipsticks, slides, plates, and microarrays. In some aspects, such substrates may be composed of a variety of materials known to be compatible with nucleic acid analysis, including, but not limited to, agarose, styrene, nylon, glass, and silica.
first solution
In some embodiments, provided herein are methods of printing arrays on a substrate using one or more liquid solutions (eg, two or more solutions containing different capture probes). In some aspects, a method of printing an array on a substrate using one or more solutions can increase the resolution of the printed array. In some aspects, the methods provided herein include spraying a first solution (e.g., a bulk solution) onto a substrate. In some aspects, the first solution (eg, the bulk solution) has a lower Reynolds number than the second solution (eg, the second solution including capture probes that attach to the substrate). The Reynolds number expresses the inverse relationship between the density and velocity of the fluid and its viscosity in a certain channel length. A more viscous, less dense and/or slower moving fluid will have a lower Reynolds number and is easier to transfer, stop, start or reverse without turbulence. In some embodiments, the first solution and the second solution are immiscible.
In some aspects, the first (eg, bulk) solution is hydrophobic. In some aspects, after applying the first (e.g., batch) solution to the slide, the first (e.g., batch) solution remains on the slide in discrete spatial regions on the slide. In some aspects, the first (eg, bulk) solution is made from a solution that does not denature the one or more probes and/or inhibit binding of the probes to the substrate. In some embodiments, bulk solutions may include aqueous solutions, high viscosity solutions, or low diffusivity nucleic acid solutions. In some aspects, the first (eg, bulk) solution is a gel. In some aspects, the first (eg, bulk) solution is a hydrogel. In some aspects, the first (eg, bulk) solution comprises natural polymers including, for example, glycerol, collagen, gelatin, sugars such as starch, alginate, and agarose, or any combination thereof. In some aspects, the first (eg, bulk) solution includes a synthetic polymer. In some aspects, the synthetic polymers are prepared by any method known in the art, including, for example, chemical polymerization methods. In some aspects, the gel or polymer is hydrophobic. In some aspects, the gel or polymer is hydrophilic. In some aspects, the gel or polymer is aqueous. In some aspects, the gel or polymer is set at room temperature. In some aspects, the gel or polymer shrinks when heated. In some aspects, the polymer is a film that shrinks when heated.
In some aspects, the first (eg, bulk) solution includes glycerol. In some aspects, glycerol is present in the first (eg, bulk) solution at a concentration of 5-100%. In some aspects, the glycerol is present in an amount of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. %.
In some aspects, the first (eg, bulk) solution includes sugar. In some aspects, the sugars are monosaccharides, disaccharides, polysaccharides, or combinations thereof. In some aspects, the sugar is glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch cellulose, or combinations thereof. In some aspects, sugar is derived from complex compounds such as molasses or other by-products of sugar refining. In some aspects, the sugar is present in the first (eg, bulk) solution at a concentration of 5-100%. In some aspects, the sugar is present in an amount of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%. %.
In some aspects, the first (e.g. bulk) solution has a multiple viscosity of about 0.1 times, 0.2 times, 0.3 times, 0.4 times, 0.5 times, 0.6 times, 0.7 times , 0.8 times , 0.9 times, 1.0 times, 1.1 times, 1.2x, 1.3x, 1.4x, 1.5x, 1.6x, 1.7x times, 1.8x, 1.9x, 2.0x, 2.5x, 3.0x, 4.0x , 5.0x, 6.0x, 7.0x, 8.0x , which is 9.0 or 10 times the viscosity of the other solution.
another solution
In some embodiments, printing an array on a substrate using two or more solutions includes using a second solution. In some embodiments, the second solution may include one or more capture probes. In some embodiments, the second solution is dispersed as droplets. Some embodiments include a plurality of other solutions, wherein each member of the plurality of other solutions includes one or more capture probes that contain a spatial barcode unique to that particular droplet. In some embodiments, the second solution is applied to a substrate covered or partially covered by the first (eg, bulk) solution. In some embodiments, the first (e.g., bulk) solution reduces or prevents the diffusion of one or more capture probes from the second solution. In some aspects, when printed on a substrate covered or partially covered by a first (eg, bulk) solution, one or more capture probes are completely retained within the second solution.
In some aspects, another solution includes one or more capture probes (eg, all capture probes disclosed herein). In some aspects, another solution includes a spatially barcoded capture probe. In some aspects, the second solution comprises at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 3000, at least 4000 or at least 5000 spatially barcoded recording probes. In some aspects, the second solution includes a compound that facilitates binding of one or more capture probes to the substrate. In some aspects, the second solution does not inhibit the binding of the one or more capture probes to the substrate and/or does not denature the one or more capture probes. In some aspects, the second solution is less viscous than the first (eg, bulk) solution. In some aspects, the second solution and the first (eg, bulk) solution are completely or substantially immiscible (eg, immiscible). In some aspects, the second solution is an aqueous solution. In some aspects, the second solution is a hydrophilic solution.
pouring
In some embodiments, printing a high feature density pattern may include sputtering oligonucleotides and/or features in the form of droplets onto a substrate surface in the presence of a bulk solution. In some embodiments, the second solution droplets (e.g., oligonucleotides and/or signature droplets) and the first (e.g., bulk) solution are substantially immiscible with each other. In some aspects, the printing methods disclosed herein include sputtering a second solution of capture probes containing one or more spatially barcoded capture probes onto a substrate in the presence of (e.g., via) a first (e.g., bulk) solution resulting in a dot size of the second solution (e.g. .the size of the cross-sectional point of the second solution on the plane of the substrate) relative to the solution in the absence of the first solution (e.g. bulk). In some aspects, after printing the second solution on the substrate in the presence of the first solution (e.g., the second solution), the spot size of the second solution (e.g., the cross-sectional spot size of the second solution in the plane of the substrate) does not increase. , in bulk) solution. In some aspects, the dot surface of the second solution remains unchanged after printing the second solution onto the substrate in the presence of the first (e.g. bulk) solution. In some aspects, printing a second solution onto a substrate in the presence of a first (eg, bulk) solution results in a desired pattern on the surface of the substrate. For example, multiple second solutions can be printed on the substrate in the presence of the first solution such that the locations where the multiple second solutions are printed produce the desired pattern on the substrate. In some embodiments, two or more members of the plurality of other solutions printed on the substrate include different populations of capture probes that are attached to the substrate, thereby creating an array of capture probes.
density
In some embodiments, the cross-sectional area of the oligonucleotide and/or signature droplet on the substrate is less than the corresponding cross-sectional area of the oligonucleotide and/or signature droplet that would undergo scattering in the absence of solution. Oligonucleotides and/or elements are then added to the surface of the substrate. In some embodiments, the cross-sectional area of the oligonucleotide and/or signature droplet on the substrate is about two times smaller than the corresponding cross-sectional area of the oligonucleotide and/or signature droplet. Created by spraying oligonucleotides and/or signature droplets onto a substrate surface in the absence of a solution.
In some aspects, the cross-sectional area of the second solution on the substrate produced by spraying the second solution onto the surface of the substrate in the presence of the first solution is less than the corresponding cross-sectional area of the second solution, the second solution The second solution will be produced by applying the second solution to the surface of the substrate in the absence of the first solution. In some aspects, the cross-sectional area of the second solution on the substrate produced by spraying the second solution onto the surface of the substrate in the presence of the first solution is about twice, about three times, about four times, or about 5 times, about 10 times, about 20 times, about 30 times , about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times - in the absence of the first solution, by adding the corresponding cross-sectional area of the second solution resulting from applying the second solution to the surface of the substrate will be about 100 times smaller than the corresponding cross-sectional area of the second solution.
maintenance
Any suitable technique or conditions can be used to solidify the first solution, the second solution, and/or the spots formed from the second solution after application of the second solution (including capture probes) to the substrate. As used herein, the term "solidification" and related terms may refer to treating a solution (eg, a first solution, a second solution, or both) with an agent and/or conditions that convert the solution from a liquid state to a solid state (eg, a matrix), where the solution retains its three-dimensional shape after the solidification process. Suitable examples of curing techniques and/or conditions include ultraviolet (UV) radiation, infrared (IR) radiation, thermal radiation, microwave radiation, visible light radiation, narrow wavelength radiation, laser light, natural light, humidity, or combinations thereof. Suitable examples of curing sources include, for example, ultraviolet light, heating devices, radiation devices, microwave devices, plasma devices, or combinations thereof.
In some embodiments, the first solution and/or the second solution are chemically cured. In some embodiments, oligonucleotides and/or signature droplets are chemically immobilized. In some embodiments, the bulk solution is chemically cured. In some aspects, the dots formed from the second solution are chemically immobilized after application of the second solution (including capture probes) to the substrate. In some embodiments, the oligonucleotides and/or the feature and bulk solution are attached to the substrate (eg, by curing), thereby creating the feature and bulk solution-matrix. In some aspects, the matrix (eg, the first and second solution-matrix) is chemically cured. Chemical curing of the solution can be achieved by any means known in the art. For example, the solution may include one or more hydrogel subunits, which may be chemically polymerized (eg, cross-linked) to form a three-dimensional (3D) hydrogel network. Elements that are distributed in the form of droplets on the surface of the substrate can be polymerized in the presence of a bulk solution. In some embodiments, the features are copolymerized with the bulk solution to create gel features in a flexible array of hydrogels, while in other embodiments, the pads or gel features are polymerized and the bulk solution is removed leaving a series of dots or beads. Non-limiting examples of hydrogel subunits include acrylamide, bisacrylamide, polyacrylamide and its derivatives, polyethylene glycol and its derivatives (eg, PEG-acrylate (PEG-DA), PEG-RGD), gelatin-methacryloyl (GelMA), methacrylated hyaluronic acid (MeHA), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polybutylene oxide, polyvinylpyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate) and poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dex trans, agarose , gelatin, alginate, protein polymers, methylcellulose, etc., or combinations thereof. Other materials and techniques that can be used to form and/or crosslink hydrogels are described in more detail here.
In some aspects, the first solution and/or the second solution are photoreactively curable. In some embodiments, the oligonucleotides and/or signature droplets are photoreactively curable. In some embodiments, the bulk solution is photoreactively cured. In some aspects, the spots formed from the second solution are photoreactively cured after applying the second solution containing the capture probes to the substrate. In some aspects, the matrices (eg, the first and second matrix solutions) are photoreactively cured. Photoreactive curing can be accomplished by any means known in the art.
In some aspects, the methods disclosed herein include curing a first (e.g., mass) and/or second solution. In some aspects, the methods disclosed herein include curing the spots formed by the second solution after applying the second solution (e.g., the second solution containing the capture probes) to the substrate. In some aspects, the methods disclosed herein include curing the first solution and the second solution after applying the first solution and the second solution to the glass object. In some embodiments, curing the first and second solutions produces matrices of the first and second solutions. In some aspects, the methods disclosed herein include curing the second solution and then removing the uncured first solution from the substrate. In some aspects, the methods disclosed herein include curing the first and second solutions on a substrate, thereby producing a matrix (eg, first and second solution-matrix).
expansion matrix
In some embodiments, prior to spraying droplets of a second solution (e.g., oligonucleotides and/or signature droplets) onto a substrate surface in the presence of a first (e.g., bulk) solution, the first (e.g., bulk) solution is solidified to form a bulk solution matrix , and the bulk solution matrix reversibly expands along one or more axes (eg, one or more axes of the matrix or substrate surface). In some embodiments, the second solution droplets may be deployed in the presence of an expanded first (eg, bulk) solution matrix.
In some embodiments, a plurality of droplets of a second solution (e.g., containing different capture probes with different spatial barcodes) are distributed on a substrate covered with a first (e.g., bulk) solution, the first solution initially together with one or more stretched axes. In some embodiments, the stretched first solution is cured. In some embodiments, the stretched first solution is partially cured. In some embodiments, the stretched first solution is uncured and is sprayed onto a self-stretched surface. In some aspects, the volume (e.g., cross-sectional area) of the second solution droplet decreases after being dispersed into the initially stretched first solution. In some aspects, disclosed herein are methods of fabricating arrays comprising (i) placing a gel or polymer (e.g., a solidified or partially solidified solution) on a substrate, (2) stretching the gel or polymer, (3) distributing droplets onto a droplet of another solution onto the substrate as the gel or polymer stretches and (4) allow the gel or polymer to relax, thereby reducing the total surface area of the second solution droplet on the substrate (For example, the cross-sectional area) of the matrix. In some aspects, the stretching step includes reversibly swelling the gel or polymer along one or more axes coplanar with the surface of the substrate.
removal solution
In some embodiments, the first (eg, bulk) solution can be removed from the substrate after the oligonucleotides and/or signature droplets are attached to the substrate. In some aspects, the first (eg, bulk) solution is removed after spraying a second solution containing one or more capture probes (eg, multiple second solutions containing different capture probes) onto the substrate. Methods of removing the first (eg bulk) solution are known in the art. In some aspects, removal of the first (e.g., bulk) solution results in complete removal of the first solution, leaving only the second solution containing one or more capture probes (e.g., multiple capture probes containing different capture probes). second solution) into the matrix. In some aspects, removal of the first (e.g., bulk) solution does not alter the surface area of the second solution (e.g., multiple second solutions including different capture probes) in contact with the substrate. In some aspects, removal of the first (e.g., bulk) solution does not change the droplet shape of the second solution (e.g., multiple second solutions containing different capture probes) in contact with the substrate. In some aspects, prior to removal of the first (e.g., bulk) solution, a second solution containing one or more probes (e.g., multiple other solutions containing different capture probes) is solidified by the methods described herein, but the first (e.g., bulk) solution it is not hardened. For example, the first and second solutions can be subjected to reagents and/or conditions under which the second solution (eg, multiple other solutions containing different capture probes) solidifies while the first (eg, pooled) solution does not solidify. In some embodiments, the second solution (e.g., multiple second solutions including different capture probes) includes one or more hydrogel subunits that can be polymerized (e.g., cross-linked) to form a three-dimensional (3D) hydrogel network, while the first (e.g., bulk) the solution does not include such one or more hydrogel subunits. After subjecting the first and second solutions to curing conditions, only the second solution will harden, allowing the first solution to be removed.
In some embodiments, the first (eg, bulk) solution is not removed from the substrate after the oligonucleotides and/or tags are attached to the substrate.
Reduce number of drops/features
In some embodiments, the extended first (e.g., bulk) solution-matrix containing the second solution (e.g., oligonucleotides and/or characteristic droplets) may be along one or more axes (e.g., or the substrate) such that the cross-sectional area of the second droplet solution (e.g. oligonucleotide and/or characteristic droplet) is smaller than the corresponding cross-sectional area of another solution droplet (e.g. oligonucleotide) Area if the first (e.g. pooled) solution matrix containing other solution droplets (e.g. oligonucleotides and/or significant droplets ) is not along one or more axes. In some embodiments, the reduced matrix (e.g., first and second solution matrices) produces a reduced matrix (e.g., reduced first and second solution matrices), wherein the second solution droplet (e.g., plural) Volume The volume of the second solution droplet into the matrix is reduced in comparison with the volume of the second drop of solution in the non-shrinking matrix. In some aspects, the reduction of two or more other solutions (eg, droplets of the other solutions) results in a higher density of the two or more other solutions. In some embodiments, the second solution droplets (eg, oligonucleotides and/or signature droplets) and the first (eg, pooled) solution matrix can be collected by any of the methods disclosed herein. In some embodiments, the shrinkage may include the removal of water. In some aspects, the resolution of the array increases as the droplet shrinks. In some aspects, shrinking the second solution droplet results in a reduction in the cross-sectional area of the droplet (eg, the in-plane cross-sectional area of the substrate onto which the second solution droplet is printed or sprayed). In some aspects, once the droplet is reduced, the concentration of probes on the substrate is increased, leading to improved sensitivity.
The spatial array is in contact with a biological sample, wherein the biological sample can be any sample described herein (eg, an FFPE tissue section). After the spatial array is placed on a biological sample, cellular and/or nuclear permeabilization reactions occur, releasing biological analytes (e.g. DNA, RNA, proteins, metabolites, small molecules, lipids, etc.) and remaining in space on the array, its spatial information is preserved. Remove the spatial arrays and determine the molecular information within them (for example, by performing next-generation sequencing library construction). Sequencing can be followed by correlation of expression values (eg analyte gene expression) with features.
In some aspects, the cross-sectional area of the second solution droplet is reduced by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, by about the same amount as printed or applied to the substrate after shrinkage . The cross-sectional area of the second solution drop is reduced by about 70%, about 80% or about 90%. In some aspects, the cross-sectional area of the second solution droplet is reduced by about 1.0 times, about 1.1 times, about 1.2 times, about 1.3 times, about 1.4 times, about 1.5 times that of the second solutions printed or distributed on the substrate. About 2 times, about 3 times, about 4 times, or about 5 times the cross-sectional area of the droplet.
In some aspects, disclosed herein are methods of making an array comprising spraying a plurality of droplets of a second solution (e.g., containing different capture probes with different spatial barcodes) onto a substrate covered with a first (e.g., bulk) solution, solidifying the first solution [ 0060] and a second solution to generate the matrix (for example, a first solution matrix and a second solution matrix), wherein the volume and shape of the plurality of droplets of the second solution in the matrix are related to the distribution of the plurality of other The solution droplets are generally the same and collect the matrix, thereby reducing the total volume (eg, cross-sectional area) of a plurality of droplets of another solution. In some aspects, shrinkage can be achieved using any of the various shrinkage agents or conditions described herein. In some aspects, shrinkage can be achieved by dehydrating the matrix.
In some aspects, after application of the second solution, the area of the second solution is contracted along one or more axes coplanar with the surface of the substrate such that the cross-sectional area of the second solution on the substrate (e.g., glass slide) is less than the corresponding cross-sectional area of the second solution that would formed so that the first solution matrix containing the second solution does not collect along one or more axes of the substrate surface.
With respect to orientation, the shrinkage need not be equal in any two orthogonal directions on the substrate. However, in some aspects, the collection of droplets of the second solution is substantially uniform. In some aspects, the second solution droplet shrinks by approximately the same amount in each direction, regardless of position on the substrate plane.
In some aspects, disclosed herein are methods for detecting an analyte (eg, a plurality of analytes) in a biological sample after preparation of a substrate with one or more capture probes (eg, comprising a spatial array of capture probes). an assay comprising (a) generating a spatial array containing a plurality of capture probes bound to a substrate; wherein (i) at least part of the substrate is coated with the first solution; (ii) in the presence of the first solution, wherein a plurality of other solutions in the form of droplets are sprayed onto the surface of the substrate, wherein the first solution and the plurality of other solutions are substantially immiscible with each other, wherein at least two members of the plurality of other solutions form one or more different probes for capturing different spatial barcodes, and wherein at least one of the one or more probes for capturing at least two members of the array of other solutions is bound to the substrate; (b) such that the biological sample is in contact with the spatial array such that analytes present in the biological sample are captured by one or more capture probes; (c) determining the spatial distribution of the captured analytes in the biological sample.
(xii) Construct an array with a linker
In some embodiments, the array includes branched or dendritic links. In some embodiments, the linker comprises a dendritic polyamine. Non-limiting examples of dendritic polyamines include symmetric and asymmetric dendrimers, nucleic acid dendrimers, polyamidoamine dendrimers, and lysine-based dendrimers. In some embodiments, the linker is a nucleic acid dendrimer. In some embodiments, the nucleic acid dendrimer comprises a single nucleic acid molecule that shares an at least partially complementary region at the position of each nucleic acid molecule at which there are at least 2 (eg, 2, 3, 4 or more) to produce a single overhang. In some embodiments, the four single-stranded overhangs are designed to interact with additional nucleic acid dendrimers or specific complementary sequences (eg, sequences on capture probes). In some embodiments, the dendritic linker (eg, nucleic acid dendrimer) is immobilized (eg, immobilized using any of the methods described herein (eg, covalent attachment or physical absorption)) to the substrate. For example, nucleic acid dendrimers are immobilized on substrates and interact with capture probes through single-stranded overhangs. In another example, a nucleic acid dendrimer is immobilized on a substrate and also interacts with another nucleic acid dendrimer. Additional nucleic acid dendrimers can also interact with capture probes via single-stranded overhangs that have not yet interacted with other nucleic acid dendrimers. In this case, each additional layer of nucleic acid dendrimer has the potential to increase the number of sites with which the capture probes can interact, thereby increasing the capture domain (eg, any of the example capture domains described herein). series.
(xiii) Sequence/Feature Preservation
In some embodiments, the biological sample can be saved for additional rounds of spatial analyte detection after completion of the signature or sequence of signature assay. In some embodiments, biological patterns, features, sequences, or any combination thereof may be preserved after spatial profiling. In some embodiments, the biological samples, features, arrays, or combinations thereof may be protected from dehydration (eg, desiccation, desiccation). In some embodiments, the biological samples, features, arrays, or combinations thereof may be protected from evaporation. Procedures for preserving and/or protecting biological samples, characteristics or sequences are known in the art. For example, without limitation, a biological pattern, feature, array, or combination thereof may be covered with a reversible sealant. Any suitable reversible sealant can be used. Reversible sealing methods are known in the art (see, e.g., WO 2019/104337, which is incorporated herein by reference). Without limitation, suitable reversible sealants may include non-porous materials, membranes, caps, or oils (eg, silicone oil, mineral oil). In a further non-limiting example, biological samples, features, arrays, or combinations thereof may be kept in an environmental chamber (eg, hermetically sealed) and removed later for additional rounds of spatial analysis.
(e) Capture of analytes
In this section, general aspects of analyte capture methods and systems are described. Various method steps and system features can be combined in many different embodiments; the particular combinations described herein are in no way limiting other combinations of steps or features.
(i) Catch conditions
Typically, when a biological sample is mixed with a substrate that includes a capture probe (e.g., a substrate with an embedded capture probe, spotted, printed on the substrate, or with a feature (e.g., bead, well) that includes probe capture, when in contact with the substrate ), the analyte can be captured.
As used herein, "contacting," "contacting," and/or "bringing into contact" a biological sample with a substrate refers to any contact (eg, directly or indirectly) such that the capture probe can interact with an analyte from substrate (eg capture) of biological samples. For example, a substrate can be near or adjacent to a biological sample without direct physical contact and still capture analytes from the biological sample. In some embodiments, the biological sample is in direct physical contact with the matrix. In some embodiments, the biological sample is in indirect physical contact with the matrix. For example, the liquid layer can be between the biological sample and the substrate. In some embodiments, the analyte diffuses through the fluidized bed. In some embodiments, capture probes diffuse through the fluidized bed. In some embodiments, the reagents can be delivered through a fluidized bed between the biological sample and the matrix. In some embodiments, the indirect physical contact may include a second substrate (eg, hydrogel, thin film, porous membrane) between the biological sample and the first substrate containing the capture probes. In some embodiments, the reagents can be delivered to the biological sample using another matrix.
In some embodiments, a cell immobilization agent can be used to contact a biological sample with a substrate (eg, by immobilizing an unaggregated or disaggregated sample on a spatially barcoded array prior to analyte capture). As used herein, a "cell immobilizing agent" can refer to a substrate-bound agent (eg, an antibody) that can bind a marker on the surface of a cell. Non-limiting examples of cell surface markers include CD45, CD3, CD4, CD8, CD56, CD19, CD20, CD11c, CD14, CD33, CD66b, CD34, CD41, CD61, CD235a, CD146, and epithelial cell adhesion molecule (EpCAM). A cell fixative may include any probe or component capable of binding (eg, fixing) a cell or tissue while on the matrix. Cell fixatives attached to the surface of the matrix can be used to bind cells bearing cell surface markers. The cell surface marker can be a ubiquitous cell surface marker where the purpose of the cell fixative is to capture a high percentage of cells in the sample. Cell surface markers can be specific or rarely expressed cell surface markers, where the purpose of the cell fixative is to capture a specific population of cells expressing the target cell surface marker. Thus, cell immobilization agents can be used to selectively capture cells expressing a target cell surface marker from a population of cells that do not share the same cell surface marker.
Capture probes on the substrate (or features on the substrate) can interact with the released analyte through the capture domain, as described elsewhere, to capture the analyte. In some embodiments, certain steps are performed to enhance the transfer or capture of analytes to array capture probes. Examples of such modifications include, but are not limited to, adjusting the conditions under which substrates are in contact with biological samples (eg time, temperature, direction, pH level, pretreatment of biological samples, etc.), using forces to transport analytes (eg electrophoresis , centrifugation, mechanical, etc.), perform amplification reactions to increase the amount of biological analyte (eg PCR amplification, in situ amplification, clonal amplification) and/or use labeled probes to detect multiples and barcodes.
In some embodiments, the array is adjusted to facilitate migration of the biological analyte. Non-limiting examples of arrays adapted to facilitate biological analyte transport include arrays with substrates comprising nanopores, nanopores, and/or microfluidic channels; arrays of porous membranes; and arrays with substrates made of hydrogels. In some cases, the array substrate is liquid permeable. In some cases, the array is a coverslip or a coverslip that includes nanowires or patterns (eg, by fabrication). In some cases where the substrate includes nanopores, nanopores, and/or microfluidic channels, these structures can facilitate exposure of the biological sample to reagents (e.g., permeabilization reagents, biological analyte capture, and/or nucleic acid elongation reactions), thereby improving analyte capture efficiency compared to with substrates that do not have such properties.
In some embodiments, analyte capture is facilitated by treating the biological sample with a permeabilization reagent. If the biological sample is not sufficiently permeabilized, the amount of analyte absorbed by the substrate may be too small for adequate analysis. In contrast, if the biological sample is too permeable, the analyte diffuses away from its source in the biological sample, leading to a loss of the relative spatial relationship of the analytes within the biological sample. Therefore, a balance needs to be struck between sufficient permeabilization of the biological sample to obtain good signal intensity, while still maintaining the spatial resolution of the analyte distribution in the biological sample. Methods of preparing biological samples to facilitate analyte capture are known in the art and can be modified depending on the biological sample and the manner in which the biological sample was prepared (eg, fresh frozen, FFPE, etc.).
(ii) Substrate support
Described herein are methods in which an array with capture probes on a substrate is in contact with a biological sample on another substrate such that the array is in contact with the biological sample (eg, the substrates are bonded together). In some embodiments, the array and the biological sample may be in contact (eg, sandwiched) without the aid of a substrate support. In some embodiments, the array and the biological sample substrate can be placed in a substrate holder (e.g., an array alignment device) designed to align the biological sample and the array. For example, a mat holder may have placeholders for two mats. In some embodiments, the array containing the capture probes can be positioned on one side of the substrate holder (eg, in the first substrate holder). In some embodiments, the biological sample can be placed on the adjacent side of the substrate holder in another user. In some embodiments, a hinge may be located between two substrate placeholders that allows the substrate placeholder to close, eg, forming a sandwich between the two substrate placeholders. In some embodiments, when the substrate holder is closed, the biological sample and the array with capture probes contact each other under conditions sufficient to allow analytes present in the biological sample to interact with the capture probes of the array. For example, dry permeabilization reagents can be applied to biological samples and rehydrated. The permeabilization solution can flow through the substrate support to permeabilize the biological sample and allow the analytes in the biological sample to interact with the capture probes. Additionally, the temperature of the substrate or permeabilization solution can be used to initiate or control the rate of permeabilization. For example, the substrate containing the array, the substrate containing the biological sample, or both can be kept at low temperatures to slow diffusion and permeabilization efficiency. Once sandwiched, in some embodiments, the substrate can be heated to initiate permeabilization and/or increase diffusion efficiency. Transcripts released from permeabilized tissue can diffuse into the array and be captured by capture probes. The sandwich can be opened and cDNA synthesis performed on the chip.
Any of the various combinations described herein, including sandwiching an array of capture probes and a biological sample on two different substrates, can be placed in a substrate holder designed to align the biological sample and the array. For example, a mat holder may have placeholders for two mats. In some embodiments, the array containing the capture probes can be positioned on one side of the substrate holder (eg, in the first substrate holder). In some embodiments, the biological sample can be placed on the adjacent side of the substrate holder in another user. In some embodiments, there may be a hinge between the two substrate holders that allows the substrate holder to close, eg, forming a sandwich between the two substrate holders. In some embodiments, when the substrate holder is closed, the biological sample and the capture probe array may contact each other under conditions sufficient to allow analytes present in the biological sample to interact with the capture probes of the spatial analysis array in any manner. . method described in this article. For example, dry permeabilization reagents can be applied to biological samples and rehydrated. Additionally, the permeabilization solution can flow through the substrate support to permeabilize the biological sample and allow the analytes in the biological sample to interact with the capture probes.
In some embodiments, the flexible arrays described herein can be placed in a substrate holder and hold a biological sample. In some embodiments, the flexible array may include barcode spatial association features. In some embodiments, the flexible array may be pre-soaked in a permeabilization reagent before being placed in the substrate holder. In some embodiments, the flexible array may be soaked with a permeabilization reagent after it is placed in the substrate holder. In some embodiments, a substrate holder containing a biological sample in a single place holder and a flexible array can be closed (eg, sandwiched) such that permeabilization reagents allow analytes present in the biological sample to interact with capture probes of the flexible array. Interactions (eg, probe capture on spatially barcoded features).
In some embodiments, the substrate support can be heated or cooled to adjust the permeability and/or diffusion efficiency.
(iii) Passive capture methods
In some embodiments, the analytes can migrate from the sample to the substrate. Methods of promoting migration can be passive (eg diffusion) and/or active (eg electrophoretic migration of nucleic acids). Non-limiting examples of passive transport may include simple diffusion and osmotic pressure created by rehydration of dehydrated objects.
Passive migration by diffusion uses a concentration gradient. Diffusion is the movement of an unbound object towards equilibrium. Therefore, when there are areas of high object concentration and areas of low object concentration, the objects (eg, capture probes, analytes, etc.) are moved to areas of lower concentration. In some embodiments, the unbound analyte travels down a concentration gradient.
In some embodiments, various reagents can be added to the biological sample such that the biological sample is rehydrated while enhancing analyte capture. In some embodiments, the biological sample can be contacted with the clamping array described herein. In some embodiments, the biological sample and/or pooled array may be rehydrated with a permeabilization reagent. In some embodiments, the biological sample and/or collected array can be rehydrated with a staining solution (eg, hematoxylin and eosin staining).
(iv) Anti-Proliferation Media/Headlines
Anti-diffusion media can be used to increase efficiency by promoting analyte diffusion onto the spatial barcode capture probes. In general, molecular diffusion of biological analytes occurs in all directions, including toward the capture probe (ie, towards the barcode spatial array) and away from the capture probe (ie, into the bulk solution). Increasing diffusion into the barcode array reduces analyte diffusion from the barcode array and increases the capture efficiency of capture probes.
In some embodiments, the biological sample is placed on top of the spatial barcode substrate, and the anti-diffusion medium is placed on top of the biological sample. For example, an anti-diffusion medium can be applied to an array that is placed in contact with a biological sample. In some embodiments, the diffusion-resistant medium and the spatial barcode array are the same component. For example, a diffusion-resistant medium can contain spatially barcoded capture probes in or on a diffusion-resistant medium (eg, a coverslip, glass slide, hydrogel, or membrane). In some embodiments, the sample is placed on a substrate and an anti-diffusion medium is placed on top of the biological sample. In addition, arrays of spatially barcoded capture probes can be placed close to a diffusion-resistant medium. For example, an anti-diffusion medium can be sandwiched between the spatial array of the barcode and the pattern on the substrate. In some embodiments, the anti-diffusion medium is cast or applied to the sample. In other embodiments, the anti-diffusion medium is placed in close proximity to the sample.
In general, the antidiffusion medium can be any material known to limit the rate of diffusion of biological analytes. For example, the antidiffusion medium can be a solid coverslip (such as a coverslip or a slide). In some embodiments, the diffusion-resistant medium can be made of glass, silicon, paper, hydrogel polymer monoliths, or other materials. In some embodiments, the glass side may be acrylic glass. In some embodiments, the anti-diffusion medium is a porous membrane. In some embodiments, the material may be naturally porous. In some embodiments, the material may have pores or pores etched into the solid material. In some embodiments, the pore volume can be manipulated to minimize loss of target analytes. In some embodiments, membrane chemistry can be manipulated to minimize loss of target analytes. In some embodiments, the diffusion-resistant medium (e.g., hydrogel) is covalently bound to a substrate (e.g., glass slide). In some embodiments, the anti-diffusion medium can be any material known to limit the diffusion of poly(A) transcripts. In some embodiments, the anti-diffusion medium can be any material known to limit the rate of protein diffusion. In some embodiments, the diffusion-resistant medium can be any material known to limit the rate of diffusion of macromolecular components.
In some embodiments, the non-diffusing medium includes one or more non-diffusing media. For example, one or more antidiffusion media can be combined in various ways, including but not limited to coating, layering, or staining, prior to contacting the media with the biological sample. As another example, a hydrogel can be placed on top of a biological sample, and then a coverslip (such as a glass slide) is placed on top of the hydrogel.
In some embodiments, forces (eg, hydrodynamic pressure, ultrasonic vibrations, solute contrast, microwave radiation, vascular circulation, or other electrical, mechanical, magnetic, centrifugal, and/or thermal forces) are applied to control diffusion and enhance analyte capture. In some embodiments, one or more forces and one or more anti-diffusion media are used to control diffusion and enhance capture. For example, centrifugal force and glass can be used simultaneously. Any of the various combinations of force and anti-diffusion media can be used to control or moderate diffusion and improve analyte capture.
In some embodiments, the anti-diffusion medium is immersed in the bulk solution along with the spatial barcode array and the pattern. In some embodiments, the bulk solution includes a permeabilizing agent. In some embodiments, the anti-diffusion medium includes at least one permeabilizing agent. In some embodiments, the anti-diffusion medium (ie, the hydrogel) is impregnated with a permeabilization reagent prior to contacting the anti-diffusion medium with the sample. In some embodiments, the anti-diffusion medium may contain pores (eg, micropores, nanopores, or picopores) that contain buffers or permeabilization reagents. In some embodiments, the anti-diffusion medium may include a permeability-enhancing agent. In some embodiments, the anti-diffusion medium may contain dry reagents or monomers for delivery of permeabilizing reagents after application of the anti-diffusion medium to the biological sample. In some embodiments, an anti-diffusion medium is added to the barcode spatial array and sample components prior to immersing the components in the solution. In some embodiments, an anti-diffusion medium is added to the barcode spatial array and sample assembly after the sample has been exposed to a permeabilizing reagent. In some embodiments, the permeabilization reagent flows through the microfluidic chamber or channel above the diffusion-resistant medium. In some embodiments, flow controls sample access to permeabilization reagents. In some embodiments, the target analyte diffuses from the sample into solution and becomes embedded in a diffusion-resistant medium in which the spatial barcode capture probes are embedded. In some embodiments, the free solution is sandwiched between the biological sample and the non-diffusing medium.
(v) Active capture method
In some of the methods described herein, analytes in a biological sample (eg, in cells or tissue sections) can be transported (eg, passively or actively) to capture probes (eg, immobilized on a substrate (eg, carrier) on the capture probes). , substrate or balls)).
For example, analytes can be transported to capture probes (eg, immobilized capture probes) using electric fields (eg, using electrophoresis), pressure, fluid flow, gravity, temperature, and/or magnetic fields. For example, analytes may use pressure gradients, chemical concentration gradients, temperature gradients, and/or pH gradients. For example, analytes can be transported to capture probes (eg, immobilized capture probes) using gels (eg, hydrogels), liquids, or permeabilized cells.
In some examples, an electrophoretic field can be applied to the analyte to facilitate the migration of the analyte into the capture probes. In some examples, a sample containing an analyte is contacted with a substrate (such as a slide, coverslip, or beads) with capture probes immobilized on the substrate, and an electrical current is applied to promote the directed migration of the charged analyte into the probe matrix for catching. An electrophoretic assembly (e.g., an electrophoresis chamber) in which the biological sample is in contact with a cathode and a capture probe (e.g., a capture probe immobilized on a substrate), and where the capture probe is in contact with the biological sample and an anode, may be used for the application of electric current.
In some embodiments, methods using active capture methods can use conductive substrates (e.g., any conductive substrate described herein). In some embodiments, the conductive substrate includes paper, hydrogel film, or glass slides with a conductive coating. In some embodiments, the conductive substrate (e.g., any conductive substrate described herein) includes one or more capture probes.
biological sample2402and the second layer2408(eg, the lawn of the capture probe) may be exposed to the permeabilization solution2410Non-limiting examples of permeabilization solutions include enzymes (such as proteinase K, pepsin, and collagenase), detergents (such as sodium dodecyl sulfate (SDS), polyethylene glycol tert-octylphenyl ether, polysorbate 80, and polysorbate 20), ribonuclease inhibitors, buffers optimized for electrophoresis, buffers optimized for permeabilization, buffers optimized for hybridization or combinations thereof. Permeabilization reagents may also include, but are not limited to, dried permeabilization reagents, permeabilization buffers, buffers without permeabilization reagents, permeabilization gels, and permeabilization solutions. In some examples, a biological sample (eg, a tissue sample) may first be permeabilized and then subjected to electrophoresis.
in step2420, after permeabilization of biological samples2402summary, sample2402Electrophoresis can be performed. During electrophoresis, biological samples2402Subjected to an electric field that can be generated by clamping the biological sample2402Between the first pad2404and the second substrate2422, connect each substrate to the cathode or anode, respectively, and allow current to flow through the substrates. The application of an electric field "-E" causes the analyte2404(eg negatively charged analytes) migrate towards the substrate2404and capture probes2416(eg positively charged capture probes) in the direction of the arrows shown in the figure
In some embodiments, the analyte2414be proteins or nucleic acids. In some embodiments, the analyte2414are negatively charged proteins or nucleic acids. In some embodiments, the analyte2414are positively charged proteins or nucleic acids. In some embodiments, a capture probe2416be proteins or nucleic acids. In some embodiments, a capture probe2416are positively charged proteins or nucleic acids. In some embodiments, a capture probe2416are negatively charged proteins or nucleic acids. In some embodiments, the analyte2414are negatively charged transcripts. In some embodiments, the analyte2414is a poly(A) transcript. In some embodiments, a capture probe2416Features to be added to the feature set. In some embodiments, a permeabilization reagent2410access to samples2402, the first pad2404another substrate2422, or any combination thereof.
In some embodiments, the permeabilization reaction is performed at freezing temperatures (eg, about 4°C). In some embodiments, performing the permeabilization reaction at freezing temperatures controls the enzymatic activity of the permeabilization reaction. In some embodiments, the permeabilization reaction is performed at freezing temperatures to prevent movement and/or diffusion of the analyte.2414From the original location (for example, locations in the cell of a biological sample2402) until the user is ready to start the permeabilization reaction. In some embodiments, the permeabilization reaction is performed at a warm temperature (eg, a temperature in the range of about 15°C to about 37°C or higher) to initiate and/or increase the rate of the permeabilization reaction. In some embodiments, the permeabilization reaction allows the analyte to migrate from its original site (e.g., a site in a cell of a biological sample) upon application of electrophoresis and/or a heated permeabilization reaction.2402) on the capture probe2416fixed on the first substrate2404.
Generally refers to
In some embodiments, methods using active capture methods may include one or more solutions between a biological sample and a matrix (eg, a matrix that includes capture probes). In some embodiments, the one or more solutions between the biological sample and the substrate containing the capture probe may include a permeabilization buffer (eg, any of the permeabilization buffers described herein). In some embodiments, one or more solutions between the biological sample and the matrix containing the capture probes may comprise an electrophoresis buffer.
In some embodiments, the active capture of the analyte may include one or more porous materials between the biological sample and the matrix containing the capture probes. In some embodiments, the one or more porous materials between the biological sample and the substrate including the capture probes may include paper or an absorbent membrane. In some embodiments, the one or more porous materials between the biological sample and the substrate including the capture probes may comprise a gel containing one or more solutions. For example, without limitation, the gel may be an SDS-PAGE gel. In some embodiments, one or more porous materials between the biological sample and the matrix containing the capture probes may comprise a permeabilization buffer. In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes may comprise an electrophoresis buffer. In some embodiments, active analyte capture may include one or more solutions and one or more porous materials interposed between the biological sample and the matrix containing the capture probes.
In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes (e.g., an array) may act as a filter to separate analytes (e.g., analytes of interest) from those present in the Separate the samples from other molecules or analytes in biological samples. In some embodiments, the analyte (eg, the analyte of interest) is an RNA transcript. In some embodiments, one or more porous materials between the biological sample and the matrix containing the capture probes may act as a filter to separate RNA transcripts from other molecules (eg, analytes) such as proteins, lipids, and/or other nucleic acids for isolation. In some embodiments, one or more porous materials between the biological sample and the matrix, including capture probes, can act as a filter to separate analytes and other molecules based on physicochemical properties. For example, without limitation, analytes can be classified according to parameters such as charge, size (eg, length, radius of gyration, effective diameter, etc.), hydrophobicity, hydrophilicity, molecular binding (eg, immunoaffinity), and combinations thereof. In some embodiments, one or more porous materials between the biological sample and the matrix including the capture probes may separate the analyte from other molecules to reduce non-specific binding in the vicinity of the capture probes and thus improve analyte and capture. Probe-to-probe binding, improving subsequent detection performance.
In some embodiments, one or more porous materials between the biological sample and the substrate including capture probes can serve as a molecular sieve matrix for electrophoretic separation of analytes. For example, without limitation, separation of analytes can occur based on physicochemical properties such as charge, size (eg, length, radius of gyration and effective diameter, etc.), electrophoretic mobility, zeta potential, isoelectric point, hydrophobicity, hydrophilicity, molecular binding (eg immunoaffinity) and their combinations. In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes may have the same pore size. In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes may have a discontinuity in pore size, which is commonly used in various gel electrophoresis protocols. In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes may have a pore size gradient. For example, one or more porous materials (eg, hydrogels) may have a pore size gradient such that the gradient separates analytes as they migrate to a substrate containing capture probes (eg, an array).
In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes can separate the analytes based on length. For example, shorter analytes will have higher electrophoretic mobility and therefore migrate faster towards the capture probes than longer analytes in the electrophoretic device. In some embodiments, one or more porous materials between the biological sample and the substrate containing the capture probes separate the analytes based on length such that only shorter analytes can migrate through the one or more porous materials to the capture probe, while longer analytes cannot reach the probe for catching.
In some embodiments, a specific subset of analytes (eg, a subset of transcripts) can be captured by applying an electrophoretic field over a period of time. In some embodiments, specific subsets of analytes (eg, subsets of transcripts) can be captured by selecting different porous materials (eg, porous materials with different compositions) between the biological sample and the matrix containing the capture probes. In some embodiments, the biological sample and the inclusion of capture probes (eg, arrays) can be detected by applying an electrophoretic field over a period of time.
In some embodiments, one or more porous materials between the biological sample and the matrix containing the capture probes may have a discontinuity in pore size, which may lead to increased concentrations (eg, "packing") of migrating analytes. For example, one or more porous materials (e.g., hydrogels) between the biological sample and the matrix including the capture probes may have a discontinuity in pore size, which may lead to increased analyte concentrations in the vicinity of the capture probes, leading to favorable binding kinetics and increased sensitivity. In some embodiments, one or more porous materials between the biological sample and the matrix containing the capture probes may have a discontinuity in pore size, which increases the separation between migrating analytes of different sizes and/or lengths. In some embodiments, the one or more porous materials between the biological sample and the substrate including the capture probes may include a first porous material and a second porous material, wherein the first porous material has more porous material than the second porous material. Porous materials with larger pore sizes. In some embodiments, the first porous material is located at or near the surface of the biological sample. In some embodiments, a second porous material (eg, a second porous material having a smaller pore size than the first porous material) may be placed on or near the surface of the first porous material. In some embodiments, as the analyte from the biological sample migrates sequentially (e.g., by electrophoretic migration) through the first porous material and the second porous material, the migrated analyte may pass between the first porous collection material (e.g., "stacking") and the second porous material at the interface .
In some embodiments, the one or more porous materials between the biological sample and the substrate containing the capture probes may include a pore size gradient for migration as the analyte passes through the reduced pore size (e.g., reduced pore size) during sequential stacking. In some embodiments, the one or more porous materials between the biological sample and the substrate including the capture probes may include a pore size gradient such that, as the analyte migrates from the biological sample to the substrate including the capture probes, the pore diameter decreases. In some embodiments, a pore size gradient can increase resolution between analytes of different sizes. In some embodiments, the pore size gradient can increase the analyte concentration in the vicinity of the capture probe. In some embodiments, the pore size gradient may continuously decrease the rate at which the analyte migrates and collects (eg, “packs”) as the analyte migrates through the gradient of reduced pore size (eg, reduced pore size).
In some embodiments, one or more porous materials between the biological sample and the matrix containing the capture probes may comprise a gradient gel to reduce the pore size as the analyte migrates through the gradient gel (eg, reduced pore size) while stacking. In some embodiments, the gradient gel may have pores that decrease in diameter as the analytes migrate toward the capture probes. In some embodiments, gradient gels can increase the resolution of separation between analytes of different sizes. In some embodiments, gradient gels can increase the concentration of analyte near the capture probes. In some embodiments, the gradient gel may continuously decrease the rate at which the analyte migrates and collects (eg, "packs") as the analyte migrates through the gradient gel with decreasing pore size (eg, decreasing diameter).
In some embodiments, the biological sample may be placed in a first matrix holder (eg, the matrix holder described herein). In some embodiments, a spatially barcoded array of capture probes (eg, capture probes, bar-coded array) can be placed on another substrate carrier (eg, the substrate carrier described herein). In some embodiments, the biological sample may be placed in a first matrix holder that also contains capture probes. In some embodiments, the first substrate holder, the second substrate holder, or both may be conductive (eg, any conductive substrate described herein). In some embodiments, the first substrate holder containing the biological sample, the second substrate holder containing the capture probe, or both can be contacted with a permeabilization reagent (eg, permeabilization buffer) and the analyte can be removed from the Biological Sample Migration. the pattern in the barcode field uses an electric field.
In some embodiments, electrophoresis can be applied to a biological sample on a barcoded array while in contact with a permeabilization buffer. In some embodiments, electrophoresis can be applied to a biological sample on a barcoded array while in contact with a liquid buffer (eg, a buffer that does not have a permeabilization reagent). In some embodiments, the permeabilization buffer can be replaced with electrophoresis buffer after a desired time. In some embodiments, electrophoresis can be administered simultaneously with permeabilization buffer or electrophoresis buffer. In some embodiments, electrophoresis can be applied after a desired contact time between the biological sample and the permeabilization buffer or liquid buffer.
In some embodiments, the biological sample can be placed on a substrate (eg, porous membrane, hydrogel, paper, etc.). In some embodiments, a biological sample placed on a substrate may have a gap (e.g., space) between the substrate and the substrate holder (e.g., a conductive substrate holder). In some embodiments, the barcode array can be placed on a substrate (eg, porous membrane, hydrogel, paper, etc.). In some embodiments, the barcode array may have a gap between the substrate and the substrate holder (eg, a conductive substrate holder). In some embodiments, the barcode array can be placed in close proximity to the biological sample or at a desired distance from the biological sample. In some embodiments, a buffer reservoir can be used between a substrate support (eg, a conductive substrate support) and a barcode sequence, a biological sample, or both. This setup allows analytes to migrate onto the barcode array without approaching electrodes such as conductive substrate supports, resulting in more stable electrophoresis.
In some embodiments, a combination of at least two buffers with different ionic compositions can be used to differentially transfer an analyte based on its ion mobility (eg, isotachophoresis (ITP)). For example, using two or more buffers with different ionic compositions can increase the concentration of the analyte before exposure to the barcode array. Isotachophoresis involves at least two buffers containing common counterions (eg, ions that have a different charge sign than the analyte) and different coions (eg, ions that have the same charge sign as the analyte) (Smejkal P., et al. ., Microfluidic isotachophoresis: A Review, Electrophoresis, 34.11 1493-1509, (2013) which is incorporated herein by reference in its entirety). In some embodiments, the buffer may contain coions that have a higher ion mobility (eg, their speed through solution in an electric field) than the analyte (eg, a "lead" buffer). In some embodiments, the second buffer may contain a coion with a lower ion mobility than the analyte (eg, a "tail" buffer). In some embodiments, the third buffer may contain co-ions with an ionic mobility between the electrical mobility of the analyte. In some embodiments, the biological sample may be placed on a first matrix support (e.g., a conductive matrix support) and the barcode array may be placed on a second matrix support (e.g., a second conductive matrix support) and communicate with the permeable matrix support the analyte is in contact with. with a buffer and an electric field can be used to move analytes from a biological sample to a barcode array. When an electric field is applied to a biological sample, analytes can be concentrated in the buffer as they migrate toward the capture probes. In some embodiments, isotachophoresis can be used with a gel-based separation (eg, any of the gel-based separations described herein).
In some embodiments, the permeabilization buffer can be applied to a region of interest in a biological sample (eg, a region of interest as described herein). In some embodiments, a permeabilization reagent (eg, a hydrogel containing a permeabilization reagent) can be applied to a region of interest in a biological sample. For example, a region of interest may be a region with a small surface area relative to the total surface area of a biological sample. In some embodiments, the permeabilization buffer or permeabilization reagent can be contacted with a biological sample and a substrate (eg, an array) that includes capture probes. In some embodiments, a biological sample may have more than one region of interest (eg, two, three). In some embodiments, a biological sample, a substrate including capture probes, or both can be placed in a conductive substrate holder. In some embodiments, analytes can be released from the region of interest and migrate from the biological sample to the capture probes using an electric field.
In some embodiments, electrophoretic transfer of analytes can be performed while preserving the relative spatial position of the analytes in the biological sample while minimizing passive diffusion of the analytes away from their location in the biological sample. In some embodiments, an analyte captured by a capture probe (eg, a capture probe on a substrate) retains the spatial location of the analyte present in the biological sample from which it was obtained (eg, the spatial location of the captured analyte). Spatial Position) Capture probes on a substrate may be more accurate or representative of the spatial location of an analyte in a biological sample when the analyte actively migrates to the capture probe by electrophoretic transfer than when the analyte does not actively migrate to the capture probe. In some embodiments, electrophoretic transport and binding processes are described by the Damkler number (Da), which is the ratio of reaction rate to mass transfer. The ratio of bound analytes and the shape of the biological sample will depend on the parameters in Da. Parameters include the electromigration rate of Uelectronic(depending on the electrophoretic mobility of the analyte µelectronicand electric field strength E), density of capture probes (eg barcoded oligonucleotides) p0, the binding rate between the probe (eg barcoded oligo) and the analyte kexist, and the thickness of the trapping region L.
Fast migration (eg, electromigration) shortens analysis time and can minimize analyte molecular diffusion.
In some embodiments, electrophoretic transfer of analytes can be performed while maintaining the relative spatial distribution of analytes in the sample. Thus, an analyte captured by a capture probe (eg, a capture probe on a substrate) retains the spatial information of the cell or biological sample from which the analyte was obtained. Application of an electrophoretic field to the analyte also results in an increase in temperature (eg heat). In some embodiments, elevated temperature (e.g., heat) may facilitate analyte migration into capture probes.
In some examples, a spatially addressable array of microelectrodes is used to spatially limit the capture of at least one charged analyte of interest by the capture probes. For example, spatially addressable microelectrode arrays may allow discrete (eg, localized) application of electric fields instead of uniform electric fields. Spatially addressable arrays of microelectrodes can be individually addressable. In some embodiments, an electric field can be applied to one or more regions of interest in a biological sample. The electrodes can be next to each other or far from each other. Microelectrode arrays can be configured to include a high density of discrete sites with small electric field application areas to facilitate migration of charged analytes of interest. For example, an array of spatially addressable microelectrodes can be used to electrophoretically capture a region of interest.
A high density of discrete sites on an array of microelectrodes can be used. The surface may contain any suitable density of discrete sites (eg, a density suitable for processing a sample on a conductive substrate within a given time). In one embodiment, the surface has a discrete spot density greater than or equal to about 500 spots per 1 mm2In some embodiments, the surface has about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 2000, about 3000, about 4000 per mm, about 5000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 20,000, about 40,000, about 60,000, about 80,000, about 100,000 or about 500,000 pages2In some embodiments, the surface has at least about 200, at least about 300, at least about 400, at least about 500, at least about 600, at least about 700, at least about 800, at least about 900, at least about 1,000, at least about 2,000, at least about 3,000, at least about 4,000, at least about 5,000, at least about 6,000, at least about 7,000, at least about 8,000, at least about 9,000, at least about 10,000 per millimeter, at least about 20,000, at least about 40,000, at least about 60,000, at least about 80,000, at least about 100,000 or at least about 500,000 pages2.
Schematic showing an electrophoretic transfer system configured to target nucleic acid analytes (eg, mRNA transcripts) to arrays of spatially barcoded capture probes, as shown
(vi) Targeted analysis
In some aspects, the array (eg, glass) includes a plurality of capture probes that bind to one or more specific biological targets in the sample. The capture probes can be directly or indirectly attached to the substrate. The capture probe can be or include, for example, DNA or RNA. In some aspects, capture probes on the array can be immobilized (eg, attached or bound) to the array via their 5' or 3' ends, depending on the chemical substrate of the array. In some aspects, the probes are linked by a 3' linker, leaving the 5' end free. In some aspects, the probes are linked by a 5' linker, leaving the 3' end free. In some aspects, the probe is immobilized indirectly. For example, probes can be attached to beads, which can be applied to a substrate. Capture probes disclosed in this section may include any of the various capture probe components provided throughout this disclosure (eg, spatial barcodes, UMIs, functional domains, cleavage domains, etc.).
In some aspects, the capture probe or probes interact with an analyte specific to a particular species or organism (eg, host or pathogen). In some aspects, the probe or probes can be used to detect viral, bacterial or plant proteins or nucleic acids. In some aspects, the capture probe or probes can be used to detect the presence of a pathogen (eg, bacteria or virus) in a biological sample. In some aspects, the capture probe or probes can be used to detect the expression of a specific nucleic acid associated with a pathogen (eg, the presence of 16S ribosomal RNA or human immunodeficiency virus (HIV) RNA in a human sample).
In some aspects, a capture domain in a capture probe can interact with one or more specific analytes (eg, an analyte or a subset of analytes in a total analyte library). The specific analyte to be detected can be any of a variety of biomolecules, including but not limited to proteins, nucleic acids, lipids, carbohydrates, ions, small molecules, subcellular targets, or multicomponent complexes containing any of the above. In some embodiments, analytes can be localized to subcellular locations including, for example, organelles such as mitochondria, Golgi apparatus, endoplasmic reticulum, chloroplasts, endocytic vesicles, exocytic vesicles, vacuoles, lysosomes, and the like. In some embodiments, analytes can be peptides or proteins, including but not limited to antibodies and enzymes.
In some aspects, an analyte from a biological sample interacts with one or more capture probes (eg, one or more capture probes immobilized directly or indirectly on a substrate), and the capture probes interact with specific analyte interactions. In some aspects, the capture probe is allowed to interact (eg, hybridize) with a particular analyte, e.g. under suitable conditions under which the oligonucleotide capture probe can hybridize to the target nucleic acid. In some aspects, analytes that do not hybridize to the capture probe (eg, analytes that do not interact with the capture domain of the capture probe) are removed. In some embodiments, removal of analytes that do not interact with capture probes can be accomplished, for example, by washing the sample to remove such analytes.
In some aspects, the capture probe or probes include a capture domain that interacts with one or more analytes present in a biological sample. In some aspects, the capture probe or probes include a capture domain that detects the presence or level (e.g., expression level) of a particular analyte or analytes of interest. The capture domains of capture probes (immobilized directly or indirectly on a substrate) are capable of selectively binding a desired subtype or subset of nucleic acids. In some aspects, for example, the capture domain binds a subset of nucleic acids in the genome or a subset of nucleic acids in the transcriptome. In some aspects, the analyte may include one or more nucleic acids. In some aspects, the probe or capture probes can be used to detect the expression of a specific transcript (eg, a specific mRNA). In some aspects, the capture probe or probes may be specific for (eg, bind to) a single change in a nucleic acid or protein (eg, a mutation or a single nucleotide polymorphism (SNP)).
In some aspects, the biological sample includes an analyte that is or includes a nucleic acid. Nucleic acid can be RNA or DNA. In some aspects, the capture probe or multiple capture probes detect the DNA copy number of a particular set of nucleic acid analytes. For example, a capture probe or a plurality of capture probes provided herein can be used to detect the copy number of DNA nucleic acids that share homology with one another.
In some aspects, the capture probe or probes include a capture domain that detects the presence or amount (eg, expression level) of one or more RNA transcripts (eg, a particular RNA transcript). In some aspects, the capture probe or probes comprise the detection of one or more non-coding RNAs (eg, microRNAs, transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), small interfering RNAs (siRNAs), and small interfering RNAs (siRNAs). Nucleolar RNA ( snoRNA).In some aspects, the probe or probes include a capture domain that detects the presence or level (eg, expression level) of one or more proteins (eg, expressed by the nucleic acid of interest).protein).
In some aspects, the probe or probes may be specific for a particular protein. In some aspects, a capture probe or multiple capture probes can be used to detect the presence of a particular protein of interest. In some aspects, the capture probe or probes can be used to detect the translation of a particular protein. In some aspects, the capture probe or probes can be combined with an active site of an enzyme, an immunoglobulin binding domain, a defined protein domain, an entire protein, a synthetic peptide, a mutated peptide, an aptamer, or any combination thereof. In some aspects, the analyte may include one or more proteins. In some aspects, the analyte may include one or more nucleic acids and one or more proteins.
In some aspects, the probe or capture probes can be used to detect a specific post-translational modification of a specific protein. In such embodiments, the analyte capture agent can detect a post-translational modification (eg, phosphorylation, glycosylation, ubiquitination, nitrosylation, methylation, acetylation, or lipidation) of a given state. ) that is specific for cell surface analytes such that the cell surface Analyte Profile may include information about post-translational modifications of one or more analytes.
In some aspects, the probe or probes may be specific for a particular set of nucleic acids (eg, nucleic acids associated with a particular cellular pathway). In some aspects, the pool of nucleic acids is DNA. In some aspects, the pool of nucleic acids is RNA. In some aspects, a set of nucleic acids have similar and/or homologous sequences. In some aspects, a pool of nucleic acids encodes analytes that function in similar cellular pathways. In some aspects, the set of nucleic acids encodes an analyte expressed in a particular pathological condition (eg, cancer, Alzheimer's disease, or Parkinson's disease). In some aspects, a set of nucleic acids encodes an analyte that is overexpressed in a particular pathological condition. In some aspects, the pool of nucleic acids encodes an analyte that is underexpressed in a particular pathological condition.
In some aspects, the capture probe or probes may be specific for the detection or expression of a particular nucleic acid or a particular set of proteins (eg, in a similar cellular pathway). In some aspects, this group of proteins has similar functional domains. In some aspects, this group of proteins functions in similar cellular pathways. In some aspects, a group of proteins is expressed in a particular pathological condition (eg, cancer, Alzheimer's disease, or Parkinson's disease). In some aspects, this group of proteins is overexpressed in certain pathological conditions. In some aspects, this group of proteins is underexpressed in certain pathological conditions.
In some embodiments, the capture probe includes a capture domain capable of binding more than one analyte. In some embodiments, the capture domains can bind one or more analytes that are about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, approximately 99% identical, 100% identical to the target analyte. In some aspects, capture probes can bind about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical, about 98% identical, or about 99% identical to each other . In some embodiments, the capture domain can bind a conserved region of one or more analytes, wherein the conserved regions are about 80% identical, about 85% identical, about 90% identical, about 95% identical, about 96% identical, about 97% identical , about 98% identical, about 99% identical, 100% identical to the target analyte.
In some aspects, the capture probe or probes interact with two or more analytes (eg, nucleic acids or proteins) that are not similar in sequence and/or do not share a conserved domain. In some embodiments, the capture probe includes two or more capture domains, wherein each capture domain interacts with a different analyte. In such embodiments, members of two or more capture domains may be adjacent to each other in a capture probe and/or members of two or more capture domains may be in a capture probe across one or more domains (eg, nucleic acid domains). For example, in some aspects, the detected set of analytes includes mutational changes in target nucleic acids or proteins. In some aspects, the capture probe or probes detect sets of nucleic acids or proteins (eg, non-homologous nucleic acids or proteins) that are individually mutated during a pathogenic condition. In some aspects, the pathogenic condition is cancer.
In some aspects, the capture probe or multiple capture probes include a capture domain that can be used to detect an analyte typically detected using a diagnostic panel. In some aspects, a capture probe or multiple capture probes are used to detect changes in one or more analytes. In some aspects, the alteration of the analyte comprises one or more of increased expression of the analyte, decreased expression of the analyte, mutated nucleic acid sequence, or any combination thereof. In some aspects, the change in the analyte correlates with and/or results in the manifestation of a pathogenic condition in the subject. In some aspects, the detected changes are compared to one or more reference analytes.
(vii) Peptide capture
Methods and materials for identifying the location of a polypeptide in a biological sample are provided herein. In some embodiments, analytes (eg, polypeptide analytes) can be captured directly on the matrix. For example, polypeptide analytes can be captured by amino groups on a functionalized substrate. In other examples, an analyte (eg, a polypeptide analyte) can be captured by an analyte-binding moiety directly attached to a substrate. In some embodiments, the substrate can be loaded with analyte moieties of interest attached directly to the substrate and spatially barcoded capture probes attached directly to the substrate. In other embodiments, an analyte (eg, a polypeptide analyte) can be captured by an analyte-binding moiety that is indirectly bound to a substrate. In one example, the substrate can be loaded with capture probes bound to an analyte capture agent, wherein the analyte capture domain of the analyte capture agent binds the capture domain of the capture probe and the analyte binding portion binds the polypeptide analyte.
In some embodiments, analytes (eg, polypeptide analytes) can be captured or immobilized directly on the matrix. Direct immobilization can be achieved by covalent coupling of the polypeptide analyte to the substrate via an amide bond between the carboxylic acid of the C-terminal amino acid residue and the surface of the functionalized substrate. For example, substrates such as glass coverslips or glass slides can be functionalized by aminosilylation with aminopropyltriethoxysilane. The surface of the substrate is further passivated by overnight incubation with polyethylene glycol (PEG)-NHS solution, and the functionalized slides can be stored in a vacuum desiccator until use. Incubate the substrate with 90% TFA (v/v in water) for 5 h before use to remove the tert-butoxycarbonyl protecting group, thus exposing free amine groups for peptide immobilization. The resulting functionalized substrates are stable to multiple cycles of Edman degradation and washing steps.
In some embodiments, the method for capturing a polypeptide in a biological sample includes administering a substrate, wherein the analyte binding moiety is immobilized directly on the substrate. In some embodiments, direct immobilization is achieved by chemical modification of the substrate and/or chemical modification of the binding moiety of the analyte. For example, substrates can be prepared with free amines on the surface. When exposed to an analyte-binding moiety with a free carboxylic acid at the C-terminal residue, the free amine can form an amide bond with the carboxylic acid, thereby covalently linking the analyte-binding moiety to the substrate. The substrate and/or analyte binding moiety can be modified in any manner that facilitates covalent binding of the analyte binding moiety to the substrate. Non-limiting examples of chemical modifications that can be used to covalently attach an analyte binding moiety to a substrate include those described herein.
In some embodiments, methods for capturing an analyte polypeptide include providing a substrate (eg, an array), wherein the analyte-binding moiety is indirectly linked to the substrate. For example, the analyte binding moiety can be indirectly linked to the substrate via an oligonucleotide (eg, a capture barcode domain or a capture barcode domain that hybridizes to a capture probe) or other domain that can bind both substrate and analyte. field. . The recording agent barcode domains are described elsewhere herein. The barcode domain of the capture agent can be modified to include a cleavage domain, which can be attached to the substrate using any of the chemistries described herein. In some embodiments, the barcode domain of the capture agent may comprise an analyte capture sequence as described herein, wherein the analyte capture sequence may hybridize to the capture domain of the capture probe. In some embodiments, a substrate (eg, an array) containing a capture probe can be modified to capture a polypeptide analyte by hybridizing the analyte capture sequence of an analyte capture agent to the capture domain of the capture probe.
In some embodiments, methods for capturing an analyte polypeptide include administering a substrate (eg, an array) and administering an analyte capture agent to a biological sample. For example, after drying and fixing the sectioned tissue samples, the tissue samples can be placed on a matrix (e.g., spatial array), rehydrated, blocked, and permeabilized (e.g., 3XSSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse inhibitor for 10 min at 4°C), followed by fluorescent primary antibody (1:100) and an analyte capture pool (in 3XSSC, 2% BSA, 0.1% Triton X, 1 U/μl RNAse) staining inhibitor at 4° C for 30 min). Biological samples can be washed, coverslipped (in glycerol + 1 U/µl RNAse inhibitor), imaged for detected analytes (eg, using a confocal microscope or other device capable of detecting fluorescence), and washed again. Analyte capture agents bound to the analyte can be released from the biological sample (eg, the biological sample can be treated with a protease, such as proteinase K) and migrate into the spatial array. The analyte capture sequence of the analyte-bound analyte capture agent can be captured by the capture domain of the capture probe, and the barcode domain of the capture agent can be extended to generate a spatially tagged analyte capture agent. Spatially labeled analyte capture agents can be processed according to the spatial workflow described here.
In some embodiments, the method for capturing an analyte polypeptide comprises providing a blocking probe to an analyte capture agent prior to introducing the analyte capture agent into a biological sample. In some embodiments, blocking probes can alternatively or additionally be provided in any of the rehydration or blocking buffers provided herein. In some embodiments, the analyte capture sequence of the analyte capture agent can be blocked using a blocking probe sequence complementary to the analyte capture sequence prior to binding to the capture domain of the capture probe. Blocking the barcode capture domain, particularly the free 3' end of the barcode capture domain (eg, the analyte capture sequence), prior to contacting the analyte capture agent with the biological sample and/or substrate prevents analyte capture. A sequence that binds the barcode domain of the capture agent, eg, prevents poly(A) tails from binding to the capture domain of the capture probe. In some embodiments, blocking the analyte capture domain of the analyte capture agent reduces nonspecific background staining. In some embodiments, the blocking probe is reversible such that the blocking probe can be removed from the analyte capture sequence during or after contacting the analyte capture agent with the biological sample. In some embodiments, blocking probes can be removed by RNAse treatment (eg, RNAse H treatment).
In some embodiments, methods for capturing polypeptides in biological samples include active transfer (eg, electrophoresis). For example, a biological sample is placed on a conductive substrate and contacted with a spatial array containing one or more analyte binding units. As described herein, an electric field can be applied to a conductive substrate to facilitate migration of the polypeptide toward the analyte-binding moiety.
In some embodiments, the method for identifying the spatial location of a polypeptide in a biological sample includes determining the sequence of the captured polypeptide. In some embodiments, the sequence of the captured polypeptide is determined by detecting amino acid residues labeled with a detectable label (eg, radioactive labeling with a fluorophore). Non-limiting examples of detectable labels that can be used to label captured polypeptides include fluorophores and radioactive labels. In some embodiments, the polypeptides are labeled only at specific amino acid residues (eg, not all amino acid residues are labeled). In some embodiments, the polypeptide is labeled prior to contacting the biological sample with the substrate. In some embodiments, the captured polypeptides are labeled with fluorophores using standard conjugation protocols (see Hernandez et al.,New Journal of Chemistry41:462-469 (2017)). For example, peptides can be labeled by reaction with Atto647N-NHS, Atto647Niodoacetamide, TMR-NHS, or JF549-NHS, to label lysine (via NHS) or cysteine (via iodoacetamide), as the case may be. Alternatively, serine or threonine phosphorylation sites can be selectively labeled by β-elimination, followed by the addition of thiol conjugates to substitute thiol-linked fluorophores for phosphates (see Stevens et al.Quick newsletter. mass spectrometry., 15: 2157-2162 (2005). The number of fluorophores incorporated into the polypeptide is any number that can be spectrally resolved. In some cases, four or more fluorophores are used.
In some embodiments, the captured polypeptide is radiolabeled. In some embodiments, specific amino acids can be isotopically labeled. Non-limiting examples of isotopes used to label amino acids include3H,14C,15,32i125I. In some embodiments, isotopes are incorporated into selected amino acids prior to incorporation into the polypeptide. In some embodiments, radiolabeled amino acids can be incorporated into the polypeptide after its formation.
In some embodiments, the sequence of the captured polypeptide is determined using Edman digestion (and in some embodiments, successive rounds of Edman digestion). In this case, the peptide is captured, and the peptide sequence can be resolved by imaging the substrate after repeated rounds of Edman digestion. For example, the substrate is imaged after each Edman reaction to capture detectable labels due to the removal of amino acids as by-products of the reaction. Information obtained from Edman degradation can be used for peptide identification. In some embodiments, the biological sample is visualized or imaged using optical or fluorescence microscopy.
(viii) Enrichment of captured analytes after capture
In some aspects, the spatial analysis of the target analyte includes an enrichment step or a post-capture step to enrich the captured analyte for the target analyte. For example, capture domains can be selected or designed to selectively capture more analytes than the practitioner wishes to analyze. In some embodiments, capture probes containing random sequences (eg, random hexamers or similar sequences) that form all or part of a capture domain can be used to capture nucleic acids from biological samples in an unbiased manner. For example, capture probes with capture domains that include random sequences can often be used to capture DNA, RNA, or both from biological samples. Alternatively, the capture probe may include a capture domain that normally captures mRNA. This may be based on hybridization to the poly-A tail of the mRNA, as is well known in the art. In some embodiments, the capture domain includes a sequence that interacts (eg, hybridizes) with the polyA tail of the mRNA. Non-limiting examples of such sequences include poly-T DNA sequences and poly-U RNA sequences. In some embodiments, random sequences can be used in combination with poly-T (or poly-T analogs, etc.) sequences. Thus, when a capture domain includes poly-T (or "poly-T-like") oligonucleotides, it may also include random oligonucleotide sequences.
In some aspects, after capturing more analytes than the practitioner wishes to analyze, the methods disclosed herein include enrichment for a particular captured analyte. In some aspects, the methods include enrichment of analytes including mutations of interest (eg, SNPs), nucleic acids of interest, and/or proteins of interest.
In some embodiments, the spatial analysis methods provided herein include the selective enrichment of one or more analytes of interest (e.g., target analytes) following analyte capture. For example, one or more analytes of interest can be enriched by adding one or more oligonucleotides to the collected pool of analytes. In some embodiments, one or more analytes of interest can be enriched by adding one or more oligonucleotides to the pool of analytes covered by the array. In some embodiments, one or more sets of sensor analytes can be enriched by adding one or more oligonucleotides to the analyte pool that has released (eg, removed) the analyte pool from the analyte array of interest. In some embodiments, one or more nucleotides may be complementary to a portion or portions thereof of the TSO and R1 sequences when the captured analyte is released from the array. In some embodiments, additional oligonucleotides include sequences for initiating polymerase reactions. For example, one or more primer sequences having sequence complementarity to one or more analytes of interest can be used to amplify one or more analytes of interest, thereby selectively enriching those analytes. In some embodiments, one or more primer sequences may be complementary to other domains on the capture probe (eg, the R1 sequence or a portion thereof as described above), but not to the analyte. In some embodiments, using a first primer that is complementary to one or more analytes of interest (or the capture probe and another domain in the TSO) or its complement, and the capture probe region or its complement. In some embodiments, the region of the capture probe or its complement is remote from the spatial barcode from the capture domain such that amplification enrichment amplifies the captured analyte(s) and their associated spatial barcode, allowing spatial analysis of the Enriched Analyte or Analytes.
In some embodiments, two or more capture probes capture two or more different analytes that are enriched (eg, simultaneously or sequentially) from a pool of captured analytes. In some embodiments, enrichment by PCR amplification involves multiple rounds of amplification. For example, enrichment by PCR amplification may involve nested PCR reactions using different primers specific for one or more analytes of interest. In some embodiments, non-PCR amplification methods can be used to enrich the amplification. A non-limiting example of a non-PCR amplification method is rolling circle amplification. Other non-PCR amplification methods are known in the art.
In some embodiments, oligonucleotides having sequence complementarity to the captured one or more analytes of interest or their complements can be used to enrich the captured one or more analytes from a captured analyte library. In some embodiments, oligonucleotides having sequence complementarity to the captured analyte or analyte of interest (or other domain of the captured probe) or its complement may include one or more functional moieties. For example, biotinylated oligonucleotides having a sequence complementary to one or more analytes of interest or their complements can bind analytes of interest and can be used in various ways known in the art. Or use biotinylation affinity to streptavidin for selection (eg, streptavidin beads). In some embodiments, oligonucleotides having a sequence complementary to one or more analytes of interest, or their complements, include magnetic moieties (eg, magnetic beads) that can be used in an enrichment process.
Additionally or alternatively, any of a number of methods can be used to select (eg, remove) one or more analytes (eg, mitochondrial DNA or RNA). In some embodiments, this low selection of analytes not of interest may result in improved capture of other types of analytes of interest. For example, probes can be applied to samples that selectively hybridize to ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. In some embodiments, this down-selection may result in improved capture of other types of RNA due to the reduction of non-specific RNA present in the sample. Additionally or alternatively, double-strand specific nuclease (DSN) treatment can deplete rRNA (see, e.g., Archer et al., Selective and Flexible Depletion of Problematic Sequences from a cDNA-Stage RNA-seq Library,BMC Genomics, 15 401, (2014), which is hereby incorporated by reference in its entirety). In some embodiments, hydroxyapatite chromatography can be used to remove abundant species (eg, rRNA).
(ix) Ligation of the RNA template
In some embodiments of the methods provided herein, ligation with an RNA sample is used to examine spatial gene expression in a biological sample (eg, FFPE tissue section). Binding to an RNA sample allows sensitive measurement of specific nucleic acid analytes of interest that might otherwise fail less sensitive analysis with intact transcriptomic approaches. It has the advantage of being compatible with common histochemical staining and is suitable for the analysis of decades-old material (eg FFPE samples) and very small microscopic tissue fragments.
In some aspects, the steps of ligation of the RNA template include: (1) hybridizing pairs of probes (eg, DNA probes) to RNA (eg, formalin-fixed RNA) within tissue sections; (2) in situ ligation adjacent annealing of pairs of probes; (3) RNase H treatment to (i) release RNA-template ligation products from the tissue (eg, into solution) for downstream analysis, and (ii) destroy unwanted DNA-template ligation products; and optionally, (4) amplification of the RNA-template ligation product (eg, multiplex PCR).
In some aspects, disclosed herein are methods for direct detection of duplex RNA meta-DNA probes without first converting the RNA to cDNA by reverse transcription. In some aspects, the ligation with the RNA sample may involve a DNA ligase. In some aspects, ligation with the RNA sample may involve RNA ligase. In some aspects, ligation with the RNA sample may involve T4 RNA ligase.
In certain aspects, ligation of an RNA template is used to detect RNA, determine RNA sequence identity, and/or monitor expression and analyze transcripts. In certain aspects, ligation of an RNA template enables the detection of specific changes in nucleic acids (eg, mutations or single nucleotide polymorphisms (SNPs)), the detection or expression of specific nucleic acids, or the detection or expression of specific groups of nucleic acids (eg, in similar cellular pathways or in specific pathologies). In some embodiments, methods involving ligation of RNA templates are used to analyze nucleic acids, e.g. genotyping, quantification of the number of copies of DNA or RNA transcripts, localization of specific transcripts within the sample and the like. In some aspects, the systems and methods provided herein that involve ligation with an RNA sample identify single nucleotide polymorphisms (SNPs). In some aspects, such systems and methods identify mutations.
In some aspects, methods of detecting RNA expression are disclosed herein that include contacting a first probe, a second probe, and a ligase (e.g., T4 RNA ligase). In some embodiments, the first probe and the second probe are designed to hybridize to a target sequence such that the 5' end of the first probe and the 3' end of the second probe are contiguous and can be ligated, wherein at least the 5'-terminal nucleotide of the first probe and at least the 3' -terminal nucleotide of the second probe is deoxyribonucleotides (DNA), and wherein the target sequence comprises (eg consists of) ribonucleosidic acid (RNA). After hybridization, a ligase (such as T4 RNA ligase) ligates the first probe to the second probe if the target sequence is present in the target sample, but not if the target sequence is not present in the target sample. The presence or absence of a target sequence in a biological sample can be determined by determining whether the first and second probes are ligated in the presence of ligase. Any of a number of methods can be used to determine whether the first and second probes have been ligated in the presence of ligase, including, but not limited to, sequencing of the ligated products, hybridization of the ligated products to the detection probe that the first and second probes are ligated only in presence of ligase, restriction enzyme analysis and other methods known in the art.
In certain aspects, two or more RNA analytes are analyzed using a method that involves ligation of an RNA template. In some aspects, when two or more analytes are analyzed, first and second probes are used that are specific for (eg, specifically hybridize to) each RNA analyte.
In some aspects, three or more probes are used in the RNA template ligation methods provided herein. In some embodiments, three or more probes are designed to hybridize to the target sequence such that the three or more probes hybridize to each other such that the 5' and 3' ends of adjacent probes can be ligated. In some embodiments, the presence or absence of a target sequence in a biological sample can be determined by determining whether three or more probes are ligated in the presence of a ligase.
In some aspects, the first probe is a DNA probe. In some aspects, the first probe is a chimeric DNA/RNA probe. In some aspects, the second probe is a DNA probe. In some aspects, the second probe is a chimeric DNA/RNA probe.
In certain aspects, RNA-template ligation methods use T4 RNA ligase 2 to efficiently ligate adjacent chimeric RNA-DNA probe pairs that hybridize in situ to immobilized RNA target sequences. Subsequent treatment with RNase H releases the ligation products of the template RNA (eg into solution) for further analysis.
(x) Region of interest
A biological sample may have regions with morphological features that may indicate the presence of a disease or the development of a disease phenotype. For example, the morphologic features of a particular site within a tumor biopsy specimen may be indicative of the aggressiveness, resistance to treatment, metastatic potential, migration, stage, diagnosis, and/or prognosis of the subject's cancer. Changes in site-specific morphologic features in tumor biopsy specimens often correlate with changes in the levels or expression of site-specific intracellular analytes, which in turn can be used to provide information on invasiveness, treatment resistance, metastatic potential, migration, staging, diagnosis, and /or or prognosis of the subject's cancer. A region or region of a biological sample that is selected for a particular analysis (eg, a region of a biological sample with a morphological feature of interest) is often described as a "region of interest".
Regions of interest in biological samples can be used to analyze specific regions of interest in biological samples, thereby targeting experiments and data collection to specific regions of biological samples (rather than the entire biological sample). This results in increased time efficiency of biological sample analysis.
Regions of interest can be identified in biological samples using a number of different techniques, such as expansion microscopy, bright field microscopy, dark field microscopy, phase contrast microscopy, electron microscopy, fluorescence microscopy, reflectance microscopy, interference microscopy, confocal microscopy and visual microscopy. recognition (eg by eye) and their combinations. For example, staining and imaging of biological samples can be performed to identify regions of interest. In some examples, the region of interest may correspond to a particular structure of the cellular structure. In some embodiments, the biological sample can be stained prior to visualization to provide contrast between different regions of the biological sample. The type of staining can be selected according to the type of biological sample and the cellular area to be stained. In some embodiments, more than one dye can be used to visualize different aspects of a biological sample, eg, different regions of the sample, specific cellular structures (eg, organelles), or different cell types. In other embodiments, the biological sample can be visualized or displayed without staining the biological sample.
In some embodiments, staining and imaging of the biological sample is performed prior to contacting the biological sample with the spatial array to select samples for spatial analysis. In some embodiments, the staining comprises the application of a fiducial marker as described herein, including a fluorescent, radioactive, chemiluminescent, or colorimetrically detectable marker. In some embodiments, staining and imaging the biological samples allows the user to identify a particular sample (or region of interest) that the user wishes to evaluate.
In some examples, an array (eg, any of the example arrays described herein) may be in contact with only a portion of a biological sample (eg, a cell, tissue portion, or region of interest). In some examples, the biological sample contacts only a portion of the array (eg, any example array described herein). In some embodiments, capture probes on an array corresponding to a region of interest (eg, near a region of interest) of a biological sample can be selectively cleaved and analyzed. For example, capture probes on an array can be deactivated or eliminated outside the regions corresponding to the regions of interest of the biological sample. In some embodiments, capture probes that contain photocleavable linkages and in regions on the array that correspond to regions of interest in the biological sample can be selectively cleaved using light. Mirrors, mirror arrays, lenses, moving stages, and/or photomasks can be used to direct light to areas of the array that correspond to regions of the biological sample outside of one or more regions of interest. Some embodiments include the use of light to inactivate or remove capture probes, e.g., capture probes containing a photocleavable bond as described herein. In some embodiments, a laser, such as a scanning laser, can be used to deactivate or remove capture probes. In some embodiments, the eliminated members of the plurality of capture probes can be washed away. In some embodiments, the regions of interest can be labeled with different heavy metals, and the laser can sequentially ablate these regions of interest prior to mass spectrometric identification. For example, lasers can deactivate or eliminate capture probes by destroying DNA with UV light, heating, inducing chemical reactions that prevent capture probes from proceeding to the next step, causing photocleavage of photocleavable bonds, or a combination thereof. In some examples, a portion of the array may be inactivated so that it does not interact with the analyte in the biological sample (eg, optically inactivated, chemically inactivated, heat inactivated, or blocked capture probes in the array (eg, using probe blocking)). In some embodiments, capture probes can be blocked (eg, masked or modified) prior to contacting the biological sample with the array. For example, the free 3' ends of the capture probes can be blocked or modified prior to contacting the biological sample with the array to avoid modification of the capture probes (eg, avoid removal or modification of the free 3' OH groups at the ends of the capture probes). In some embodiments, capture probes can be blocked prior to contact of the biological sample with the array. In some embodiments, a blocking probe is used to block or modify the free 3' end of the capture domain of the capture probe. In some embodiments, the blocking probe can be hybridized to a capture probe. In some embodiments, the free 3' end of the capture domain can be blocked by chemical modification.
In some examples, a region of interest can be removed from a biological sample, and the region of interest can then be contacted with an array (eg, any array described herein). Regions of interest can be removed from biological samples using microsurgery, laser microdissection, blocking, microtome, cutting, trypsinization, fluorescence-assisted labeling and/or cell sorting, among others. In some embodiments, the biological sample is dissected using laser microdissection, one or more biological samples are retained for analysis, and/or one or more biological samples are discarded. In some embodiments, biological samples are dissected on an array. In some embodiments, one or more regions of interest are selected using an array of spatially addressable microelectrodes.
In some examples, the region of interest may be permeabilized or lysed, while regions outside the region of interest are not permeabilized or lysed (eg, Kashyap et al.representative of science2016;6:29579, which is incorporated herein by reference in its entirety). For example, in some embodiments, the region of interest can be contacted with a hydrogel containing a permeabilization or lysis reagent. In some embodiments, regions other than regions of interest are not in contact with the hydrogel containing permeabilization or lysis reagents. In some embodiments, eliminated members of the plurality of capture probes are washed away after permeabilization of the biological sample.
(f) bulkhead
As noted above, in some embodiments, the sample can optionally be separated into individual cells, populations of cells, or other fragments/fragments smaller than the original, continuous sample. Each of these smaller portions of the sample can be analyzed to obtain spatially resolved information about the sample analyte.
For samples that are separated into smaller fragments—especially samples that are broken, dissociated, or otherwise separated into individual cells—one method of fragment analysis involves separating the fragments into individual partitions (eg, droplets) and then analyzing the contents of the partition. Typically, each partition separates its own contents from the contents of other partitions. For example, a barrier can be a droplet in an emulsion.
The baffles can flow in the fluid stream. The baffle may comprise, for example, a microbubble with an outer barrier surrounding an inner fluidic center or core. In some cases, the separator may include a porous matrix capable of entraining and/or retaining material within its matrix. The partition may be a droplet of the first phase within the second phase, whereby the first and second phases do not mix. For example, the partition may be a droplet of an aqueous liquid within a non-aqueous continuous phase (eg, an oil phase). In another example, the partition may be a droplet of a non-aqueous liquid within an aqueous phase. In some examples, the distribution can be provided in a water-in-oil emulsion or an oil-in-water emulsion. A variety of different containers are described, for example, in US Patent No. Application Publication No. 2014/0155295, the entire contents of which are incorporated herein by reference. Emulsion systems for generating stable droplets in nonaqueous or oily continuous phases are described, for example, in U.S. Pat. Patent No. Application Publication No. 2010/0105112, the entire contents of which are incorporated herein by reference.
For droplets in emulsions, individual particles can be divided into discrete compartments, for example, by introducing a stream of particles in an aqueous fluid into a stream of nonaqueous fluid so that the droplets are in both streams. Fluid properties (e.g., fluid flow rate, fluid viscosity, etc.), particle properties (e.g., volume fraction, particle volume, particle concentration, etc.), microfluidic structure (e.g., channel geometry, etc.) and other parameters can be adjusted to control the resulting occupancy of partitions (eg number of analytes per partition, number of beads per partition, etc.). For example, zone occupancy can be controlled by providing a flow of water of a specific concentration and/or analyte flow rate.
To create individual analyte partitions, the relative flow rates of the immiscible liquids can be chosen such that, on average, each compartment can contain less than one analyte to ensure that these occupied compartments are predominantly self-occupied. In some cases, a partition of the plurality of partitions may contain at most one analyte. In some embodiments, various parameters (eg, fluid properties, particle properties, microfluidic structure, etc.) can be selected or adjusted so that most partitions are occupied, eg, only a small percentage of unoccupied partitions is allowed. The flow and channel architecture can be controlled to provide a certain number of singly occupied partitions, less than a certain level of unoccupied partitions, and/or less than a certain level of multiply occupied partitions.
The channel segments described herein may be connected to any of a number of different fluid sources or receivers, including reservoirs, pipes, manifolds, or other fluid components of the system. It should be kept in mind that microfluidic channel structures can have different geometries. For example, a microfluidic channel structure may have one or more channel junctions. As another example, microfluidic channel structures may have 2, 3, 4, or 5 channel segments, each carrying particles that meet at channel junctions. Fluid may be directed to flow along one or more channels or reservoirs via one or more fluid flow cells. A fluid flow unit may include a compressor (eg to provide positive pressure), a pump (eg to provide negative pressure), an actuator, etc. to control fluid flow. Fluids can also or otherwise be controlled by applied pressure differentials, centrifugal force, electrical pumping, vacuum, capillary and/or gravity flow.
In addition to cells and/or analytes, the compartments may also contain additional components, in particular one or more beads. The compartments may include single gel beads, single cell beads, or single cell beads and single gel beads. Various beads can be installed in the partitions. In some embodiments, for example, non-barcoded beads may be incorporated into the compartments. For example, the beads may be non-barcoded beads when the biological particles (eg, cells) into which the compartments are embedded carry one or more barcodes (eg, spatial barcodes, UMIs, and combinations thereof).
In some embodiments, balls bearing bar codes can be incorporated into the compartments. In general, a single bead can be coupled to any number of individual nucleic acid molecules, eg, from one to tens of thousands to hundreds of thousands or even millions of individual nucleic acid molecules. Appropriate barcodes for individual nucleic acid molecules may include common sequence segments or relatively common sequence segments as well as variable or unique sequence segments between different individual nucleic acid molecules attached to the same bead. For example, nucleic acid molecules (eg, oligonucleotides) can be attached to the beads via releasable bonds (eg, disulfide bonds), wherein the nucleic acid molecules can be or include barcodes. For example, barcodes can be injected into the droplets before, after, or simultaneously with the formation of the droplets. The transfer of barcodes to specific partitions enables later attribution of the characteristics of individual bioparticles to specific partitions. Barcodes can be delivered to partitions, eg, nucleic acid molecules (eg, oligonucleotides), by any suitable mechanism. Barcoded nucleic acid molecules can be delivered to compartments via microcapsules. In some cases, the microcapsules may contain beads. The same bead can be linked (eg, via a releasable bond) to one or more other nucleic acid molecules.
In some embodiments, microcapillary arrays with spatially labeled beads can be generated. Multiple spatially barcoded beads can flow into the channels on the microcapillary array so that each microcapillary channel can be filled with a single spatially barcoded bead. In some embodiments, the spatially barcoded microcapillary bead array can be contacted with a biological sample for subsequent spatial analysis of biological analytes within the biological sample. In some embodiments, the channels of the microcapillary array can mechanically compress biological samples to form fluid-isolated reaction chambers. In some embodiments, reagents (eg, enzymes, nucleic acids) are introduced into the reaction chamber. Reagents can be sealed (eg, silicone oil, mineral oil) within the reaction chamber and incubated to allow the cell and/or nuclear permeabilization reaction. In some embodiments, biological analytes (eg, DNA, RNA, proteins, metabolites, small molecules, and lipids) are released and captured onto spatially barcoded microcapillary arrays, thereby preserving their spatial information. In some embodiments, spatial analysis using spatially barcoded microcapillary arrays can be used to obtain spatial information of analytes of a biological sample at single cell resolution.
Nucleic acid molecules can include functional domains that can be used in post-processing. For example, a functional domain may include one or more sequencing-specific flow cell attachment sequences (eg, a P5 sequence for the Illumina® sequencing system) and a sequencing primer sequence (eg, an R1 primer for the Illumina® sequencing system). A nucleic acid molecule may contain a barcode sequence (eg, DNA, RNA, protein, etc.) to barcode the sample. In some cases, the barcode sequence may be bead-specific such that the barcode sequence is common to all nucleic acid molecules attached to the same bead. Alternatively or additionally, the barcode sequence may be partition specific such that the barcode sequence is common to all nucleic acid molecules associated with one or more beads partitioned into the same partition. A nucleic acid molecule may include a specific primer, such as an mRNA-specific primer (eg, a poly(T) sequence), a targeted primer, and/or a random primer. A nucleic acid molecule may include an anchor sequence to ensure that a specific primer sequence hybridizes to the end of the sequence (eg, mRNA). For example, anchor sequences can include random short nucleotide sequences such as 1-mer, 2-mer, 3-mer or longer sequences, which can ensure that poly(T) fragments are more likely to be in poly(A) mRNA end sequence.
A nucleic acid molecule can include a unique molecular recognition sequence (eg, a unique molecular identifier (UMI)). In some embodiments, a unique molecular recognition sequence may comprise about 5 to about 8 nucleotides. Alternatively, the unique molecular recognition sequence may comprise less than about 5 or more than about 8 nucleotides. A unique molecular recognition sequence may be a unique sequence that varies between individual nucleic acid molecules coupled to a single bead. In some embodiments, the unique molecular recognition sequence can be a random sequence (eg, such as a random N-mer sequence). For example, UMI can provide a unique identifier for captured starting mRNA molecules to allow quantification of the amount of raw expressed RNA.
The partition may also include one or more reagents. Unique identifiers, such as barcodes, can be introduced into the droplets before, after or simultaneously with the formation of the droplets, for example via microcapsules (eg beads). Networks of microfluidic channels (eg on-chip) can be used to generate partitions. Alternative mechanisms can also be used to separate individual biological particles, including porous membranes through which aqueous mixtures of cells are forced into non-aqueous fluids.
In some embodiments, the barcoded nucleic acid molecules can be initially associated with microcapsules and then released from the microcapsules. The release of barcoded nucleic acid molecules can be passive (eg, by diffusion from microcapsules). Additionally or alternatively, release from the microcapsules can be performed after administration of a stimulus that allows the barcoded nucleic acid molecule to be detached or released from the microcapsules. This stimulation can disrupt the microcapsules, the interaction that binds the nucleic acid molecules to the barcode on the microcapsules or within the microcapsules, or both. Such stimuli may include, for example, thermal stimuli, optical stimuli, chemical stimuli (eg changes in pH or use of reducing agents), mechanical stimuli, radiation stimuli; biological stimuli (eg enzymes) or any combination thereof.
In some embodiments, one or more barcodes (eg, spatial barcodes, UMIs, or combinations thereof) may be introduced into the compartments as part of the analyte. As previously mentioned, the barcode may be directly bound to the analyte or may form part of a capture probe or analyte capture means that is hybridized, conjugated, or otherwise bound to the analyte so that when the analyte is introduced into the compartment, it is introduced and bar code. As stated above,
another liquid1710does not mix with aqueous liquids1707(eg oil) can be delivered to the connection point1706from each channel segment1703i1704.When encountering water-based liquids1707from each channel segment1701i1702and the other liquid1710from each channel segment1703i1704at the passage junction1706, Aqueous solution1707It can be segmented into discrete droplets1711in another liquid1710and flow away from the joint1706Along the waterway1705.Part of the channel1705Discrete droplets can be delivered to an outlet container fluidly connected to the channel segment1705, where it can be harvested.
Alternatively, a channel segment1701i1702It can be met at another intersection upstream from the intersection1706.At such a junction, balls and bioparticles can form a mixture that is led along another channel to the junction1706they produce droplets1711The mixture can give balls and bioparticles alternately, so that, for example, a droplet contains one ball and one bioparticle.
another liquid1710It may include oils, such as fluorinated oils, which include fluorosurfactants to stabilize the formed droplets, eg to inhibit subsequent coalescence of the formed droplets1711.
The partitions described herein may include small volumes, such as less than about 10 microliters (μL), 5 μL, 1 μL, 900 picoliters (pL), 800 pL, 700 pL, 600 pL, 500 pL, 400 pL, 300 pL, 200 pl, 100 pl, 50 pl, 20 pl, 10 pl, 1 pl, 500 nanoliters (nL), 100 nL, 50 nL or less.
In the previous discussion, globular droplets were formed at the interface of different fluid streams. In some embodiments, the droplets may be formed by gravity-based spray methods.
watery liquid1760Contains more particles1756It can be transported along parts of the channelin 1752enter the intersectionin 1758encounter another liquid1762It does not mix with aqueous liquids (eg oils, etc.)1760in the tankin 1754they form water droplets1764watery liquid1760into the tankin 1754.at the crossroadsin 1758of which a watery liquid1760and the other liquid1762When they meet, droplets form according to factors such as hydrodynamic forces at the interfacein 1758, the relative velocity of the two fluids1760,1762, fluid properties and certain geometric parameters of the channel structure (eg Δh, etc.)in 1750.More drops can be collected in the containerin 1754by continuous injection of aqueous liquid1760Segment by channelin 1752at the crossroadsin 1758.
The resulting discrete droplets may contain one or more of a plurality of particles1756As described elsewhere herein, the particle can be any particle, such as a bead, cell bead, gel bead, bioparticle, macromolecular component of a bioparticle, or other particle. Alternatively, the resulting discrete droplets may not contain any particles.
In some cases, aqueous liquids1760It may have a generally uniform concentration or frequency of particles1756.As described elsewhere in this paper, particles1756(eg balls) can be introduced into channel segmentsin 1752from a separate channel (not shown in
In some cases, another liquid1762It must not be subject to and/or direct flow into or out of the tankin 1754For example, another liquid1762It can be basically stationary in the tankin 1754.In some cases another liquid1762the flow inside the reservoir can be affectedin 1754, but not in and out of the tankin 1754, for example by applying pressure to the reservoirin 1754and/or affected by the inflow of aqueous fluids1760at the crossroadsin 1758.or, another liquid1762can withstand and/or direct flow into or out of the reservoirin 1754.for example, a tankin 1754it may be a channel leading to another fluid1762The resulting droplets are transferred from upstream to downstream.
channel structurein 1750at or near the junctionin 1758they may have certain geometric features that determine, at least in part, the volume and/or shape of the droplets formed by the channel structurein 1750.Part of the channelin 1752can have a first cross-sectional height,H1, and the reservoirin 1754It can have a different cross-sectional height,H2.the first section height,H1, and the second section height,H2, can be different, such that at the junctionin 1758, with a height difference of Δh. the height of the second part,H2, can be greater than the first height of the cross section,H1.In some cases, the reservoir may then gradually increase in cross-sectional height, for example, the further away from the junctionin 1758.In some cases, the cross-sectional height of the container can be increased according to the expansion angle β at or near the connectionin 1758.The height difference Δh and/or the angle of expansion β allow the tongue (part of the aqueous humor1760leave the channel segmentin 1752at the crossroadsin 1758and into the reservoirin 1754before droplet formation) to increase the depth and facilitate the reduction of the curvature of the droplets formed in the middle. For example, droplet volume may decrease with increasing height difference and/or increasing spreading angle.
The height difference Δh can be at least about 1 μm. Alternatively, the height difference may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 microns or more. Alternatively, the height difference can be up to about 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15 , 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 µm or less. In some cases, the expansion angle β may range between about 0.5° to about 4°, about 0.1° to about 10°, or about 0° to about 90°. For example, the spreading angle may be at least about 0.01°, 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°, 0 ,8°, 0.9°, 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, 10°, 15°, 20°, 25°, 30° , 35°, 40°, 45°, 50°, 55°, 60° , 65° , 70°, 75°, 80°, 85° or more. In some cases, the spread angle can be up to about 89°, 88°, 87°, 86°, 85°, 84°, 83°, 82°, 81°, 80°, 75°, 70°, 65°, 60 °, 55°, 50°, 45°, 40°, 35°, 30°, 25°, 20°, 15°, 10°, 9°, 8°, 7°, 6°, 5°, 4°, 3°, 2°, 1°, 0.1°, 0.01° or less.
In some cases, the flow rate of the aqueous fluid1760enter the intersectionin 1758It can be between about 0.04 microliters (μL)/minute (min) and about 40 μL/min. In some cases, the flow rate of the aqueous fluid1760enter the intersectionin 1758It can be between about 0.01 microliters (μL) per minute (min) and about 100 μL/min. Alternatively, the flow rate of the aqueous fluid1760enter the intersectionin 1758It can be less than about 0.01 µL/min. Alternatively, the flow rate of the aqueous fluid1760enter the intersectionin 1758It can be greater than about 40 µL/min, such as 45 µL/min, 50 µL/min, 55 µL/min, 60 µL/min, 65 µL/min, 70 µL/min, 75 µL/min, 80 µL / min, 85 µL/min, 90 µL/min, 95 µL/min, 100 µL/min, 110 µL/min, 120 µL/min, 130 µL/min, 140 µL/min, 150 µL/min or more. At lower flow rates, such as approximately less than or equal to 10 µl/min, the droplet radius may not depend on the flow rate of the aqueous liquid1760enter the intersectionin 1758.another liquid1762It can be dormant or mostly dormant in the tankin 1754.or, another liquid1762It can flow, eg, at the flow rates described above for aqueous liquids1760.
although
It should be noted that while the example workflow is in progress
For example, in the case of analyzing an RNA sample such as
Within any given partition, all cDNA transcripts for a single mRNA molecule may contain a common barcode sequence segment. However, transcripts made from different mRNA molecules within a given partition may vary in segments of a unique molecular recognition sequence, such as UMI segments. Usefully, even after any subsequent amplification of the contents of a given partition, the number of distinct UMIs can indicate the amount of mRNA originating from a given partition. Transcripts can be amplified, purified and sequenced to identify the sequence of cDNA transcripts for mRNA, as well as sequence barcoded fragments and UMI fragments, as described above. Although poly(T) primers are described, other targeted or random primers can also be used to initiate the reverse transcription reaction. Likewise, although described as the release of barcoded oligonucleotides into compartments, in some cases bead-bound nucleic acid molecules can be used to hybridize and capture mRNA on the solid phase of the bead, for example, to facilitate binding of RNA to other cellular contents are separated .
In some embodiments, the barriers include precursors that contain functional groups that are reactive or can be activated to become reactive and can be polymerized with other precursors to produce activated or activatable functionalized Gel beads. This functional group can then be used to attach other substances (eg, disulfide bonds, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors with carboxylic acid (COOH) groups can be copolymerized with other precursors to form beads that also contain COOH functional groups. In some cases, acrylic acid (a substance containing free COOH groups), acrylamide, and bis(acryloyl)cystamine can be copolymerized together to produce beads with free COOH groups. The COOH groups of the beads can be activated (for example, with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or 4-(4,6-dimethoxy-1,3,5-triazine -2-yl)-4-methylmorpholine chloride (DMMTMM)), making them reactive (eg, reactive towards amine functions when activated with EDC/NHS or DMTMM). The activated COOH groups can then be reacted with a suitable material containing a bead-binding moiety (eg, a material containing an amine functional group wherein the carboxylic acid group is activated to react with the amine functional group).
In some embodiments, the beads can be formed from materials that include degradable chemical cross-linkers such as BAC or cystamine. Degradation of such degradable cross-linkers can be achieved by different mechanisms. In some examples, the beads may come into contact with chemical degradants that may cause oxidation, reduction, or other chemical changes. For example, the chemical degradation agent may be a reducing agent such as dithiothreitol (DTT). Other examples of reducing agents may include beta-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof. The reducing agent can break down the disulfide bonds formed between the bead-forming gel precursors and thus break down the beads.
Degradable beads may include one or more substances with labile bonds such that when the bead/substance is exposed to an appropriate stimulus, the bonds break and the beads degrade within the compartments. A labile bond can be a chemical bond (eg covalent bond, ionic bond) or it can be another type of physical interaction (eg van der Waals interaction, dipole-dipole interaction, etc.). In some embodiments, the cross-linking agents used to form the beads may include labile linkages. When exposed to the right conditions, the unstable bonds break and the grains degrade. For example, polyacrylamide beads with cystamine disulfide attached to barcode sequences can bind to reducing agents in water-in-oil emulsion droplets. Within the droplet, the reducing agent can break the various disulfide bonds, causing degradation of the bead and release of the barcode sequence into the aqueous internal environment of the droplet. In another example, heating a droplet with a barcode sequence attached to the bead in an alkaline solution also results in degradation of the bead and release of the associated barcode sequence into the internal aqueous environment of the droplet. Free substances (eg, oligonucleotides, nucleic acid molecules) can interact with other reagents contained in the compartments.
Degradable beads can be used to rapidly release bound substances (eg, nucleic acid molecules, barcode sequences, primers, etc.) from the beads without degradation when an appropriate stimulus is applied to the beads. For example, for a substance bound to the inner surface of a porous bead or in the case of an encapsulated substance, the substance may have greater mobility and accessibility to other substances in solution as the bead degrades. In some embodiments, the substances can also be attached to the degradable beads via degradable linkages (eg, disulfide linkages). The degradable linker may respond to the same stimulus as the degradable seed, or the two degradable substances may respond to different stimuli. For example, barcode sequences can be disulfide-linked to cystamine-containing polyacrylamide beads. When the barcode beads are exposed to a reducing agent, the beads are degraded and the barcode sequence is released after breaking the disulfide bond between the barcode sequence and the bead, as well as the cystamine disulfide bond in the bead.
Any suitable number of substances (eg, primers, barcoded oligonucleotides) can be bound to the beads so that upon release from the beads, the substances (eg, primers, eg, barcoded oligonucleotides) are present in the compartments for a predetermined concentration time. Such predefined concentrations can be chosen to facilitate certain reactions within the partitions for generating sequencing libraries, such as amplification. In some cases, a predefined concentration of primers can be limited by the manufacturing process of nucleic acid molecules (eg, oligonucleotides) with beads.
It will be clear from the above description that although referred to as bead degradation, in many embodiments, degradation may refer to the separation of bound or entrapped species from the bead, structurally degrading the physical bead itself, rather than degrading the physical bead itself. For example, trapped substances can be released from the beads due to differences in osmotic pressure, for example, due to changes in the chemical environment. For example, changes in bead pore volume due to differences in osmotic pressure can often occur without structural degradation of the bead itself. In some cases, the increase in pore volume due to osmotic swelling of the beads may allow the release of trapped species within the beads. In some embodiments, osmotic shrinkage of the beads may result in the beads better retaining trapped species due to shrinkage of pore volume.
A number of chemical triggers can be used to initiate the breakdown of beads within the barrier. Examples of these chemical changes may include, but are not limited to, pH-mediated changes in the integrity of components within the spheres, degradation of the sphere components by breaking cross-links, and deaggregation of the sphere components.
In certain embodiments, a change in the pH of the solution, such as an increase in pH, can cause degradation of the beads. In other embodiments, exposure to an aqueous solution, such as water, can cause hydrolytic degradation, thereby degrading the beads. In some cases, any combination of stimuli can trigger bead degradation. For example, a change in pH can make a chemical such as DTT an effective reducing agent.
The beads can also be induced to release their contents when a heat stimulus is applied. Changes in temperature cause different changes to the balls. For example, heat can cause solid beads to liquefy. The change in heat causes the beads to melt, which degrades part of the bead. In other cases, the heat increases the internal pressure of the pellet's ingredients, causing the pellet to crack or explode. Heat can also be applied to heat-sensitive polymers used as bead building materials.
In addition to beads and analytes, the formed partitions can contain a number of different reagents and substances. For example, when a lysis reagent is present within the compartment, the lysis reagent may facilitate the release of the analyte within the compartment. Examples of lysing agents include biologically active agents such as lytic enzymes used to lyse various cell types, e.g. gram-positive or negative bacteria, plants, yeasts, mammals, etc., such as lysozyme, leukopeptidase, lysostaphin labialase, catalase, lyase, and various other lyases available from, for example, Sigma-Aldrich, Inc. (St. Louis, MO), as well as other commercially available lyases. Other lysis agents may additionally or alternatively partition to cause analyte release into compartments. For example, surfactant-based lysis solutions can be used to lyse cells in some cases, although they are not ideal for emulsion-based systems because surfactants can interfere with stable emulsions. In some embodiments, the lysis solution may include nonionic surfactants, such as Triton X-100 and Tween 20. In some embodiments, the lysis solution may include ionic surfactants, such as sarkosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical disruption of cells can also be used in certain embodiments, e.g. non-emulsion-based dispersion, such as analyte encapsulation, which can complement or replace droplet dispersion, where at any encapsulant pore size the nucleic acid fragments are small enough to retain a certain volume after cell disruption.
Examples of other substances that may be shared with the analytes in the compartment include, but are not limited to, DNase and RNase inactivators or inhibitors, such as proteinase K, chelating agents, such as EDTA, and other substances used to remove or remove or removal of another substance.reagent. The negative activity or influence of various components of cell lysates on the subsequent processing of nucleic acids is otherwise reduced. Additional reagents can also be assigned together, including endonucleases for DNA fragmentation, DNA polymerases, and dNTPs for amplifying nucleic acid fragments and attaching barcoded molecular tags to the amplified fragments.
Additional reagents may also include reverse transcriptase, including enzymes with terminal transferase activity, primers and oligonucleotides, and replacement oligonucleotides (also referred to herein as "replacement oligonucleotides" or "template replacement oligonucleotides"). In some embodiments, template shifting can be used to increase the length of the cDNA. Template switching can be used to add predefined nucleic acid sequences to cDNA. In one example of template switching, cDNA can be generated from reverse transcription of a template, such as cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, such as a poly(C) pathway. A replacement oligonucleotide may include a sequence complementary to an additional nucleotide, such as poly(G). Additional nucleotides on the cDNA (eg, poly(C)) can be hybridized to additional nucleotides on the oligonucleotide replacement (eg, poly(G)), whereby the oligonucleotide replacement can be read by reverse transcriptase, which is used as a template for further cDNA elongation.
A template-changing oligonucleotide may include a hybridization region and a template region. A hybridizing region can include any sequence capable of hybridizing to a target. In some cases, the hybridization region includes a series of G bases that complement the overhanging C bases at the 3' end of the cDNA molecule. A series of G bases may include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. Template sequences can include any sequence that is incorporated into cDNA. In some cases, the template region includes at least 1 (eg, at least 2, 3, 4, 5, or more) tag sequences and/or functional sequences. Replacement oligonucleotides may include deoxyribonucleic acid; ribonucleic acid; bridging nucleic acid, modified nucleic acid including 2-aminopurine, 2,6-diaminopurine (2-Amino-dA), inverted dT, 5-methyl dC, 2'-deoxyinosine, Super T (5-hydroxybutynyl-2'-deoxyuridine) , Super G (8-aza-7-deazaguanosine), Locked Nucleic Acids (LNA), Unlocked Nucleic Acids (UNA, eg UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso -dC, 2' fluorobases (eg fluoroC, fluoroU, fluoroA and fluoroG) and the above combination.
In some embodiments, the beads separated from the analyte may include different types of oligonucleotides bound to the beads, where different types of oligonucleotides bind to different types of analyte. For example, the bead may include one or more first oligonucleotides (eg, which may be capture probes), which may bind or hybridize to a first type of analyte, such as mRNA, and one or more other oligonucleotides. A nucleotide (which can be, for example, a capture probe) can bind or hybridize to another type of analyte, such as gDNA. The partitions may also include lysis agents that facilitate the release of nucleic acids from the copartitioned cells, and may also include reagents (eg, reducing agents) that can degrade the beads and/or break the covalent bonds between the oligonucleotide and the beads, so that the oligonucleotide released into the compartment. The released barcoded oligonucleotides (which may also be barcoded) can hybridize to mRNA released from the cell or to gDNA released from the cell.
The barcode constructs formed by hybridization may therefore include a first type of construct containing a sequence corresponding to the original barcode sequence from the bead and a sequence corresponding to a transcript from the cell, and a second type of construct, including sequences corresponding to the original barcode from the bead and sequences corresponding to the genomic DNA from cells. The barcoded constructs can then be released/removed from the partitions and, in some embodiments, further processed to add additional sequences. The resulting constructs can then be sequenced, the sequencing data processed, and the results used for the spatial characterization of mRNA and gDNA in cells.
In another example, the partition comprises a bead containing a first type of oligonucleotide (eg, a first capture probe) having a first barcode sequence, a poly(A) poly(A) tail that can hybridize to an mRNA start sequence (T). transcripts and UMI barcode sequences that uniquely identify a given transcript. The bead also includes a second type of oligonucleotide (e.g., a second capture probe) having a second barcode sequence, which may be specific for a third barcode oligonucleotide (e.g., an analyte capture agent) coupled to an antibody. the surface of the partitioned cells. The third barcoded oligonucleotide includes a UMI barcode sequence that uniquely identifies the antibody (and the specific cell surface feature to which it binds).
In this example, the first and second barcode oligonucleotides include the same spatial barcode sequence (eg, the first and second barcode sequences are identical), which enables downstream linkage of the barcode nucleic acid to the partition. However, in some embodiments, the first and second barcode strings are different.
Partitions also include lysis agents that help release the nucleic acids from the cells, and may also include agents (eg, reducing agents) that can degrade the beads and/or break the covalent bonds between the barcoded oligonucleotides and the beads, releasing them to enter the partition. The first released barcode oligonucleotide can be hybridized with mRNA released from the cell, and the second released barcode oligonucleotide can be hybridized with a third barcode oligonucleotide to form a barcode construct.
The first barcode construct includes a spatial barcode sequence corresponding to the first barcode sequence from the bead and a sequence corresponding to the UMI barcode sequence from the first type of oligonucleotide, which recognizes the cellular transcript. Another type of barcode construct includes a spatial barcode sequence corresponding to a second barcode sequence from a second type of oligonucleotide, and a spatial barcode sequence corresponding to a third type of oligonucleotide (eg, an analyte capture agent) and is used to identify the cell. Surface features of the UMI barcode sequence. The barcoded constructs can then be released/removed from the partitions and, in some embodiments, further processed to add additional sequences. The resulting constructs are then sequenced, the sequencing data processed, and the results used to characterize the cells for mRNA and cell surface characteristics.
The previous discussion referred to two specific examples of beads carrying oligonucleotides for the analysis of two different analytes within a partition. More generally, split beads may have any of the structures described above and may include any combination of oligonucleotides described for the assay of two or more (eg, three or more, four or more, five or more, six or more), eight or more , ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more) division of different types of analytes. Examples of beads with combinations of different types of oligonucleotides (eg, capture probes) for simultaneous analysis of different combinations of analytes within a partition include, but are not limited to: (a) genomic DNA and cell surface features (eg, use of analyte capture reagents described herein); (b) mRNA and lineage tracing structures; (c) mRNA and cellular methylation status; (d) mRNA and available chromatin (eg ATAC-seq, DNase-seq and/or MNase-seq); (e) mRNA and cell surface or intracellular proteins and/or metabolites; (f) barcoded analyte capture agents (e.g., MHC multimers as described herein) and immune cell receptors ((e.g., V(D)J sequence T cell receptor); (g) mRNA and perturbants (e.g., CRISPR crRNA/ sgRNAs, TALENs, zinc finger nucleases, and/or antisense oligonucleotides as described herein; ).In some embodiments, the perturbing agent can be a small molecule, antibody, drug, aptamer, miRNA, physical environment (eg, temperature change), or any other a known disturbing agent.
Further, in some embodiments, non-aggregated cells or dissociated cells introduced and processed within a baffle or droplet as described herein can be removed from the baffle, contacted with a spatial array, and spatially barcoded according to the methods described herein. For example, individual cells of a non-aggregated cell sample can be partitioned into compartments or droplets as described herein. The baffles or droplets may contain cell permeabilization reagents, cellular analytes targeted to the barcoded cell barcode, and amplification of barcoded analytes. The baffles or droplets may be in contact with any of the spatial arrays described herein. In some embodiments, the baffles can be dissolved by contacting the contents of the baffles with spatially distributed capture probes. A spatial array of capture probes can then capture target analytes from broken partitions or droplets and process them through the spatial workflow described herein.
(g) Analysis of captured analytes (i) remove samples from the array
In some embodiments, after contacting the biological sample with the substrate containing the capture probes, a removal step may optionally be performed to remove all or part of the biological sample from the substrate. In some embodiments, the removal step includes enzymatic and/or chemical degradation of the cells of the biological sample. For example, the removal step may include treating the biological sample with an enzyme (eg, a protease, such as proteinase K) to remove at least a portion of the biological sample from the substrate. In some embodiments, the removal step may include tissue ablation (eg, laser ablation).
In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting the location of an analyte, e.g., a biological analyte) from (e.g., present in) a biological sample, the method comprising: (a) selectively staining and/or displaying the biological sample on substrate; (b) permeabilizing (eg, providing a solution containing a permeabilizing reagent) the biological sample on the substrate; contacting the plurality of capture probes, wherein the plurality of capture probes capture the biological analyte; (d) analyzing the captured biological analyte, thereby spatially detecting the biological analyte; whereby the biological sample is completely or partially removed from the substrate.
In some embodiments, the biological sample is not removed from the substrate. For example, the biological sample is not removed from the substrate until the capture probes (eg, analyte-bound capture probes) are released from the substrate. In some embodiments, such release involves cleavage of the capture probe from the substrate (eg, by a cleavage domain). In some embodiments, such release does not involve release of the capture probe from the substrate (eg, a copy of the capture probe bound to the analyte can be made, and the copy can be released from the substrate, eg, by denaturation). In some embodiments, after the analyte is released from the substrate, the biological sample is not removed from the substrate until the analyte binds to the capture probes. In some embodiments, the biological sample remains on the substrate during removal of the capture probes from the substrate and/or analysis of the analyte bound to the capture probes after release of the capture probes from the substrate. In some embodiments, the biological sample remains on the substrate during removal (eg, by denaturation) of copies of capture probes (eg, complement). In some embodiments, the capture probe from the substrate can be combined without subjecting the biological sample to enzymatic and/or chemical cell degradation (eg, permeabilized cells) or tissue ablation (eg, laser ablation). Analyze the bound analyte. .
In some embodiments, at least a portion of the biological sample is not removed from the substrate. For example, a portion of the biological sample may remain on the substrate prior to release of the capture probes from the substrate (eg, capturing evidence of binding to the analyte) and/or analysis of the analyte bound to the capture probes released from the substrate. In some embodiments, at least a portion of the biological sample being analyzed is bound to capture probes from the substrate.
In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting an analyte, e.g., location of a biological analyte) from (e.g., present in a biological sample) a biological sample comprising: (a) selectively staining and/or imaging the biological sample on substrate; (b) permeabilizing (eg, administering a solution containing a permeabilizing reagent) the biological sample to the substrate; where multiple capture probes capture the biological analyte; (d) analyzing the captured biological analyte, whereby the biological analyte is spatially detected; whereby the biological sample is not removed from the matrix.
In some embodiments, provided herein are methods for spatially detecting a biological analyte of interest from a biological sample comprising: (a) staining and imaging the biological sample on a substrate; A solution containing a permeabilization reagent is given to the biological sample; (c) ) contacting the biological sample with the array on the substrate, wherein the array contains one or more sets of capture probes, thereby enabling the one or more capture probes to capture the organism of interest in the Analyte; (d) Analysis of captured biological analytes for spatial detection of biological analytes of interest; where the biological sample is not removed from the matrix.
In some embodiments, the method further includes selecting a region of interest in the biological sample for spatial transcriptomic analysis. In some embodiments, one or more of the one or more capture probes includes a capture domain. In some embodiments, one or more of the plurality of one or more capture probes comprises a unique molecular identifier (UMI). In some embodiments, one or more of the plurality of one or more capture probes comprises a cleavage domain. In some embodiments, the cleavage domain comprises a uracil-DNA glycosylase, an apurinic/apyrimidinic (AP) endonuclease (APE1), a Uuracil-specific excision reagent (USER), and/or a nucleic acid sequence recognized and cleaved by endonuclease VIII. In some embodiments, the one or more capture probes do not contain a cleavage domain and are not cleaved from the array.
(ii) Extended capture probes
In some embodiments, the capture probe can be extended ("extended capture probe", e.g., as described herein (e.g., Section II(b)(vii))). For example, extension of the capture probe may involve generating cDNA from the captured (hybridized) RNA. The process involves the synthesis of complementary strands of hybridized nucleic acids, e.g. generation of cDNA based on the captured RNA template (RNA that hybridizes to the capture domain of the capture probe). Therefore, in the initial step of extending the capture probe, e.g. generation of cDNA, captured (hybridized) nucleic acid, e.g. RNA, serves as a template for the extension step (eg, reverse transcription).
In some embodiments, capture probes are amplified using reverse transcription. For example, reverse transcription involves the synthesis of cDNA (complementary or copied DNA) from RNA such as (messenger RNA) by reverse transcriptase. In some embodiments, reverse transcription is performed while the tissue is still in situ, generating an analyte library, wherein the analyte library includes spatial barcodes from adjacent capture probes. In some embodiments, the capture probe is extended by one or more DNA polymerases.
In some embodiments, the capture domain of the capture probe includes a primer to generate the complementary strand of the nucleic acid to which the capture probe hybridizes, such as a DNA polymerase and/or reverse transcription primer. Nucleic acids, eg, DNA and/or cDNA molecules, which are formed by the extension reaction, include capture probe sequences. Extension of capture probes, eg, DNA polymerase reaction and/or reverse transcription, can be performed using a variety of suitable enzymes and protocols.
In some embodiments, full-length DNA molecules (eg, cDNA) are produced. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if a nucleic acid (eg, RNA) is partially degraded in a tissue sample, the captured nucleic acid molecule will not be the same length as the original RNA in the tissue sample. In some embodiments, the 3' end of the extended probe (eg, the first strand of the cDNA molecule) is modified. For example, linkers or adapters can be attached to the 3' ends of extension probes. This can be accomplished using a single-stranded ligase such as T4 RNA ligase or Circligase™ (available from Lucigen, Middleton, WI). In some embodiments, template-switching oligonucleotides are used to extend cDNA to generate full-length cDNA (or as close to full-length cDNA as possible). In some embodiments, a second-strand synthesis helper probe (a partially double-stranded DNA molecule capable of hybridizing to the 3' end of the extension capture probe) can be ligated to the 3' end of the extension probe, such as first-strand cDNA, using double-stranded ligase molecules as which is T4 DNA ligase. Other enzymes suitable for the ligation step are known in the art and include, for example, Tth DNA ligase, Taq DNA ligase,Thermococcussp. (strain 9°N) DNA Ligase (9°N™ DNA Ligase, New England Biolabs), Ampligase™ (available from Lucigen, Middleton, WI) and SplintR (available from New England Biolabs, Ipswich, MA). In some embodiments, a polynucleotide tail, e.g., a poly(A) tail, is incorporated into the 3' end of the extended probe molecule. In some embodiments, the polynucleotide tail is incorporated by a terminally active transferase enzyme.
In some embodiments, double-stranded extended capture probes are treated to remove any unextended capture probes prior to amplification and/or analysis, e.g. sequence analysis. This can be achieved in various ways, for example, by using enzymes to degrade unextended probes, such as exonucleases or purification columns.
In some embodiments, the extended capture probes are amplified to obtain sufficient amounts for analysis, eg, by DNA sequencing. In some embodiments, the first strand of an extended capture probe (eg, DNA and/or cDNA molecule) serves as a template for an amplification reaction (eg, polymerase chain reaction).
In some embodiments, the amplification reaction includes an affinity group to an extended capture probe (eg, an RNA-cDNA hybrid) using a primer containing the affinity group. In some embodiments, the primer includes an affinity group and the extended capture probe includes an affinity group. The affinity group may correspond to any of the previously described affinity groups.
In some embodiments, an extended capture probe containing an affinity group can be coupled to a substrate specific for the affinity group. In some embodiments, the substrate may include an antibody or antibody fragment. In some embodiments, the substrate includes avidin or streptavidin and the affinity group includes biotin. In some embodiments, the substrate comprises maltose and the affinity group comprises a maltose-binding protein. In some embodiments, the substrate comprises a maltose-binding protein and the affinity group comprises maltose. In some embodiments, amplification of the extended capture probe can function to release the extended probe from the surface of the substrate, until copies of the extended probe are immobilized on the substrate.
In some embodiments, the extended capture probe or its complement or amplicon is released. The step of releasing the extended capture probe or its complement or amplicon from the substrate surface can be accomplished in a number of ways. In some embodiments, the extended capture probes or their complements are released from the array by nucleic acid cleavage and/or denaturation (eg, heating to denature double-stranded molecules).
In some embodiments, the extended capture probe or its complement or amplicon is physically released from the surface of the substrate (e.g., the array). For example, where the extended capture probes are indirectly immobilized on the array substrate, for example by hybridization with surface probes, this may be sufficient to disrupt the interaction between the extended capture probes and the surface probes. Methods for disrupting interactions between nucleic acid molecules, including denaturing double-stranded nucleic acid molecules, are known in the art. A simple approach to release the DNA molecule (ie, stripping the array of extended probes) is to use a solution that disrupts the hydrogen bonding of the double-stranded molecules. In some embodiments, the extended capture probe is heated by applying a temperature of at least 85°C, such as at least 90, 91, 92, 93, 94, 95, 96, 97, 95, 96, 97, 98, or 99°C. . In some embodiments, a solution of salts, surfactants, etc. is added. which can further disrupt interactions between nucleic acid molecules to release probes with extended capture from the substrate.
In some embodiments, when the extended capture probe includes a cleavage domain, the extended capture probe is released from the surface of the substrate by cleavage. For example, the cleavage domain of an extended capture probe can be cleaved by any of the methods described herein. In some embodiments, prior to the step of amplifying the extended capture probes, the extended capture probes are released from the substrate surface, e.g., by cleaving the cleavage domain in extended capture assays.
In some embodiments, the probe that is complementary to the extended capture probe may contact the substrate. In some embodiments, the biological sample may be in contact with the substrate while the probe is in contact with the substrate. In some embodiments, the biological sample may be removed from the substrate prior to contacting the substrate with the probes. In some embodiments, the probe may be labeled with a detectable label (eg, any detectable label described herein). In some embodiments, probes that do not specifically bind (eg, hybridize to) the extended capture probe can be washed away. In some embodiments, probes complementary to extended capture probes can be detected on a substrate (eg, imaging, any of the detection methods described herein).
In some embodiments, the probe that is complementary to the extended capture probe can be about 4 nucleotides to about 100 nucleotides in length. In some embodiments, probes (eg, detectable probes) that are complementary to the extended capture probes can be about 10 nucleotides to about 90 nucleotides in length. In some embodiments, probes (eg, detectable probes) that are complementary to extended capture probes can be about 20 nucleotides to about 80 nucleotides in length. In some embodiments, probes (eg, detectable probes) that are complementary to extended capture probes can be about 30 nucleotides to about 60 nucleotides in length. In some embodiments, probes (eg, detectable probes) that are complementary to extended capture probes can be about 40 nucleotides to about 50 nucleotides in length. In some embodiments, the probes (e.g., detectable probes) complementary to the extension capture probes can be about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 , about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , around 32 around 33 around 34 around 35 around 36 around 37 around 38 around 39 around 40 around 41 around 42 around 43 around 44 around 45 around 46 around 47 around 48 , around 49, around 50, around 51, around 52, around 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, around 70, around 71, around 72, around 73, around 74, around 75, around 76, around 77, around 78, around 79, around 80, around 81, around 82 around 83 around 84 around 85 around 86 around 87 around 88 about 89 about 90 about 91 about 92 about 93 about 94 about 95 about 96 about 97 about 98, and about 99 nucleotides in length.
In some embodiments, from about 1 to about 100 probes can contact the substrate and specifically bind (eg, hybridize) to the extended capture probes. In some embodiments, about 1 to about 10 probes can contact the substrate and specifically bind (eg, hybridize) to the extended capture probe. In some embodiments, about 10 to about 100 probes can contact the substrate and specifically bind (eg, hybridize) to the extended capture probes. In some embodiments, about 20 to about 90 probes can contact the substrate and specifically bind (eg, hybridize) to the extended capture probes. In some embodiments, about 30 to about 80 probes (eg, detectable probes) can contact the substrate and specifically bind (eg, hybridize) to the extended capture probes. In some embodiments, about 40 to about 70 probes can contact the substrate and specifically bind (eg, hybridize) to the extended capture probes. In some embodiments, about 50 to about 60 probes can contact the substrate and specifically bind (eg, hybridize) to the extended capture probes. In some embodiments, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, around 34 around 35 around 36 around 37 around 38 around 39 around 40 around 41 around 42 around 43 around 44 around 45 around 46 around 47 around 48 around 49 around 50 , around 51, around 52, around 53, around 54, around 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72 , About 73, About 74, About 75, About 76, About 77, About 78, About 79, About 80, About 81, About 82, About 83, About 84, About 85, About 86, About 87, About 88 , about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98 and about 99 probes can contact the substrate and specifically bind (eg, hybridize) to the elongated capture probes.
In some embodiments, the probe may be complementary to an analyte (eg, a gene). In some embodiments, the probe may be complementary to one or more analytes (eg, analytes within a gene family). In some embodiments, a probe (eg, a detectable probe) can target a set of genes associated with a disease (eg, cancer, Alzheimer's disease, Parkinson's disease).
(iii) Cleavage domain
A capture probe may optionally include a "cleavage domain", wherein one or more segments or regions of the capture probe (eg, a spatial barcode and/or UMI) can be detached, cleaved, or reversibly attached to a feature or other substrate such that that the spatial barcodes and/or UMIs can be released or released by cleaving the bond between the capture probe and the feature or by degrading the underlying substrate or chemical substrate, whereby the spatial barcodes and/or UMIs can the cleaved capture probe is accessible or accessible to other reagents, or both. Non-limiting aspects of the cleavage domains are described herein (eg, in Section II(b)(ii)).
In some embodiments, the capture probe is attached to the feature (eg, via a disulfide bond). In some embodiments, the capture probe is attached to the feature (eg, spacer C3) via a propylene group. Reducing agents can be added to cleave the various disulfide bonds, resulting in the release of capture probes containing the spatial barcode arrays. In another example, heating can also cause degradation and release of attached capture probes. In some embodiments, heating is achieved with a laser (eg, laser ablation) and features may be degraded at certain locations. In addition to heat-cleavable bonds, disulfide bonds, photosensitive bonds, and UV-sensitive bonds, other non-limiting examples of labile bonds that can be attached to capture probes (eg, steric barcodes) include ester bonds (eg, acids, bases, or hydroxylamine), vicinal diol bonds (e.g., cleavable by sodium periodate), Diels-Alder bonds (e.g., cleavable by thermal), sulfonic bonds (e.g., cleavable by base), silyl ether bonds (e.g., cleavable by acids), glycosidic bonds (e.g., cleavable by amylases), peptide bonds (eg, cleavable by proteases), or phosphodiester bonds (eg, cleavable by nucleases (eg, DNase cleavage)).
In some embodiments, the cleavage domain comprises a polynucleotide cleavable by a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER™ enzyme).(U) sequences. In some embodiments, the cleavage domain can be a single U. In some embodiments, the cleavage domain can be an abasic site, which can be cleaved by an abasic site-specific endonuclease (eg, endonuclease IV or endonuclease VIII).
In some embodiments, the cleavage domain of the capture probe is a nucleotide sequence within the capture probe that is specifically cleaved, e.g., physically, chemically, or enzymatically using light or heat. The location of the cleavage domain within the capture probe will depend on whether the capture probe is immobilized on a substrate with a free 3' end that can function as an extension primer (eg, via its 5' or 3' end). For example, if the capture probe is immobilized via its 5' end, the cleavage domain will be located 5' to the spatial barcode and/or UMI, and cleavage of said domain results in the release of a portion of the capture probe, including the spatial barcode and/or The UMI and sequence 3' to the spatial barcodes, and optionally part of the cleavage domain, are derived from the signature. Alternatively, if the capture probe is immobilized via its 3' end, the cleavage domain will be located 3' to the capture domain (and the spatial barcode), and cleavage of said domain results in the release of a portion of the capture probe, including the spatial barcode and from the sequence 3' of the characteristic spatial barcode. In some embodiments, cleavage results in partial removal of the cleavage domain. In some embodiments, cleavage results in complete removal of the cleavage domain, particularly when the capture probes are immobilized via their 3' ends, since the presence of a portion of the cleavage domain interferes with hybridization and/or its subsequent extensions.
(iv) Classification
After the analyte from the sample is hybridized or otherwise bound to a capture probe, analyte capture agent, or other barcoded oligonucleotide sequence, the hybridization/association is analyzed by sequencing to identify the analyte.
In some embodiments, when a sample is directly barcoded by hybridization with a capture probe or analyte capture agent that hybridizes, binds or associates with, or is introduced into, the cell surface, the entire sample can be sequenced, as described above. Alternatively, if the barcoded sample is separated into fragments, cell populations or single cells, as described above, then the individual fragments, cell populations or cells can be sequenced. As described above, for analytes barcoded by bead splitting, individual analytes (eg, cells or cell content after cell lysis) can be extracted from the partitions by breaking the partitions and then analyzed by sequencing to identify the analytes.
Barcoded analyte constructs can be analyzed using a variety of different sequencing methods. Typically, the sequenced polynucleotide may be, for example, a nucleic acid molecule such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (eg, single-stranded DNA or DNA/RNA hybrids and glycoside analogs).
Polynucleotide sequencing can be performed with various commercial systems. More generally, nucleic acid amplification, polymerase chain reaction (PCR) (eg digital PCR and digital droplet PCR (ddPCR), quantitative PCR, real-time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR can be use) for sequencing and/or isothermal amplification.
Other examples of methods for sequencing genetic material include, but are not limited to, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion methods, Sanger sequencing methods, next-generation sequencing methods (e.g., single molecule real-time sequencing), nanopore sequencing and Polony sequencing), ligation methods and microarray methods. Other examples of sequencing methods that can be used include targeted sequencing, real-time single molecule sequencing, exome sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole genome sequencing , hybrid sequencing, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, low denaturation Coamplification-PCR at temperature (COLD- PCR), reversible dye-terminator sequencing, paired-end sequencing, short-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, synthesis sequencing, real-time sequencing, reverse terminator sequencing, nanopore sequencing, MS-PET sequencing and any combination thereof.
Sequence analysis of nucleic acid molecules, including barcoded nucleic acid molecules or derivatives thereof, may be direct or indirect. Thus, a sequence analysis substrate (which may be considered a molecule subjected to a sequence analysis step or process) may be a barcoded nucleic acid molecule or may be a molecule derived from it (eg, its complement). Thus, for example, in the sequence analysis step of a sequencing reaction, the template for sequencing may be a barcoded nucleic acid molecule or may be a molecule derived from it. For example, the first and/or second strand DNA molecules may be directly sequenced (e.g., sequenced), that is, may directly participate in a sequence analysis reaction or process (e.g., a sequencing reaction or process, or sequence or otherwise identify the molecules ). Alternatively, the barcoded nucleic acid molecule may undergo a second-strand synthesis or amplification step prior to sequence analysis (eg, sequencing or identification by another technique). The substrate for sequence analysis (eg, template) can therefore be an amplicon or second strand of a barcoded nucleic acid molecule.
In some embodiments, both strands of the double-stranded molecule can be analyzed (eg, sequenced). In some embodiments, single-stranded molecules (eg, barcoded nucleic acid molecules) can be analyzed (eg, sequenced). Nucleic acid strands can be modified at the 3' end for single molecule sequencing.
Massively parallel pyrosequencing technology can be used to sequence nucleic acids. In pyrosequencing, nucleic acids are amplified within droplets of water in an oil solution (emulsion PCR), with each droplet containing a nucleic acid template attached to primer-coated beads, which then form clonal colonies. The sequencing system contains many wells of picoliter volume, each containing a bead and sequencing enzyme. Pyrosequencing uses luciferase to generate light to detect individual nucleotides added to nascent nucleic acids and uses the combined data to generate sequence reads.
As another example of a pyrosequencing application, released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP levels can be detected by photons generated by luciferase, as described by Ronaghi et al. . ,anus. biochemical242(1), 84-9 (1996);genome research11(1), 3-11 (2001); Ronaji et al.science281 (5375), 363 (1998) and US Pat. LOUSE. patents no. 6,210,891, 6,258,568 and 6,274,320, the entire contents of which are incorporated herein by reference.
In some embodiments, massively parallel sequencing technologies may be based on reversible dye terminators. For example, DNA molecules are first attached to primers on a glass or silicon substrate and then replicated, resulting in the formation of localized clonal colonies (bridge amplification). Four types of ddNTPs are added and unincorporated nucleotides are washed away. Unlike pyrosequencing, DNA can only be extended one nucleotide at a time due to the presence of blocking groups (for example, 3' blocking groups present on the sugar residue of ddNTP). The detector acquires images of fluorescently labeled nucleotides, and the dye is then chemically removed from the DNA, along with the terminal 3' blocking group, as a precursor for subsequent cycles. This process can be repeated until the data on the desired sequence is obtained.
In some embodiments, sequencing is performed by detecting hydrogen ions released during DNA polymerization. Microwells containing template DNA strands to be sequenced can be filled with one type of nucleotide. If the incoming nucleotide is complementary to the leader nucleotide of the template, it is incorporated into the growing complementary strand. This causes hydrogen ions to be released, activating the ultra-sensitive ion sensor, which indicates that a reaction has occurred. If there are homopolymeric repeats in the template sequence, more nucleotides will be incorporated into one cycle. This results in a corresponding number of released hydrogen ions and a proportionally higher electronic signal.
In some embodiments, sequencing can be performed in situ. In situ sequencing methods are particularly useful, for example, when biological samples remain intact after analytes have been barcoded on the surface of the sample (eg cell surface analytes) or within the sample (eg intracellular analytes). In situ sequencing typically involves the incorporation of labeled nucleotides (eg, fluorescently labeled mono- or dinucleotides) or labeled primers (eg, labeled random hexamers) in a sequential, template-dependent manner. Hybridization with a nucleic acid template allows the identity (ie, nucleotide sequence) of the incorporated nucleotides or labeled products of the starting products to be determined, and thus the nucleotide sequence of the corresponding template nucleic acid. For example, Mitra et al., (2003) describe aspects of in situ sequencinganus. biochemical320, 55-65 iplumi on., (2014.)science, 343(6177), 1360-1363, the entire contents of each article are incorporated herein by reference.
In addition, PCT patent application no. WO2014/163886, WO2018/045181, WO2018/045186 and U.S. patents no. Examples of in situ sequencing techniques include, but are not limited to, STARmap (as described in Wang et al., (2018))science, 361(6499) 5691), SIRENA (e.g. described in Moffitt, (2016)enzymatic method, 572, 1-49) and FISSEQ (described, for example, in US Patent Application Publication No. 2019/0032121). The entire contents of each of the above references are incorporated herein by reference.
For cleavage barcoded analytes, barcoded nucleic acid molecules or derivatives thereof (eg, barcoded nucleic acids to which one or more functional sequences have been added or from which one or more features have been removed) can be pooled and processed. molecules) for subsequent analysis, such as sequencing on a high-throughput sequencer. Association can be achieved using barcode sequences. For example, barcoded nucleic acid molecules for a particular partition may have the same barcode, which is different from barcodes for other spatial partitions. Alternatively, barcoded nucleic acid molecules of different partitions can be processed separately for subsequent analysis (eg, sequencing).
In some embodiments, when the capture probe does not contain a spatial barcode, the spatial barcode can be added after the capture probe captures the analyte from the biological sample and before the analyte is analyzed. When spatial barcoding is added after analyte capture, barcoding can be added after analyte amplification (eg, RNA reverse transcription and polymerase amplification). In some embodiments, analyte analysis utilizes direct sequencing of one or more captured analytes, e.g., direct sequencing of hybridized RNA. In some embodiments, direct sequencing is performed after reverse transcription of the hybridized RNA. In some embodiments, direct sequencing is performed after reverse transcription amplification of the hybridizing RNA.
In some embodiments, direct sequencing of the captured RNA is performed by sequencing by synthesis (SBS). In some embodiments, the sequencing primer is complementary to a sequence in one or more domains (eg, functional domains) of the capture probe. In such embodiments, sequencing by synthesis may involve reverse transcription and/or amplification to generate template sequences (eg, functional domains) from which primer sequences can be ligated.
SBS may involve hybridizing appropriate primers (sometimes called sequencing primers) to the nucleic acid template to be sequenced, extending the primers, and detecting the nucleotides used to extend the primers. Preferably, the nucleic acid used to extend the primer is detected prior to the addition of additional nucleotides to the growing nucleic acid strand, thereby allowing in situ base-by-base sequencing of the nucleic acid. Detection of incorporated nucleotides is facilitated by including one or more labeled nucleotides in the primer extension reaction. To allow suitable sequencing primers to hybridize to the template nucleic acid to be sequenced, the template nucleic acid should generally be in single-stranded form. If the nucleic acid templates that make up the nucleic acid signature exist in double-stranded form, those nucleic acid templates can be converted to single-stranded nucleic acid templates using methods well known in the art, such as denaturation, cleavage, and the like. Oligonucleotides that hybridize to the nucleic acid template and are used for primer extension are preferably short oligonucleotides, eg, 15 to 25 nucleotides in length. Sequencing primers can be provided in solution or in immobilized form. After the sequencing primers are annealed to the template nucleic acid to be sequenced by subjecting the template nucleic acid and the sequencing primer to appropriate conditions, primer extension is performed, for example using a nucleic acid polymerase and the provided nucleotides, at least some of which are provided in labeled form , if provided. The corresponding nucleotides are suitable for the primer extension conditions.
It is desirable to include a washing step after each primer extension step to remove unincorporated nucleotides that may interfere with subsequent steps. After the primer extension step is performed, the nucleic acid colonies are monitored to determine if the labeled nucleotides have been incorporated into the extended primers. The primer extension step can then be repeated to determine the next and subsequent nucleotides incorporated into the extended primer. If the sequence to be determined is unknown, the nucleotides are usually applied to a given colony in a selected order and then repeated during the analysis, e.g. dATP, dTTP, dCTP, dGTP.
SBS technology that can be used is, for example, but not limited to, patent application no. bar. US Patent No. 2007/0166705. application. bar. no. 2006/0188901, US Patent 7,057,026, US Pat. application. bar. US patent no. 2006/0240439. application. bar. 2006/0281109, PCT patent application. bar. WO 05/065814, US Pat. application. bar. PCT patent application no. 2005/0100900. bar. WO 06/064199, PCT patent application. bar. no. WO07/010,251, US Pat. application. bar. US Patent No. 2012/0270305. application. bar. 2013/0260372 and US Pat. application. bar. no. 2013/0079232, each of which is incorporated herein by reference in its entirety.
In some embodiments, direct sequencing of the captured RNA is performed by sequential fluorescence hybridization (eg, hybridization sequencing). In some embodiments, the hybridization reaction in which the RNA hybridizes to the capture probe is performed in situ. In some embodiments, the captured RNA is not amplified prior to hybridization with sequencing probes. In some embodiments, RNA is amplified (eg, reverse transcription to cDNA and cDNA amplification) prior to hybridization to sequencing probes. In some embodiments, amplification is performed using unimolecular chain reaction hybridization. In some embodiments, the amplification is performed using rolling chain amplification.
Sequential fluorescence hybridization may include sequential hybridization of probes that contain detectable degenerate primer sequences. A degenerate primer sequence is a short oligonucleotide sequence capable of hybridizing to any nucleic acid fragment independent of the sequence of the nucleic acid fragment. For example, such a method may include the steps of: (a) providing a mixture of four probes, each containing A, C, G or T at the 5'-end, and 5 to 5 degenerate nucleosides. The acid sequence is 11 nucleotides in length and further includes a functional domain (eg fluorescent molecule) that is different for probes with A, C, G or T at the 5'-end; (b) conversion step (a) the probes are linked to the target polynucleotide sequence, whose sequence requirements will be determined by the method; (c) the activity of the four functional domains is measured and the relative spatial location of the activity is recorded; (d) the relative spatial location of the activity is recorded from the target polynucleotide; Remove the reagents in steps (a)-(b) from the sequence; repeat steps (a)-(d) for n cycles until the nucleotide sequence of the spatial domain of each bead is determined, modified for use in step (a) The oligonucleotide is complementary to a portion of the target polynucleotide sequence and positions 1 through n flank the portion of the sequence. Since the barcode sequences differ, in some embodiments these additional flanking sequences are degenerate sequences. The fluorescence signal from cycle 1 to cycle n of each spot on the array can be used to determine the sequence of the target polynucleotide sequence.
In some embodiments, direct sequencing of the captured RNA using sequential fluorescence hybridization is performed in vitro. In some embodiments, the captured RNA is amplified (eg, reverse transcription to cDNA and cDNA amplification) prior to hybridization to sequencing probes. In some embodiments, the capture probe containing the captured RNA is exposed to a sequencing probe that targets the coding region of the RNA. In some embodiments, one or more sequencing probes target each coding region. In some embodiments, sequencing probes are designed to hybridize with sequencing reagents (eg, dye-labeled readout oligonucleotides). Sequencing probes can then be hybridized with sequencing reagents. In some embodiments, the output from the sequencing reaction is shown. In some embodiments, specific cDNA sequences are separated from images of sequencing reactions. In some embodiments, reverse transcription of the captured RNA is performed prior to hybridization with the sequencing probe. In some embodiments, the sequencing probes are designed to target the complement of the RNA coding region (e.g., the target cDNA).
In some embodiments, the captured RNA is directly sequenced using a nanopore-based approach. In some embodiments, direct sequencing is performed using direct nanopore RNA sequencing, in which the captured RNA is translocated through the nanopore. Nanopore currents can be recorded and converted to base sequences. In some embodiments, the captured RNA remains bound to the substrate during nanopore sequencing. In some embodiments, the captured RNA is released from the substrate prior to nanopore sequencing. In some embodiments, when the analyte of interest is a protein, the protein can be sequenced directly using nanopore-based methods. Examples of nanopore-based sequencing methods that can be used are described in Deamer et al.,Trend Biotech18, 14 7-151 (2000); Deamer et al.cumulative chemistry. reservoir35:817-825 (2002); Li et al.,night. alma mater2:611-615 (2003); Sony et al.,clinical. Chemical.53, 1996-2001 (2007); Healey et al.,Nanomedicine2, 459-481 (2007); Cockcroft et al.,J. Am. Chemical. society.130, 818-820 (2008); in US patents. 7,001,792. The entire contents of each of the above references are incorporated herein by reference.
In some embodiments, direct sequencing of the captured RNA is performed by ligation using single molecule sequencing. These techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides often have different labels that correlate with the identity of specific nucleotides in the sequence to which the oligonucleotide hybridizes. For example, aspects and features involved in sequencing by ligation are described in Shendure et al.science(2005), 309: 1728-1732, and US Pat. 5,599,675; 5,750,341; 6,969,488; 6,172,218; and 6,306,597, each of which is incorporated herein by reference in its entirety.
In some embodiments, nucleic acid hybridization can be used for sequencing. These methods use labeled nucleic acid decoder probes that are complementary to at least part of the barcode sequence. Multiplexed decoding can be performed using a set of many different probes with distinct labels. Non-limiting examples of nucleic acid hybridization sequencing are described, e.g., in U.S. Pat. patent no. 8,460,865, in Gunderson et al.,genome research14:870-877 (2004), each of which is incorporated herein by reference in its entirety.
In some embodiments, commercial high-throughput digital sequencing technologies can be used to analyze barcode sequences, where DNA templates are prepared for sequencing, not one-by-one, but in a batch process, and where many sequences are preferably read in parallel, or using serial very high-throughput processes that are inherently parallel. Examples of such technologies include Illumina®Sequencing (eg, flow cell-based sequencing by synthesis), using modified nucleotides (eg, commercialized in HiSeq™ and other sequencing technology instruments from Illumina, Inc., San Diego, CA), Helicos Biosciences Corporation, Cambridge, HeliScope™ MA, and PacBio RS from Pacific Biosciences of California, Inc., Menlo Park, CA), ion detection technology sequencing (Ion Torrent, Inc., South San Francisco, CA), and DNA nanosphere sequencing (Complete Genomics, Inc., Mountain View, CA ).
In some embodiments, detection of protons released upon incorporation of nucleotides into extension products is employed in the methods described herein. For example, no. US patent application, publication no. 2009/0026082, 2009/0127589, 2010/0137143 and 2010/0282617 can be used for direct barcode sequencing.
In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET), as described by Levene et al.,science(2003), 299, 682-686, Lundquist et al.,choose. Wright(2008), 33, 1026-1028 i Korlach et al.,procedure. national team. college. know america(2008), 105, 1176-1181. The entire contents of each of the above references are incorporated herein by reference.
(v) Weather analysis
In some embodiments, the methods described herein can be used to assess analyte levels and/or expression in cells or biological samples over time (eg, before or after treatment with an agent or at different stages of differentiation). In some examples, the methods described herein may be performed at different time points (e.g., before or after treatment with an agent, at different stages of differentiation, at different stages of disease progression, at different ages of the subject, before or after a physical disorder, before or after treatment with a disruptor as described herein, either before or after the development of resistance to the agent). As described herein, a "disturbing agent" or "disturbing reagent" can be a small molecule, antibody, drug, aptamer, nucleic acid (eg, miRNA), CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, antisense oligonucleotide Acidic physical environment (eg temperature change) and/or any other known perturbing agent where the agent alters equilibrium or homeostasis.
In some embodiments, the methods described herein can be performed 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times on a plurality of similar biological samples or cells obtained from a subject. For example, multiple similar biological samples can be replicate samples from the same subject, the same tissue, the same organoid, the same cell suspension, or any other biological sample described herein. In some embodiments, the methods described herein can be performed at different time points (e.g., before or after treatment with a perturbing agent, at different stages of differentiation, at different stages of disease progression, at different ages of the subject, or in response to an agent). before or after resistance to drug). In some embodiments, the perturbing agents can be small molecules, antibodies, nucleic acids, peptides, and/or other external stimuli (eg, temperature changes). In some embodiments, the biological sample is contacted with a different array at each time point.
In some embodiments, the sample can be placed in a controlled environment that allows cell growth and/or maintenance and/or prevents hypoxia. In some embodiments, the controlled environment enables analysis of samples at different time points. The barcode array can be placed adjacent to (eg, on top of) the sample and imaged using a microscope or other suitable instrument to record the relative position of the biological sample relative to the barcode array, optionally using optically encoded fiducial marks. An electric field can be applied for a period of time so that biological analytes (eg, DNA, RNA, proteins, metabolites, small molecules, lipids, etc.) are released from the sample and captured by spatial capture probes. A series of barcodes, saving the spatial information of the sample. Barcoded arrays can be removed and their spatial and molecular information determined (for example, by library construction for next-generation sequencing or in situ sequencing). Computational analysis can be performed after sequencing to correlate molecular information (for example, gene expression values with spatial barcodes). These steps can be repeated one or more times to capture information about the analyte space at different time points.
In some embodiments, the methods described herein can be combined with cell migration assays. Cell migration assays can include one or more microprinted lines or suspended 3D nanofibers, on which cells migrate. Migration using these assays can be measured by recording cell migration and/or exposing migrated cells to spatially labeled arrays. Arrays for cell migration assays may contain one or more channels on the array substrate, e.g. to limit cell migration to one dimension along the substrate. In addition, the channel may direct the cell's migration so that it does not contact another cell in a row (e.g., the channels do not overlap), and in some embodiments the channel is approximately the same width or wider than the cell (e.g., for mammals. In animal cells, the channels they can have a width of about 2 µm to about 10 µm). Any of the methods described here can be used to identify cell locations on a spatial barcode array.
In some embodiments, cells can be arrayed and allowed to migrate as described herein. Cell migration in cell migration assays can be used to measure phenotypes of interest (eg, invasive phenotypes). In some embodiments, the distance of cell migration can be measured and correlated with a biological analyte. Reagents can be added to the array to stimulate cell migration. For example, arrays can be prepared with one or more extracellular matrix (ECM) components (eg, basement membrane extract (BME), laminin I, collagen I, collagen IV, fibronectin, vitronectin, elastin protein), cell culture medium, chemoattractants , chemorepellents or their combinations. In some embodiments, agents such as chemoattractants or chemorepellents may be distributed over only a portion of the array, present as a gradient along one or more axes or channels of the array, or a combination thereof.
(vi) Spatially resolved analyte information
In some embodiments, a look-up table (LUT) may be used to relate one characteristic of a feature to another characteristic. These attributes include, for example, location, barcodes (eg, nucleic acid barcode molecules), spatial barcodes, optical tags, molecular tags, and other attributes.
In some embodiments, a lookup table can associate nucleic acid barcode molecules with features. In some embodiments, optical labeling of a feature may allow the feature to be associated with a biological particle (eg, a cell or nucleus). Association of a feature with a biological particle may also enable association of the nucleic acid sequence of the nucleic acid molecule of the biological particle with one or more physical characteristics of the biological particle (eg, cell type or cell location). For example, based on barcodes and optical tags. An optical tag can be used to determine the location of a feature, thereby associating the location of the feature with the barcode sequence of the feature. Subsequent analysis (eg, sequencing) can correlate the barcode sequence with the analyte in the sample. Thus, based on the relationship between the location and the barcode sequence, the location of the biological analyte (eg, in a specific cell type or in cells at a specific location in a biological sample) can be determined.
In some embodiments, a feature can have multiple nucleic acid barcode molecules linked thereto. A plurality of nucleic acid barcode molecules may include a barcode sequence. Multiple nucleic acid molecules associated with a particular feature may have the same barcode sequence or two or more different barcode sequences. Different barcode sequences can be used to provide improved spatial localization accuracy.
Analytes obtained from the samples, such as RNA, DNA, peptides, lipids and proteins, can be further processed as described above. In particular, the contents of individual cells from a sample can be equipped with a unique spatial barcode sequence so that analyte characterization can be attributed to the analyte originating from the same cell. More generally, spatial barcoding can be used to assign analytes to appropriate spatial locations in a sample. For example, hierarchical spatial localization of multiple spatial barcodes can be used to identify and characterize analytes at specific spatial regions of a sample. In some embodiments, the spatial region corresponds to a previously identified specific spatial region of interest, such as a previously identified specific cellular structure. In some embodiments, spatial regions correspond to small structures or populations of cells that cannot be seen with the naked eye. In some embodiments, unique molecular identifiers can be used to identify and characterize analytes at the single-cell level.
The analytes may include nucleic acid molecules, which may be barcoded by the nucleic acid barcode molecule sequence. In some embodiments, the barcoded analyte can be sequenced to obtain a nucleic acid sequence. In some embodiments, the nucleic acid sequence may include genetic information associated with the sample. The nucleic acid sequence may contain the barcode sequence or its complement. Barcoded sequences of nucleic acid sequences or their complements can be electronically linked to analyte properties (eg, color and/or intensity) using LUTs to identify associated features in the sequence.
(vii) Proximity Capture
In some embodiments, two-dimensional or three-dimensional spatial analysis of one or more analytes present in a biological sample can be performed using a proximity capture reaction, which is the detection of two assays that are spatially close to each other and/or interact. Things react to each other. For example, proximity capture reactions can be used to detect DNA sequences that are spatially close to each other, e.g. DNA sequences can be within the same chromosome but separated by about 700 bp or less. As another example, proximity capture reactions can be used to detect protein binding, e.g. two interacting proteins. Proximity capture reactions can be performed in situ to detect two analytes that are in spatial proximity and/or intercellularly interacting. Non-limiting examples of proximity capture reactions include DNA nanomicroscopy, DNA microscopy, and chromosome conformation capture methods. Chromosome conformation capture (3C) and experimental derivation procedures can be used to estimate spatial proximity between different genomic elements. Non-limiting examples of chromatin capture methods include chromosome conformation capture (3-C), on-chip conformation capture (4-C), 5-C, ChIA-PET, Hi-C, targeted chromatin capture (T2C). Examples of such methods are described e.g. in Miele et al.,Methods Molecular biology(2009), 464, Simon et al.,Wet. Genette.(2006), 38(11): 1348-54, Raab et al.,Embo. j.(2012), 31(2): 330-350, in Eagen et al.,Trends in biochemistry. science.(2018) 43(6): 469-478, each of which is incorporated herein by reference in its entirety.
In some embodiments, proximity capture reactions include proximity ligation. In some embodiments, proximate ligation may involve the use of antibodies with attached DNA strands that may participate in ligation, replication, and sequence decoding reactions. For example, a proximity ligation reaction may involve oligonucleotides attached to pairs of antibodies that can be joined by ligation if the antibodies are brought into close proximity to each oligonucleotide, for example by binding to the same target protein (complex) Yes, and the ligation DNA product is then used for PCR amplification sample as described by Söderberg et al.,method(2008), 45(3): 227-32, which is hereby incorporated by reference in its entirety. In some embodiments, proximity splicing may include methods of capturing chromosome conformation. In some embodiments, the proximity capture response is within a distance of about 400 nm (eg, about 300 nm, about 200 nm, about 150 nm, about 100 nm, about 50 nm, about 25 nm, about 10 nm, or about 5 nm). nm) from each other. In general, proximity capture reactions can be reversible or irreversible.
(viii) remove features from the array
A spatial array of barcodes can be associated with a biological sample to spatially detect analytes present in the biological sample. In some embodiments, features (eg, gel pads, beads) can be removed from the substrate surface for additional analysis (eg, imaging, sequencing, or quantification). For example, the features on the substrate delivered by the droplet manipulation systems described herein can be removed from the surface of the substrate. In some embodiments, features (eg, gel pads, beads) can be removed mechanically (eg, by scraping), enzymatic reactions, or chemical reactions. In some embodiments, features (eg, gel pads, beads) can be aspirated. In some embodiments, after removing the features (by any method), the features can be combined with unique barcoded beads. In some embodiments, the oligonucleotides within the feature can bind or hybridize to the barcode sequence on the barcode bead. For example, a spatial barcode oligonucleotide within a feature can be linked to a barcode sequence on a barcode bead. In addition, capture probes can be attached to barcode sequences on barcode beads. In some embodiments, the features and balls may be segmented. In some embodiments, features (eg, gel pads, beads) and uniquely labeled beads can be distributed into vesicles. In some embodiments, the vesicle may have a lipid bilayer. In some embodiments, the features and beads may be encapsulated. In some embodiments, the features and beads may be encapsulated in an oil emulsion. In some embodiments, the features and beads may be encapsulated in a water-in-oil emulsion. Once sectioned features (eg gel pads, beads) can be processed for further analysis (eg quantification, amplification or sequencing) according to any of the methods described herein.
(ix) Other applications
The spatial analysis methods described here can be used to detect and characterize the spatial distribution of one or more haplotypes in a biological sample. As used in this publication, haplotype is used to describe one or more mutations, DNA variations, polymorphisms in a given segment of the genome, which can be used to classify genetic segments or alleles A collection of nucleotide polymorphisms (SNPs) or genetic segments that make up one the genome. Haplotype association studies are used to better understand biological conditions. For example, identifying and characterizing haplotype variants at or associated with putative disease loci in humans can provide a basis for mapping the genetic causes of disease susceptibility. As used in the art, the term "locus" (plural "locus") can be a fixed location on a chromosome, including the location of a gene or genetic marker, which can contain multiple haplotypes, including alleles and SNPs.
Haplotype variant detection is a technique used to identify heterozygous cells in single-cell studies. In combination with spatial analysis, the detection of haplotype variants can further provide new information on the distribution of heterozygous cells in biological samples (eg, tissues) affected or exhibited by different biological conditions. These data can reveal causal relationships between haplotype variants and disease outcomes, help identify disease-associated variants, or reveal heterogeneity in biological samples.
In some embodiments, haplotype variant detection is a technique that can be used in conjunction with, in addition to, or as part of the spatial analysis methods described herein. Briefly, the discovery of haplotype variants may involve providing input data to run an algorithm on a computer system and performing analysis to identify and determine the spatial distribution of haplotypes. One input can be a plurality of sequence reads obtained from a two-dimensional spatial array contacted with a biological sample and then aligned to the genome. A sequence read can also contain spatial barcodes with positional information so that the sequence reads can be mapped to locations in the biological sample. Other inputs may include electronic data files of genetic sequence variants or haplotypes and reference genomes. For each locus, the corresponding sequence reads and variant haplotypes were aligned to determine the haplotype identity of each sequence read. The haplotype identities and spatial barcodes of the sequence reads are then classified to determine the spatial distribution of haplotypes within the biological sample. As mentioned above, this spatial distribution can be used to characterize the biological state of the sample. In some embodiments, sequence reads are obtained by in situ sequencing of a two-dimensional array of sites on a substrate, and in some embodiments, sequence reads are obtained by high-throughput sequencing. In some embodiments, other methods are used to generate the sequence reads described herein, such as paired-end sequencing.
In some embodiments, the respective of the plurality of loci are biallelic and the respective set of haplotypes for the respective loci consists of a first allele and a second allele. In some such embodiments, the corresponding locus comprises a heterozygous single nucleotide polymorphism (SNP), a heterozygous insertion, or a heterozygous deletion.
In some embodiments, analytes captured by any of the spatial analysis methods described herein can be analyzed (eg, sequenced) by in situ sequencing methods. For example, a substrate containing a plurality of capture probes (e.g., an array), connected directly or indirectly (e.g., via a feature), that includes a spatial barcode and a capture domain. In some embodiments, the capture domain can be configured to interact (eg, hybridize) with an analyte (eg, mRNA). In some embodiments, the biological sample can be contacted with the array such that the capture domains of the capture probes interact with (eg, hybridize) the analyte. In some embodiments, capture probes can be used as templates for hybridization or ligation reactions with captured analytes. For example, any of the reverse transcriptase examples described herein can be used to perform a reverse transcription reaction to extend the 3' end of a capture probe that hybridizes to an analyte, thereby creating an extended capture probe (e.g., an extended capture probe that includes spaced barcode and sequence complementary to the sequence in the analyte). After synthesis of the extended capture probe, a second strand complementary to the extended capture probe can be synthesized. In some embodiments, the synthesis of the second strand can be performed by any of the methods described herein. In some embodiments, amino-modified nucleotides can be used to generate extended capture probes or second strands, or both. For example, the amino-modified nucleotide can be aminoallyl (aa)-dUTP, aa-dCTP, aa-dGTP and/or aa-dATP.
In some embodiments, the second strand, or both the extended capture probe and/or the second strand, may be released from the substrate surface after the extended capture probe is generated. For example, the extended capture probe and/or second strand can be released by any of the methods described herein (eg, by heating or cleavage by a cleavage domain). In some embodiments, amino-modified nucleotides incorporated into extended capture probes can be cross-linked to a substrate surface or to a biological sample using their amino-modified nucleotides. In some embodiments, the surface of the substrate may be coated with a hydrogel. In some embodiments, the surface of the substrate may be coated with a protein matrix. In some embodiments, the cross-linking may be irreversible. In some embodiments, the crosslinked extended capture probe and/or second strand may be circularized. For example, templated circular ligation can be performed with a DNA ligase (eg, T4 DNA ligase), or template-free circular ligation can be performed with a template-independent ligase (eg, CircLigase). In some embodiments, the extended capture probe is circularized with CircLigase. In some embodiments, the circularized extended capture probes can be amplified. For example, amplification of the rolling circle can be performed with the appropriate DNA polymerase (eg, phi29). In some embodiments, capture probes include functional domains (eg, sequencing adapters). In some embodiments, rolling circle amplification can be performed with primers (eg, sequencing adapters) that are complementary to functional domains. In some embodiments, rolling circle amplification can be performed to generate two or more amplicons (eg, one or more amplicons containing any of the amine-modified nucleotides described herein). In some embodiments, two or more amplicons produced by rolling circle amplification can be cross-linked to a substrate surface and/or cross-linked to a biological sample. In some embodiments, two or more amplicons can be sequenced in situ. In situ sequencing can be performed by any of the methods described herein (see, Lee, J.H., Fluorescence In Situ RNA Sequencing for Gene Expression Profiling (FISSEQ), p.natural agreement., 10(3): 442-458, doi:10.1038/nprot.2014.191 (2015), incorporated herein by reference). In some embodiments, two or more amplicons may be displayed.
In some embodiments, ribosomal RNA (rRNA) can be spatially analyzed by any of the methods described herein, including endogenous ribosomal RNA (eg, native in a biological sample) and/or exogenous RNA (eg, microbial ribosomal RNA). Somatic RNA and/or viral RNA are also present in biological samples). As used herein, "metagenomics" may refer to the study of exogenous nucleic acids (eg, DNA, RNA, or other nucleic acids described herein) present in a biological sample. As used herein, "spatial metagenomics" may refer to the study of the spatial location of exogenous nucleic acids present in a biological sample. Spatial metagenomics can also refer to the study of identifying one or more species (eg, viruses or microorganisms) present in a biological sample and/or identifying patterns of proximity between species (eg, colocalization).
In some embodiments, microbial rRNA can be spatially detected, quantified and/or amplified from a biological sample. In some embodiments, rRNA (eg, 16S ribosomal RNA) can be associated with a particular microbial species. For example, microbial ribosomal RNA (e.g., 16S ribosomal RNA) can be used to identify one or more microorganisms present in a biological sample (see, e.g., Kolbert, C.P. and Persing, D.H., Ribosomal DNA Sequencing as a Tool for Identification of Bacterial Pathogens,Current Opinion in Microbiology2(3): 299-305. doi: 10.1016/S1369-5274(99)80052-6. PMID 10383862 (1999), incorporated herein by reference). In some embodiments, the identification of microbial species in the vicinity of one or more other microbial species can be identified.
In some embodiments, the biological sample is covered (eg, coated) or embedded with a photocrosslinked coating (eg, a conditionally soluble polymer, eg, a DTT-sensitive hydrogel). A biological sample can come into contact with a substrate coated with photocrosslinking. In some embodiments, the biological sample and the photocrosslinkable substrate are assembled in a flow cell, and the photocrosslinkable polymer can be incubated with the biological sample. Biological samples can be crosslinked into hydrogel voxels of defined size using a light source and a photomask. In some embodiments, the flow cell can be disassembled and washed to remove unpolymerized hydrogel. Photocrossovers can be treated with DTT to generate single cell fractions or near single cell fractions.
In some embodiments, single-cell or near-single-cell fractions can be encapsulated in vesicles. Vesicles may contain barcoded features (such as beads) and barcoded features may contain capture domains. In some embodiments, the capture domain can specifically bind microbial rRNA (eg, microbial 16S rRNA). In some embodiments, the captured microbial rRNA can be amplified and analyzed (eg, sequenced) by any of the methods described herein. In some embodiments, the amplified and sequenced microbial rRNA can identify microbial species and/or proximity patterns (eg, colocalization) of one or more species.
Alternatively, spatial analysis can be performed on exogenous rRNA (eg, microorganisms or viruses) with multiple capture probes on a substrate (eg, an array), where the capture probes include spatial barcodes and capture domains. In some embodiments, the capture domain can be configured to interact with (eg, hybridize with) a microbial rRNA present in a biological sample. Capture probes can be configured to interact with any microbial rRNA. In some embodiments, the capture probe is configured to interact with microbial 16S rRNA. A biological sample can be treated (eg, permeabilized) so that the capture domain and the analyte (eg, microbial rRNA) interact (eg, hybridize). In some embodiments, the captured analyte (eg, microbial rRNA) can be reverse transcribed to create an extended capture probe, followed by a second strand complementary to the extended capture probe described herein. The extended capture probe and/or second strand may comprise part or all of the capture probe sequence, or its complement. The capture probe sequence or its complement may comprise a spatial barcode or its complement. In some embodiments, the first cDNA strand and optionally the second cDNA strand can be amplified by any of the methods described herein. Amplified capture probes and analyte can be analyzed (eg, sequenced) by any of the methods described herein. Spatial information from spatial barcode features can be used to determine the spatial location of a captured analyte (eg, microbial rRNA) within a biological sample or portion thereof. In some embodiments, the captured analytes can identify the microbial species present in the biological sample or a portion thereof. In some embodiments, spatial information and properties of microbial species present in a biological sample can be correlated, revealing whether certain microbial species may be in close proximity to each other (eg, colocalize) in the biological sample.
In exemplary embodiments, provided herein are methods for detecting nucleic acids within a portion of a biological sample, comprising: (a) immobilizing the biological sample in a gel matrix to produce an embedded biological sample; (b) immobilization of the embedded biological sample separated into fractions; (c) lysed cells present in the fraction; (d) fractions from step (c) are encapsulated with beads having attached capture probes containing spatial barcodes and a capture domain that specifically binds nucleic acid in the fraction; (e) determining (i) all or part of the spatial barcode sequence or its complement, and (ii) all or part of the nucleic acid sequence or its complement, and using the determined sequences (i) and (ii) to detect nucleic acids within a portion of the biological sample . In some embodiments of these methods, the nucleic acid comprises microbial ribosomal RNA (rRNA). In some embodiments of these methods, the microbial rRNA comprises 16S rRNA. In some embodiments of these methods, the method includes detecting 16S rRNA from at least two different microorganisms within a portion of the biological sample. In some embodiments of these methods, the nucleic acid is mRNA.
Methods for the spatial analysis of analytes in biological samples are listed here. A profile of a biological sample (eg, a single cell, a population of cells, a tissue section, etc.) can be compared to profiles of other cells (eg, a "normal" or "healthy" biological sample). In some embodiments of any of the analyte spatial profiling methods described herein, the method can provide a diagnosis of a disease (eg, cancer, Alzheimer's disease, Parkinson's disease). In some embodiments of any of the methods described herein for the spatial analysis of analytes, the method can be used in drug screening. In some embodiments of any of the methods described herein for the spatial analysis of analytes, the method can be used for organoid drug screening. In some embodiments of any of the methods described herein for the spatial analysis of an analyte, the method can be used to detect changes in (eg, altered) cellular signaling. In some embodiments of any of the methods described herein for the spatial analysis of an analyte, the method may include introducing a pathogen into a biological sample and assessing the response of the biological sample to the pathogen. In some embodiments of any of the analyte spatial profiling methods described herein, the method includes exposing the biological sample to a perturbing agent (eg, any perturbing agent described herein) and assessing the response of the biological sample to the perturbing agent. In some embodiments of any of the methods described herein for the spatial analysis of an analyte, the method includes monitoring cellular differentiation in a biological sample (eg, an organoid). In some embodiments of any of the methods described herein for the spatial analysis of an analyte, the method includes analyzing tissue morphogenesis. In some embodiments of any of the methods described herein for spatially profiling an analyte, the method includes identifying spatial heterogeneity in a biological sample (eg, identifying different cell types or populations in a biological sample). In some embodiments of any of the methods described herein for the spatial analysis of an analyte, the method includes analyzing a spatiotemporal sequence (e.g., time) of molecular events. For example, a method for spatial analysis of an analyte may involve monitoring expression levels during a disease process.
The methods provided herein may also be used to determine a subject's relative level of inflammation (eg, to determine an inflammation score) or the subject's response or development of resistance to treatment. The methods described herein may also be used to identify candidate targets for potential therapeutic intervention and/or to identify biomarkers associated with various disease states in a subject.
(h) Quality control (i) Control samples
As used herein, the term "control sample" generally refers to a matrix that is insoluble in an aqueous liquid and allows accurate and traceable localization of the analyte of interest on the matrix. The terms "control sample" and "test substrate" are used interchangeably herein. The control sample can be any suitable substrate known to those skilled in the art. Exemplary control samples include semi-porous materials. Non-limiting examples of semiporous materials include nitrocellulose membranes, hydrogels, and nylon filters.
The control sample or test substrate may be of any suitable size or volume (eg, size or shape). In some embodiments, the control pattern is a regular shape (eg, square, circular, or rectangular). In some embodiments, the control pattern surface has any suitable shape or format. For example, the surface of the control sample may be flat or curved (eg, convexly or concavely curved towards the area where the interaction between the substrate and the control sample occurs). In some embodiments, the control patterns have rounded corners (eg for added security or robustness). In some embodiments, the control sample has one or more cut corners (eg, for use with slide holders or cross stands).
A control sample may contain multiple test analytes. In some embodiments, members of a plurality of test analytes are deposited on the substrate in known amounts and at known locations. For example, multiple test analytes are placed in known amounts at one or more locations on a control sample. In some embodiments, a plurality of test analytes are distributed on a substrate in a defined pattern (eg, an x-y grid pattern). In some embodiments, the defined pattern includes one or more locations or points.
In some embodiments, each site contains multiple test analytes of the same type. In some embodiments, each site contains a plurality of one or more different types of test analytes. In some embodiments, each spot on the control sample represents a different region of the biological sample, e.g., a tissue sample. In some embodiments, areas on the control sample that do not contain a plurality of test analytes represent areas where there is no biological sample.
In some embodiments, the plurality of test analytes includes one or more test analytes,
For example, a first test analyte, a second test analyte, a third test analyte, a fourth test analyte, etc. In some embodiments, the plurality of test analytes comprise nucleic acids. In some embodiments, each position or feature includes a population of nucleic acid sequences. In some embodiments, the nucleic acid sequence of the first test analyte differs from the nucleic acid sequence of the second test analyte by one nucleic acid residue. In some embodiments, each location or feature comprises a set of RNA transcripts and one or more specific surface marker proteins or one or more CRISPR guide RNAs. In some embodiments, the plurality of test analytes comprises a bacterial artificial chromosome (BAC). In some embodiments, each spot on the control sample contains a unique mixture of BACs. In some embodiments, proteins are cross-linked to BACs, for example, to mimic histone binding to DNA.
In some embodiments, the concentration of the first test analyte is different from the concentration of the second test analyte at another location or feature on the control sample. In some embodiments, the first test analyte and the second test analyte comprise the same nucleotide sequence.
Control samples can be used to determine process variations. A bar code array can be placed on top of a control sample containing multiple test analytes, wherein members of the multiple test analytes can be distributed in known amounts and at known positions on the substrate. The array can then be removed, and the molecular information within it can be determined by performing next-generation sequencing library construction, followed by computational analysis to correlate the expression values of the test analytes with barcodes (eg, spatial barcodes) on the array. Sequencing data can be compared to known amounts and known locations of multiple test analytes to determine if the spatial analysis workflow accurately detects the presence, amount, location, or combination of test analytes to determine process bias space. Analytical workflow.
(ii) RNA Spatial Integrity Number (sRIN)
As used herein, the term "spatial RNA integrity number" or "sRIN" refers to an in situ indication of RNA quality based on an integrity score. Higher sRIN scores correlate with higher data quality in the spatial analysis described here. For example, a first biological sample with a high sRIN score will have higher data quality than a second biological sample with a lower sRIN score than the first biological sample. In some embodiments, the sRIN is calculated for a tissue portion, one or more regions of a tissue portion, or a single cell.
In some embodiments, one or more sRINs for a given biological sample (e.g., a tissue section, one or more tissue regions, or a single cell) are calculated by: (a) providing (i) a spatial array containing multiple A capture probes on a substrate, wherein the capture probe comprises a capture domain and (ii) tissue stained with a histological stain (eg, any stain described herein); (b) combining the spatial array with the contact of a biological sample (eg tissue); (c) capturing a biological analyte (eg 18S rRNA molecule) from a biological sample (eg tissue) with a capture domain; (d) generation of cDNA molecules; (e) hybridizing one or more labeled oligonucleotide probes to the cDNA; (f) imaging the labeled cDNA and a histological stain (e.g., any of the stains described herein), and (g) generating an RNA spatial integrity number for a position in the spatial array, wherein the RNA spatial integrity number includes an image of the labeled cDNA and a histological stain (e.g., any which stain described here) position image.
In some embodiments, the biological sample (eg, tissue) is stained with a histological dye. As used herein, a "histological stain" can be any stain described herein. For example, biological samples can be stained with the IF/IHC dyes described herein. For example, a biological sample (eg, tissue) can be stained with hematoxylin and eosin ("H&E"). In some embodiments, the biological sample (e.g., tissue) is stained with a histological dye (e.g., any dye described herein) before, concurrently with, or after labeling the cDNA with a labeled oligonucleotide probe. In some embodiments, the stained biological sample can be optionally destained (eg, washed 1, 2, 3, 4, 5 or more times in a low pH acid (eg, HCl)). For example, hematoxylin from H&E stains can be optionally removed from biological specimens by washing in dilute HCl (0.001 M to 0.1 M) before further processing. In some embodiments, the stained biological sample can be optionally destained after imaging and prior to permeabilization.
In some embodiments, the spatial array includes a plurality of capture probes immobilized on a substrate, wherein the capture probes include at least one capture domain. In some embodiments, the capture domain includes a poly(T) sequence. For example, capture domains include poly(T) sequences capable of capturing 18S rRNA transcripts from biological samples.
In some embodiments, calculating one or more spatial RNA integrity values for a biological sample includes combining at least one (eg, at least two, at least three, at least four, or at least five) labeled oligonucleotide probes. Hybridization with cDNA generated from biological samples. 18s rRNA. In some embodiments, the labeled oligonucleotide probe includes a sequence that is complementary to a portion of the 18S cDNA. In some embodiments, four labeled oligonucleotide probes (P1-P4) are designed to hybridize to four different locations throughout the body of the 18S rRNA gene. In some embodiments, a labeled oligonucleotide probe can include any of the detectable labels described herein. For example, probes labeled with oligonucleotides may include a fluorescent label (eg, Cy3). In some embodiments, one or more labeled oligonucleotide probes designed to be complementary to different positions within the 18S cDNA sequence include the same detectable label. For example, four labeled oligonucleotide probes (P1-P4), each designed to be complementary to a different position in the 18S cDNA sequence, can all have the same detectable label (eg, Cy3). In some embodiments, one or more labeled oligonucleotide probes designed to be complementary to different locations within the 18S cDNA sequence include different detectable labels. For example, each of the four labeled oligonucleotide probes (P1-P4) designed to be complementary to a different site within the 18S cDNA sequence can include a different detectable label.
In some embodiments, determining the spatial value of RNA integrity of a biological sample (eg, a tissue section, one or more tissue regions, or a single cell) includes analysis from spatial arrays and histological staining (eg, any of the dyes described herein). ) for the same location. For example, for spatial arrays, all images are generated by laser scanning (eg, wavelength 532 nm) after hybridization of fluorescently labeled (eg, Cy3) oligonucleotide probes to 18S cDNA. One image is generated for each probe (P1-P4) and one image (P0) is generated when no fluorescently labeled probe is hybridized. Normalization of fluorescence unit (FU) data was performed by subtracting the autofluorescence recorded at P0 and dividing by P1. After alignment, five images (one image from each probe, P1-P4, and one image from the area without bound probes) were loaded into the script. The script generates two different plots, a heatmap of spatial RIN values and an image alignment error plot that combines histologically stained (eg, any of the stains described here) images. The image alignment error map is used to visualize which pixels and locations should be excluded from the analysis due to alignment errors between images P0-P4.
3. General methods of spatial cellular analysis (a) Barcoded biological samples
In some embodiments, provided herein are methods for attaching and/or introducing molecules (e.g., peptides, lipids, or nucleic acid molecules) having barcodes (e.g., spatial barcodes) to a biological sample (e.g., cells in a biological sample) for spatial analysis. In some embodiments, a plurality of molecules (eg, a plurality of nucleic acid molecules) having a plurality of barcodes (eg, a plurality of spatial barcodes) are introduced into a biological sample (eg, a plurality of cells in a biological sample) for spatial analysis.
In some embodiments, a plurality of molecules (eg, a plurality of lipid or nucleic acid molecules) having a plurality of barcodes (eg, a plurality of spatial barcodes) are introduced into a biological sample (eg, a plurality of cells of a plurality of cells). biological samples) for spatial analysis, where multiple molecules are introduced into the biological sample in sequence. In some embodiments, a plurality of molecules (eg, a plurality of lipid or nucleic acid molecules) having a plurality of barcodes are provided in any array of arrays on a matrix (eg, any of the various arrays described herein). ) as described herein, and the biological sample contacts the molecules on the substrate so that the molecules are introduced into the biological sample. In some embodiments, the molecule introduced into the biological sample can be cleaved to the substrate and upon contact with the biological sample is cleaved from the substrate and released into the biological sample. In some embodiments, the molecule introduced into the biological sample is covalently bound to the substrate prior to cleavage. In some embodiments, molecules introduced into the biological sample are non-covalently bound to the matrix (eg, by hybridization) and are released from the matrix into the biological sample when contacted with the biological sample.
In some embodiments, a plurality of molecules (eg, a plurality of lipid or nucleic acid molecules) having a plurality of barcodes (eg, a plurality of spatial barcodes) migrate or are transferred from the matrix into the cells of the biological sample. In some embodiments, migration of the plurality of molecules from the matrix into cells of the biological sample involves applying a force (eg, mechanical, centrifugal, or electrophoretic force) to the matrix and/or biological sample to facilitate migration of the plurality of molecules. molecules from the matrix into the biological sample.substrate.
In some embodiments of any of the spatial analysis methods described herein, physical forces are used to facilitate attachment or introduction of barcoded molecules (eg, nucleic acid molecules) (eg, spatial barcodes) to a biological sample (eg, biological sample). , cells present in biological samples). As used herein, "physical force" refers to the use of physical force to overcome cell membrane barriers to facilitate intracellular delivery of molecules. Examples of physical force instruments and methods that may be used in accordance with the materials and methods described herein include the use of needles, ballistic DNA, electroporation, sonication, photoporation, magnetofection, hydroporation, and combinations thereof.
(i) Introduction of station marking means to the station surface
In some embodiments, a biological sample (e.g., cells in a biological sample) can be labeled with a cell labeling agent that facilitates the introduction of molecules (e.g., nucleic acid molecules) with a barcode (e.g., a spatial barcode) into the biological sample (e.g., cells in a biological sample). As used herein, the term "cell tagging agent" refers to a molecule having a moiety that can bind to a cell surface (eg, thereby attaching a barcode to the cell surface) and/or penetrate and pass through a cell membrane (eg .by which the barcode is introduced into the cell) internal). In some embodiments, the cell tagging means includes a barcode (eg, a spatial barcode). The bar code of the barcoded cell marker can be any of the array of bar codes described herein. In some embodiments, the barcode of the barcode cell labeling means is a spatial barcode. In some embodiments, the cell labeling agent comprises a nucleic acid molecule that comprises a barcode (eg, a spatial barcode). In some embodiments, barcode cell labeling agents identify associated molecules, each barcode being associated with a specific molecule. In some embodiments, one or more molecules are applied to the sample. In some embodiments, the nucleic acid molecule comprising the barcode is covalently linked to a cell labeling agent. In some embodiments, the nucleic acid molecule comprising the barcode is non-covalently linked to a cell labeling agent. A non-limiting example of non-covalent binding includes hybridization of a nucleic acid molecule containing a barcode to a nucleic acid molecule on a cell labeling agent (the nucleic acid molecule on a cell labeling agent can bind the cell labeling agent covalently or non-covalently). In some embodiments, the nucleic acid molecule associated with a cell-marking agent comprising a barcode (eg, a spatial barcode) further comprises one or more additional domains. Such additional domains include, but are not limited to, PCR handles, sequencing start sites, domains for hybridization to another nucleic acid molecule, and combinations thereof.
In some embodiments, the cell labeling agent is attached to the surface of the cell. When the cell labeling agent includes a barcode (eg, a nucleic acid including a spatial barcode), the barcode is also attached to the surface of the cell. In some embodiments of any of the spatial analysis methods described herein, the cell labeling agent is covalently bound to the cell surface to facilitate introduction of the spatial analysis reagent. In some embodiments of any of the spatial analysis methods described herein, the cell labeling agent is non-covalently bound to the cell surface to facilitate introduction of the spatial analysis reagent.
In some embodiments, after one or more cells in the biological sample have been spatially labeled with a cell labeling agent, spatial analysis of analytes present in the biological sample is performed. In some embodiments, such spatial analysis includes separating spatially labeled cells of the biological sample (or a subset of spatially labeled cells of the biological sample) and analyzing the analyte present in those cells on a cell-by-cell basis. Any of a number of methods can be used to analyze analytes present in cells on a cell-by-cell basis. Non-limiting examples include any of the various methods described herein and the methods described in PCT application no. WO 2019/113533A1, the contents of which are incorporated herein by reference in their entirety. For example, spatially labeled cells can be encapsulated with beads containing one or more nucleic acid molecules (eg, emulsions) with barcodes (eg, cellular barcodes). The nucleic acid present on the bead may have a domain that hybridizes to a domain on the nucleic acid present on the labeled cell (eg, a domain on the nucleic acid linked to a cell labeling agent), thus linking the cellular barcode of the cell spatially to the barcode on the bead. Once the spatial barcode of the cell and the cellular barcode of the bead are linked, the analyte present in the cell can be analyzed using a capture probe (eg, a capture probe present on the bead). This allows (using these methods) nucleic acids produced from specific cells to be individually amplified and sequenced (eg in separate compartments or droplets).
In some embodiments, spatial analysis of analytes present in a biological sample is performed after one or more cells in the biological sample are spatially labeled with a cell labeling agent, wherein the cells of the biological sample are not dissociated into individual cells. In such embodiments, various spatial analysis methods may be used, such as any of those provided herein. For example, when one or more cells in a biological sample are spatially labeled with a cell labeling agent, analytes in the cells can be captured and analyzed. In some embodiments, the cell labeling means includes a spatial barcode and a capture domain capable of capturing an analyte present in the cell. For example, cell markers including spatial barcodes and capture domains can be introduced into the cells of a biological sample in such a way that the location of the cell markers is known (or can be determined after being introduced into the cells). cell). A non-limiting example of introducing a cell marker into a biological sample is to provide a cell marker in an array (e.g., on any substrate such as the various substrates and arrays provided herein), where the location of the cell marker on the array is known at the time of introduction (or can be determined after introduction). . Cells can be permeabilized if necessary (e.g., using permeabilization agents and methods described herein), and cells can be supplied with reagents for analyte analysis (e.g., reverse transcriptase, polymerase, nucleotides, etc., where the analyte is absent) is a nucleic acid bound to the capture probe) and the analyte can be analyzed. In some embodiments, the analyte under test (and/or a copy thereof) can be released from the matrix and analyzed. In some embodiments, the tested analyte (and/or a copy thereof) is tested in situ.
Non-limiting examples of cell labeling agents and systems that may be used in accordance with the materials and systems that attach to the cell surface (eg, by introducing the cell labeling agent and any barcodes attached thereto to the outside of the cell) and the methods for use herein in biological samples. Methods for spatial analysis of one or more analytes include: lipid-labeled primers/lipophilic-labeled moieties, positive or neutral oligo-conjugated polymers, antibody-labeled primers, streptavidin-conjugated oligonucleotides, dye-labeled oligonucleotides, click chemistry, receptor-ligand systems, covalent binding systems via amine or thiol functional groups and their combination.
(ii) Introduction of station marking means inside the stations
Materials and methods that may be provided herein for the spatial analysis of one or more analytes in a biological sample include: cell-penetrating agents (eg, cell-penetrating peptides), nanoparticles, liposomes, polymers, vesicles, peptide-based chemical delivery, electroporation , sonoporation, lentiviral vectors, retroviral vectors and their combinations.
In some embodiments, the cell-labeling agent includes a cell-penetrating agent (described below). In some embodiments, the cell-penetrating agent transports the cell-labeling agent into the cells of the biological sample. When the cell labeling agent contains a barcode (for example, a nucleic acid containing a spatial barcode), the barcode also penetrates the cell. In some embodiments, the plurality of cell markers is cleaved (eg, photocleaved) from the array by the cleavage domain, thereby releasing the cell markers from the array and allowing at least one probe to capture the cell marker array to pass through. Cell-labeling agents can then interact with intracellular biological analytes through the capture domain. In some embodiments, the plurality of capture probes migrate from the array into the cells of the biological sample with the aid of a cell-penetrating agent. In some embodiments, migration of the plurality of capture probes from the array into the cells of the biological sample involves applying a force (eg, mechanical, centrifugal, or electrophoretic force) to the biological sample.
In some embodiments, the biological sample is treated with one or more reagents to facilitate the migration of a plurality of free (eg, cleaved) capture probes into the cells of the biological sample. In one embodiment, an organic solvent (eg, methanol or acetone) can be used to permeabilize the cells of the biological sample. In another embodiment, detergents (eg, saponin, Triton X-100™ or Tween-20™) can be used to permeabilize the cells of the biological sample. In yet another embodiment, an enzyme (eg, trypsin) can be used to permeabilize the cells of the biological sample. The methods disclosed herein may be practiced using any suitable cell permeabilization method. In some embodiments, the biological sample may be incubated with a cell permeabilization reagent after contacting the array with the biological sample. In some embodiments, biological samples can be fixed according to the methods described herein.
In some embodiments, migration of the plurality of released (eg, cleaved) capture probes into cells of the biological sample involves passive migration (eg, diffusion). In some embodiments, migration of the plurality of released (eg, cleaved) capture probes into cells of the biological sample involves active migration (eg, electrophoretic migration). In some embodiments, migration of the plurality of released (eg, cleaved) capture probes into cells of the biological sample involves antibodies. In some embodiments, migration of the plurality of released (eg, cleaved) capture probes into cells of the biological sample involves transfection (eg, chemical, biological, physical, viral vector).
1. Cell breakers
In some embodiments of any of the spatial analysis methods described herein, cell-by-cell penetration facilitates the identification of a biological analyte by a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) and capture of an agent domain. In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and a capture domain is linked to a cell-penetrating agent, and the cell-penetrating agent allows the molecule to interact with an analyte inside the cell. As used herein, a "cell-penetrating agent" may refer to an agent capable of facilitating the introduction of a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and a capture domain into a cell. Biological samples (see, e.g., Lovatt et al. Nat Methods. 2014 Feb;11(2):190-6, which is incorporated herein by reference in its entirety). In some embodiments, the cell penetrating agent is a cell penetrating peptide. As used herein, a "cell-penetrating peptide" refers to a peptide that has the ability to pass through cell membranes (eg, a short peptide, eg, a peptide that typically does not exceed 30 residues). In some embodiments, the cell-penetrating agent or cell-penetrating peptide can be covalently or non-covalently attached to a molecule (eg, a barcoded nucleic acid molecule), possibly at the 5' end of the molecule. Cell-penetrating peptides can target barcoded nucleic acid molecules to specific organelles.
In some embodiments of any of the spatial analysis methods described herein, a cell-penetrating peptide associated with a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) and a capture domain may use an energy-dependent or energy-independent mechanism . For example, cell-penetrating peptides may undergo direct translocation by physical perturbation of the plasma membrane, endocytosis (eg, clathrin-mediated), adaptive translocation, pore formation, electroporation-like permeabilization, and/or into microcellular membranes, domain boundaries. Non-limiting examples of cell-penetrating peptides include: penetratin, tat peptide, pVEC, transporter, MPG, Pep-1, polyarginine peptide, MAP, R6W3, (D-Arg)9, Cys(Npys)-(D-Arg)9, Anti-BetaGamma (MPS - phosducin - protein C terminus), Cys(Npys) antenapedia, Cys(Npys)-(Arg)9, Cys(Npys)-TAT (47-57), HIV-1 Tat (48- 60) , KALA, mastoparan, penetratin-Arg, pep-1-cysteamine, TAT(47-57)GGG-Cys(Npys), Tat-NR2Bct, transdermal peptide, SynB1, SynB3, PTD -4, PTD-5, FHV Coat- (35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), R9-tat, SBP, FBP, MPG, MPG(ΔNLS), Pep-2, MTS, plsl and polylysine peptides (see, e.g., Bechara et al. FEBS Lett. 2013 Jun 19;587(12):1693-702, which is incorporated herein by reference in its entirety).
In some embodiments, the cell penetrating peptide (CPP) conjugation can have two orientations. For example, one orientation may be (N-terminal)-CPP-Cys-(C-terminal)-linker-NH2C6-5'-oligo-3'; 3'-oligo-5'-NH2C6-linker-(N-terminal)-Cys-CPP-(C-end). The methods outlined here can be performed with other CPP conjugations and orientations.
In some embodiments, the cell-labeling agent further comprises a cell-penetrating marker. As used herein, a "cell-penetrating label" refers to an agent that can be detected within a cell. In some embodiments, the cell-penetrating label includes a fluorophore. In some embodiments, the cell-penetrating label is selected from the group consisting of: Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, Fluorescein (6-FAM), DyLight, Alexa Fluor®, and Tetramethylrhodamine ( TAMRA ) azide.
In some embodiments, a cell-penetrating label is detected. In some embodiments, the cell-penetrating labels are detected after the plurality of capture probes are released from the array and after the array is removed from the biological sample. In some embodiments, the introduction of a cell-labeling agent into a cell is determined by detecting the presence of a cell-permeable label in the cell.
In some embodiments, the cell labeling agent can optionally include an intracellular cleavage domain, wherein one or more segments or regions of the capture probe (e.g., capture domain, spatial barcode, and/or UMI) can be released and bound to the cell a permeabilizing agent that can be cleaved or cleaved so that the capture domain, spatial barcode, and/or UMI can be released. In some embodiments, cleavage of the linkage between the capture domain, spatial barcode, and/or UMI and the cell-penetrating agent is induced in an intracellular environment (eg, cleavage of the intracellular cleavage domain upon introduction of the cell-labeling agent into the cell). In some embodiments, the intracellular cleavage domain comprises disulfide bonds. For example, the intracellular cleavage domain may be a disulfide bond that is cleaved by reducing conditions in the cell. Any other suitable linker can be used to release or cleave the intracellular cleavage domain of the capture probe.
2. Nanoparticles
In some embodiments of any of the spatial analysis methods described herein, the biological analyte is captured by an inorganic particle (eg, a nanoparticle) using a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and an imaging domain. In some embodiments, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) and a capture domain is linked to an inorganic particle (e.g., a nanoparticle), and the molecule (e.g., a nucleic acid has a barcode (e.g. .spatial barcodes) and molecular domain capture) use nanoparticles to capture analytes inside cells. Non-limiting examples of nanoparticles that can be used in embodiments herein to deliver molecules (e.g., nucleic acid molecules) having barcodes (e.g., spatial barcodes) and capture domains into cells and/or cell beads include inorganic nanoparticles made of Metals (such as is iron, gold and silver), inorganic salts and ceramics (such as phosphates or carbonates of calcium, magnesium or silicon). The surface of nanoparticles can be coated to facilitate binding of molecules (e.g., nucleic acid molecules) with barcodes (e.g., spatial barcodes) and capture domains, or the surface can be chemically modified to facilitate binding of molecules (e.g., nucleic acid molecules ).molecule) has a barcode (eg a spatial barcode) and a recording domain. Magnetic nanoparticles (eg supermagnetic iron oxide), fullerenes (eg soluble carbon molecules), carbon nanotubes (eg cylindrical fullerenes), quantum dots and supramolecular systems can also be used.
3. Liposomes
In some embodiments of any of the spatial analysis methods described herein, liposomes facilitate capture of biological analytes by molecules (eg, nucleic acid molecules) having a barcode (eg, spatial barcode) and a capture domain. Various types of lipids can be used for liposomal delivery, including cationic lipids. In some cases, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) and capture domains are delivered to cells via lipid nanoemulsions. A lipid emulsion refers to the dispersion of one immiscible liquid in another liquid that is stabilized by an emulsifier. Cell labeling may involve the use of solid lipid nanoparticles.
4. Polymers
In some embodiments of any of the spatial analysis methods described herein, the polymer facilitates the capture of a biological analyte by a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., spatial barcode) and a capture domain. In some embodiments, a molecule (e.g., a nucleic acid molecule) with a barcode (e.g., a spatial barcode) and a capture domain is contained within a polymersome, and the molecule (e.g., a nucleic acid molecule) with a barcode (e.g., a spatial barcode) ). , spatial barcodes) and capture domains are used by polymersomes to capture analytes inside cells. A "polymersome" as referred to herein is an artificial vesicle. For example, the polymersome may be a liposome-like vesicle but with a membrane containing an amphiphilic synthetic block copolymer (see, e.g., Rideau et al. Chem. Soc. Rev., 2018, 47, 8572-8610, which is incorporated herein by reference entirely). In some embodiments, the polymersome comprises a di-(AB) or triblock copolymer (eg, ABA or ABC), where A and C are hydrophilic blocks and B is a hydrophobic block. In some embodiments, the polymersome contains poly(butadiene)-b-poly(ethylene oxide), poly(ethylvinyl)-b-poly(ethylene oxide), polystyrene-b-poly(ethylene oxide), poly(2-vinylpyridine) - b -poly(ethylene oxide), polydimethylsiloxane -b-poly(ethylene oxide), polydimethylsiloxane Siloxane-g-poly(ethylene oxide), polycaprolactone-b-poly(ethylene oxide), polyisobutylene-b-poly(ethylene oxide), polystyrene - b-polyacrylic acid, polydimethylsiloxane-b-poly-2-methyl-2-oxazoline or combinations thereof (where b=block and g=grafted).
5. Peptide-based chemical carriers
In some embodiments of any of the spatial analysis methods described herein, a peptide-based chemical carrier, e.g., a cationic peptide-based chemical carrier. Cationic peptides can be rich in basic residues such as lysine and/or arginine. In some embodiments of any of the spatial analysis methods described herein, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and a capture domain captures a biological analyte through a polymer-based chemical carrier. Cationic polymers, when mixed with molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) and capture domains, can form nanoscale complexes called polyplexes. Polymer-based carriers can contain natural proteins, peptides and/or polysaccharides. Polymer-based carriers may contain synthetic polymers. In some embodiments, the polymer-based carrier comprises polyethyleneimine (PEI). PEI can condense DNA into positively charged particles that bind to anionic cell surface residues and enter cells through endocytosis. In some embodiments, the polymer-based chemical carrier comprises poly(L)-lysine (PLL), poly(DL-lactic acid) (PLA), poly(DL-lactide-co-glycoside) (PLGA), polyornithine, polyarginine, histone , protamine or their combinations. A polymer-based carrier may contain a mixture of polymers such as PEG and PLL. Other non-limiting examples of polymers include dendrimers, chitosan, synthetic amino derivatives of dextran, and cationic acrylic polymers.
6. Electroporation
In some embodiments of any of the spatial analysis methods described herein, the capture of a biological analyte by a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and a capture domain is facilitated by electroporation. By electroporation, a biological analyte having a barcode (eg spatial barcode) and a molecule (eg nucleic acid molecule) with a capture domain can enter the cell through one or more pores in the cell membrane formed by the application of electrical energy. . Membrane pores can be reversible based on applied field strength and pulse duration.
7. Sound hole
In some embodiments of any of the spatial analysis methods described herein, the capture of a biological analyte by a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and a capture domain is facilitated by sonoporation. Cell membranes can be temporarily permeabilized using sound waves, allowing cellular uptake of biological analytes by molecules with barcodes (eg, spatial barcodes) and capture domains (eg, nucleic acid molecules).
8. Lentivirus and retrovirus vectors
In some embodiments of any of the spatial analysis methods described herein, the capture of a biological analyte by a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) and a capture domain is facilitated by a carrier. For example, a vector described herein can be an expression vector, wherein the expression vector includes a promoter sequence and a capture domain operably linked to a sequence encoding a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode ) and recording domain. Non-limiting examples of vectors include plasmids, transposons, cosmids, and virus-derived vectors (eg, any adenovirus (AV)-derived vector, cytomegalovirus (CMV)-derived vector, simian virus-derived vector (SV40), adenovirus-associated vector viral ( AAV), lentiviral and retroviral vectors) and any Gateway® vector. For example, a vector may include sufficient cis-acting expression elements, where other expression elements may be provided by a mammalian host cell or an in vitro expression system. The skilled practitioner will be able to select appropriate vectors and mammalian cells for introducing any of the spatial analysis reagents described herein.
9. Additional methods and means for labeling cells for intracellular introduction of molecules
In some embodiments of any of the spatial analysis methods described herein, the biological analyte is captured by a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) and a capture domain, e.g. for injection (eg microinjection), particle bombardment, photoporation, magnetofection and/or hydroporation. For example, by particle bombardment, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) and capture domains can be coated with heavy metal particles and delivered to cells at a high rate. In photoporation, laser pulses can be used to create transient pores in the cell membrane, allowing cellular uptake of molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) and capture domains. In magnetofection, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) and capture domains can be coupled to the magnetic field of magnetic particles (eg, magnetic nanoparticles, nanowires, etc.). In hydrogenation, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) and capture domains can be delivered to cells and/or cell granules by hydrodynamic pressure.
(iii) Lipid labeling primer/lipophilic labeling moiety
In some embodiments of any of the spatial analysis methods described herein, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) is bound to a lipophilic molecule. In some embodiments, the lipophilic molecule is capable of delivering the lipophilic molecule to the cell membrane or nuclear membrane. In some embodiments, molecules (eg, nucleic acid molecules) having barcodes (eg, spatial barcodes) attached to lipophilic molecules can bind and/or insert into lipid membranes, such as cell membranes and nuclear membranes. In some cases, insertions may be reversible. In some cases, the link between the lipophilic molecule and the cells may allow for subsequent processing of the cells (eg, distribution, cell permeabilization, expansion, contraction, etc.). In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) linked to a lipophilic molecule can enter the intracellular space and/or the nucleus.
Non-limiting examples of lipophilic molecules useful in the embodiments described herein include sterol lipids such as cholesterol, tocopherol, sterols, palmitate, lignoceric acid, and derivatives thereof. In some embodiments, the lipophilic molecule is a neutral lipid conjugated to a hydrophobic moiety such as cholesterol, squalene or a fatty acid (see Raouane et al.bioconjugate chemistry., 23(6):1091-1104 (2012), which is incorporated herein by reference in its entirety). In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be linked to a lipophilic moiety via a linker using a covalent bond or a direct bond. In some embodiments, the linker is a tetraethylene glycol (TEG) linker. Other examples of linkers include, but are not limited to, a C6 amino linker, a C12 amino linker, a C3 spacer, a C6 spacer, a C12 spacer, a 9 spacer, and a 18 spacer. In some embodiments, a molecule (e.g., a nucleic acid molecule) having a barcode ( eg spatial barcode) is linked indirectly (eg by hybridization or ligand-ligand interaction, such as biotin-streptavidin) to avidin lipid molecules. In some embodiments, a lipophilic moiety can be attached to a capture probe, spatial barcode, or other DNA sequence at the 5' or 3' end of a particular DNA sequence. In some embodiments, lipophilic moieties can be linked to capture probes, spatial barcodes, or other DNA sequences in a lipid-dependent manner. Other lipophilic molecules that can be used in accordance with the methods provided herein include amphiphilic molecules in which the head group (e.g., charge, aliphatic content, and/or aromatic content) and/or fatty acid chain length (e.g., C12, C14, C16, or C18 ) may vary. For example, fatty acid side chains (eg, C12, C14, C16, or C18) can be linked to glycerol or glycerol derivatives (eg, 3-tert-butyldiphenylsilylglycerol), which can also contain, for example, a cationic head group. In some embodiments, molecules (eg, nucleic acid molecules) having barcodes (eg, spatial barcodes) disclosed herein can then be linked (directly or indirectly) to these amphiphilic molecules. In some embodiments, molecules (eg, nucleic acid molecules) having barcodes (eg, spatial barcodes) attached to amphiphilic molecules can bind and/or insert into membranes (eg, cells, cell beads, or nuclear membranes). In some cases, the amphiphilic or lipophilic moiety can cross the cell membrane and deliver the barcoded molecule (eg, nucleic acid molecule) (eg, spatial barcode) into the cell and/or the inner region of the cell sphere.
In some embodiments, additives may be added to supplement the lipid-based modifications. In some embodiments, the additive is low-density lipoprotein (LDL). In some embodiments, the additive is a cholesterol transfer inhibitor U-18666A. In some embodiments, U-18666A inhibits the transport of cholesterol from micromolar concentrations of late endosomes and/or nanomolar concentrations of lysosomes to the endoplasmic reticulum (ER). In some embodiments, U-18666A can inhibit oxidized squalene cyclase, a key enzyme in the cholesterol biosynthetic pathway, at sufficiently high concentrations (eg, at or about >0.5 mM).
In some embodiments, where the molecule (e.g., having a nucleic acid sequence) has an amino group within the molecule, the molecule (e.g., nucleic acid molecule) having a bar code (e.g., a spatial barcode) and the amino group can be conjugated to an amine-reactive lipophilic molecule. For example, molecules (e.g., nucleic acid molecules) with barcodes (e.g., spatial barcodes) and amino groups can be conjugated to DSPE-PEG(2000)-cyanide chloride (1,2-distearoyl-sn-glycerol)-3 - phosphoethanolamine -N-[cyano(polyethyleneglycol)-2000]).
In some embodiments, cell labeling agents can be attached to the cell surface via a combination of lipophilic and covalent bonds. For example, cell labeling agents can include lipid-linked oligonucleotides to target the oligonucleotides to cell membranes and amine groups that can be covalently attached to cell surface proteins by the various chemical procedures described herein. In these embodiments, lipids can increase the surface concentration of oligonucleotides and can facilitate covalent reactions.
As used herein, "anchor oligonucleotides" and/or "co-anchor oligonucleotides" may include lipid-conjugated oligonucleotides in which the lipids are capable of intercalating within cell membranes. In some embodiments, lipids that can be intercalated into cell membranes include, but are not limited to, sterol lipids such as cholesterol, tocopherol, sterol, palmitate, lignoceric acid, and derivatives thereof. In some embodiments, the sterol lipid of the anchored oligonucleotide and/or co-anchored oligonucleotide may be attached to the 5' or 3' end of the oligonucleotide portion. In some embodiments, the anchor oligonucleotide and/or the auxiliary anchor oligonucleotide can be integrated into the cell membrane of the cells in the biological sample (eg, the anchor oligonucleotide and/or the auxiliary anchor oligonucleotide acidic sterol lipids).
In some embodiments, a sterol lipid (eg, lignoceric acid) anchors the oligonucleotide to the 5' end of the oligonucleotide. In some embodiments, the anchor oligonucleotides may have a constant sequence. In some embodiments, the constant sequence of the anchored oligonucleotide can be about 15 to about 30 nucleotides in length. In some embodiments, the anchor oligonucleotide may have an additional 3' constant sequence domain. In some embodiments, the additional domain may be an adapter sequence (eg, a sequencing adapter). In some embodiments, the adapter sequence can be about 15 to about 35 nucleotides in length.
In some embodiments, a lipid (eg, a sterol lipid) that co-anchors an oligonucleotide (eg, palmitic acid) is attached to the 3' end of the oligonucleotide. In some embodiments, the co-anchored oligonucleotide may have a constant sequence. For example, the constant sequence of the co-anchor oligonucleotide may be the reverse complement of the constant sequence from the anchor oligonucleotide. In some embodiments, the constant sequence of the anchored oligonucleotide and the constant sequence of the co-anchored oligonucleotide can bind (eg, hybridize) to each other. In some embodiments, the lipids (eg, sterol lipids) of anchored oligonucleotides and co-anchored oligonucleotides can be integrated into cell membranes in a biological sample and the corresponding constant sequences can hybridize to each other simultaneously. In some embodiments, barcoded oligonucleotides, which may include multiple domains, can be introduced into integrating anchor oligonucleotides and co-anchor oligonucleotides that hybridize with each other. Barcoded oligonucleotides may include a functional domain (eg, a sequencing adapter domain), a unique molecular identifier, a pattern barcode, another unique molecular identifier, and an inverted constant sequence in the 5' to 3' direction of the complementary sequence. for example, after cells are labeled with any of the cell labeling agents described herein, the cells can be separated (eg, encapsulated in vesicles) with barcoded characteristics (eg, beads). In some embodiments, the reverse complement of the constant sequence of the barcoded oligonucleotide can react (eg, hybridize) with the constant sequence (eg, a portion of the sequence) on the barcoded feature.
(iv) intracellular cleavage domain
As used herein, a capture probe may optionally include an "intracellular cleavage domain", wherein one or more segments or regions of the capture probe (eg, capture domain, spatial barcode, and/or UMI) may be released. Further segments or regions, such as cell penetrants, are cleavable or linked to one or more capture probes such that the capture domain, spatial barcode, and/or UMI can be cleaved by cleavage between the capture domain, spatial barcode releasable code or the link between, and/or the UMI and the cell penetration agent and/or cell penetration tag. In some embodiments, cleavage of the bond between the capture domain, spatial barcode, and/or UMI and the cell-penetrating agent is induced in the intracellular environment (eg, after the capture probe is introduced into the cell, the cellular endolytic domain is cleaved) Cells ). For example, the bond between the capture domain, spatial barcode, and/or UMI and the cell penetrating agent may be a disulfide bond, which is cleaved by reducing conditions in the cell, eg, when the intracellular cleavage domain contains a disulfide bond. Any other suitable linker can be used to release or cleave the intracellular cleavage domain of the capture probe.
(v) Positive or neutral oligomeric conjugated polymers
In some embodiments of any of the spatial analysis methods described herein, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be coupled to a glycol chitosan derivative. In some embodiments, the chitosan glycol derivative can be linked to two or more molecules (eg, nucleic acid molecules) that have barcodes (eg, spatial barcodes). In some embodiments, the ethylene glycol chitosan derivative can be combined with about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10 or more molecular bonds. Glycol chitosan derivatives (e.g. glycol chitosan-cholesterol) can be used as hydrophobic anchors (see Wang et al.J. Alma mater. Chemical. Other., 30:6165 (2015), which is incorporated herein by reference in its entirety). Non-limiting examples of chitosan derivatives that can be conjugated to molecules (e.g., nucleic acid molecules) with barcodes (e.g., spatial barcodes) can be found in Cheung et al.,marine medicine, 13(8): 5156-5186 (2015), the entire contents of which are incorporated herein by reference.
(vi) Labeling of bifunctional NHS adapter cells
In some embodiments of any of the spatial analysis methods described herein, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) can be coupled to a bifunctional NHS linker. In some embodiments, fused bifunctional NHS adapters (e.g., bifunctional adapters and barcoded molecules) can facilitate attachment of spatial barcodes to the cell surface. In some embodiments, after facilitating binding to the cell surface, the excess NHS linker can be removed (eg, washed away). In some embodiments, the process of conjugating the barcode molecule can be performed under anhydrous conditions to maintain the activity of the unreacted bifunctional NHS. In some embodiments, the anhydrous conditions may be in the presence of DMSO. In some embodiments, the anhydrous conditions may be in the presence of DMF.
(vii) Antibody labeling primers
In some embodiments of any of the spatial analysis methods described herein, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) can be coupled to an antibody or fragment thereof that binds to an antigen having a barcode (e.g. . , spatial barcode) in a way that facilitates attachment. , spatial barcode) molecules (eg nucleic acid molecules) attached to the cell surface. In some embodiments, facilitating attachment to a cell surface facilitates introduction of a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) into the cell. In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be conjugated to an antibody directed against an antigen present on the surface of a cell. In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be associated with a molecule (eg, a nucleic acid molecule) present in a plurality of cells (eg, a plurality of cells in a biological sample). In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be conjugated to an antibody directed against an antigen present on the surface of a cell, a plurality of cells, or almost all present in a biological sample of a cell. In some embodiments, the barcoded antibody is directed against an intracellular antigen. Any of the exemplary methods described herein may be used to couple a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) to another molecule (eg, an antibody or antigenic fragment thereof) described herein.
(viii) Streptavidin-conjugated oligonucleotides
In some embodiments of any of the spatial analysis methods described herein, molecules (eg, nucleic acid molecules) having barcodes (eg, spatial barcodes) can be attached to the cell surface using biotin-streptavidin. In some embodiments, primary amines in the side chains of cell surface polypeptide lysine residues are labeled with an NHS-activated biotin reagent. For example, the N-terminus of a peptide can react with an NHS-activated biotin reagent to form a stable amide bond. In some embodiments, the cell labeling agent comprises a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) conjugated to streptavidin. In some cases, streptavidin can be conjugated to a molecule (eg, a nucleic acid molecule) with a barcode (eg, a spatial barcode) using click chemistry (eg, maleimide modification) as described herein. In some embodiments, cells containing NHS-activating biotin incorporated into the side chain of a protein lysine on the cell surface form a non-covalent bond with streptavidin conjugated to barcodes) molecules (eg, nucleic acid molecules) barcodes). In some embodiments, the molecule (eg, nucleic acid molecule) having a barcode (eg, spatial barcode) conjugated to streptavidin is itself part of the cell labeling agent.
(ix) Dye-labeled oligonucleotides
In some embodiments of any of the spatial analysis methods described herein, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) is directly associated with a detectable label. In some embodiments, the detectable label is any of the detectable labels described herein. In some embodiments, the detectable label is a fluorescent label. In some embodiments, the physical properties of fluorescent labels (eg, fluorescent labels with hydrophobic properties) can overcome the hydrophilicity of molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes). For example, in some embodiments where the molecule is a nucleic acid molecule, a fluorescent label (eg, BODIPY, Cy3, Atto 647N, and Rhodamine Red C2) can be attached to the 5' end of the nucleic acid molecule using a barcode (eg, spatial barcode). In some embodiments, where the molecule is a nucleic acid molecule, any fluorescent label having hydrophobic properties can be coupled to the nucleic acid molecule having a barcode (eg, a spatial barcode) in a manner that overcomes the hydrophilic nature of the nucleic acid molecule. properties Non-limiting examples of labels include BODIPY, Cy3, Atto 647N and Rhodamine Red C2.
(x) Click chemistry
In some embodiments of any of the spatial analysis methods described herein, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) is linked to the chemical portion of the click. As used herein, the term "click chemistry" generally refers to modular, broad-spectrum, high-yield, by-product (e.g., those that can be removed by non-chromatographic methods) and stereospecific (but not necessarily enantioselective) responses (see, e.g., Angew .Chemical. int. ed., 2001, 40(11):2004-2021, the entire contents of which are incorporated herein by reference). In some cases, click chemistry can describe pairs of functional groups that selectively react with each other under mild aqueous conditions.
An example of a click chemical reaction is the Huisgen 1,3-dipolar cycloaddition of azide and alkyne, that is, the copper-catalyzed reaction of an azide with an alkyne to form a 5-membered heteroatomic ring 1,2,3-tri Azole. This reaction is also known as Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC), Cu(I) click chemistry, or Cu+ click chemistry. Click chemistry catalysts include, but are not limited to, Cu(I) salts or Cu(I) salts prepared by in situ reduction of Cu(II) reagents to Cu(I) reagents with reducing agents (drug research2008, 25(10): 2216-2230, which is hereby incorporated by reference in its entirety). Known Cu(II) click chemistry reagents may include, but are not limited to, Cu(II)-(TBTA) complex and Cu(II)(THPTA) complex. TBTA, tris-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, also known as tris-(benzyltriazolylmethyl)amine, can be used as a stable Cu(I) ligand salt. THPTA, tris(hydroxypropyltriazolylmethyl)amine, is another example of a Cu(I) stabilizer. Other conditions can also be used to construct 1,2,3-triazole rings from azides and alkynes using copper-free click chemistry, such as azide-alkyne click chemistry (SPAAC) (see, e.g.,Chemical. Community., 2011, 47:6257-6259 and Nature, 2015, 519(7544):486-90, each of which is incorporated herein by reference in its entirety).
In some embodiments of any of the spatial assays described herein, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) is coupled to a click chemistry moiety in the absence of copper (e.g., a copper-free click chemistry). An example of a copper-free click chemistry approach involves the reaction between cyclooctyne and phenylazide, which produces the product 1-phenyl-4,5,6,7,8,9-hexahydro-1H-cycloocta[ d ][1 ,2,3]triazole (see , e.g., 2010, Akeroyd, N., et al., Click Chemistry for the Preparation of Advanced Macromolecular Structures, Ph.D. Dissertation, hereby incorporated by reference in its entirety.). Additional copper-free click chemistry methods are known to those skilled in the art (see, e.g., 2009, Click Chemistry for Biotechnology and Materials Science, Ed. Jeorg Lahann, published by John Wiley & Sons, Ltd., p. 410).
(xi) Receptor-ligand system
In some embodiments of any of the spatial analysis methods described herein, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be coupled to a ligand, wherein the ligand is part of a receptor-ligand cell surface Interaction. For example, molecules (e.g., nucleic acid molecules) with barcodes (e.g., spatial barcodes) can be linked to ligands that selectively interact with cell surface receptors to target molecules with the following characteristics (e.g., nucleic acid molecules) station-specific barcodes (eg spatial barcodes). Non-limiting examples of receptor-ligand systems that may be used include integrin receptor-ligand interactions, GPCR receptor-ligand interactions, RTK receptor-ligand interactions, and TLR-ligand interactions (cf. Juliano,nucleic acid research., 44(14): 6518-6548 (2016), which is hereby incorporated by reference in its entirety). Any of the methods described herein can be used to link molecules (e.g., spatial barcodes) having barcodes (e.g., spatial barcodes) to ligands (e.g., any of the methods described herein regarding linking molecules (e.g., molecules nucleic acids) Acid molecules with barcodes for antibodies (eg spatial barcodes).
(xii) Covalent bonds between amines and thiols
In some embodiments of any of the spatial analysis methods described herein, molecules (e.g., nucleic acid molecules) having barcodes (e.g., spatial barcodes) may include reactive functional groups at sites within the molecule (e.g., with nucleic acid sequences). . In this case, reactive functional groups can facilitate conjugation on ligands and/or surfaces. In some embodiments, barcoded molecules (e.g., nucleic acid molecules) (e.g., spatial barcodes) may include sulfur compounds designed to react with a wide range of activation receptor moieties (e.g., maleimide and gold microspheres).alcohol modifier. For example, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) with thiol modifications can interact with maleimide-conjugated peptides, resulting in labeling of the peptide. In some embodiments, the maleimide-conjugated peptide is present on the cell surface and thus interacts with a thiol-modified molecule (e.g., a nucleic acid molecule) that has a barcode (e.g., a spatial barcode) that has a barcode (e.g., a spatial barcode). which (eg nucleic acid molecules) Acid molecules with barcodes (eg spatial barcodes) are attached to the cell surface. Non-limiting examples of thiol modifiers include: 5' thiol modifier C6 S-S, 3' thiol modifier C3 S-S, dithiol, oxo 3' thiol modifier 6-S-S, and dithiol serinol.
In some embodiments of any of the spatial analysis methods described herein, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) may include an amine modifier, e.g. an amine modifier designed to link to another molecule in the presence of an acylating agent. In some embodiments, barcoded molecules (e.g., nucleic acid molecules) (e.g., spatial barcodes) may include amine modifiers that are designed to bind to a wide variety of linking groups (e.g., carbonyl amides, thioureas, sulfonamides, and formamides). . For example, barcoded molecules (eg, nucleic acid molecules) (eg, spatial barcodes) and amine modifiers can react with sulfonamide-conjugated peptides, resulting in labeling of the peptide. In some embodiments, the sulfonamide-conjugated peptide is present on the cell surface and thereby interacts with an amino-modified molecule (e.g., a nucleic acid molecule) that has a barcode (e.g., a spatial barcode) that has a barcode (e.g., nucleic acid molecule) that has a bar code (eg spatial barcode) acid molecule) attached to the cell surface. Non-limiting examples of amine modifiers include: DMS(O)MT-amino-modifier-C6, amino-modifier-C3-TFA, amino-modifier-C12, amino-modifier-C6-TFA, amino-dT, amino-modifier -5, amino-modifier-C2-dT, amino-modifier-C6-dT and 3'-amino-modifier-C7.
As another example, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) can include reactive functional groups such as N-hydroxysuccinimide (NHS). In some embodiments, an amine (e.g., an amine-containing peptide) is present on the cell surface and thus interacts with an NHS-modified molecule (e.g., a nucleic acid molecule) that has a barcode (e.g., a spatial barcode) that has a barcode (e.g. spatial barcodes) (e.g. nucleic acid molecules) attached to the cell surface. In some embodiments, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) is reacted with a bifunctional NHS linker to form an NHS-modified molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a molecule nucleic acids). For example, spatial barcodes).
In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) can be linked to a biocompatible anchor (BAM) of a cell membrane. For example, BAMs may include molecules containing oleyl and PEG. The oil base can facilitate the anchoring of molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) into cells, and PEG can increase water solubility. In some embodiments, oleyl-PEG-NHS can be linked to molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) using NHS chemistry.
(xiii) Sustavi na bazi azida
In some embodiments, where the molecule (e.g., having a nucleic acid sequence) includes a reactive functional group at a site within the molecule, the molecule (e.g., nucleic acid molecule) having a barcode (e.g., a spatial barcode) can be coupled to a cell surface Azido . In some embodiments, the reactive functional group is an alkynyl group. In some embodiments, click chemistry as described herein can be used to attach an alkyne-modified molecule (eg, a nucleic acid molecule) with a barcode (eg, a spatial barcode) to an azide group on a cell surface. Azido groups can be attached to the cell surface by various methods. For example, NHS chemistry can be used to attach azide groups to the cell surface. In some embodiments, tetraacylation of N-azidoacetylmannosamine (Ac4ManNAz) containing an azido group can react with sialic acid on the cell surface to attach the azido group to the cell surface. In some embodiments, the azide is linked to the cell surface by bioorthogonal expression of the azide. For example, azide is incubated with cells. In some embodiments, an alkyne-modified molecule (eg, a nucleic acid molecule) with a barcode (eg, a spatial barcode) can be attached to the cell surface via an azide group in the presence of copper. In some embodiments, an alkyne-modified molecule (eg, a nucleic acid molecule) with a barcode (eg, a spatial barcode) can be attached to the cell surface via an azide group in the absence of copper.
(xiv) Lectin-based systems
In some embodiments of any of the spatial analysis methods described herein, a molecule (e.g., a nucleic acid molecule) having a barcode (e.g., a spatial barcode) can be linked to a lectin acid molecule that facilitates attachment of the molecule (e.g., nucleic acid molecule) to barcodes on the surface of the station (eg spatial barcodes). Lectins can bind glycans, such as those on the surface of cells. In some embodiments, molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes) have embedded reactive functional groups, such as azido groups. In some embodiments, a molecule (eg, a nucleic acid molecule) having a barcode (eg, a spatial barcode) and an azide group is reacted with a modified lectin, eg, using an NHS chemically modified lectin to introduce an azide reactive group. In some embodiments, living cells are labeled with a lectin-modified molecule (eg, a nucleic acid molecule) that has a barcode (eg, a spatial barcode). In some embodiments, the fixed cells are labeled with a lectin-modified molecule (eg, a nucleic acid molecule) that has a barcode (eg, a spatial barcode). In some embodiments, the permeabilized cells are labeled with a lectin-modified molecule (eg, a nucleic acid molecule) that has a barcode (eg, a spatial barcode). In some embodiments, organelles in the secretory pathway can be labeled with lectin-modified molecules (eg, nucleic acid molecules) with barcodes (eg, spatial barcodes).
(b) Methods for separating samples into single cells or cell populations
Some embodiments of any of the methods described herein may involve isolating a biological sample into single cells, cell populations, cell types, or one or more regions of interest. For example, a biological sample can be separated into single cells, cell populations, cell types, or one or more regions of interest prior to contact with one or more capture probes. In other examples, a biological sample is first contacted with one or more capture probes and then separated into individual cells, cell populations, cell types, or one or more regions of interest.
In some embodiments, pixelization can be used to separate a biological sample into parts. Pixelization may include the steps of providing a biological sample and extruding one or more portions of the biological sample. The excised portion of the biological sample can then be used to perform any of the methods described herein. In some embodiments, the excised portion of the biological sample may be a random sample or a designed sample. In some embodiments, the excised portion of the biological sample can be focused on a region of interest or a subcellular structure in the biological sample.
In some embodiments, prior to performing any of the spatial analysis methods described herein, a biological sample (eg, a tissue sample or tissue portion) is divided into smaller portions compared to the original size of the biological sample ("block"). In some embodiments, the method may include spatially barcoding the FFPE "block" with a barcode applied in a spatially well-defined pattern (eg, array printing). To link spatial barcodes to specific "blocks" of biological samples, the barcodes (e.g., spatial barcodes) can be of sufficient length to prevent spread of the barcodes in subsequent steps, or the spatial barcodes can be covalently applied to the FFPE samples. In some embodiments, the spatial barcode is unique for each FFPE block. In some embodiments, spatial barcodes can be embedded on FFPE slides (eg, within a matrix such as wax or hydrogel). In some embodiments, the FFPE slide is heated (eg, the wax is heated) prior to adding the spatial barcode. In some embodiments, after adding the spatial barcode, the FFPE slide can be cooled and cut or disassembled. Methods of blocking (eg cutting) biological samples are known in the art. For example, in one non-limiting example, blocking of a biological sample can be performed by various means, such as laser microdissection, mechanical means, acoustic (eg, sonication) means, or any other method described herein. In some embodiments, fluorophores/Qdots, etc., can be incorporated into blocks to preserve spatial information about biological samples. Barcoding in this step enables massively parallel encapsulation of blocks while preserving local spatial information (eg, tumor vs. normal/healthy cells). In some embodiments, comminution of a biological sample (e.g., a tissue section) may result in single-celled portions of the biological sample. In other embodiments, comminution of the biological sample may be performed to obtain portions corresponding to diseased portions of the biological sample. In another embodiment, blocking of the biological sample can be performed to obtain discrete blocks of the biological sample corresponding to diseased or healthy portions of the biological sample. In some embodiments, blocking of a biological sample can be performed to obtain blocks corresponding to specific cell types in the biological sample (eg, based on fluorescence or chemiluminescence imaging of antibodies bound to a target protein).
In some embodiments, the spatial barcode blocks may be further processed. For example, spatially barcoded blocks can be individually encapsulated (eg, in matrices, emulsions, or hydrogels). In some embodiments, the spatially barcoded blocks may be encapsulated in compartments (eg, wells, droplets, channels, or vesicles). In some embodiments, the spatially barcoded moieties may be encapsulated in vesicles. In some embodiments, the vesicle may comprise a lipid bilayer. In some embodiments, spatially barcoded FFPE blocks can be encapsulated with unique barcoded beads. In some embodiments, uniquely barcoded beads may have functional domains, cleavage domains, unique molecular identifiers, and capture domains, or combinations thereof. In some embodiments, the encapsulated spatially barcoded FFPE block and uniquely barcoded beads can be heated to deparaffinize the FFPE sample. In some embodiments, the encapsulated spatially barcoded FFPE blocks and uniquely barcoded beads can be treated with xylene to deparaffinize the FFPE samples. In some embodiments, deparaffinized samples can be treated to remove crosslinked methylene bridges in one step. In some embodiments, additional steps may be performed when, for example, the crosslinking chemistry is incompatible with the barcoding or library preparation steps. In some embodiments, nucleic acids derived from or present in the blocks can bind the uniquely barcoded beads after the methylene bridges are cleaved. In some embodiments, once the spatial barcode is bound to a unique barcode bead, the encapsulation can be disrupted (eg, lysed, dissolved, or removed) and the barcode bead can be collected. In some embodiments, the collected barcoded beads may be washed and repackaged. In some embodiments, nucleic acids (eg, spatial barcodes, unique barcodes, analyte transcripts) associated with beads can be amplified (eg, PCR amplified) and processed (eg, sequenced) according to any of the methods described herein.
In some embodiments, laser microdissection (eg, highly multiplexed laser microdissection) can be used to divide or segment a biological sample.
(c) Analyte release and amplification
In some embodiments, a lysis reagent may be added to the sample to facilitate release of the analyte from the sample. Examples of lysing agents include, but are not limited to, biologically active agents, such as lytic enzymes for lysing various cell types, such as Gram-positive or negative bacteria, plants, yeasts, mammals, such as lysozyme, leukopeptidase, lysostaphin coccycin, labiasis, kitalase, lyase and various other commercially available lyases. Other lysis agents may additionally or alternatively be co-distributed with the biological sample to induce release of the sample contents into the compartments. In some embodiments, surfactant-based lysis solutions can be used to lyse cells, although they may be less than ideal for emulsion-based systems where surfactants interfere with stable emulsions. The lysis solution may include ionic surfactants such as sarkosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical cell disruption can also be used in some embodiments, eg non-emulsion-based spraying, such as biomaterial encapsulation, which can complement or replace droplet spraying, where any encapsulation pore volume is small enough . To retain nucleic acid fragments of a certain size after cell destruction.
In addition to permeabilizing agents, other reagents may be added to interact with biological samples, including, for example, DNase and RNase inactivators or inhibitors or chelating agents, such as EDTA, and other reagents to allow post-processing of analytes from the samples. In other embodiments, nucleases, such as DNase or RNAse, or proteases, such as pepsin or proteinase K, are added to the sample.
Other reagents that can be added to the sample include, for example, endonucleases for DNA fragmentation, DNA polymerases, and dNTPs for nucleic acid amplification. Other enzymes that may also be added to the sample include, but are not limited to, polymerases, transposases, ligases, proteinase K, and DNase, among others. Other reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers, and replaceable oligonucleotides. In some embodiments, template alteration can be used to increase the length of the cDNA, for example, by adding a predefined nucleic acid sequence to the cDNA.
If tissue samples are not sufficiently permeabilized, the amount of analyte absorbed by the substrate may be too small for adequate analysis. Conversely, if the tissue sample is too permeable, the analyte diffuses away from its source in the tissue sample, leading to a loss of the relative spatial relationship of the analytes within the tissue sample. Therefore, a balance needs to be struck between sufficient permeabilization of the tissue sample to obtain good signal intensity, while still maintaining the spatial resolution of the analyte distribution in the tissue sample.
In some embodiments, when the biological sample includes living cells, the permeabilization conditions can be modified so that the living cells undergo only short-term permeabilization (eg, by applying short, repeated pulses of an electric field), allowing for one or more assays. Migration of substances from living cells transfers cells to the matrix while preserving cell viability.
In some embodiments, after contacting the biological sample with the substrate containing the capture probes, a removal step is performed to remove all or part of the biological sample from the substrate. In some embodiments, the removal step includes enzymatic or chemical degradation of the permeabilized cells of the biological sample. For example, the removal step may include treating the biological sample with an enzyme (eg, proteinase K) to remove at least a portion of the biological sample from the first substrate. In some embodiments, the removal step may include tissue ablation (eg, laser ablation).
In some embodiments, when RNA is captured from cells in a sample, one or more RNA species of interest can be selectively enriched. For example, one or more RNAs of interest can be selected by adding one or more oligonucleotides. Any of a number of methods can be used to selectively down-select (eg, remove) one or more RNAs. For example, probes can be applied to samples that selectively hybridize to ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. Subsequent application of capture probes to the sample improves RNA capture due to the reduction of non-specific RNA present in the sample. In some embodiments, the additional oligonucleotide is a polymerase priming sequence. For example, one or more primer sequences having sequence complementarity to one or more RNAs of interest can be used to amplify one or more RNAs of interest, thereby selectively enriching for those RNAs. In some embodiments, oligonucleotides having a sequence complementary to the complementary strand of the captured RNA (eg, cDNA) can bind to the cDNA. In one non-limiting example, a biotinylated oligonucleotide having a sequence complementary to one or more cDNAs of interest is ligated to the cDNA and the biotinylated strand can be used in a variety of methods known in the art. Affinity of mycoavidin for selection (eg streptavidin beads).
In some embodiments, any of the spatial analysis methods described herein may include modulating the rate of interaction between a biological analyte from a biological sample and capture probes on the array. In some embodiments, the rate of interaction can be adjusted by adjusting the biological sample (eg, by adjusting temperature or pH). In some embodiments, adjusting the rate of interaction involves the use of an external stimulus. Non-limiting examples of external stimuli that can be used to modulate the rate of interaction include light, temperature, small molecules, enzymes and/or activators. In one example, light can be used to activate polymerases in nucleic acid extension reactions. In another example, temperature can be used to regulate hybridization between two complementary nucleic acid molecules.
Nucleic acid analytes can be amplified using polymerase chain reaction (eg, digital PCR, quantitative PCR, or real-time PCR), isothermal amplification, or any nucleic acid amplification or extension reaction described herein or known in the art.
(d) undergraduate
As noted above, in some embodiments, the sample can optionally be partitioned into individual cells, populations of cells (eg, based on cell subtypes or gene expression profiles), or other fragments/fragments that are smaller than the original sample. Each of these smaller portions of the sample can be analyzed to obtain spatially resolved information about the analyte from the sample. This article describes a non-restrictive partitioning method.
For samples that have been separated into smaller fragments—especially samples that have been disaggregated, dissociated, or otherwise separated into individual cells—one method of fragment analysis involves separating the fragments into individual partitions (eg, droplets) and then analyzing the contents of the partition. Typically, each partition separates its own contents from the contents of other partitions. For example, a barrier can be a droplet in an emulsion.
The methods described here allow cells (e.g., cells) from a sample to be divided or partitioned into discrete compartments or voxels. As used herein, each "voxel" represents a 3-dimensional volume unit. In some embodiments, a voxel keeps its own content separate from the content of other voxels. A voxel can be a partition of an array of volume partitions. For example, a voxel can be one of a set of discrete partitions into which a 3D object is divided. As another example, members of a plurality of photo-crosslinkable polymer precursors can be cross-linked into voxels that are part of an array of photo-cross-linkable polymers covering a substrate or portion of a substrate. A unique identifier, such as a barcode, can be delivered to cells before, after, or at the same time to allow later attribution of cell characteristics to specific voxels. In some embodiments, voxels have defined dimensions. In some embodiments, a voxel includes a single cell.
For example, substrates can be coated with DTT-sensitive hydrogels and then contacted with biological samples. Optionally, the capture probes attached to the substrate are released from the substrate such that the released capture probes are introduced into the biological sample and the at least one released capture probe passes through the capture domain with at least one biological assay present in the biological sample. interaction of matter. Biological samples and matrices can be assembled into flow cells and photo-crosslinkable polymer precursors added. The cells of the biological sample can then be cross-linked into hydrogel voxels of a defined size using a light source. The flow cell can be disassembled and cleaned to remove unpolymerized polymer precursors. The coatings can be treated with DTT to generate single-cell compartments for further applications. The capture probe/biological analyte can be analyzed and the spatial information of the spatial barcode features can be used to determine the spatial location of the captured biological analyte in the biological sample.
In addition to the analytes, the compartments may also contain additional components, in particular one or more beads. The compartments may include single gel beads, single cell beads, or single cell beads and single gel beads.
The partition may also include one or more reagents. Unique identifiers, such as barcodes, can be introduced into the droplets before, after or simultaneously with the formation of the droplets, for example via microcapsules (eg beads). Networks of microfluidic channels (eg on-chip) can be used to generate partitions. Alternative mechanisms can also be used to separate individual biological particles, including porous membranes through which aqueous mixtures of cells are forced into non-aqueous fluids.
The baffles can flow in the fluid stream. The baffle may comprise, for example, a microbubble with an outer barrier surrounding an inner fluidic center or core. In some cases, the separator may include a porous matrix capable of entraining and/or retaining material within its matrix. The partition may be a droplet of the first phase within the second phase, whereby the first and second phases do not mix. For example, the partition may be a droplet of an aqueous liquid within a non-aqueous continuous phase (eg, an oil phase). In another example, the partition may be a droplet of a non-aqueous liquid within an aqueous phase. In some examples, the distribution can be provided in a water-in-oil emulsion or an oil-in-water emulsion. A variety of different containers are described, for example, in US Patent No. Application Publication No. 2014/0155295, the entire contents of which are incorporated herein by reference. Emulsion systems for generating stable droplets in nonaqueous or oily continuous phases are described, for example, in U.S. Pat. Patent No. Application Publication No. 2010/0105112, the entire contents of which are incorporated herein by reference.
For droplets in emulsions, individual particles can be divided into discrete compartments, for example, by introducing a stream of particles in an aqueous fluid into a stream of nonaqueous fluid so that the droplets are in both streams. Fluid properties (e.g., fluid flow rate, fluid viscosity, etc.), particle properties (e.g., volume fraction, particle size, particle concentration, etc.), microfluidic structure (e.g., channel geometry, etc.) and other parameters can be tuned to control resulting split occupancies (eg, number of analytes per partition, number of beads per partition, etc.). For example, zone occupancy analytes can be controlled by ensuring water flow in concentration and/or flow rate.
To create individual analyte partitions, the relative flow rates of the immiscible liquids can be chosen such that, on average, each compartment can contain less than one analyte to ensure that these occupied compartments are predominantly self-occupied. In some cases, a partition of the plurality of partitions may contain at most one analyte. In some embodiments, various parameters (eg, fluid properties, particle properties, microfluidic structure, etc.) can be selected or adjusted so that most partitions are occupied, eg, only a small percentage of unoccupied partitions is allowed. The flow and channel architecture can be controlled to provide a certain number of singly occupied partitions, less than a certain level of unoccupied partitions, and/or less than a certain level of multiply occupied partitions.
The channel segments described herein may be connected to any of a number of different fluid sources or receivers, including reservoirs, pipes, manifolds, or other fluid components of the system. It should be kept in mind that microfluidic channel structures can have different geometries. For example, a microfluidic channel structure may have one or more channel junctions. As another example, microfluidic channel structures may have 2, 3, 4, or 5 channel segments, each carrying particles that meet at channel junctions. Fluid may be directed to flow along one or more channels or reservoirs via one or more fluid flow cells. A fluid flow unit may include a compressor (eg to provide positive pressure), a pump (eg to provide negative pressure), an actuator, etc. to control fluid flow. Fluids can also or otherwise be controlled by applied pressure differentials, centrifugal force, electrical pumping, vacuum, capillary and/or gravity flow.
Partitions can include one or more unique identifiers, such as barcodes. The barcodes can be delivered in advance, later or simultaneously to the compartments containing the compartmentalized bioparticles. For example, barcodes can be injected into the droplets before, after, or simultaneously with the formation of the droplets. The transfer of barcodes to specific partitions enables later attribution of the characteristics of individual bioparticles to specific partitions. Barcodes can be delivered to partitions, eg, nucleic acid molecules (eg, oligonucleotides), by any suitable mechanism. Barcoded nucleic acid molecules can be delivered to compartments via microcapsules. In some cases, the microcapsules may contain beads.
In some embodiments, the barcoded nucleic acid molecules can be initially associated with microcapsules and then released from the microcapsules. The release of barcoded nucleic acid molecules can be passive (eg, by diffusion from microcapsules). Additionally or alternatively, release from the microcapsules can be performed after administration of a stimulus that allows the barcoded nucleic acid molecule to be detached or released from the microcapsules. This stimulation can disrupt the microcapsules, the interaction that binds the nucleic acid molecules to the barcode on the microcapsules or within the microcapsules, or both. Such stimuli may include, for example, thermal stimuli, optical stimuli, chemical stimuli (eg changes in pH or use of reducing agents), mechanical stimuli, radiation stimuli; biological stimuli (eg enzymes) or any combination thereof.
In some embodiments, one or more barcodes (eg, spatial barcodes, UMIs, or combinations thereof) may be introduced into the compartments as part of the analyte. As previously mentioned, the barcode may be directly bound to the analyte or may form part of a capture probe or analyte capture means that is hybridized, conjugated, or otherwise bound to the analyte so that when the analyte is introduced into the compartment, it is introduced and bar code.
As stated above,
A variety of different balls can be incorporated into the compartments as described above. In some embodiments, for example, non-barcoded beads may be incorporated into the compartments. For example, the beads may be non-barcoded beads when the biological particles (eg, cells) into which the compartments are embedded carry one or more barcodes (eg, spatial barcodes, UMIs, and combinations thereof).
In some embodiments, balls bearing bar codes can be incorporated into the compartments. For example, nucleic acid molecules, such as oligonucleotides, can be attached to the beads by releasable bonds, such as disulfide bonds. The same bead can be linked (eg, via a releasable bond) to one or more other nucleic acid molecules. A nucleic acid molecule can be or include a barcode. As described elsewhere herein, a barcode structure may include a number of string elements.
Nucleic acid molecules can include functional domains that can be used in post-processing. For example, a functional domain may include one or more sequencing-specific flow cell attachment sequences (eg, a P5 sequence for the Illumina® sequencing system) and a sequencing primer sequence (eg, an R1 primer for the Illumina® sequencing system). A nucleic acid molecule may contain a barcode sequence (eg, DNA, RNA, protein, etc.) to barcode the sample. In some cases, the barcode sequence may be bead-specific such that the barcode sequence is common to all nucleic acid molecules attached to the same bead. Alternatively or additionally, the barcode sequence may be partition specific such that the barcode sequence is common to all nucleic acid molecules associated with one or more beads partitioned into the same partition. A nucleic acid molecule may include a specific primer, such as an mRNA-specific primer (eg, a poly(T) sequence), a targeted primer, and/or a random primer. A nucleic acid molecule may include an anchor sequence to ensure that a specific primer sequence hybridizes to the end of the sequence (eg, mRNA). For example, anchor sequences can include random short nucleotide sequences such as 1-mer, 2-mer, 3-mer or longer sequences, which can ensure that poly(T) fragments are more likely to be in poly(A) mRNA end sequence.
A nucleic acid molecule may include a unique sequence for molecular recognition (eg, a unique molecular identifier (UMI)). In some embodiments, a unique molecular recognition sequence can comprise about 5 to about 8 nucleotides. Alternatively, a unique molecular recognition sequence may comprise less than about 5 or more than about 8 nucleotides. A unique molecular recognition sequence can be a unique sequence that varies between individual nucleic acid molecules attached to a single bead.
In some embodiments, the unique molecular recognition sequence can be a random sequence (eg, such as a random N-mer sequence). For example, UMI can provide a unique identifier for captured starting mRNA molecules to allow quantification of the amount of raw expressed RNA.
In general, a single bead can be coupled to any number of individual nucleic acid molecules, eg, from one to tens of thousands to hundreds of thousands or even millions of individual nucleic acid molecules. Appropriate barcodes for individual nucleic acid molecules may include common sequence segments or relatively common sequence segments as well as variable or unique sequence segments between different individual nucleic acid molecules attached to the same bead.
Within any given partition, all cDNA transcripts for a single mRNA molecule may contain a common barcode sequence segment. However, transcripts made from different mRNA molecules within a given partition may vary in segments of a unique molecular recognition sequence, such as UMI segments. Usefully, even after any subsequent amplification of the contents of a given partition, the number of distinct UMIs can indicate the amount of mRNA originating from a given partition. Transcripts can be amplified, purified and sequenced to identify cDNA transcript sequences for mRNA, as well as sequence barcoded fragments and UMI fragments, as described above. Although poly(T) primers are described, other targeted or random primers can also be used to initiate the reverse transcription reaction. Likewise, although described as the release of barcoded oligonucleotides into compartments, in some cases bead-bound nucleic acid molecules can be used to hybridize and capture mRNA on the solid phase of the bead, for example, to facilitate binding of RNA to other cellular contents are separated .
In some embodiments, precursors containing functional groups that are reactive or can be activated to become reactive can be polymerized with other precursors to produce gel beads that contain activated or activating functional groups. This functional group can then be used to attach other substances (eg, disulfide bonds, primers, other oligonucleotides, etc.) to the gel beads. For example, some precursors with carboxylic acid (COOH) groups can be copolymerized with other precursors to form beads that also contain COOH functional groups. In some cases, acrylic acid (a substance containing free COOH groups), acrylamide, and bis(acryloyl)cystamine can be copolymerized together to produce beads with free COOH groups. The COOH groups of the beads can be activated (for example, with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) or 4-(4,6-dimethoxy-1,3,5-triazine -2-yl)-4-methylmorpholine chloride (DMMTMM)), making them reactive (eg, reactive towards amine functions when activated with EDC/NHS or DMTMM). The activated COOH groups can then be reacted with a suitable material containing a bead-binding moiety (eg, a material containing an amine functional group wherein the carboxylic acid group is activated to react with the amine functional group).
In some embodiments, degradable beads can be introduced into the compartments such that the beads are degraded within the compartments and any associated substances (eg, oligonucleotides) are released within the droplets when an appropriate stimulus is applied. Free substances (eg, oligonucleotides, nucleic acid molecules) can interact with other reagents contained in the compartments. For example, polyacrylamide beads with cystamine disulfide attached to barcode sequences can bind to reducing agents in water-in-oil emulsion droplets. Within the droplet, the reducing agent can break the various disulfide bonds, causing the bead to degrade and release the barcode sequence into the aqueous internal environment of the droplet. In another example, heating a droplet with a barcode sequence attached to the bead in an alkaline solution also results in degradation of the bead and release of the associated barcode sequence into the internal aqueous environment of the droplet.
Any suitable number of substances (eg, primers, barcoded oligonucleotides) can be bound to the beads so that upon release from the beads, the substances (eg, primers, eg, barcoded oligonucleotides) are present in the compartments for a predetermined concentration time. Such predefined concentrations can be chosen to facilitate certain reactions within the partitions for generating sequencing libraries, such as amplification. In some cases, a predefined concentration of primers can be limited by the manufacturing process of nucleic acid molecules (eg, oligonucleotides) with beads.
A degradable grain may contain one or more substances with labile bonds so that when the grain/substance is exposed to an appropriate stimulus, the bond breaks and the grain degrades. A labile bond can be a chemical bond (eg covalent bond, ionic bond) or it can be another type of physical interaction (eg van der Waals interaction, dipole-dipole interaction, etc.). In some embodiments, the cross-linking agents used to form the beads may include labile linkages. When exposed to the right conditions, the unstable bonds break and the grains degrade. For example, when polyacrylamide gel beads containing a cystamine crosslinker are exposed to a reducing agent, the cystamine disulfide bonds are broken and the beads are degraded.
Degradable beads can be used to rapidly release bound substances (eg, nucleic acid molecules, barcode sequences, primers, etc.) from the beads without degradation when an appropriate stimulus is applied to the beads. For example, for a substance bound to the inner surface of a porous bead or in the case of an encapsulated substance, the substance may have greater mobility and accessibility to other substances in solution as the bead degrades. In some embodiments, the substances can also be attached to the degradable beads via degradable linkages (eg, disulfide linkages). The degradable linker may respond to the same stimulus as the degradable seed, or the two degradable substances may respond to different stimuli. For example, barcode sequences can be disulfide-linked to cystamine-containing polyacrylamide beads. When the barcode beads are exposed to a reducing agent, the beads are degraded and the barcode sequence is released after breaking the disulfide bond between the barcode sequence and the bead, as well as the cystamine disulfide bond in the bead.
It will be clear from the above description that although referred to as bead degradation, in many embodiments, degradation may refer to the separation of bound or entrapped species from the bead, structurally degrading the physical bead itself, rather than degrading the physical bead itself. For example, trapped substances can be released from the beads due to differences in osmotic pressure, for example, due to changes in the chemical environment. For example, changes in bead pore volume due to differences in osmotic pressure can often occur without structural degradation of the bead itself. In some cases, the increase in pore volume due to osmotic swelling of the beads may allow the release of trapped species within the beads. In some embodiments, osmotic shrinkage of the beads may result in the beads better retaining trapped species due to shrinkage of pore volume.
A number of chemical triggers can be used to initiate the breakdown of beads within the barrier. Examples of these chemical changes may include, but are not limited to, pH-mediated changes in the integrity of components within the spheres, degradation of the sphere components by breaking cross-links, and deaggregation of the sphere components.
In some embodiments, the beads can be formed from materials that include degradable chemical cross-linkers such as BAC or cystamine. Degradation of such degradable cross-linkers can be achieved by different mechanisms. In some examples, the beads may come into contact with chemical degradants that may cause oxidation, reduction, or other chemical changes. For example, the chemical degradation agent may be a reducing agent such as dithiothreitol (DTT). Other examples of reducing agents may include beta-mercaptoethanol, (2S)-2-amino-1,4-dimercaptobutane (dithiobutylamine or DTBA), tris(2-carboxyethyl)phosphine (TCEP), or a combination thereof. The reducing agent can break down the disulfide bonds formed between the bead-forming gel precursors and thus break down the beads.
In certain embodiments, a change in the pH of the solution, such as an increase in pH, can cause degradation of the beads. In other embodiments, exposure to an aqueous solution, such as water, can cause hydrolytic degradation, thereby degrading the beads. In some cases, any combination of stimuli can trigger bead degradation. For example, a change in pH can make a chemical such as DTT an effective reducing agent.
The beads can also be induced to release their contents when a heat stimulus is applied. Changes in temperature cause different changes to the balls. For example, heat can cause solid beads to liquefy. The change in heat causes the beads to melt, which degrades part of the bead. In other cases, the heat increases the internal pressure of the pellet's ingredients, causing the pellet to crack or explode. Heat can also be applied to heat-sensitive polymers used as bead building materials.
In addition to beads and analytes, the formed partitions can contain a number of different reagents and substances. For example, when a lysis reagent is present within the compartment, the lysis reagent may facilitate the release of the analyte within the compartment. Examples of lysing agents include biologically active agents such as lytic enzymes used to lyse various cell types, e.g. gram-positive or negative bacteria, plants, yeasts, mammals, etc., such as lysozyme, leukopeptidase, lysostaphin labialase, catalase, lyase, and various other lyases available from, for example, Sigma-Aldrich, Inc. (St. Louis, MO), as well as other commercially available lyases. Other lysis agents may additionally or alternatively partition to cause analyte release into compartments. For example, surfactant-based lysis solutions can be used to lyse cells in some cases, although they are not ideal for emulsion-based systems because surfactants can interfere with stable emulsions. In some embodiments, the lysis solution may include nonionic surfactants, such as Triton X-100 and Tween 20. In some embodiments, the lysis solution may include ionic surfactants, such as sarkosyl and sodium dodecyl sulfate (SDS). Electroporation, thermal, acoustic or mechanical disruption of cells can also be used in some embodiments, for example, non-emulsion spraying, such as analyte encapsulation, which can complement or replace droplet spraying, where the encapsulated pore volume is small. sufficient to retain nucleic acid fragments of a given size after cell disruption.
Examples of other substances that may be shared with the analytes in the compartments include, but are not limited to, DNase and RNase inactivators or inhibitors or chelators, such as EDTA, and to remove or otherwise reduce negative activity or various cell lysates Effect of components on subsequent processing of nucleic acids. Additional reagents can also be assigned together, including endonucleases for DNA fragmentation, DNA polymerases, and dNTPs for amplifying nucleic acid fragments and attaching barcoded molecular tags to the amplified fragments.
Additional reagents may also include reverse transcriptase, including enzymes with terminal transferase activity, primers and oligonucleotides, and replacement oligonucleotides (also referred to herein as "replacement oligonucleotides" or "template replacement oligonucleotides"). In some embodiments, template shifting can be used to increase the length of the cDNA. Template switching can be used to add predefined nucleic acid sequences to cDNA. In one example of template switching, cDNA can be generated from reverse transcription of a template, such as cellular mRNA, where a reverse transcriptase with terminal transferase activity can add additional nucleotides, such as a poly(C) pathway. A replacement oligonucleotide may include a sequence complementary to an additional nucleotide, such as poly(G). Additional nucleotides on the cDNA (eg, poly(C)) can be hybridized to additional nucleotides on the oligonucleotide replacement (eg, poly(G)), whereby the oligonucleotide replacement can be read by reverse transcriptase, which is used as a template for further cDNA extension.
A template-changing oligonucleotide may include a hybridization region and a template region. A hybridizing region can include any sequence capable of hybridizing to a target. In some cases, the hybridization region includes a series of G bases that complement the overhanging C bases at the 3' end of the cDNA molecule. A series of G bases may include 1 G base, 2 G bases, 3 G bases, 4 G bases, 5 G bases, or more than 5 G bases. Template sequences can include any sequence that is incorporated into cDNA. In some cases, the template region includes at least 1 (eg, at least 2, 3, 4, 5, or more) tag sequences and/or functional sequences. Replacement oligonucleotides may include deoxyribonucleic acid; ribonucleic acid; modified nucleic acids including 2-aminopurine, 2,6-diaminopurine (2-Amino-dA), inverted dT, 5-methyl dC, 2'-deoxyinosine, Super T (5-hydroxybutynl -2'-deoxyuridine), Super G ( 8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, such as UNA-A, UNA-U, UNA-C, UNA-G), Iso-dG, Iso-dC, 2' fluorobases (eg fluoroC, fluoroU, fluoroA and fluoroG) and combinations of the above.
In some embodiments, the beads separated from the analyte may include different types of oligonucleotides bound to the beads, where different types of oligonucleotides bind to different types of analyte. For example, the bead may include one or more first oligonucleotides (eg, which may be capture probes), which may bind or hybridize to a first type of analyte, such as mRNA, and one or more other oligonucleotides. A nucleotide (which can be, for example, a capture probe) can bind or hybridize to another type of analyte, such as gDNA. The partitions may also include lysis agents that facilitate the release of nucleic acids from the copartitioned cells, and may also include reagents (eg, reducing agents) that can degrade the beads and/or break the covalent bonds between the oligonucleotide and the beads, so that the oligonucleotide released into the compartment. The released barcoded oligonucleotides (which may also be barcoded) can hybridize to mRNA released from the cell or to gDNA released from the cell.
The barcode constructs formed by hybridization may therefore include a first type of construct containing a sequence corresponding to the original barcode sequence from the bead and a sequence corresponding to a transcript from the cell, and a second type of construct, including sequences corresponding to the original barcode from the bead and sequences corresponding to the genomic DNA from cells. The barcoded constructs can then be released/removed from the partitions and, in some embodiments, further processed to add additional sequences. The resulting constructs can then be sequenced, the sequencing data processed, and the results used for the spatial characterization of mRNA and gDNA in cells.
In another example, the partition comprises a bead containing a first type of oligonucleotide (eg, a first capture probe) having a first barcode sequence, a poly(A) poly(A) tail that can hybridize to an mRNA start sequence (T). transcripts and UMI barcode sequences that uniquely identify a given transcript. The bead also includes a second type of oligonucleotide (e.g., a second capture probe) having a second barcode sequence, which may be specific for a third barcode oligonucleotide (e.g., an analyte capture agent) coupled to an antibody. the surface of the partitioned cells. The third barcoded oligonucleotide includes a UMI barcode sequence that uniquely identifies the antibody (and the specific cell surface feature to which it binds).
In this example, the first and second barcode oligonucleotides include the same spatial barcode sequence (eg, the first and second barcode sequences are identical), which enables downstream linkage of the barcode nucleic acid to the partition. However, in some embodiments, the first and second barcode strings are different.
Partitions also include lysis agents that help release the nucleic acids from the cells, and may also include agents (eg, reducing agents) that can degrade the beads and/or break the covalent bonds between the barcoded oligonucleotides and the beads, releasing them to enter the partition. The first released barcode oligonucleotide can be hybridized with mRNA released from the cell, and the second released barcode oligonucleotide can be hybridized with a third barcode oligonucleotide to form a barcode construct.
The first barcode construct includes a spatial barcode sequence corresponding to the first barcode sequence from the bead and a sequence corresponding to the UMI barcode sequence from the first type of oligonucleotide, which recognizes the cellular transcript. Another type of barcode construct includes a spatial barcode sequence corresponding to a second barcode sequence from a second type of oligonucleotide, and a spatial barcode sequence corresponding to a third type of oligonucleotide (eg, an analyte capture agent) and is used to identify the cell. Surface features of the UMI barcode sequence. The barcoded constructs can then be released/removed from the partitions and, in some embodiments, further processed to add additional sequences. The resulting constructs are then sequenced, the sequencing data processed, and the results used to characterize the cells for mRNA and cell surface characteristics.
The previous discussion referred to two specific examples of beads carrying oligonucleotides for the analysis of two different analytes within a partition. More generally, split beads may have any of the structures described above and may include any combination of oligonucleotides described for the assay of two or more (eg, three or more, four or more, five or more, six or more), eight or more , ten or more, 12 or more, 15 or more, 20 or more, 25 or more, 30 or more, 40 or more, 50 or more) division of different types of analytes. Examples of beads with combinations of different types of oligonucleotides (eg, capture probes) for simultaneous analysis of different combinations of analytes within a partition include, but are not limited to: (a) genomic DNA and cell surface features (eg, use of analyte capture reagents described herein); (b) mRNA and lineage tracing structures; (c) mRNA and cellular methylation status; (d) mRNA and available chromatin (eg ATAC-seq, DNase-seq and/or MNase-seq); (e) mRNA and cell surface or intracellular proteins and/or metabolites; (f) barcoded analyte capture agents (e.g., MHC multimers as described herein) and immune cell receptors ((e.g., V(D)J sequence T cell receptor); (g) mRNA and perturbants (e.g., CRISPR crRNA/ sgRNAs, TALENs, zinc finger nucleases, and/or antisense oligonucleotides as described herein; ).In some embodiments, the perturbing agent can be a small molecule, antibody, drug, aptamer, nucleic acid (e.g., miRNA), physical environment (e.g., change temperature) or any other known disturbing agent.
(e) Sequencing analysis
After the analyte from the sample is hybridized or otherwise bound to a capture probe, analyte capture agent, or other barcoded oligonucleotide sequence, the hybridization/association is analyzed by sequencing to identify the analyte.
In some embodiments, when a sample is directly barcoded by hybridization with a capture probe or analyte capture agent that hybridizes, binds or associates with, or is introduced into, the cell surface, the entire sample can be sequenced, as described above. Alternatively, if the barcoded sample is separated into fragments, cell populations or single cells, as described above, then the individual fragments, cell populations or cells can be sequenced. As described above, for analytes barcoded by bead splitting, individual analytes (eg, cells or cell content after cell lysis) can be extracted from the partitions by breaking the partitions and then analyzed by sequencing to identify the analytes.
Barcoded analyte constructs can be analyzed using a variety of different sequencing methods. Typically, the sequenced polynucleotide may be, for example, a nucleic acid molecule such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including variants or derivatives thereof (eg, single-stranded DNA or DNA/RNA hybrids and glycoside analogs).
Polynucleotide sequencing can be performed with various commercial systems. More generally, nucleic acid amplification, polymerase chain reaction (PCR) (eg digital PCR and digital droplet PCR (ddPCR), quantitative PCR, real-time PCR, multiplex PCR, PCR-based singleplex methods, emulsion PCR can be use) for sequencing and/or isothermal amplification.
Other examples of methods for sequencing genetic material include, but are not limited to, DNA hybridization methods (e.g., Southern blotting), restriction enzyme digestion methods, Sanger sequencing methods, next-generation sequencing methods (e.g., single molecule real-time sequencing), nanopore sequencing and Polony sequencing), ligation methods and microarray methods. Other examples of sequencing methods that can be used include targeted sequencing, real-time single molecule sequencing, exome sequencing, electron microscopy-based sequencing, panel sequencing, transistor-mediated sequencing, direct sequencing, random shotgun sequencing, Sanger dideoxy termination sequencing, whole genome sequencing , hybrid sequencing, pyrosequencing, capillary electrophoresis, gel electrophoresis, duplex sequencing, cycle sequencing, single base extension sequencing, solid phase sequencing, high throughput sequencing, massively parallel signature sequencing, low denaturation Coamplification-PCR at temperature (COLD- PCR), reversible dye-terminator sequencing, paired-end sequencing, short-term sequencing, exonuclease sequencing, ligation sequencing, short read sequencing, single molecule sequencing, synthesis sequencing, real-time sequencing, reverse terminator sequencing, nanopore sequencing, MS-PET sequencing and any combination thereof.
Sequence analysis of nucleic acid molecules, including barcoded nucleic acid molecules or derivatives thereof, may be direct or indirect. Thus, a sequence analysis substrate (which can be considered a molecule undergoing a sequence analysis step or process) may be a directly barcoded nucleic acid molecule or may be a molecule derived from it (eg, its complement). Thus, for example, in the sequence analysis step of a sequencing reaction, the sequencing template may be a barcoded nucleic acid molecule or may be a molecule derived from it. For example, the first and/or second strand DNA molecules may be directly sequenced (e.g., sequenced), that is, may directly participate in a sequence analysis reaction or process (e.g., a sequencing reaction or process, or sequence or otherwise identify the molecules ). Alternatively, the barcoded nucleic acid molecule may undergo a second-strand synthesis or amplification step prior to sequence analysis (eg, sequencing or identification by another technique). The substrate for sequence analysis (eg, template) can therefore be an amplicon or second strand of a barcoded nucleic acid molecule.
In some embodiments, both strands of the double-stranded molecule can be analyzed (eg, sequenced). In some embodiments, single-stranded molecules (eg, barcoded nucleic acid molecules) can be analyzed (eg, sequenced). Nucleic acid strands can be modified at the 3' end for single molecule sequencing.
In some embodiments, massively parallel pyrosequencing technology can be used to sequence nucleic acids. In pyrosequencing, nucleic acids are amplified within droplets of water in an oil solution (emulsion PCR), with each droplet containing a nucleic acid template attached to primer-coated beads, which then form clonal colonies. The sequencing system contains many wells of picoliter volume, each containing a bead and sequencing enzyme. Pyrosequencing uses luciferase to generate light to detect individual nucleotides added to nascent nucleic acids and uses the combined data to generate sequence reads.
As another example of a pyrosequencing application, released PPi can be detected by its immediate conversion to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP levels can be detected by photons generated by luciferase, as described by Ronaghi et al. . ,anus. biochemical242(1), 84-9 (1996);genome research.11(1), 3-11 (2001); Ronaji et al.science281 (5375), 363 (1998) and US Pat. LOUSE. patents no. 6,210,891, 6,258,568 and 6,274,320, the entire contents of which are incorporated herein by reference.
As noted above, massively parallel sequencing techniques can be used to sequence nucleic acids. In one embodiment, the massively parallel sequencing technology may be based on reversible dye terminators. For example, DNA molecules are first attached to primers on, for example, glass or silicon substrates and then amplified to form localized clonal colonies (eg, by bridge amplification). Four types of ddNTPs are added and unincorporated nucleotides are washed away. Unlike pyrosequencing, DNA can only be extended one nucleotide at a time due to the presence of blocking groups (for example, 3' blocking groups present on the sugar residue of ddNTP). The detector acquires images of fluorescently labeled nucleotides, and the dye is then chemically removed from the DNA, along with the terminal 3' blocking group, as a precursor for subsequent cycles. This process can be repeated until the data on the desired sequence is obtained.
In some embodiments, sequencing is performed by detecting hydrogen ions released during DNA polymerization. Microwells containing template DNA strands to be sequenced can be filled with one type of nucleotide. If the incoming nucleotide is complementary to the leader nucleotide of the template, it is incorporated into the growing complementary strand. This causes hydrogen ions to be released, activating the ultra-sensitive ion sensor, which indicates that a reaction has occurred. If there are homopolymeric repeats in the template sequence, more nucleotides will be incorporated into one cycle. This results in a corresponding number of released hydrogen ions and a proportionally higher electronic signal.
In some embodiments, sequencing can be performed in situ. In situ sequencing methods are particularly useful, for example, when biological samples remain intact after analytes have been barcoded on the surface of the sample (eg cell surface analytes) or within the sample (eg intracellular analytes). In situ sequencing typically involves the incorporation of labeled nucleotides (eg, fluorescently labeled mono- or dinucleotides) or labeled primers (eg, labeled random hexamers) in a sequential, template-dependent manner. Hybridization with a nucleic acid template allows the identity (ie, nucleotide sequence) of the incorporated nucleotides or labeled products of the starting products to be determined, and thus the nucleotide sequence of the corresponding template nucleic acid. For example, Mitra et al., (2003) describe aspects of in situ sequencinganus. biochemical320, 55-65 and Lee et al., (2014.)science, 343(6177), 1360-1363, the entire contents of each article are incorporated herein by reference.
In addition, PCT patent application no. WO2014/163886, WO2018/045181, WO2018/045186 and U.S. patents no. Examples of in situ sequencing techniques include, but are not limited to, STARmap (e.g. described in Wang et al., (2018)science, 361(6499) 5691), SIRENA (e.g. described in Moffitt, (2016)enzymatic method, 572, 1-49) and FISSEQ (described, for example, in US Patent Application Publication No. 2019/0032121). The entire contents of each of the above references are incorporated herein by reference.
For cleavage barcoded analytes, barcoded nucleic acid molecules or derivatives thereof (eg, barcoded nucleic acids to which one or more functional sequences have been added or from which one or more features have been removed) can be pooled and processed. molecules) for subsequent analysis, such as sequencing on a high-throughput sequencer. Association can be achieved using barcode sequences. For example, barcoded nucleic acid molecules for a particular partition may have the same barcode, which is different from barcodes for other spatial partitions. Alternatively, barcoded nucleic acid molecules of different partitions can be processed separately for subsequent analysis (eg, sequencing).
In some embodiments, when the capture probe does not contain a spatial barcode, the spatial barcode can be added after the capture probe captures the analyte from the biological sample and before the analyte is analyzed. When spatial barcoding is added after analyte capture, barcoding can be added after analyte amplification (eg, RNA reverse transcription and polymerase amplification). In some embodiments, analyte analysis utilizes direct sequencing of one or more captured analytes, e.g., direct sequencing of hybridized RNA. In some embodiments, direct sequencing is performed after reverse transcription of the hybridized RNA. In some embodiments, direct sequencing is performed after reverse transcription amplification of the hybridizing RNA.
In some embodiments, direct sequencing of the captured RNA is performed by sequencing by synthesis (SBS). In some embodiments, the sequencing primer is complementary to a sequence in one or more domains (eg, functional domains) of the capture probe. In such embodiments, sequencing by synthesis may involve reverse transcription and/or amplification to generate template sequences (eg, functional domains) from which primer sequences can be ligated.
SBS may involve hybridizing appropriate primers (sometimes called sequencing primers) to the nucleic acid template to be sequenced, extending the primers, and detecting the nucleotides used to extend the primers. Preferably, the nucleic acid used to extend the primer is detected prior to the addition of additional nucleotides to the growing nucleic acid strand, thereby allowing in situ base-by-base sequencing of the nucleic acid. Detection of incorporated nucleotides is facilitated by including one or more labeled nucleotides in the primer extension reaction. To allow suitable sequencing primers to hybridize to the template nucleic acid to be sequenced, the template nucleic acid should generally be in single-stranded form. If the nucleic acid templates that make up the nucleic acid signature exist in double-stranded form, those nucleic acid templates can be converted to single-stranded nucleic acid templates using methods well known in the art, such as denaturation, cleavage, and the like. Oligonucleotides that hybridize to the nucleic acid template and are used for primer extension are preferably short oligonucleotides, eg, 15 to 25 nucleotides in length. Sequencing primers can also be longer than 25 nucleotides. For example, a sequencing primer may be about 20 to about 60 nucleotides in length, or greater than 60 nucleotides in length. Sequencing primers can be provided in solution or in immobilized form. After the sequencing primers are annealed to the template nucleic acid to be sequenced by subjecting the template nucleic acid and the sequencing primer to appropriate conditions, primer extension is performed, for example using a nucleic acid polymerase and the provided nucleotides, at least some of which are provided in labeled form , if provided. The corresponding nucleotides are suitable for the primer extension conditions.
It is desirable to include a washing step after each primer extension step to remove unincorporated nucleotides that may interfere with subsequent steps. After the primer extension step is performed, the nucleic acid colonies are monitored to determine if the labeled nucleotides have been incorporated into the extended primers. The primer extension step can then be repeated to determine the next and subsequent nucleotides incorporated into the extended primer. If the sequence to be determined is unknown, the nucleotides are usually applied to a given colony in a selected order and then repeated during the analysis, e.g. dATP, dTTP, dCTP, dGTP.
SBS technology that can be used is, for example, but not limited to, patent application no. bar. US Patent No. 2007/0166705. application. bar. no. 2006/0188901, US Patent 7,057,026, US Pat. application. bar. US patent no. 2006/0240439. application. bar. 2006/0281109, PCT patent application. bar. WO 05/065814, US Pat. application. bar. PCT patent application no. 2005/0100900. bar. WO 06/064199, PCT patent application. bar. no. WO07/010,251, US Pat. application. bar. US Patent No. 2012/0270305. application. bar. 2013/0260372 and US Pat. application. bar. no. 2013/0079232, each of which is incorporated herein by reference in its entirety.
In some embodiments, direct sequencing of the captured RNA is performed by sequential fluorescence hybridization (eg, hybridization sequencing). In some embodiments, the hybridization reaction in which the RNA hybridizes to the capture probe is performed in situ. In some embodiments, the captured RNA is not amplified prior to hybridization with sequencing probes. In some embodiments, RNA is amplified (eg, reverse transcription to cDNA and cDNA amplification) prior to hybridization to sequencing probes. In some embodiments, amplification is performed using unimolecular chain reaction hybridization. In some embodiments, the amplification is performed using rolling chain amplification.
Sequential fluorescence hybridization may include sequential hybridization of probes that contain detectable degenerate primer sequences. A degenerate primer sequence is a short oligonucleotide sequence capable of hybridizing to any nucleic acid fragment independent of the sequence of the nucleic acid fragment. For example, such a method may include the steps of: (a) providing a mixture of four probes, each containing A, C, G or T at the 5'-end, and 5 to 5 degenerate nucleosides. The acid sequence is 11 nucleotides in length and further includes a functional domain (eg fluorescent molecule) that is different for probes with A, C, G or T at the 5'-end; (b) conversion step (a) the probes are linked to the target polynucleotide sequence, whose sequence requirements will be determined by the method; (c) the activity of the four functional domains is measured and the relative spatial location of the activity is recorded; (d) the relative spatial location of the activity is recorded from the target polynucleotide; Remove the reagents in steps (a)-(b) from the sequence; repeat steps (a)-(d) for n cycles until the nucleotide sequence of the spatial domain of each bead is determined, modified for use in step (a) The oligonucleotide is complementary to a portion of the target polynucleotide sequence and positions 1 through n flank the portion of the sequence. Since the barcode sequences differ, in some embodiments these additional flanking sequences are degenerate sequences. The fluorescence signal from cycle 1 to cycle n of each spot on the array can be used to determine the sequence of the target polynucleotide sequence.
In some embodiments, direct sequencing of the captured RNA using sequential fluorescence hybridization is performed in vitro. In some embodiments, the captured RNA is amplified (eg, reverse transcription to cDNA and cDNA amplification) prior to hybridization to sequencing probes. In some embodiments, the capture probe containing the captured RNA is exposed to a sequencing probe that targets the coding region of the RNA. In some embodiments, one or more sequencing probes target each coding region. In some embodiments, sequencing probes are designed to hybridize with sequencing reagents (eg, dye-labeled readout oligonucleotides). Sequencing probes can then be hybridized with sequencing reagents. In some embodiments, the output from the sequencing reaction is shown. In some embodiments, specific cDNA sequences are separated from images of sequencing reactions. In some embodiments, reverse transcription of the captured RNA is performed prior to hybridization with the sequencing probe. In some embodiments, the sequencing probes are designed to target the complement of the RNA coding region (e.g., the target cDNA).
In some embodiments, the captured RNA is directly sequenced using a nanopore-based approach. In some embodiments, direct sequencing is performed using direct nanopore RNA sequencing, in which the captured RNA is translocated through the nanopore. Nanopore currents can be recorded and converted to base sequences. In some embodiments, the captured RNA remains bound to the substrate during nanopore sequencing. In some embodiments, the captured RNA is released from the substrate prior to nanopore sequencing. In some embodiments, when the analyte of interest is a protein, the protein can be sequenced directly using nanopore-based methods. Examples of nanopore-based sequencing methods that can be used are described in Deamer et al.,Trend Biotech18, 14 7-151 (2000); Deamer et al.cumulative chemistry. reservoir35:817-825 (2002); Li et al.,night. alma mater2:611-615 (2003); Sony et al.,clinical. Chemical53, 1996-2001 (2007); Healey et al.,Nanomedicine2, 459-481 (2007); Cockcroft et al.,J. Am. Chemical. social responsibility130, 818-820 (2008); in US patents. 7,001,792. The entire contents of each of the above references are incorporated herein by reference.
In some embodiments, direct sequencing of the captured RNA is performed by ligation using single molecule sequencing. These techniques use DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. Oligonucleotides often have different labels that correlate with the identity of specific nucleotides in the sequence to which the oligonucleotide hybridizes. For example, aspects and features involved in sequencing by ligation are described in Shendure et al.science(2005), 309: 1728-1732, and US Pat. 5,599,675; 5,750,341; 6,969,488; 6,172,218; and 6,306,597, each of which is incorporated herein by reference in its entirety.
In some embodiments, nucleic acid hybridization can be used for sequencing. These methods use labeled nucleic acid decoder probes that are complementary to at least part of the barcode sequence. Multiplexed decoding can be performed using a set of many different probes with distinct labels. Non-limiting examples of nucleic acid hybridization sequencing are described, e.g., in U.S. Pat. patent no. 8,460,865, in Gunderson et al.,genome research14:870-877 (2004), each of which is incorporated herein by reference in its entirety.
In some embodiments, commercial high-throughput digital sequencing technologies can be used to analyze barcode sequences, where DNA templates are prepared for sequencing, not one-by-one, but in a batch process, and where many sequences are preferably read in parallel, or using serial very high-throughput processes that are inherently parallel. Examples of such technologies include Illumina® sequencing (eg, flow cell-based sequencing technology), sequencing by synthesis using modified nucleotides (eg, commercially available as TruSeq™ and HiSeq™ technologies from Illumina, Inc., San Diego, CA] . HeliScope™ Helicos Biosciences Corporation, Cambridge, MA and PacBio RS from Pacific Biosciences of California, Inc., Menlo Park, CA), ion detection sequencing technology (Ion Torrent, Inc., South San Francisco, CA) and DNA sequencing nanospheres (Complete Genomics , Inc., Mountain View, CA).
In some embodiments, detection of protons released upon incorporation of nucleotides into extension products is employed in the methods described herein. For example, no. US patent application, publication no. 2009/0026082, 2009/0127589, 2010/0137143 and 2010/0282617 can be used for direct barcode sequencing. The entire contents of each of the above references are incorporated herein by reference.
In some embodiments, real-time monitoring of DNA polymerase activity can be used during sequencing. For example, nucleotide incorporation can be detected by fluorescence resonance energy transfer (FRET), as described by Levene et al.,science(2003), 299, 682-686, Lundquist et al.,choose. Wright(2008), 33, 1026-1028 i Korlach et al.,procedure. national team. college. know america(2008), 105, 1176-1181. The entire contents of each of the foregoing references are incorporated herein by reference.
4. Reuse (a) General reuse
In various embodiments of the spatial analysis described herein, the features may include different types of capture probes for analyzing internal and external information of individual cells. For example, the features may include one or more of the following: 1) a capture probe characterized by a capture domain that binds to one or more endogenous nucleic acids in the cell; 2) a capture probe characterized by a capture domain that binds to the cell One or more exogenous nucleic acids (e.g. nucleic acids from microorganisms (e.g. viruses, bacteria)) that infect cells, nucleic acids introduced into cells (e.g. plasmids or nucleic acids derived therefrom, etc.), gene editing nucleic acids (eg, crRNA, guide RNA, etc. CRISPR-associated RNA); 3) capture probes with capture domains that bind to analyte capture agents (eg, antibodies linked to oligonucleotides that include probes with capture domains that bind barcode capture agents to capture target sequence domains), and 4) a unit for capture has a domain that binds to a protein that infects the cell (eg, a foreign protein expressed in the cell, a protein from a microorganism (eg, virus, bacteria)), or cellular protein binding partners (eg, immune cell receptor antigens).
In some embodiments of any spatial analysis method as described herein, spatial profiling involves the simultaneous analysis of two different types of analytes. The feature may be a bead of gel coupled (eg, reversibly coupled) to one or more capture probes. Capture probes can include spatial barcode sequences and poly(T) primer sequences that can hybridize to the poly(A) tails of mRNA transcripts. Capture probes can also include UMI sequences that can uniquely identify a given transcript. Capture probes can also include spatial barcode sequences and random N-mer primers capable of random hybridization to gDNA. In this configuration, the capture probes can contain the same spatial barcode sequence, allowing the association of downstream sequenced reads with signatures.
In some embodiments of any spatial analysis method as described herein, the feature may be a gel bead coupled (eg, reversibly coupled) to a capture probe. Capture probes can include spatial barcode sequences and poly(T) primer sequences that can hybridize to the poly(A) tails of mRNA transcripts. Capture probes can also include UMI sequences that can uniquely identify a given transcript. A capture probe may include a spatial barcode sequence and a capture domain capable of specific hybridization with an analyte capture agent. Analyte capture agents can include oligonucleotides that include an analyte capture sequence that interacts with a capture domain associated with a feature. Analyte capture reagent oligonucleotides can be conjugated to antibodies that bind to the cell surface. The oligonucleotide includes a barcode sequence (eg, the barcode of the analyte-binding portion) that uniquely recognizes the antibody (and thus the specific feature of the cell surface to which it binds). In this configuration, the capture probes include the same spatial barcode sequence, which allows downstream linking of nucleic acids to barcoded positions on the spatial array. In some embodiments of any of the spatial analysis methods described herein, the analyte capture agent may be produced by any suitable means, including for example the conjugation scheme described elsewhere herein.
In some embodiments of any of the spatial analysis methods described herein, other combinations of two or more biological analytes that can be measured simultaneously include, but are not limited to: (a) genomic DNA and cell surface features (e.g., by binding to cell surface features), (b) mRNA and lineage tracing constructs, (c) mRNA and cellular methylation status, (d) mRNA and available chromatin (eg, ATAC-seq, DNase-seq and/or MNase-seq), (e) mRNA and cellular surface or intracellular proteins and/or metabolites, (f) mRNA and chromatin (spatial organization of chromatin in cells), (g) analyte capture reagents (eg, any of the MHC multimers described above), and V(D)J sequences of immune receptors cell (eg, T cell receptors), (h) mRNA and perturbants (eg, CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease and/or antisense oligonucleotides as described herein), (i) genomic DNA and perturbator, (j) analyte capture and perturber, (k) available chromatin and perturber, (l) protein staining (e.g. chromatin in a cell) and perturbants, and (m) surface or intracellular proteins and/or metabolites and perturbants (eg, any perturbators described herein), or any combination thereof.
In some embodiments of any of the spatial analysis methods described herein, the first analyte may include a J sequence encoding an immune cell V(D) receptor (eg, TCR or BCR). In some embodiments, the nucleic acid molecule having a nucleic acid sequence encoding at least a portion of the V(D)J sequence of an immune cell receptor is the first cDNA generated from reverse transcription of the corresponding mRNA using a poly(T) primer comprising . The resulting cDNA can then be encoded using primers having a spatial barcode sequence (and optionally a UMI sequence) that hybridizes to at least part of the generated cDNA. In some embodiments, template-switching oligonucleotides in combination with terminal transferase or reverse transcriptase with terminal transferase activity can be used to generate primer regions on cDNA to which barcode primers can hybridize during cDNA production. For example, terminal transferase activity can add a poly(C) tail to the 3' end of a cDNA so that a template-switching oligonucleotide can be ligated across the poly(G) primer and the 3' end of the cDNA can be extended. by further binding. The original mRNA template and template-altering oligonucleotides can then be denatured from the cDNA, and barcode primers containing sequences complementary to at least part of the starting region generated on the cDNA can then hybridize to the cDNA and a barcode construct containing the barcode sequence (as well as any available selected UMI sequence) and the complement of the generated cDNA. Other methods and compositions suitable for barcoding cDNAs generated from mRNA transcripts, including those encoding V(D)J regions of immune cell receptors and/or methods and compositions for barcoding, including template-switching oligonucleotides, for example, PCT Patent Application Publication no. WO 2018/075693 and US Pat. Application publication no. 2018/0105808, the entire contents of each of which are incorporated herein by reference.
In some embodiments, V(D)J analysis can be performed using methods similar to those described herein. For example, V(D)J analysis can be performed using one or more analyte capture agents that bind to specific surface features of immune cells and are associated with barcode sequences (eg, analyte binding moiety barcodes). One or more analyte capture agents may contain MHC or MHC multimers. Barcoded oligonucleotides coupled to beads ready for V(D)J analysis. Oligonucleotides are attached to the beads via releasable bonds such as disulfide bonds. Oligonucleotides can include functional sequences that can be used in downstream processing, such as functional sequences, which can include sequencer-specific flow cell attachment sequences, such as P5 sequences, and functional sequences, which can include sequences a sequencing primer, such as the R1 binding sequence primer site. In some embodiments, the sequence may include a P7 sequence and an R2 primer binding site. A barcode sequence can be included in the template polynucleotide barcoding construct. Functional sequences can be chosen to be compatible with a variety of different sequencing systems, such as Ion Torrent Proton or PGM, Illumina sequencing instruments, etc., and their requirements. In some embodiments, barcode sequences, functional sequences (eg, flow cell attachment sequences), and additional sequences (eg, sequencing primer sequences) may be common to all oligonucleotides bound to a given bead. Barcoded oligonucleotides may also include sequences that facilitate template switching (eg, poly(G) sequences). In some embodiments, the additional sequence provides a unique molecular identifier (UMI) sequence fragment, as described elsewhere herein.
In an exemplary method for analyzing cellular polynucleotides using barcoded oligonucleotides, cells are incubated with barcoded oligonucleotides and additional reagents such as reverse transcriptase, primers, oligonucleotides (e.g., template-switching oligonucleotides), dNTPs, and reducing agents enter the partition ( eg, droplets in an emulsion). Within a compartment, cells can be lysed to produce a plurality of template polynucleotides (eg, DNA such as genomic DNA, RNA such as mRNA, etc.).
A reaction mixture characterized by a template polynucleotide from the cell and (i) a primer having the 3' end of a sequence that hybridizes to the template polynucleotide (eg, poly(T)) and (ii) a template-changing oligonucleotide An acid containing the first oligonucleotide toward the 5' end can undergo an amplification reaction to produce the first amplification product. In some embodiments, the template polynucleotide is an mRNA with a poly(A) tail, and the primer that hybridizes to the template polynucleotide includes a poly(T) sequence toward the 3' end that is complementary to the poly(A) segment. The first oligonucleotide may include at least one of an adapter sequence, a barcode sequence, a unique molecular identifier (UMI) sequence, a primer binding site, and a primer binding site, or any combination thereof. In some cases, the first oligonucleotide is a sequence that may be common to all partitions in a plurality of partitions. For example, the first oligonucleotide may include a flow cell attachment sequence, an amplification primer binding site, or a sequencing primer binding site, and the first amplification reaction facilitates attachment of the oligonucleotide to the template polynucleotide from the cell. In some embodiments, the first oligonucleotide includes a primer binding site. In some embodiments, the first oligonucleotide includes a sequencing primer binding site.
The sequence towards the 3' end of the primer (eg, poly(T)) hybridizes to the template polynucleotide. In the first amplification reaction, extension reaction reagents such as reverse transcriptase, nucleoside triphosphates, cofactors (e.g. Mg2+town eat2+), are also split, and the primer sequence can be extended using the cellular nucleic acid as a template to generate a transcript, such as a cDNA, that has a segment complementary to the cellular nucleic acid strand to which the primer anneals. In some embodiments, the reverse transcriptase has terminal transferase activity and the reverse transcriptase adds additional nucleotides, such as poly(C), to the cDNA in a template-independent manner.
Template-switching oligonucleotides, such as those containing poly(G) sequences, can hybridize to cDNA and facilitate template switching in the first amplification reaction. Thus, the transcript may include a primer sequence, a sequence complementary to the template polynucleotide from the cell, and a sequence complementary to the template replacement oligonucleotide.
In some embodiments of any of the spatial analysis methods described herein, after the first amplification reaction, the first amplification product or transcript may be subjected to a second amplification reaction to produce a second amplification product. In some embodiments, additional sequences (eg, functional sequences such as flow cell junction sequences, primer sequencing sequences, barcode sequences, etc.) are appended. The first and second amplification reactions can be performed in the same volume, e.g. in a droplet. In some embodiments, the first amplification product is subjected to a second amplification reaction in the presence of a barcode oligonucleotide to produce a second amplification product having a barcode sequence. The barcode string can be unique for a partition, that is, each partition can have a unique barcode string. A barcoded oligonucleotide may contain at least one segment of a template-changing oligonucleotide and at least the sequence of another oligonucleotide. A template-change oligonucleotide segment on a barcoded oligonucleotide may facilitate hybridization of the barcoded oligonucleotide to a transcript, e.g. cDNA, to facilitate the production of a second amplification product. In addition to the barcode sequence, the barcode oligonucleotide may include another oligonucleotide, such as at least one of an adapter sequence, a unique molecular identifier (UMI) sequence, a primer binding site, and a sequencing primer binding site. species or any combination thereof.
In some embodiments of any of the spatial analysis methods described herein, the second amplification reaction uses the first amplification product as a template and the barcoded oligonucleotides as primers. In some embodiments, the template-changing oligonucleotide segment on the barcoded oligonucleotide can be complementary to cDNA or cDNA having a sequence complementary to the template-changing oligonucleotide or a sequence replicated from the template-changing oligonucleotide. Partial hybridization of fragments. In the second amplification reaction, extension reaction reagents such as polymerase, nucleoside triphosphates, cofactors (e.g. Mg2+town eat2+), also copartitioned, can use the first amplified product as a template for extending the primer sequence. Another amplification product may include a second oligonucleotide, a template sequence of a polynucleotide fragment (eg, mRNA), and a sequence complementary to the primer.
In some embodiments of any of the spatial analysis methods described herein, the second amplification product uses the barcoded oligonucleotide as a template and at least a portion of the first amplification product as a primer. A segment of the first amplification product (eg, cDNA) having a sequence complementary to the template-switching oligonucleotide can be hybridized to a barcoded oligonucleotide segment that contains at least the sequence of the template-switching oligonucleotide segment. In the second amplification reaction, extension reaction reagents such as polymerase, nucleoside triphosphates, cofactors (e.g. Mg2+town eat2+), also shared, can use barcoded oligonucleotides as templates for the extension of primer sequences (eg, first amplification products). Another amplification product may include a primer sequence, a sequence complementary to a polynucleotide sequence (eg, mRNA), and a sequence complementary to another oligonucleotide.
In some embodiments of any of the spatial analysis methods described herein, three or more classes of biological analytes can be measured simultaneously. For example, the features can include capture probes that can be incorporated into the determination of at least three different types of analytes through three different capture domains. The beads can be coupled to barcoded oligonucleotides that include a capture domain that includes a poly(T) primer for mRNA analysis; barcoded oligonucleotides that include capture domains, The capture domain contains a random N-mer initiation sequence for gDNA analysis; and a barcode oligonucleotide that includes a capture domain that can specifically bind an analyte capture agent (eg, with antibodies to spatial barcodes).
In some embodiments of any of the spatial analysis methods described herein, other combinations of three or more biological analytes that can be measured simultaneously include, but are not limited to: (a) mRNA, lineage tracing constructs, and cell surface and/or intracellular proteins and /or metabolites; (b) mRNA, available chromatin (eg, ATAC-seq, DNase-seq and/or MNase-seq) and cell surface and/or intracellular proteins and/or metabolites; (c) mRNA, genomic DNA, and perturbation reagents (eg, CRISPR crRNA/sgRNA, TALENs, zinc finger nucleases, and/or antisense oligonucleotides as described herein); (d) mRNA, and chromatin and perturbation reagents available; (e) mRNA, analyte capture reagents (eg, any of the MHC multimers described herein), and perturbation reagents; (f) mRNA, cell surface and/or intracellular proteins and/or metabolites and perturbing agents; (g) mRNA, V(D)J sequences of immune cell receptors (such as T-cell receptors) and disruption reagents; (h) mRNA, analyte scavengers and immune cell receptors (i) V(D) sequences of cell surface and/or intracellular proteins and/or metabolites, analyte scavengers (such as MHC multimers as described herein), and Immune cell receptor J-sequence; (j) methylation status, mRNA and cell surface and/or intracellular proteins and/or metabolites; (k) mRNA, chromatin (eg, spatial organization of chromatin in a cell) and perturbation reagents; (l) V(D)J chromatin sequences of immune cell receptors (eg spatial organization of chromatin in cells); and perturbing agents; (m) mRNA, V(D)J sequences of immune cell receptors, and chromatin staining (eg, the spatial organization of chromatin in a cell), or any combination thereof.
In some embodiments of any of the spatial analysis methods described herein, four or more classes of biological analytes can be measured simultaneously. The feature could be a bead connected to barcoded primers, each of which could be involved in determining a different type of analyte. This feature is linked (eg, reversibly linked) to a capture probe that includes a capture domain that includes a poly(T) primer for mRNA analysis, and is also linked (eg, reversibly linked) to capture probes that include a capture domain which includes a random N-mer initiation sequence for gDNA analysis. Additionally, this feature is coupled (eg, reversibly coupled) to a capture probe that binds via its capture domain to the analyte capture sequence of the analyte capture agent. This feature can also be coupled (eg, reversibly coupled) to capture probes that can specifically bind nucleic acid molecules that can act as perturbants (eg, CRISPR crRNA/sgRNA, TALENs, zinc finger nucleases and/or antisense molecules) such as oligonucleotides as described here), through their capture domains.
In some embodiments of any of the spatial analysis methods described herein, each of the different spatial barcode capture probes present on a given feature or on a given grain includes the same spatial barcode sequence. In some embodiments, each barcoded capture probe can be released from the feature in a manner suitable for analysis of the corresponding analyte. For example, barcoded constructs A, B, C, and D can be generated and analyzed as described herein. The barcode construct A may include a sequence corresponding to the barcode sequence from the bead (eg, a spatial barcode) and a DNA sequence corresponding to the target mRNA. Barcode construct B may include sequences corresponding to barcode sequences from beads (eg, spatial barcodes) and sequences corresponding to genomic DNA. The barcode construct C may include a sequence corresponding to a barcode sequence from the bead (eg, a spatial barcode) and a sequence corresponding to a barcode sequence associated with an analyte capture agent (eg, an analyte binding group barcode). The barcode construct D may include a sequence corresponding to a barcode sequence from a bead (eg, a spatial barcode) and a sequence corresponding to a CRISPR nucleic acid (in some embodiments, also includes a barcode sequence). Each construct can be analyzed (eg, by any of a variety of sequencing methods), and the results can be linked to a specific cell of origin for the various analytes. Barcoded constructs (or even non-barcoded) can be adapted to analyze any analyte associated with a nucleic acid that can bind to such a construct.
In some embodiments of any of the spatial analysis methods described herein, other combinations of four or more biological analytes that can be measured simultaneously include, but are not limited to: (a) mRNA, lineage tracing constructs, cell surface and/or intracellular proteins and/ or metabolites and gDNA; (b) mRNA, available chromatin (eg ATAC-seq, DNase-seq and/or MNase-seq), surface and/or intracellular proteins and/or metabolites, and perturbants (eg CRISPR crRNA/sgRNA, TALENs, zinc nucleases fingerprint and/or antisense oligonucleotides as described herein); (c) mRNA, cell surface and/or intracellular proteins and/or metabolites, analyte capture agents (such as MHC multimers described herein) and immune cell receptors (such as V(D)J sequence receptors for T cells); (d) mRNA, genomic DNA, perturbation reagents and available chromatin; (e) mRNA, cell surface and/or intracellular proteins and/or metabolites, analyte capture reagents (eg, MHC multimers described herein) and perturbation reagents; (f) V(D)J mRNA sequences, cell surface and/or intracellular proteins and/or metabolites, perturbing agents and immune cell receptors (eg, T-cell receptors); (g) mRNA, perturbing agents, V(D)J sequences of analyte capture agents (eg, MHC multimers described herein) and immune cell receptors (eg, T-cell receptors); (h) mRNA, chromatin (eg, chromatin in cells, spatial organization of chromatin in cells) and perturbing reagents; (i) V(D)J chromatin sequences of immune cell receptors (eg spatial organization of chromatin in cells); and disturbing reagents; (j) mRNA, immune cell receptor (k) mRNA, V(D)J sequences of immune cell receptors, chromatin (eg spatial organization of chromatin) and perturbing reagents or any combination thereof.
(b) Construction of spatial arrays for the analysis of multiple analytes
The present disclosure also provides methods and materials for constructing spatial arrays capable of analyzing multiple analytes. In some embodiments, the spatial array comprises a plurality of features on the substrate, wherein one or more members of the plurality of features comprises a plurality of oligonucleotides having a first type of functional sequence and a second, different type of functional oligonucleotide sequence. In some embodiments, a feature may include oligonucleotides with two types of functional sequences. Features can be conjugated to oligonucleotides containing TruSeq functional sequences or to oligonucleotides containing Nextera functional sequences. In some embodiments, functional sequences may include sequencer-specific flow cell attachment sequences, such as P5 sequences, and functional sequences, which may include sequencing primer sequences, such as R1 primer binding sites. In some embodiments, one or more members of the plurality of features include both types of functional sequences. In some embodiments, one or more members of the plurality of features comprise a first type of functional sequence. In some embodiments, one or more members of the feature array comprise another type of functional sequence. In some embodiments, additional oligonucleotides can be added to the functional sequence to generate a complete oligonucleotide, wherein the complete oligonucleotide includes a spatial barcode sequence, an optional UMI sequence, a primer sequence, and a capture domain. Linking of these sequences can be by ligation, including udlanes as described in US Patent Application Publication No. 20140378345, the entire contents of which are incorporated herein by reference, or by any other convenient means. As discussed herein, oligonucleotides can be hybridized to groove sequences that facilitate the construction of complete intact oligonucleotides (eg, oligonucleotides capable of spatial analysis).
In some embodiments, oligonucleotides that hybridize to functional sequences located on features (eg, TruSeq and Nextera) include oligonucleotides capable of capturing different types of analytes (eg, mRNA, genomic DNA, cell surface proteins, or available chromatin).) capture domain. In some examples, an oligonucleotide that can bind to a TruSeq functional sequence can include a capture domain that includes a poly(T) capture sequence. In addition to poly(T) capture sequences, oligonucleotides that can incorporate TruSeq functional groups can include capture domains that include random N-mer sequences for capturing genomic DNA (eg, or as described herein). any other sequence or domain) capable of capturing any of the biological analytes described herein). In this case, spatial arrays can be constructed by applying a ratio of TruSeq-poly(T) to TruSeq-N-mer oligonucleotides to features containing functional TruSeq sequences. This can generate spatial arrays where one part of the oligonucleotide captures mRNA and the other part captures genomic DNA. In some embodiments, one or more feature array members include TruSeq and Nextera functional sequences. In this case, features containing both types of functional sequences allow binding of oligonucleotides specific for each functional sequence. For example, oligonucleotides that bind TruSeq functional sequences can be used to deliver oligonucleotides that include poly(T) capture domains, and oligonucleotides that bind Nextera functional sequences can be used to deliver oligonucleotides that include N-capture domains of genomic DNA.mer for catching. One skilled in the art will understand that any combination of capture domains (eg, a capture domain having any of the various capture sequences described herein that are capable of binding any of the various types of analytes described herein) can be combined with Nextera functional sequence oligonucleotides are combined to create spatial arrays.
In some embodiments, an oligonucleotide comprising a capture domain (eg, an oligonucleotide capable of conjugating an analyte) or an analyte capture agent may comprise an oligonucleotide sequence capable of binding or linking to a probe primer. Adapters may allow capture probes or analyte capture agents to be attached to any suitable assay primers and used in any suitable assay. Test primers may include a primer region and a sequence capable of binding or ligation to the adapter. In some embodiments, the adapter can be a non-specific primer (eg, a 5' overhang), and the test primer can include a 3' overhang that can be ligated to a 5' overhang. The primer region on the test primer can be any primer described herein, such as a poly(T) primer, a random N-mer primer, a target-specific primer, or a capture sequence of an analyte capture agent.
In some examples, the oligonucleotide may include an adapter, such as a 5' overhang of 10 nucleotides. Adapters can be ligated to test primers, each containing a 3' overhang with 10 nucleotides complementary to the adapter's 5' overhang. Capture probes can be used in any assay by connecting to assay primers designed for that assay.
Adapters and assay primers can be used to allow capture probes or analyte capture agents to be attached to any suitable assay primers and used in any suitable assay. Capture probes containing spatial barcodes can be attached to beads containing poly(dT) sequences. Capture probes containing spatial barcodes and poly(T) sequences can be used to assay a variety of biological analytes as generally described herein (e.g., biological analytes containing poly(A) sequences or conjugated or otherwise linked to analytes containing poly(A) sequences as capture agents for the analyte capture sequence).
Indented oligonucleotides with poly(A) sequences can be used to facilitate conjugation for capture probes that include spatial barcodes and a second conjugation-facilitating sequence for probe primers. Probe primers include sequences complementary to the second strand oligonucleotide sequence and probe-specific sequences that determine the function of the assay primer (eg, poly(T) primers, random N-mer primers, target-specific primers, or analyte capture as described herein Reagent capture sequence ).
In some embodiments of any of the spatial analysis methods described herein, the feature may include a capture probe comprising a spatial barcode comprising a surrogate oligonucleotide, e.g. which has a 3' terminal 3rG. For example, features with spatial barcodes functionalized with 3rG sequences (eg, gel beads) can be used to enable template switching (eg, reverse transcriptase template switching), but are not specific for any particular assay. In some embodiments, assay primers added to the reaction can determine which type of analyte is being analyzed. For example, probe primers may include binding domains capable of binding target biological analytes (eg, poly(T) to mRNA, N-mer to genomic DNA, etc.). Capture probes (eg, oligonucleotides capable of spatial analysis) can be generated by reverse transcriptase/polymerase extension, followed by template transfer to barcode adapter oligonucleotides to include barcodes and other functional sequences. In some embodiments, probe primers include capture domains capable of binding poly(T) sequences for mRNA analysis, random primers for genomic DNA analysis, or capable of binding nucleic acid molecules conjugated to the capture sequence of analyte binding moieties (eg, nucleic acid molecules). , an analyte capture sequence of an analyte capture agent) or a nucleic acid molecule that can function as a perturbing agent (eg, CRISPR crRNA/sgRNA, TALEN, zinc finger nuclease, and/or antisense oligonucleotides as described herein).
5. System for sample analysis
The methods described above for the analysis of biological samples can be implemented using different hardware components. Examples of such components are described in this section. However, it should be understood that in general, a variety of different devices and system components may be used to perform the various steps and techniques discussed herein, not all of which are specifically set forth.
tank2216Associated with the internal volume of the sample compartment2202through the liquid inlet2218.fluid outlet2220Also connected to the internal volume of the sample chamber2202, in valve2222.In turn, the valve2222connected to the waste pool2224And, optionally, analytical instruments2226.managing unit2228Electrically connected to another carrier2210, to the valve2222, to the waste water tank2224, and the reservoir2216.
During equipment operation2200, any of the above reagents, solutions and other biochemical components can be delivered to the sample compartment2202from the reservoir2216through the liquid inlet2218.Control unit2228, connected to the tank2216, can control the delivery of reagents, solutions and components and adjust the volume and flow rate according to the programmed analysis protocol for different sample types and analysis procedures. In some embodiments, a liquid reservoir2216It includes a pump that can be controlled by a control unit2228, to facilitate entry of the substance into the sample chamber2202.
In some embodiments, the reservoir2216It consists of several chambers, each of which is connected to the liquid inlet2218through the manifold (not shown). Control unit2228It can selectively deliver substances from one or more chambers to the sample chamber2202Ensure that the selected chamber is fluidly connected to the fluid inlet by adjusting the manifold2218.
In general, the control unit2228It can be configured to introduce substances from a container2216Enter the sample room2202before, after or both before and after the sample2208on the first substrate2206has interacted with a number of features2214on the first substrate2212Many examples of such substances have already been described. Examples of such substances include, but are not limited to, permeabilizers, buffers, fixatives, staining solutions, washing solutions, and solutions of various biological reagents (eg, enzymes, peptides, oligonucleotides, primers).
Trigger interactions between samples2208and a number of features2214, patterns and sequences are brought into spatial proximity. To facilitate this step, the second holder2210- under the control of the management unit2228– It can translate another base2212In any x, y and z coordinate direction. especially the control unit2228Can direct another holder2210translation second substrate2212In the z direction, the sample2208contact or near contact, a series of features2214.
In some embodiments, the device2200Optionally, an alignment subsystem can be included2230, can be electrically connected to the control unit2228.alignment subsystem2230function to ensure that samples2208and a number of features2214Align in the x-y plane before translating the second pad2212In the z direction, the sample2208contact or near contact, a series of features2214.
alignment subsystem2230This can be done in several ways. In some embodiments, for example, an alignment subsystem2230including an imaging unit that acquires one or more images showing reference marks on the first substrate2206and/or other substrate2212.Control unit2218Analyze the image to determine the appropriate translation for the other substrate2212in x and/or y coordinates to ensure that the samples2208and a number of features2214Align before translating in the z-coordinate direction.
In some embodiments, the control unit2228Optional adjustable removal of substances in the sample chamber2202.for example, a control unit2228Optional adjustable valve2222for introducing substances into the sample chamber2202from the reservoir2216sent to the waste disposal site2224In some embodiments, a waste container2224May include a reduced pressure source electrically connected to the control unit (not shown)2228.Control unit2228The liquid pressure at the liquid outlet can be adjusted2220Controls the rate at which liquid is removed from the sample chamber2202access to waste storage2224.
In some embodiments, the analyte from the sample2208or from a range of traits2214Optionally, it can be delivered to analytical instruments2226Correct valve adjustment2222through the control unit2228As noted above, in some embodiments, the analysis device2226including a source of reduced pressure electrically connected to the control unit (not shown)2228, so that the control unit2228The rate of analyte delivery to the analytical instrument can be adjusted2226.So, liquid output2220It effectively acts as an analyte collector while analyte analysis is performed by the analytical device2226.It should be noted that not all workflows and methods described in this paper are implemented with analytical settings2226For example, in some embodiments, an analyte affected by a number of features2214remain concatenated with the array (ie not cut from the array) and represent arrays2214Direct analysis for identification of specifically bound sample components.
In addition to the listed components, the device2200Optionally include other features. In some embodiments, for example, a sample chamber2202It includes a heating subsystem2232Electrically connected to the control unit2228.Control unit2228The heating subsystem can be activated2232heated sample2208and/or a series of features2214, which helps facilitate certain steps of the methods described here.
In some embodiments, a sample chamber2202includes an electrode2234Electrically connected to the control unit2228.Control unit2228The possibility of activating the electrodes2234, thereby establishing an electric field between the first substrate and the second substrate. For example, such fields can be used to facilitate the migration of analytes in a sample2208A series of orientation features2214.
In some of the methods described herein, one or more images of a pattern and/or array of features are obtained. The imaging equipment used to obtain such images can often be implemented in a variety of ways.
During operation of the recording device2250, a light source2252produce light. In general, the light produced by a light source2252Light in any or more of the ultraviolet, visible, and/or infrared regions of the electromagnetic spectrum may be included. A variety of different light source elements can be used to generate light, including but not limited to light emitting diodes, laser diodes, laser sources, fluorescent sources, incandescent sources, and glow discharge sources.
light produced by a light source2252Received with optics for light conditioning2254.In general, light conditioning optics2254Modify the light produced by the light source2252for specific imaging applications. For example, in some embodiments, light conditioning optics2254Modifying the spectral properties of light, for example, by filtering certain wavelengths of light. For this purpose, light conditioning optics2254Various spectroscopic optics such as filters, gratings, prisms and color beam splitters can be included.
In some embodiments, light conditioning optics2254It modifies the spatial properties of the light produced by the light source2252Examples of components that can be used for this purpose include (but are not limited to) apertures, phase masks, apodized elements, and diffusers.
After transforming the dimming optics2254, the light is received by the light transmission optics2256and point to the pattern2208or array of features2214, any of which sits on a carrier2258Dimming optics2254It is typically used to collect light and direct it onto the surface of a sample or array. A number of different optical elements can be used for this purpose, and examples of such elements include, but are not limited to, lenses, mirrors, beam splitters, and various other non-zero optical power elements.
the light emitted by the sample2208or array of features2214Collected by light gathering optics2260.Light collection optics in general2260may include elements similar to any of the above in relation to light delivery optics2256.The collected light can then be selectively modified by light conditioning optics2262, may generally include any of the elements described above in connection with light conditioning optics2254.
The detection subsystem then detects the light2264.Typically, a detection subsystem2264function to generate one or more sample images2208or array of features2214By detecting light from a pattern or array of features. A number of different imaging elements are available for the detection subsystem2264, including CCD detectors and other imaging devices.
Each of the above components can optionally be connected to the control unit2228as the picture shows
Recording equipment2250Images are generally available in a number of different imaging modalities. In some embodiments, for example, the image is a transmitted light image such as
In general, the control unit2228Any step of the method described herein that does not explicitly require user intervention can be performed by sending appropriate control signals to the sample processing device components.2200and/or recording equipment2250.To perform these steps, the control unit2228It usually consists of software instructions that, when executed, cause the control unit to2228Take concrete steps. In some embodiments, the control unit2228Included are an electronic processor and software instructions that can be read by the electronic processor and cause the processor to perform the steps described herein. In some embodiments, the control unit2228It consists of one or more application-specific integrated circuits that have a circuit configuration effective as software instructions.
Control unit2228This can be done in several ways.
memory unit2282store information. In some embodiments, the storage unit2282is a computer readable medium. memory unit2282Volatile memory and/or non-volatile memory can be included. storage device2284Mass storage and, in some embodiments, computer-readable media can be provided. In some embodiments, a storage device2284It can be a floppy disk drive, hard disk, optical disk, tape, solid state drive, or other type of writable media.
input/output interface2286Implement input/output operations. In some embodiments, an input/output interface2286Includes keyboard and/or pointing device. In some embodiments, an input/output interface2286A display unit is included for displaying a graphical user interface and/or displaying information.
executes and causes the instructions of the control unit2228Implementations that perform any of the steps or processes described herein may be implemented in digital electronic circuitry or in computer hardware, firmware, or a combination thereof. These instructions may be implemented in a computer program product tangibly embodied in an information carrier, for example, in a machine-readable storage device for a programmable processor (for example, a processor2280). Computer programs may be written in any form of programming language, including translated or interpreted languages, and may be used in any form, including in a computer environment, as a stand-alone program or as a module, component, subroutine, or other suitable unit of use. Storage devices suitable for tangible embodiment of computer program instructions and data include all forms of non-volatile memory, including, for example, semiconductor storage devices such as EPROM, EEPROM, and flash memory devices; magnetic disks, such as embedded hard disks and removable disks, magneto-optical disks, and CD-ROM and DVD-ROM disks. Processors and memory can be supplemented or combined with ASICs (Application Specific Integrated Circuits).
processor2280Any one or more arrays of suitable processors may be included. Suitable processors for executing program instructions include, by way of example, general purpose and special purpose microprocessors, and a single processor or one of multiple processors of any type of computer or computing device.
exemplary embodiment
In some non-limiting examples of the workflow described herein, samples can be immersed in 100% chilled methanol and incubated at -20°C for 30 minutes. After 20 minutes, the samples can be removed and rinsed in ultra-pure water. After washing the sample, prepare a fresh eosin solution and the sample can be poured in isopropanol. After incubating the sample in isopropanol for 1 minute, the reagent can be removed by holding the slide at an angle so that the bottom edge of the slide is in contact with a lab tissue and allowing it to air dry. Samples can be covered evenly with hematoxylin solution and incubated at room temperature for 7 minutes. After incubating the specimen in hematoxylin for 7 minutes, the reagent can be removed by holding the slide at an angle so that the bottom edge of the slide can come into contact with a lab cloth. The sample slide can be immersed in water and excess liquid removed. Afterwards, the samples can be covered with blue buffer and incubated for 2 minutes at room temperature. The slide in question filled with samples can be re-immersed in water, evenly covered with eosin solution and incubated at room temperature for 1 minute. Slides can be air-dried for a maximum of 30 minutes and incubated at 37°C for 5 minutes. Specimens can be imaged using a bright field imaging setup.
Additionally, biological samples can be processed to permeabilize the sample and generate cDNA using the following exemplary steps. Samples can be exposed to permeabilase and incubated at 37°C for a predetermined permeabilization time (which is tissue type specific). Permeabilase can be removed and samples prepared for analyte capture by adding 0.1X SSC buffer. Samples can then be subjected to a thermocycling protocol before equilibration (eg, coverslip temperature and pre-equilibration at 53 °C, reverse transcription at 53 °C for 45 min, then hold at 4 °C) and the SSC buffer can be removed. A master mix containing nuclease-free water, reverse transcriptase reagents, template-changing oligonucleotides, reducing agents, and reverse transcriptase can be added to biological samples and substrates, and samples with the master mix can be used for thermocycling protocols (eg, reverse transcription at 53 C for 45 minutes and keep at 4 °C). Second-strand synthesis can be performed on the substrate by subjecting the substrate to a thermal cycling protocol (eg, pre-equilibration at 65°C, second-strand synthesis at 65°C for 15 minutes, then holding at 4°C). The Master Mix reagent can be removed from the samples by adding 0.8 M KOH and incubating at room temperature for 5 minutes. KOH can be removed and wash buffer can be added and removed from the sample. A second-strand mixture including second-strand reagents, second-strand primers, and second-strand enzyme can be added to the sample, and the sample can be sealed and incubated. At the end of the incubation, the reagent can be removed and the elution buffer can be added and removed from the sample, 0.8 M KOH can be added back to the sample and the sample can be incubated at room temperature for 10 minutes. Tris-HCl can be added and the reagents mixed. Samples can be transferred to new tubes, vortexed and placed on ice.
Additionally, biological samples can be processed using the following example steps for cDNA amplification and quality control. The qPCR Mix, including nuclease-free water, qPCR Master Mix and cDNA primers, can be prepared and pipetted into the wells of the qPCR plate. A small amount of sample can be added to the qPCR mixture on the plate and thermally cycled according to a predetermined thermal cycling protocol (eg step 1: 98°C for 3 minutes, step 2: 98°C for 5 seconds, step 3: 63°C for 30 seconds, fourth step: record the amplification signal, fifth step: repeat 98°C for 5 seconds, 63°C for 30 seconds, a total of 25 cycles). After completion of the thermal cycle, the cDNA amplification mix including the amplification mix and the cDNA primer can be prepared and combined and mixed with the remaining sample. Samples can then be incubated and thermocycled (eg, lid temperature at 105°C for approximately 45-60 minutes; step 1: 98°C for 3 minutes, step 2: 98°C for 15 seconds, step 3: 63°C for 20 seconds, fourth step: 1 minute at 72°C, fifth step: [cycle number determined by qPCR Cq value], sixth step: 1 minute at 72°C, seventh step: hold at 4°C). Samples can then be stored at 4°C for up to 72 hours or -20°C for up to 1 week, or resuspended in 0.6X SPRIselect reagent and pipetted to ensure proper mixing. Samples can then be incubated at room temperature for 5 minutes and cleaned by placing the samples on a magnet (eg with the magnet on high). The supernatant can be removed and 80% ethanol can be added to the pellet and incubated for 30 seconds. The ethanol can be removed and the bead can be washed again. The sample can then be centrifuged and placed on a magnet (eg magnet in low position). Any remaining ethanol can be removed and the sample air-dried for a maximum of 2 minutes. The magnet can be removed, the elution buffer is added to the sample, mixed and incubated at room temperature for 2 minutes. The sample can then be placed on the magnet (eg in the down position) until the solution becomes clear. Samples can be transferred to new test tube strips and stored at 4°C for up to 72 hours or at -20°C for up to 4 weeks. A portion of the sample can be tested on an Agilent Bioanalyzer High Sensitivity chip, where a region can be selected and the cDNA concentration measured to calculate the total cDNA yield. Alternatively, quantification can be determined using an Agilent Bioanalyzer or an Agilent TapeStation.
Additionally, biological samples can be processed using the following example steps to construct a spatial gene expression library. A fragmentation mixture including fragmentation buffer and fragmentation enzyme can be prepared on ice. Elution buffer and residue mixture can be added to each sample, mixed and centrifuged. The sample mixture can then be placed in a thermocycler and cycled according to a predetermined protocol (eg, lid temperature at 65 °C for approximately 35 min, block precooling at 4 °C, followed by lysis at 32 °C for 5 min, end fix and A-tailing at 65°C for 30 minutes, incubation at 4°C). 0.6X SPRIselect reagent can be added to samples and incubated at room temperature for 5 minutes. Samples can be placed on a magnet (eg high level) until the solution is clear, then the supernatant can be transferred to a new strip of tubing. 0.8X SPRIselect reagent can be added to samples, mixed and incubated at room temperature for 5 minutes. The sample can be placed on a magnet (eg high) until the solution becomes clear. The supernatant can be removed and 80% ethanol can be added to the pellet, the pellet can be incubated for 30 seconds and the ethanol can be removed. Ethanol washing can be repeated and the sample placed on the m