Generating capture probes for spatial analysis

Abstract

The present disclosure relates to generating capture probes for spatial analysis. The present disclosure relates to compositions and methods for producing capture probes on a substrate for identifying the position of an analyte in a biological sample.

Description

[0001] This application is a divisional application of Chinese patent application No. 201980091742.0.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 777,521 filed on December 10, 2018, U.S. Provisional Patent Application No. 62 / 779,342 filed on December 13, 2018, U.S. Provisional Patent Application No. 62 / 779,348 filed on December 13, 2018, U.S. Provisional Patent Application No. 62 / 788,867 filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,871 filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,885 filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,897 filed on January 6, 2019, U.S. Provisional Patent Application No. 62 / 788,905, U.S. Provisional Patent Application No. 62 / 788,906 filed January 6, 2019, U.S. Provisional Patent Application No. 62 / 811,495 filed February 27, 2019, U.S. Provisional Patent Application No. 62 / 812,219 filed February 28, 2019, U.S. Provisional Patent Application No. 62 / 819,439 filed March 15, 2019, U.S. Provisional Patent 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The contents of each of these applications are incorporated herein by reference in their entirety. Background Art

[0004] Cells within a subject's tissue differ in cell morphology and / or function due to different analyte levels (e.g., gene and / or protein expression) within different cells. The specific location of a cell within a tissue (e.g., the location of the cell relative to neighboring cells or the location of the cell relative to the tissue microenvironment) can affect, for example, the cell's morphology, differentiation, fate, viability, proliferation, behavior, and signal transduction and crosstalk with other cells in the tissue.

[0005] Spatial heterogeneity has been previously studied using techniques that provide data for only a few analytes in intact tissue or partial tissue contacts, or provide data for a large number of analytes in single cells, but cannot provide information about the location of single cells in the maternal biological sample (e.g., tissue sample).

[0006] Arrays for capturing spatial information of analytes in biological samples can be prepared in a variety of ways. Arrays can be designed with multiple capture probes. Capture probes can be designed to capture a large number of analytes (e.g., mRNA) or to capture specific (e.g., gene-specific) analytes. The methods described herein provide for preparing arrays with capture probes and means for capturing analytes. Summary of the Invention

[0007] Provided herein is a method for producing a spatial array, comprising: (a) providing an array comprising a plurality of oligonucleotides, wherein the 3′ ends of the oligonucleotides of the plurality of oligonucleotides are attached to a substrate, (b) providing a plurality of primers, wherein a primer in the plurality of primers is substantially complementary to a portion of the oligonucleotide, (c) extending the primer using the oligonucleotide as a template, thereby producing a first oligonucleotide having a free 3′ end, (d) extending the first oligonucleotide to produce a 3′ overhang, (e) providing a splint oligonucleotide that hybridizes to the 3′ end of the first oligonucleotide, and (f) attaching a second oligonucleotide to the 3′ end of the first oligonucleotide, wherein the second oligonucleotide comprises a sequence that is substantially complementary to the portion of the splint oligonucleotide, and wherein the second oligonucleotide comprises a capture domain, thereby producing the spatial array.

[0008] In some embodiments of the method for producing spatial array, the oligonucleotide in a plurality of oligonucleotide comprises a spatial barcode. In some embodiments, the oligonucleotide in a plurality of oligonucleotide comprises a first constant sequence. In some embodiments of the method for producing spatial array, the oligonucleotide in a plurality of oligonucleotide also comprises a second constant sequence. In some embodiments of the method for producing spatial array, the oligonucleotide in a plurality of oligonucleotide has a length of 20 to 60 nucleotide.

[0009] In some embodiments of the method for producing spatial arrays, the second oligonucleotide also includes a constant sequence, and wherein the constant sequence is substantially complementary to the portion of the splint oligonucleotide. In some embodiments of the method for producing spatial arrays, the second oligonucleotide also includes a unique molecular identifier. In some embodiments of the method for producing spatial arrays, the second oligonucleotide also includes a spatial barcode.

[0010] In some embodiments of the method of producing a spatial array, the capture domain comprises a poly-T sequence. In some embodiments of the method of producing a spatial array, the capture domain comprises a gene-specific sequence.

[0011] In some embodiments of the method for producing a spatial array, the 3′ overhang of step (d) is produced by an enzyme. In some embodiments of the method for producing a spatial array, the enzyme is a template-independent polymerase. In some embodiments of the method for producing a spatial array, the template-independent polymerase is terminal deoxynucleotidyl transferase (TdT) or poly (A) polymerase.

[0012] On the other hand, provided herein are methods for determining the location of an analyte in a biological sample, comprising: (a) contacting the biological sample with a spatial array produced by any one of the various methods described herein, (b) permeabilizing the biological sample to allow the analyte in the biological sample to specifically bind to the capture domains on the spatial array, and (c) identifying the analyte that specifically binds to the capture domain, thereby determining the spatial location of the analyte in the biological sample.

[0013] In some embodiments of the method for determining the location of an analyte in a biological sample, a second analyte in the biological sample interacts with a second capture domain on the spatial array, and step (c) includes identifying the second analyte that specifically binds to the second capture domain, thereby determining the spatial location of the analyte in the second biological sample.

[0014] On the other hand, provided herein is a method for preparing a spatial array, comprising (a) contacting an array comprising one or more sets of first oligonucleotides with a hydrogel comprising one or more sets of second oligonucleotides, wherein the first oligonucleotides of the one or more sets of first oligonucleotides comprise a capture domain, the second oligonucleotides of the one or more sets of second oligonucleotides comprise a capture domain, or both, (b) attaching the first oligonucleotides to the second oligonucleotides to produce a capture probe comprising a capture domain, and (c) reducing the size of the hydrogel comprising the capture probes by removing water to form a shrinking hydrogel comprising the capture probes, thereby producing a spatial array.

[0015] In some embodiments of the method for preparing the spatial array, one or more groups of second oligonucleotides are fixed in a hydrogel. In some embodiments of the method for preparing the spatial array, the fixed second oligonucleotides are divided so as to separate a first population of the fixed second oligonucleotides from another population of the fixed second oligonucleotides.

[0016] In some embodiments of the method for the preparation of spatial array, the second oligonucleotide fixing is divided into a plurality of second oligonucleotide partitions, thereby the different populations of the second oligonucleotide fixing are separated from each other. In some embodiments of the method for the preparation of spatial array, the first fixing second oligonucleotide of the partition colony of the second oligonucleotide fixing is different from the second fixing second oligonucleotide of another partition colony of the second oligonucleotide fixing. In some embodiments of the method for the preparation of spatial array, the first fixing second oligonucleotide, the second fixing second oligonucleotide or both comprise spatial bar code.

[0017] In some embodiments of the method for preparing a spatial array, a first immobilized second oligonucleotide of a partitioned population of immobilized second oligonucleotides comprises a spatial barcode that is different from a spatial barcode of a second immobilized second oligonucleotide of another partitioned population of immobilized second oligonucleotides.

[0018] In some embodiments of the method for preparing a spatial array, one or more second oligonucleotide partitions comprises a population of immobilized capture probes comprising a spatial barcode that is different from the spatial barcode of the population of immobilized capture probes of another second oligonucleotide partition.

[0019] In some embodiments of the method for preparing a spatial array, one or more groups of first oligonucleotides are partitioned. In some embodiments of the method for preparing a spatial array, one or more groups of first oligonucleotides are partitioned into first oligonucleotide partitions. In some embodiments of the method for preparing a spatial array, one or more first oligonucleotide partitions include a first oligonucleotide population that is different from the first oligonucleotide in another first oligonucleotide partition. In some embodiments of the method for preparing a spatial array, the first oligonucleotide of the first oligonucleotide population in one or more first oligonucleotide partitions includes a spatial barcode. In some embodiments of the method for preparing a spatial array, the first oligonucleotide of the first oligonucleotide population in one or more first oligonucleotide partitions includes a spatial barcode that is different from the spatial barcode of the first oligonucleotide of the first oligonucleotide population in another first oligonucleotide partition. In some embodiments of the method for preparing a spatial array, the spatial barcode of the first oligonucleotide of the first oligonucleotide population in one or more first oligonucleotide partitions is different from the spatial barcode of the second oligonucleotide of the second oligonucleotide population in one or more second oligonucleotide partitions. In some embodiments of the method for preparing a spatial array, the first oligonucleotide partition includes a first population of the first oligonucleotide, the spatial barcode it includes is different from the spatial barcode of the second population of the first oligonucleotide in another first oligonucleotide partition.

[0020] In some embodiments of the method for preparing a spatial array, the reduction in size of the hydrogel in step (c) is equidistant. In some embodiments of the method for preparing a spatial array, the removal of water in step (c) comprises a dehydrating solvent. In some embodiments of the method for preparing a spatial array, the solvent comprises at least one of alcohol, ketone, aldehyde, acetonitrile, or a combination thereof. In some embodiments of the method for preparing a spatial array, the solvent comprises at least one of glycerol, ethanol, acetonitrile, acetone, 2-propanol, and a combination thereof. In some embodiments, the removal of water in step (c) comprises the use of salt. In some embodiments of the method for preparing a spatial array, the salt comprises an inorganic salt. In some embodiments of the method for preparing a spatial array, the removal of water in step (c) comprises an acid. In some embodiments of the method for preparing a spatial array, the removal of water in step (c) comprises a dehydration process.

[0021] In some embodiments of the method for preparing a spatial array, the shrunk hydrogel is about 2 to about 10 times smaller in linear dimension than the size of the hydrogel before shrunk hydrogel (pre-shrunk hydrogel) of step (c). In some embodiments of the method for preparing a spatial array, the shrunk hydrogel is at least 2 times smaller in linear dimension than the pre-shrunk hydrogel. In some embodiments of the method for preparing a spatial array, the shrunk hydrogel is at least 4 times smaller in linear dimension than the pre-shrunk hydrogel. In some embodiments of the method for preparing a spatial array, the shrunk hydrogel is at least 8 times smaller in linear dimension than the pre-shrunk hydrogel.

[0022] In some embodiments of the method for preparing the spatial array, attachment includes polymerase chain reaction. In some embodiments of the method for preparing the spatial array, the hydrogel includes DNA polymerase, multiple primers and multiple nucleotides. In some embodiments of the method for preparing the spatial array, members of multiple first oligonucleotides are cross-linked with the hydrogel. In some embodiments of the method for preparing the spatial array, the capture probe is amplified. In some embodiments of the method for preparing the spatial array, the capture probe is amplified before reducing the size of the hydrogel.

[0023] In some embodiments of the method for preparing a spatial array, the hydrogel is prepared from hydrogel subunits comprising at least one of acrylamide, bisacrylamide, poly(ethylene glycol), polyaliphatic polyurethane, polyether polyurethane, polyester polyurethane, polyethylene copolymer, polyamide, polyvinyl alcohol, polypropylene glycol, polytetrahydrofuran, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and combinations thereof.

[0024] In some embodiments of the method for preparing a spatial array, the first oligonucleotide in one or more groups of first oligonucleotides comprises a spatial barcode. In some embodiments of the method for preparing a spatial array, the first oligonucleotide in one or more groups of first oligonucleotides comprises a unique molecular identifier. In some embodiments of the method for preparing a spatial array, the first oligonucleotide in one or more groups of first oligonucleotides comprises a spatial barcode and a capture domain.

[0025] On the other hand, provided herein are methods for determining the location of an analyte in a biological sample, comprising (a) providing a spatial array of any one of the methods described herein, (b) contacting the biological sample with a spatial array of any one of the spatial arrays described herein under conditions that allow the analyte to specifically bind to the capture probes of the spatial array, and (c) determining the identity of the analyte that specifically binds to the capture probe, thereby determining the location of the analyte in the biological sample.

[0026] In some embodiments of the method for determining the location of an analyte in a biological sample, the biological sample is a tissue sample. In some embodiments of the method for determining the location of an analyte in a biological sample, the tissue sample is a fresh frozen tissue sample. In some embodiments of the method for determining the location of an analyte in a biological sample, the tissue sample comprises tumor cells. In some embodiments of the method for determining the location of an analyte in a biological sample, the tissue sample comprises a tissue section.

[0027] In some embodiments, the method for determining the location of an analyte in a biological sample further comprises migrating the analyte in the biological sample to the capture probe. In some embodiments of the method for determining the location of an analyte in a biological sample, the migration comprises passive migration. In some embodiments of the method for determining the location of an analyte in a biological sample, the migration comprises active migration.

[0028] In some embodiments of the method for determining the location of an analyte in a biological sample, the analyte comprises at least one of RNA, DNA, a protein, a lipid, a peptide, a metabolite, a small molecule, and an analyte-capture agent.

[0029] On the other hand, provided herein is a method for producing an array comprising (a) providing a substrate comprising a plurality of features fixed to the substrate, wherein a feature in the plurality of features comprises a first oligonucleotide and wherein the first oligonucleotide comprises a first spatial barcode, (b) delivering a second oligonucleotide to the first oligonucleotide, wherein the second oligonucleotide comprises a second spatial barcode and a capture domain, and (c) attaching the first oligonucleotide and the second oligonucleotide, thereby producing a capture probe comprising the first spatial barcode, the second spatial barcode, and the capture domain.

[0030] In some embodiments of the method of generating an array, the first oligonucleotide is associated with one of a plurality of different first spatial barcodes. In some embodiments of the method of generating an array, the second oligonucleotide is associated with one of a plurality of different second spatial barcodes.

[0031] In some embodiments of the method for producing an array, step (c) comprises connecting the first oligonucleotide and the second oligonucleotide. In some embodiments of the method for producing an array, connecting comprises using a ligase. In some embodiments of the method for producing an array, the first oligonucleotide and the second oligonucleotide are attached to each other by a chemical coupling reaction.

[0032] In some embodiments of the method for producing an array, the first oligonucleotide includes a 3' end masked by a blocking group. In some embodiments of the method for producing an array, the blocking group is removed before the second oligonucleotide is delivered to the first oligonucleotide. In some embodiments of the method for producing an array, the blocking group is a photo-cleavable blocking group. In some embodiments of the method for producing an array, the photo-cleavable blocking group is removed by exposing the first oligonucleotide to light. In some embodiments of the method for producing an array, the photo-cleavable blocking group coupled to the first oligonucleotide is protected by applying a photomask.

[0033] In some embodiments of the method of producing an array, the first feature is separated from other features in the set by one or more physical barriers, wherein the one or more physical barriers form one or more chambers, and wherein the first oligonucleotide is delivered to a chamber of the one or more chambers.

[0034] In some embodiments of the method of producing an array, the one or more physical barriers comprise a hydrophobic surface. In some embodiments of the method of producing an array, the one or more physical barriers comprise one or more chamber walls having a tapered end in contact with the substrate. In some embodiments of the method of producing an array, the first oligonucleotide comprises a unique molecular identifier.

[0035] On the other hand, provided herein is a method for producing an array, comprising (a) providing a substrate comprising two or more partitions, wherein a first partition of the two or more partitions comprises a feature fixed to the substrate, wherein the feature comprises a first oligonucleotide, and wherein the first oligonucleotide comprises a first spatial barcode, (b) delivering a second oligonucleotide to the first partition, wherein the second oligonucleotide comprises a second spatial barcode and a capture domain, and (c) attaching the first oligonucleotide and the second oligonucleotide in the first partition, thereby generating a capture probe in the first partition comprising the first spatial barcode, the second spatial barcode, and the capture domain.

[0036] In some embodiments, the method of producing an array further comprises (d) delivering a third oligonucleotide to a second partition of two or more partitions, wherein the third oligonucleotide comprises a third spatial barcode and a second capture domain, wherein the second partition comprises a second feature, wherein the second feature comprises a fourth oligonucleotide comprising the first spatial barcode, and (e) connecting the third oligonucleotide and the fourth oligonucleotide, thereby producing a second capture probe in the second partition comprising the first spatial barcode, the third spatial barcode, and the second capture domain.

[0037] In some embodiments of the method for generating an array, two or more partitions are separated by one or more physical barriers. In some embodiments of the method for generating an array, one or more physical barriers are printed on a substrate. In some embodiments of the method for generating an array, one or more physical barriers form one or more chambers comprising one or more chamber walls. In some embodiments of the method for generating an array, one or more chamber walls comprise a hydrophobic surface. In some embodiments, one or more chamber walls comprise a tapered end in contact with the substrate. In some embodiments of the method for generating an array, a microfluidic device is used to deliver the second oligonucleotide.

[0038] On the other hand, the present invention provides a method for enriching a population of capture probes, comprising: (a) providing a plurality of capture probes, wherein: (i) members of the plurality of capture probes comprise a plurality of different spatial barcodes, and (ii) a target capture probe of the population of capture probes to be enriched comprises a target spatial barcode, and (b) enriching a population of target capture probes comprising a target spatial barcode from the plurality of capture probes.

[0039] On the other hand, provided herein is a method for determining the location of an analyte in a biological sample, comprising (a) providing a substrate comprising a plurality of capture probes, wherein a subset of the plurality of capture probes is attached at a location on the substrate, and wherein two or more capture probes of the subset of capture probes comprise a first common spatial barcode, (b) contacting the biological sample with the substrate such that the capture probes of the subset of capture probes specifically bind to an analyte from the biological sample, wherein the analyte specifically bound to the capture probes comprises the first common spatial barcode or its complement, (c) identifying a region of interest in the biological sample, wherein the region of interest comprises the analyte specifically bound to the capture probe, and (d) enriching the analyte specifically bound to the capture probes to determine the location of the analyte in the biological sample.

[0040] In some embodiments of the method for determining the location of an analyte in a biological sample, the substrate further comprises a second subset of capture probes attached at a second location on the substrate, wherein two or more capture probes of the second subset of capture probes comprise a second common spatial barcode that is different from the first common spatial barcode.

[0041] In some embodiments of the method for determining the location of an analyte in a biological sample, the substrate further comprises a third subset of capture probes attached to a third location on the substrate, wherein two or more capture probes of the third subset of capture probes comprise a third common spatial barcode that is different from the first common spatial barcode and the second common spatial barcode. In some embodiments of the method for determining the location of an analyte in a biological sample, the capture probe comprises a capture domain.

[0042] In some embodiments of the method for determining the position of an analyte in a biological sample, the analyte comprises at least one of RNA, DNA, and an analyte capture agent. In some embodiments of the method for determining the position of an analyte in a biological sample, the analyte capture agent comprises an antigen binding molecule. In some embodiments of the method for determining the position of an analyte in a biological sample, the analyte capture agent comprises a region configured to couple or specifically bind to a capture domain of a capture probe.

[0043] In some embodiments of the method for determining the location of an analyte in a biological sample, the region of interest is identified by shallow sequencing at least a subset of the plurality of analytes that specifically bind to the capture probes and identifying a first common spatial barcode associated with the region of interest. In some embodiments of the method for determining the location of an analyte in a biological sample, the region of interest is identified by at least one of immunostaining, immunohistochemistry, microscopy, visual inspection, and combinations thereof.

[0044] In some embodiments of the method for determining the location of an analyte in a biological sample, step (d) comprises performing a nucleic acid extension reaction using (i) an analyte that specifically binds to a capture probe and (ii) a primer that has sequence specificity to a first common spatial barcode or its complement to produce an enriched plurality of analytes that specifically bind to one or more capture probes that include the first common barcode or its complement.

[0045] In some embodiments of the method for determining the location of an analyte in a biological sample, the analyte is amplified by performing a polymerase chain reaction (PCR).

[0046] In some embodiments, the method for determining the location of an analyte in a biological sample further includes enriching a population of capture probes present in a subregion of interest (a), wherein the subregion of interest is located within the region of interest, wherein the subregion of interest includes a sub-plurality of capture probes, and wherein: (i) two or more capture probes of the sub-plurality of capture probes include a first common subspace barcode, and (ii) the first common subspace barcode sequence is associated with the subregion of interest, and (b) enriching the population of capture probes comprising the first common subspace barcode.

[0047] In some embodiments, the method for determining the location of an analyte in a biological sample further comprises (a) enriching a population of capture probes present in a sub-subregion of interest, wherein the sub-subregion of interest is within the sub-subregion of interest, wherein the sub-subregion of interest comprises a sub-sub-plurality of capture probes, and wherein: (i) two or more capture probes in the sub-subgroup of capture probes comprise a first common sub-subspace barcode, and (ii) the first common sub-subspace barcode is associated with the sub-subregion of interest, and (b) enriching the population of capture probes comprising the first sub-subspace barcode.

[0048] In some embodiments of the method for determining the location of an analyte in a biological sample, the first common spatial barcode is associated with a single cell of the biological sample.

[0049] In some embodiments of the method for determining the location of an analyte in a biological sample, the analyte comprises mRNA. In some embodiments of the method for determining the location of an analyte in a biological sample, the analyte comprises a protein. In some embodiments of the method for determining the location of an analyte in a biological sample, the analyte comprises an antibody. In some embodiments of the method for determining the location of an analyte in a biological sample, the analyte comprises DNA. In some embodiments of the method for determining the location of an analyte in a biological sample, the DNA encodes a T cell receptor. In some embodiments of the method for determining the location of an analyte in a biological sample, the DNA comprises one or more chromatin regions in the biological sample.

[0050] On the other hand, provided herein is a method for producing a spatial array, comprising (a) providing a substrate comprising a plurality of first oligonucleotides, wherein a first oligonucleotide of the plurality of first oligonucleotides is attached to a region of the substrate via its 3′-end or its 5′-end, and (b) attaching a second oligonucleotide to the unattached end of the first oligonucleotide, wherein the second oligonucleotide comprises a spatial barcode.

[0051] In some embodiments, the method for generating a spatial array further comprises, after (b), (c) attaching a third oligonucleotide segment to the unattached end of the second oligonucleotide.

[0052] In some embodiments of the method for producing a spatial array, a first oligonucleotide is attached to a substrate via its 5′-end, and a second oligonucleotide is produced at the 3′-end of the first oligonucleotide using spatially directed in situ synthesis. In some embodiments of the method for producing a spatial array, a first oligonucleotide is attached to a substrate via its 3′-end, and a second oligonucleotide is produced at the 5′-end of the first oligonucleotide using spatially directed in situ synthesis.

[0053] In some embodiments of the method for generating a spatial array, the phosphodiester and exocyclic amine moieties of the first oligonucleotide are protected.

[0054] In some embodiments of the method for generating a spatial array, the length of the second oligonucleotide is selected from 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides or 30 nucleotides.

[0055] In some embodiments of the method for producing a spatial array, the in situ synthesis is a light-directed synthesis.

[0056] In some embodiments of the method for producing a spatial array, the area of ​​the substrate is less than about 2500 μm 2 , less than about 1600μm 2 , less than about 400μm 2 , less than about 100μm 2 , less than about 50μm 2 , less than about 25μm 2 , less than about 15μm 2 , less than about 10μm 2 or less than about 5 μm 2 .

[0057] In some embodiments of the method for generating a spatial array, two or more second oligonucleotides are attached to the two or more first oligonucleotides, wherein a member of the two or more second oligonucleotides is different from another member of the two or more second oligonucleotides.

[0058] In some embodiments of the method for generating a spatial array, the first oligonucleotide comprises protected phosphodiester and exocyclic amine moieties.

[0059] In some embodiments of the method for producing a spatial array, the substrate further comprises a substantially complementary oligonucleotide that hybridizes to the first oligonucleotide.

[0060] In some embodiments of the method for generating a spatial array, the first oligonucleotide is at least about 2 nucleotides, 5 nucleotides, 8 nucleotides, 10 nucleotides, 15 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 25 nucleotides, 27 nucleotides, 30 nucleotides, 33 nucleotides, or 35 nucleotides in length. In some embodiments of the method for generating a spatial array, the first oligonucleotide is about 2 to 45 nucleotides, 5 to 40 nucleotides, 15 to 40 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, or 27 to 33 nucleotides in length.

[0061] In some embodiments of the method for producing a spatial array, the first oligonucleotide is prepared by synthesizing off a substrate and subsequently attaching the first oligonucleotide to the substrate.

[0062] In some embodiments of the method for producing a spatial array, the phosphodiester and exocyclic amine moieties of the first oligonucleotide are protected before the first oligonucleotide is attached to the substrate. In some embodiments of the method for producing a spatial array, the phosphodiester and exocyclic amine moieties of the first oligonucleotide are protected after the first oligonucleotide is attached to the substrate.

[0063] In some embodiments of the method for producing spatial array, the first oligonucleotide is prepared by in situ synthesis on substrate. In some embodiments of the method for producing spatial array, in situ synthesis is not light-guided synthesis. In some embodiments of the method for generating spatial array, in situ synthesis is not spatially oriented synthesis.

[0064] In some embodiments of the method for producing spatial array, the first oligonucleotide comprises a sequence containing a cleavage site. In some embodiments of the method for producing spatial array, the first oligonucleotide is attached to a substrate by a joint. In some embodiments of the method for producing spatial array, the chemical group comprises: silane, amine, peptide, polyethylene glycol, biotin, desthiobiotin, streptavidin, thiol, acrylate, azide, digoxin, hexynyl or its combination. In some embodiments of the method for producing spatial array, the joint is a branched joint or a dendritic joint.

[0065] In some embodiments of the method for generating a spatial array, the third oligonucleotide is attached to the second oligonucleotide by ligation.

[0066] In some embodiments of the method for generating a spatial array, any protected moieties of the first and second oligonucleotides are deprotected prior to ligating the third oligonucleotide to the second oligonucleotide.

[0067] In some embodiments of the method for generating a spatial array, the plurality of third oligonucleotides is at least about 5 nucleotides, 7 nucleotides, 9 nucleotides, 13 nucleotides, 20 nucleotides, 25 nucleotides, 27 nucleotides, 30 nucleotides, 33 nucleotides, or 35 nucleotides in length. In some embodiments of the method for generating a spatial array, the plurality of third oligonucleotides is about 5 to 40 nucleotides, 5 to 20 nucleotides, 5 to 10 nucleotides, 10 to 35 nucleotides, 15 to 30 nucleotides, 25 to 35 nucleotides, or 27 to 33 nucleotides in length.

[0068] In some embodiments of the method for generating a spatial array, the third oligonucleotide comprises a capture domain at the 3′-end that is at least 6 nucleotides in length. In some embodiments of the method for generating a spatial array, the capture domain comprises a poly-T sequence. In some embodiments of the method for generating a spatial array, the third oligonucleotide comprises a random sequence that is 6 to 20 nucleotides in length.

[0069] In another aspect, provided herein is a composition comprising a plurality of oligonucleotides attached to a plurality of regions on a substrate, wherein the oligonucleotides in the plurality of oligonucleotides include: a first oligonucleotide having a length of 2 to 50 nucleotides attached to the substrate via its 3′-end or its 5′-end, and a second oligonucleotide having a length of 8 to 30 nucleotides attached to the unattached end of the first oligonucleotide, wherein the second oligonucleotide comprises a spatial barcode.

[0070] In another aspect, provided herein are compositions wherein the first oligonucleotide and the second oligonucleotide comprise protected phosphodiester and exocyclic amine moieties.

[0071] In another aspect, provided herein are compositions wherein a first oligonucleotide is attached to a substrate via its 5'-end and a second oligonucleotide is attached to the 3'-end of the first oligonucleotide.

[0072] In another aspect, provided herein are compositions wherein a first oligonucleotide is attached to a substrate via its 3 '-end and a second oligonucleotide is attached to the 5 '-end of the first oligonucleotide.

[0073] In some embodiments, the composition wherein a first oligonucleotide is attached to a substrate via its 3′-end and a second oligonucleotide is attached to the 5′-end of the first oligonucleotide further comprises a third oligonucleotide of 5 to 40 nucleotides in length attached to the unattached end of the second oligonucleotide, wherein the third oligonucleotide comprises a capture domain.

[0074] In some embodiments, in the composition wherein the first oligonucleotide is attached to the substrate via its 3′-end and the second oligonucleotide is attached to the 5′-end of the first oligonucleotide, the length of the second oligonucleotide is selected from 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.

[0075] In some embodiments, in a composition wherein a first oligonucleotide is attached to a substrate via its 3′-end and a second oligonucleotide is attached to the 5′-end of the first oligonucleotide, the region is less than about 2500 μm 2 , less than about 1600μm 2 , less than about 400μm 2 , less than about 100 μm 2 , less than about 50μm 2 , less than about 25μm 2 , less than about 15μm 2 , less than about 10 μm 2 , less than about 5μm 2 , or less than about 1 μm 2 .

[0076] In some embodiments, in a composition where a first oligonucleotide is attached to a substrate via its 3′-end and a second oligonucleotide is attached to the 5′-end of the first oligonucleotide, the oligonucleotides in each region are at least 15 nucleotides, at least 20 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or at least 60 nucleotides in length.

[0077] In some embodiments, in a composition where the first oligonucleotide is attached to the substrate via its 3′-end and the second oligonucleotide is attached to the 5′-end of the first oligonucleotide, each region is 15 to 100 nucleotides, 20 to 100 nucleotides, 40 to 100 nucleotides, 50 to 90 nucleotides, or 60 to 80 nucleotides.

[0078] On the other hand, the present invention provides a method for preparing a spatial array, comprising: (a) providing a substrate, (b) fixing multiple sets of primers on the substrate, wherein the first set of the multiple sets of primers includes a first primer and a second primer, (c) providing a template oligonucleotide, which includes a reverse complement of the spatial barcode domain, a reverse complement of the capture domain, a first hybridization domain capable of hybridizing with the first primer, and a second hybridization domain, wherein the reverse complement of the second hybridization domain is capable of hybridizing with the second primer, (d) hybridizing the template oligonucleotide with the first primer, (e) using the template oligonucleotide as a template to extend the first primer to produce a capture probe including the first primer, the spatial barcode and the capture domain, wherein the capture probe is fixed on the substrate, and (f) releasing the template oligonucleotide from the capture probe, thereby producing a spatial array.

[0079] In some embodiments, the method of making a spatial array further comprises amplifying the capture probes to produce clusters of one or more capture probes.

[0080] In some embodiments of the method for preparing a spatial array, amplification includes (a) hybridizing the capture probe with a second primer, (b) extending the second primer using the capture probe as a template to produce a copy of the template oligonucleotide fixed on the substrate, (c) hybridizing the copy of the template oligonucleotide with a first primer of a second group of multiple primers, and (d) using the copy of the template oligonucleotide as a template to extend the first primer of the second group of multiple primers to produce a copy of the capture probe fixed on the substrate, wherein the cluster of capture probes includes the capture probe and the copy of the capture probe.

[0081] In some embodiments of the method of making a spatial array, the template oligonucleotide comprises the reverse complement of a unique molecular identifier (UMI), and wherein the capture probes generated comprise the UMI.

[0082] In some embodiments of the method for preparing a spatial array, the first primer is attached to a substrate at the 5' end of the first primer. In some embodiments of the method for preparing a spatial array, the first primer comprises a sequence complementary to a template oligonucleotide. In some embodiments of the method for preparing a spatial array, the first primer comprises a poly (A) domain. In some embodiments of the method for preparing a spatial array, the first primer comprises a cleavage domain.

[0083] In some embodiments of the method of making a spatial array, the second primer is attached to the substrate at the 5' end of the second primer. In some embodiments of the method of making a spatial array, the second primer includes a cleavage domain.

[0084] In some embodiments of the method of making a spatial array, copies of the template oligonucleotide are released by cleavage of the cleavage domain after copies of the capture probe are produced. In some embodiments of the method of making a spatial array, the template oligonucleotide is attached to a bead.

[0085] In some embodiments of the method for preparing a spatial array, the beads are separated from the template oligonucleotide after the step of extending the first primer using the template oligonucleotide as a template to produce a capture probe comprising the first primer, a spatial barcode and a capture domain, wherein the capture probe is immobilized on a substrate.

[0086] In some embodiments of the method for preparing a spatial array, the beads are gel beads. In some embodiments of the method for preparing a spatial array, the diameter of the beads is 2 microns or less. In some embodiments of the method for preparing a spatial array, the diameter of the beads is 1 micron or less. In some embodiments of the method for preparing a spatial array, the beads are removed before the step of extending the first primer. In some embodiments of the method for preparing a spatial array, the beads are removed after the step of extending the first primer.

[0087] In some embodiments of the method of making a spatial array, all or part of the sequence of the generated capture probes is determined. In some embodiments of the method of making a spatial array, all or part of the sequence of the generated capture probes is determined after releasing the template oligonucleotide.

[0088] In some embodiments of the method for preparing spatial arrays, all or part of the sequence of the capture probes produced is determined in situ. In some embodiments of the method for preparing spatial arrays, all or part of the sequence of the capture probes produced is determined by in situ sequencing. In some embodiments of the method for preparing spatial arrays, in situ sequencing is performed by synthesis sequencing (SBS), sequential fluorescence hybridization, ligation sequencing, nucleic acid hybridization or high-throughput digital sequencing technology.

[0089] In some embodiments of the method of making a spatial array, the capture domain comprises a sequence complementary to the analyte of interest. In some embodiments of the method of making a spatial array, the capture domain comprises a poly(T) domain.

[0090] In some embodiments of the method of making a spatial array, the substrate comprises a glass slide. In some embodiments of the method of making a spatial array, the glass slide is a treated glass slide.

[0091] On the other hand, provided herein is a method for determining the location of an analyte in a biological sample, comprising (a) preparing a spatial array using any of the various methods described herein, (b) decoding one or more capture probes by determining all or part of the sequence of the spatial barcode of the one or more capture probes, thereby associating the spatial barcode of the one or more capture probes with a location on the spatial array, (c) contacting the biological sample with the spatial array under conditions in which the analyte specifically binds to the capture probes, (d) extending the one or more capture probes by a polymerization reaction using the analyte as a template to produce extended one or more capture probes, which include one or more capture probes and the reverse complement of the analyte, and (e) determining the sequence of the extended one or more capture probes and associating the spatial barcode of the one or more capture probes with the analyte, thereby determining the location of the analyte in the biological sample.

[0092] In some embodiments of the method for determining the position of an analyte in a biological sample, the one or more capture probes comprise a template oligonucleotide and beads. In some embodiments, the method for determining the position of an analyte in a biological sample further comprises separating the beads from the template oligonucleotide after extending the one or more capture probes to produce extended one or more capture probes comprising the one or more capture probes and the reverse complement of the analyte by a polymerization reaction using the analyte as a template.

[0093] In some embodiments of the method for determining the location of an analyte in a biological sample, the ratio of the first primer to the second primer is between 1:1.5 and 1.5: 1. In some embodiments of the method for determining the location of an analyte in a biological sample, the first primer and the second primer are less than 5 μm apart.

[0094] In another aspect, provided herein are spatial arrays produced by any of the methods described herein.

[0095] In another aspect, provided herein is a spatial array comprising (a) a substrate, and (b) multiple sets of primers immobilized on the substrate, wherein a first set of the multiple sets of primers comprises a first primer and a second primer, and wherein the first primer comprises a sequence complementary to the capture probe.

[0096] In some embodiments, the spatial array further comprises a template oligonucleotide comprising a reverse complement of a unique molecular identifier (UMI), and wherein the capture probe comprises the UMI.

[0097] In some embodiments of the spatial arrays provided herein, the first primer is attached to a substrate at the 5' end of the first primer. In some embodiments of the spatial arrays provided herein, the first primer includes a sequence complementary to a template oligonucleotide. In some embodiments of the spatial arrays provided herein, the first primer includes a poly (A) domain. In some embodiments of the spatial arrays provided herein, the first primer includes a cleavage domain.

[0098] In some embodiments of the spatial arrays provided herein, the second primer is attached to the substrate at the 5' end of the second primer. In some embodiments of the spatial arrays provided herein, the second primer comprises a cleavage domain.

[0099] In some embodiments of the spatial array provided herein, the template oligonucleotide is attached to the bead. In some embodiments of the spatial array provided herein, the bead is a gel bead. In some embodiments of the spatial array provided herein, the diameter of the bead is 2 microns or less. In some embodiments of the spatial array provided herein, the diameter of the bead is 1 micron or less.

[0100] In some embodiments of the spatial arrays provided herein, the substrate comprises a glass slide. In some embodiments of the spatial arrays provided herein, the glass slide is a treated glass slide.

[0101] In some embodiments of the spatial arrays provided herein, the ratio of the first primer to the second primer is between 1 : 1.5 and 1.5: 1. In some embodiments of the spatial arrays provided herein, the first primer and the second primer are less than 5 μm apart.

[0102] On the other hand, provided herein is a method for producing a spatial array, comprising (a) providing a substrate comprising an arrayed plurality of acceptor oligonucleotides, wherein the acceptor oligonucleotides of the plurality of acceptor oligonucleotides comprise a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate, (b) providing a splint oligonucleotide comprising a sequence complementary to the first ligation handle and a sequence complementary to a donor oligonucleotide, and (c) connecting a donor oligonucleotide comprising a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, thereby generating a spatial array.

[0103] In some embodiments of the method of generating a spatial array, the acceptor oligonucleotide further comprises a cleavage domain, a unique molecular identifier, a functional sequence, or any combination thereof.

[0104] In some embodiments of the method for generating a spatial array, the sequence complementary to the capture domain of the splint oligonucleotide is a poly (A) sequence. In some embodiments, the sequence complementary to the capture domain of the splint oligonucleotide is a gene specific sequence. In some embodiments, the gene specific sequence is a Penk specific sequence. In some embodiments, the sequence complementary to the capture domain of the splint oligonucleotide is a random sequence. In some embodiments, the random sequence is a hexamer.

[0105] In some embodiments, the method of generating a spatial array further comprises contacting the biological sample with the spatial array after step (c). In some embodiments, the method of generating a spatial array further comprises permeabilizing the biological sample to allow analytes in the biological sample to interact with the capture probes.

[0106] In some embodiments, the method for generating a spatial array further comprises migrating the analyte in the biological sample to a plurality of capture probes. In some embodiments of the method for generating a spatial array, migration comprises passive migration. In some embodiments of the method for generating a spatial array, migration comprises active migration.

[0107] On the other hand, provided herein is a method for producing a spatial array, comprising (a) providing a substrate comprising an arrayed plurality of acceptor oligonucleotides, wherein the acceptor oligonucleotides of the plurality of acceptor oligonucleotides comprise a spatial barcode and a first ligation handle, and wherein the 5′ end of the acceptor oligonucleotide is attached to the substrate, (b) providing a splint oligonucleotide comprising a sequence complementary to the first ligation handle and a sequence complementary to the second ligation handle, and (c) connecting a donor oligonucleotide comprising a second ligation handle and a capture domain to the 3′ end of the acceptor oligonucleotide to generate a capture probe, thereby generating a spatial array.

[0108] In some embodiments of the method of generating a spatial array, the acceptor oligonucleotide further comprises a cleavage domain, a unique molecular identifier, a functional sequence, or any combination thereof.

[0109] In some embodiments of the method for generating spatial arrays, the capture domain includes a poly (T) sequence. In some embodiments of the method for generating spatial arrays, the capture domain includes a gene specific sequence. In some embodiments of the method for generating spatial arrays, the gene specific sequence is a Penk specific sequence. In some embodiments of the method for generating spatial arrays, the capture domain includes a random sequence. In some embodiments of the method for generating spatial arrays, the random sequence is a hexamer.

[0110] In some embodiments, the method of generating a spatial array further comprises contacting the biological sample with the spatial array after step (c). In some embodiments, the method of generating a spatial array further comprises permeabilizing the biological sample to allow analytes in the biological sample to interact with the capture probes.

[0111] In some embodiments, the method for generating a spatial array further comprises migrating the analyte in the biological sample to the capture probe. In some embodiments of the method for generating a spatial array, migration comprises passive migration. In some embodiments of the method for generating a spatial array, migration comprises active migration.

[0112] In another aspect, provided herein is a method for producing capture probes bound to a substrate, comprising: dispensing a second solution onto a portion of a substrate surface coated with a first solution, wherein after dispensing, the molecules in the first solution and the second solution do not substantially mix with each other, wherein the second solution includes the capture probes, and the dispensing causes the capture probes to attach to the substrate surface.

[0113] In some embodiments, the method of producing capture probes bound to a substrate further comprises solidifying the second solution and removing the first solution from the substrate, wherein the solidified second solution remains on the surface of the substrate.

[0114] In some embodiments of the method for producing substrate-bound capture probes, the area of ​​the substrate surface to which the capture probes are attached is smaller than a corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution. In some embodiments of the method for producing substrate-bound capture probes, the area of ​​the substrate surface to which the capture probes are attached is about 2-fold smaller than a corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution.

[0115] In some embodiments, the method of producing a capture probe bound to a substrate further comprises curing the first and second solutions on the substrate, thereby producing a matrix. In some embodiments of the method of producing a capture probe bound to a substrate, the first and second solutions are cured chemically. In some embodiments of the method of producing a capture probe bound to a substrate, the first and second solutions are cured photoreactively.

[0116] In some embodiments, the method of producing capture probes bound to a substrate further comprises shrinking the substrate, thereby producing a shrunken substrate having regions to which the capture probes are attached, wherein the area in the shrunken substrate is smaller than a corresponding area in the substrate.

[0117] In some embodiments, the method of producing capture probes bound to a substrate further comprises, before the step of dispensing the second solution: solidifying the first solution to form a first solution matrix, wherein the first solution matrix can reversibly swell along one or more axes thereof. In some embodiments of the method of producing capture probes bound to a substrate, the step of dispensing the second solution onto a portion of the substrate comprises dispensing the second solution onto the surface of the first solution matrix in a swollen state. In some embodiments, the method of producing capture probes bound to a substrate further comprises, after the dispensing step: contracting the first solution matrix along one or more axes of the surface of the substrate.

[0118] In some embodiments of the method of producing capture probes bound to a substrate, the viscosity of the first solution is 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, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or 10 times greater than the viscosity of the second solution.

[0119] In some embodiments of the method of producing capture probes bound to a substrate, the first solution has a lower diffusion rate for nucleic acids than the second solution.

[0120] In some embodiments of the method for producing a capture probe bound to a substrate, the capture probe comprises a spatial barcode. In some embodiments, the capture probe comprises a unique molecular identifier. In some embodiments of the method for producing a capture probe bound to a substrate, the capture probe comprises a functional domain. In some embodiments of the method for producing a capture probe bound to a substrate, the capture probe comprises a cleavage domain, a spatial barcode, a unique molecular identifier, a functional domain, and a capture domain.

[0121] In another aspect, provided herein is a method for producing an array of capture probes bound to a substrate, comprising dispensing droplets of a second solution onto a portion of a substrate surface coated with a first solution, wherein after dispensing, the molecules in the droplets of the first solution and the second solution do not substantially mix with each other, wherein the second solution includes the capture probes, and the dispensing causes the capture probes to attach to the substrate surface.

[0122] In some embodiments, the method of producing a capture probe array bound to a substrate further comprises solidifying the second solution and removing the first solution from the substrate, wherein the solidified second solution remains on the surface of the substrate.

[0123] In some embodiments of the method of producing an array of capture probes bound to a substrate, the area of ​​the substrate surface to which the capture probes are attached is smaller than a corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution. In some embodiments of the method of producing an array of capture probes bound to a substrate, the area of ​​the substrate surface to which the capture probes are attached is about 2-fold smaller than a corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution.

[0124] In some embodiments, the method of producing a capture probe array bound to a substrate further comprises curing the first and second solutions on the substrate to produce a matrix. In some embodiments of the method of producing a capture probe array bound to a substrate, the first solution and the second solution are chemically cured. In some embodiments of the method of producing a capture probe array bound to a substrate, the first and second solutions are photocured.

[0125] In some embodiments, the method of producing an array of capture probes bound to a substrate further comprises shrinking the substrate, thereby producing a shrunken substrate having regions to which the capture probes are attached, wherein the area in the shrunken substrate is smaller than the corresponding area in the substrate.

[0126] In some embodiments, the method of producing a capture probe array bound to a substrate further comprises, prior to the step of dispensing the second solution: solidifying the first solution to form a first solution matrix, wherein the first solution matrix is ​​reversibly swellable along one or more axes thereof. In some embodiments of the method of producing a capture probe array bound to a substrate, the step of dispensing the second solution onto a portion of the substrate comprises dispensing the second solution onto a surface of the first solution matrix in a swollen state.

[0127] In some embodiments, the method of producing a capture probe array bound to a substrate further comprises, after the dispensing step: shrinking the first solution matrix along one or more axes of the substrate surface.

[0128] In some embodiments of the method of producing an array of capture probes bound to a substrate, the viscosity of the first solution is 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, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or 10 times greater than the viscosity of the second solution.

[0129] In some embodiments of the method of producing a substrate-bound capture probe array, the first solution has a lower nucleic acid diffusion rate than the second solution.

[0130] In some embodiments of the method of generating an array of capture probes bound to a substrate, the capture probes comprise a spatial barcode. In some embodiments of the method of generating an array of capture probes bound to a substrate, at least one of the capture probes comprises a unique molecular identifier. In some embodiments of the method of generating an array of capture probes bound to a substrate, the capture probes comprise a functional domain. In some embodiments of the method of generating an array of capture probes bound to a substrate, the capture probes comprise a cleavage domain, a spatial barcode, a unique molecular identifier, a functional domain, and a capture domain.

[0131] In another aspect, provided herein is a method for spatially analyzing analytes in a biological sample, comprising (a) dispensing droplets of a second solution onto a portion of a surface of a substrate coated with a first solution, wherein after dispensing, molecules in the first solution and in the droplets of the second solution do not substantially mix with each other, wherein the second solution includes capture probes, and the dispensing causes the capture probes to attach to the surface of the substrate, and (b) contacting the biological sample with a spatial array such that analytes present in the biological sample are captured by the capture probes, thereby determining a spatial distribution profile of the captured analytes.

[0132] In some embodiments of the method for spatially profiling an analyte in a biological sample, at least one capture probe comprises a cleavage domain, a spatial barcode, a unique molecular identifier, a functional domain, and a capture domain.

[0133] All publications, patents, and patent applications available on the Internet mentioned in this specification are incorporated herein by reference as if each individual publication, patent, patent application, and information item was specifically and individually incorporated herein by reference. To the extent that publications, patents, patent applications, and information items incorporated by reference conflict with the disclosure contained in this specification, it is intended that this specification control and / or take precedence over any conflicting material.

[0134] Where values ​​are described in a range, it should be understood that the description includes disclosure of all possible sub-ranges within that range as well as disclosure of specific values ​​within that range, regardless of whether a specific value or specific sub-range is explicitly stated.

[0135] The term "each" when referring to a group of items is intended to identify an individual item in the group, but not necessarily every item in the group unless expressly stated otherwise or unless the context of usage clearly indicates otherwise.

[0136] Various embodiments of the features of the present invention are described herein. However, it should be understood that these embodiments are provided as examples only, and that many variations, changes, and substitutions may be made by those skilled in the art without departing from the scope of the present disclosure. It should also be understood that various alternatives to the specific embodiments described herein are also within the scope of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0137] The following drawings illustrate certain embodiments of the features and advantages of the present invention. These embodiments are not intended to limit the scope of the appended claims in any way. The same reference numerals in the drawings represent the same elements.

[0138] Figure 1 An exemplary spatial analysis workflow is shown.

[0139] Figure 2 An exemplary spatial analysis workflow is shown.

[0140] Figure 3 An exemplary spatial analysis workflow is shown.

[0141] Figure 4 An exemplary spatial analysis workflow is shown.

[0142] Figure 5 An exemplary spatial analysis workflow is shown.

[0143] Figure 6 is a schematic diagram showing an example of a barcoded capture probe as described herein.

[0144] Figure 7 is a schematic diagram illustrating a cleavable capture probe, wherein the cleaved capture probe can enter a non-permeabilized cell and bind to a target analyte within a sample.

[0145] Figure 8 is a schematic diagram of an exemplary multiple spatial barcoding feature.

[0146] Figure 9 is a schematic diagram of an exemplary analyte-capture agent.

[0147] Figure 10 is a schematic diagram depicting an exemplary interaction between a feature-immobilized capture probe 1024 and an analyte-capture agent 1026.

[0148] Figure 11A 、 11B and 11C are schematic diagrams illustrating how streptavidin cell tagging can be used in an array-based system to generate spatially barcoded cells or cell contents.

[0149] Figure 12is a schematic diagram showing the arrangement of barcoding features within an array.

[0150] Figure 13 is a schematic diagram showing a side view of an anti-diffusion medium (eg, a cover).

[0151] Figure 14A and 14B This diagram shows the development of the electrophoretic transfer system. Figure 14A and side view Figure 14B Schematic diagram of the electrophoretic transfer system configured to direct transcript analytes to a spatially barcoded capture probe array.

[0152] Figure 15 is a schematic diagram illustrating an exemplary workflow scheme utilizing an electrophoretic transfer system.

[0153] Figure 16 An example of a microfluidic channel structure 1600 for partitioning a dissociated sample, such as biological particles or individual cells from a sample, is shown.

[0154] Figure 17A An example of a microfluidic channel structure 1700 for delivering beads carrying spatial barcodes to droplets is shown.

[0155] Figure 17B A cross-sectional view of another example of a microfluidic channel structure 1750 having geometric features for controlled partitioning is shown.

[0156] Figure 17C An example of a workflow diagram is shown.

[0157] Figure 18 Schematic diagram depicting cell tagging using covalent coupling of analyte-binding moieties to the cell surface or non-covalent interactions with cell membrane components.

[0158] Figure 19 is a schematic diagram depicting cell spiking using a cell-penetrating peptide or delivery system.

[0159] Figure 20A is a schematic diagram of a workflow illustrating exemplary, non-limiting, non-exhaustive steps for "pixelating" a sample, wherein the sample is cut, stamped, microdissected, or transferred by a hollow needle or microneedle to move a small portion of the sample into individual partitions or wells.

[0160] Figure 20B A schematic depicting multi-needle pixilation, in which a set of needles penetrates a sample on a scaffold and into a nanopore below containing gel beads and reagents. Once the needles enter the nanopores, the cells pop out.

[0161] Figure 21 A workflow diagram showing exemplary, non-limiting, non-exhaustive steps for dissociating spatially barcoded samples for analysis by droplet or flow cell analysis methods.

[0162] Figure 22A is a schematic diagram illustrating an exemplary sample processing device that can be used to implement the various steps and methods described herein.

[0163] Figure 22B is a schematic diagram illustrating an example imaging device that can be used to obtain images of biological samples, analytes, and feature arrays.

[0164] Figure 22C yes Figure 22A and Figure 22B Schematic diagram of an example of a control unit of a device.

[0165] Figure 23A Shown is a tissue section of invasive ductal carcinoma annotated by a pathologist.

[0166] Figure 23B Shown are histograms with spots colored by unsupervised clustering.

[0167] Figure 23C is a tSNE plot of the blobs colored by unsupervised clustering.

[0168] Figure 23D Gene expression heatmaps of the most varying genes among the nine clusters are shown.

[0169] Figure 23E Shown are the expression levels of genes corresponding to human epidermal growth factor receptor 2 (Her2), estrogen receptor (ER), and progesterone receptor (PR) in tissue sections.

[0170] Figure 23F Gene expression levels of the most differentially expressed genes from each of the nine clusters on each plot are shown.

[0171] Figure 23G Gene expression levels of the most differentially expressed genes from each of the nine clusters on a single plot are shown.

[0172] Figure 23H is a graph of the expression levels of the most differentially expressed genes from each of the nine clusters in invasive ductal cell carcinoma (IDC) and normal breast tissue.

[0173] Figure 23I The expression of KRT14 in IDC and matched normal tissues is shown.

[0174] Figure 23Jis a graph of the expression levels of extracellular matrix genes in IDCs and normal tissues.

[0175] Figure 24 is a schematic diagram showing a method for generating arrays of capture probes for spatial analysis of analytes in biological samples.

[0176] Figure 25 is a schematic diagram showing a method for capturing analytes with a capture domain and performing reverse transcription to obtain complementary cDNA sequences.

[0177] Figure 26 Shown is a schematic diagram depicting the steps involved in generating high-density spatially barcoded arrays with increased resolution.

[0178] Figure 27 is a schematic diagram illustrating an exemplary two-step barcode construction process for constructing an array.

[0179] Figure 28 is a schematic diagram illustrating an exemplary two-step barcode construction process for constructing an array.

[0180] Figure 29 is a schematic diagram illustrating an exemplary spatial array preparation process.

[0181] Figure 30 is a schematic diagram showing an exemplary design of a fluid isolation chamber.

[0182] FIG31 depicts an illustrative embodiment of a method for making a spatial array of the present disclosure. Figure 31A Depicted is a substrate provided with a first oligonucleotide attached thereto in a 5' to 3' direction, wherein the phosphodiester and exocyclic amine groups are protected. Figure 31B Depicted is the 5' to 3' photo-directed spatial synthesis of a second oligonucleotide onto the free 3'-end of a first oligonucleotide on a substrate, wherein the second oligonucleotide has different sequences in two different regions. Figure 31C Ligation of a third oligonucleotide to the free 3'-ends of two different second oligonucleotides is depicted.

[0183] FIG32 depicts an illustrative embodiment of a method for making a spatial array of the present disclosure. Figure 32A Depicted is the ex situ solid phase synthesis of a first oligonucleotide in the 3' to 5' direction followed by cleavage from a feature having protected phosphodiester and exocyclic amine groups. Figure 32B Depicted is the coupling of an ex situ synthesized first oligonucleotide to a substrate in a 5' to 3' direction. Figure 32C Depicted is the 5' to 3' photo-directed spatial synthesis of a second oligonucleotide onto the free 3'-end of a first oligonucleotide on a substrate, wherein the second oligonucleotide has different sequences in two different regions.

[0184] FIG33 depicts an illustrative embodiment of a method for making a spatial array of the present disclosure. Figure 33A Depicted is a first oligonucleotide having phosphodiester and exocyclic amine protecting groups attached to a substrate in a 5' to 3' direction, wherein the first oligonucleotide has been synthesized in situ on the surface of the substrate. Figure 33B Depicted is the 5' to 3' photo-directed spatial synthesis of a second oligonucleotide onto the free 3'-end of a first oligonucleotide (with in situ first segment phosphodiester and exocyclic amine protecting groups) on a substrate, wherein the second oligonucleotide has different sequences in two distinct regions.

[0185] FIG34 depicts an illustrative embodiment of a method for making a spatial array of the present disclosure. Figure 34A Depicted are a first oligonucleotide phosphodiester and exocyclic amine protecting group attached to a substrate in a 5' to 3' direction, wherein the first oligonucleotide has been synthesized on the substrate surface using 5' to 3' photodirected spatial synthesis. Figure 34B Depicted is the 5' to 3' photodirected spatial synthesis of a second oligonucleotide onto the free 3'-end of a first oligonucleotide (with in situ first oligonucleotide phosphodiester and exocyclic amine protecting groups) wherein the second oligonucleotide has different sequences in two distinct regions.

[0186] FIG35 depicts an illustrative embodiment of a method for making a spatial array of the present disclosure. Figure 35A Depicted is the coupling of a first oligonucleotide synthesized ectopically in the 3' to 5' direction without phosphodiester and exocyclic amine group protection. Figure 35B In situ protection of the phosphodiester and exocyclic amine groups of the first oligonucleotide is depicted. Figure 35C Depicted is the 5' to 3' photo-directed spatial synthesis of a second oligonucleotide onto the free 3'-end of a first oligonucleotide on a substrate, wherein the second oligonucleotide has different sequences in two different regions.

[0187] FIG36 depicts an illustrative embodiment of a method for making a spatial array of the present disclosure. Figure 36A Depicted is the coupling of a first oligonucleotide synthesized ectopically in the 3' to 5' direction without phosphodiester and exocyclic amine group protection. Figure 36B Depicted is the in situ hybridization of a complementary oligonucleotide to a first oligonucleotide, thereby protecting the phosphodiester and exocyclic amine groups of the first oligonucleotide. Figure 36C Described is the photo-directed spatial synthesis of a second oligonucleotide 5' to 3' from the free 3' end of the first oligonucleotide on a substrate when the first oligonucleotide is hybridized to a complementary oligonucleotide, wherein the synthesized second oligonucleotide has different sequences in two different regions.

[0188] Figure 37An example of a spatially hierarchical spatial array is shown, where one region is divided into nine subregions, which are further divided into fifteen sub-subregions. The individual spatial barcodes of the 135 sub-subregions include a regional spatial barcode, a subregional spatial barcode, and a sub-subregional spatial barcode.

[0189] Figure 38 An example of a library structure and amplification scheme including secondary spatial barcodes is shown, where the spatial barcodes include a regional spatial barcode prefix and a specific spatial barcode suffix.

[0190] Figure 39 is a schematic diagram of a workflow showing exemplary, non-limiting, non-exhaustive steps for preparing spatial arrays and decoding clusters on a slide.

[0191] Figure 40 is a schematic diagram showing the workflow of oligonucleotide construction.

[0192] Figure 41 A method for generating capture probes is shown.

[0193] Figure 42 is a graph showing detection of cDNAs with linked and unlinked poly-T and gene (eg, Penk gene) specific capture domains.

[0194] Figure 43 For display Figure 41 Fluorescence images of Penk mRNA detected by Penk-specific capture probes generated by the method shown are compared with control tissue-optimized slides.

[0195] Figure 44 An exemplary ligation-based method for generating capture probes using two ligation handles is shown. DETAILED DESCRIPTION

[0196] I. Introduction

[0197] The present invention describes devices, systems, methods and compositions for spatial analysis of biological samples. This section describes certain general terms, analytes, sample types and preparation steps that will be referred to in later sections of the present invention.

[0198] (a) Spatial Analysis

[0199] Tissues and cells can be obtained from any source. For example, tissues and cells can be obtained from unicellular or multicellular organisms (e.g., mammals). Tissues and cells obtained from mammals (e.g., humans) typically have different analyte levels (e.g., gene and / or protein expression), which can lead to differences in cell morphology and / or function. The location of cells in a tissue can affect the fate, behavior, morphology, and signal transduction and crosstalk of cells with other cells in the tissue. Information about the differences in analyte levels (gene and / or protein expression) in different cells of mammalian tissue can also help doctors select or administer effective treatments for unicellular or multicellular organisms (e.g., mammals) based on the differences in analyte levels in different cells of the detected tissue. The differences in analyte levels in different cells of mammalian tissue can also provide information on how tissues (e.g., healthy and diseased tissues) function and / or develop. The differences in analyte levels in different cells of mammalian tissue can also provide different information on the pathogenesis of diseases in tissues and information on therapeutic mechanisms of action in tissues. The differences in analyte levels in different cells of mammalian tissue can also provide information on drug resistance mechanisms and the development of drug resistance mechanisms in mammalian tissues. The differences in the presence or absence of an analyte in different cells in a tissue of a multicellular organism (eg, a mammal) can provide information on the mechanisms of drug resistance and its development in the tissue of the multicellular organism.

[0200] The spatial analysis methods provided herein are used to detect differences in analyte levels (e.g., gene and / or protein expression) in different cells in mammalian tissue or in a single cell of a mammal. For example, spatial analysis methods can be used to detect differences in analyte levels (e.g., gene and / or protein expression) in different cells in a tissue section sample, and data therefrom can be recombined to generate a three-dimensional graph of analyte levels (e.g., gene and / or protein expression) of a tissue sample obtained from a mammal, e.g., with a certain degree of spatial resolution (e.g., single cell resolution).

[0201] Spatial heterogeneity in developmental systems is typically investigated using RNA hybridization, immunohistochemistry, fluorescent reporters, purification or induction of predefined subpopulations, and subsequent genomic profiling (e.g., RNA-seq). However, such approaches rely on a relatively small set of predefined markers and therefore introduce selection bias that limits discovery. These previous methods also rely on a priori knowledge. Traditionally, spatial RNA analysis has relied on staining of a limited number of RNA species. In contrast, single-cell RNA sequencing allows for deep profiling of cellular gene expression, including noncoding RNAs, but established methods isolate cells from their native spatial context.

[0202] Current spatial analysis methods provide a large amount of analyte levels and / or expression data for a variety of analytes in a sample with high spatial resolution, for example, while retaining the natural spatial environment. Spatial analysis methods include, for example, using a capture probe comprising a spatial barcode (e.g., a nucleic acid sequence providing information about the position of the capture probe in a cell or tissue sample (e.g., a mammalian cell or mammalian tissue sample)) and a capture domain capable of binding to an analyte (e.g., protein and / or nucleic acid) produced by a cell or present in a cell. As described herein, a spatial barcode can be a nucleic acid with 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 agent. The capture domain can be any reagent capable of binding to an analyte produced by a cell and / or present in a cell (e.g., a nucleic acid capable of hybridizing with: a nucleic acid from a cell (e.g., mRNA, genomic DNA, mitochondrial DNA or miRNA), a substrate comprising an analyte, a binding partner of an analyte, or an antibody specifically bound to an analyte). The capture probe can also include a nucleic acid sequence complementary to a universal forward and / or universal reverse primer sequence. The capture probe can also include a cleavage site (eg, a cleavage recognition site for a restriction endonuclease), a photolabile bond, a heat-sensitive bond, or a chemical-sensitive bond.

[0203] Binding of the analyte to the capture probe can be detected using a variety 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 by and / or present in a cell (e.g., a mammalian cell).

[0204] The capture probe can be, for example, attached to a surface, such as a solid array, a bead, or a coverslip. In some examples, the capture probe is not attached to a surface. In some examples, the capture probe can be encapsulated in, embedded in, or layered on a surface of a permeable composition (e.g., any substrate described herein). For example, the capture probe can be encapsulated or arranged in permeable beads (e.g., gel beads). In some examples, the capture probe can be encapsulated in, embedded in, or layered on a surface of a substrate (e.g., any exemplary substrate described herein, such as a hydrogel or a porous membrane).

[0205] In some embodiments, cells or tissue samples comprising cells are contacted with capture probes attached to a substrate (e.g., a surface of a substrate), and the cells or tissue samples are permeabilized to release analytes from the cells and bind to the capture probes attached to the substrate. In some examples, analytes released from cells can be actively guided to the capture probes attached to the substrate using a variety of methods (e.g., electrophoresis, chemical gradients, pressure gradients, fluid flow, or magnetic fields).

[0206] In other examples, a variety of methods can be used to direct the capture probe to interact with the cell or tissue sample, including, for example, lipid anchors in the capture probe, reagents that can specifically bind to or form covalent bonds with membrane proteins in the capture probe, fluid flow, pressure gradients, chemical gradients, or magnetic fields.

[0207] 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, U.S. Patent No. 10,002,316, U.S. Patent No. 9,727,810, U.S. Patent Application Publication No. 2017 / 0016053, Rodriques et al., Science 363(6434):1463-1467, 2019; WO 2018 / 045186, Lee et al., Nat. Protoc. 10(3):442-458, 2015; WO 2016 / 007839, WO 2018 / 045181, WO 2014 / 163886, Trejo et al., PLoS ONE 14(2):e0212031, 2019, U.S. Patent Application Publication No. 2018 / 0245142, Chen et al., Science 348(6233):aaa6090, 2015, Gao et al., BMC Biol. 15:50, 2017, WO 2017 / 144338, WO 2018 / 107054, WO 2017 / 222453, WO 2019 / 068880, WO 2011 / 094669, U.S. Patent No. 7,709,198, U.S. Patent No. 8,604,182, U.S. Patent No. 8,951,726, U.S. Patent No. 9,783,841, U.S. Patent No. 10,041,949, WO 2016 / 057552, WO 2017 / 147483, WO 2018 / 022809, WO 2016 / 166128, WO 2017 / 027367, WO 2017 / 027368, WO 2018 / 136856, WO 2019 / 075091, U.S. Patent No. 10,059,990, WO 2018 / 057999, WO 2015 / 161173, and Gupta et al., Nature Biotechnol. 36: 1197-1202, 2018, and may be used in any combination herein. Further non-limiting aspects of the spatial analysis method are described herein.

[0208] (b) General terms

[0209] Specific terminology is used throughout this disclosure to explain various aspects of the described devices, systems, methods, and compositions. This section contains explanations of certain terms that appear in subsequent sections of this disclosure. If the descriptions in this section clearly conflict with usage in other sections of this disclosure, the definitions in this section will prevail.

[0210] (i) Barcode

[0211] A "barcode" is a label or identifier that conveys or is capable of conveying information (e.g., information about an analyte, bead, and / or capture probe in a sample). A barcode can be part of an analyte or can be independent of the analyte. A barcode can be attached to an analyte. A particular barcode can be unique relative to other barcodes.

[0212] Barcodes can come in a variety of different forms. For example, barcodes can include polynucleotide barcodes, random nucleic acid and / or amino acid sequences, and synthetic nucleic acid and / or amino acid sequences. Barcodes can be attached to an analyte or another part or structure in a reversible or irreversible manner. For example, a barcode can be added to a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before or during sequencing of the sample. Barcodes can allow identification and / or quantification of individual sequencing reads (e.g., a barcode can be or can include a unique molecular identifier or "UMI").

[0213] A barcode can spatially resolve molecular components present in a biological sample, e.g., at single-cell resolution (e.g., a barcode can be or can include a "spatial barcode"). In some embodiments, a barcode includes both a UMI and a spatial barcode. In some embodiments, a barcode includes two or more sub-barcodes that together function as a single barcode. For example, a polynucleotide barcode can include two or more polynucleotide sequences (e.g., sub-barcodes) separated by one or more non-barcode sequences.

[0214] (ii) Nucleic acids and nucleotides

[0215] The terms "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and include naturally occurring substances or functional analogs thereof. Particularly useful functional analogs of nucleic acids are capable of hybridizing to nucleic acids in a sequence-specific manner (e.g., capable of hybridizing to two nucleic acids so that a connection can occur between the two hybridized nucleic acids), or can be used as a template for replicating a specific nucleotide sequence. Naturally occurring nucleic acids typically have a backbone containing phosphodiester bonds. Analog structures can have other backbone connections, including any of a variety of connections known in the art. Naturally occurring nucleic acids typically have deoxyribose (e.g., found in deoxyribonucleic acid (DNA)) or ribose (e.g., found in ribonucleic acid (RNA)).

[0216] Nucleic acids can include nucleotides having any of the various analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural nucleotides. In this regard, natural deoxyribonucleic acids can have one or more bases selected from adenine (A), thymine (T), cytosine (C) or guanine (G), and ribonucleic acids can have one or more bases selected from uracil (U), adenine (A), cytosine (C) or guanine (G). Useful non-natural bases that can be included in nucleic acids or nucleotides are known in the art.

[0217] (iii) Probes and targets

[0218] "Probe" or "target," when applied to a nucleic acid or nucleic acid sequence, is intended to serve as a semantic identifier for 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 or sequence beyond that explicitly indicated.

[0219] (iv) Oligonucleotides and polynucleotides

[0220] The terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to single-stranded polymers of nucleotides having a length of about 2 to about 500 nucleotides. Oligonucleotides can be synthesized, enzymatically prepared (e.g., by polymerization), or prepared using a "split-pool" method. Oligonucleotides can include ribonucleotide monomers (i.e., oligoribonucleotides) and / or deoxyribonucleotide monomers (i.e., oligodeoxyribonucleotides). In some examples, an oligonucleotide can include a combination of deoxyribonucleotide monomers and ribonucleotide monomers in an oligonucleotide (e.g., a random or ordered combination of deoxyribonucleotide monomers and ribonucleotide monomers). For example, the length of the oligonucleotide 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 250, 250 to 300, 300 to 350, 350 to 400, or 400 to 500 nucleotides. The oligonucleotide may include (e.g., covalently or non-covalently) one or more functional moieties attached to the polymer structure. For example, the oligonucleotide may include one or more detectable labels (e.g., radioisotopes or fluorophores).

[0221] (v) Object

[0222] A "subject" is an animal, such as a mammal (eg, a human or non-human ape), or avian (eg, a bird), or other 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 (i.e., humans or non-human primates); plants, such as Arabidopsis thaliana, corn, sorghum, oats, wheat, rice, rapeseed, or soybeans; algae, such as Chlamydomonas reinhardtii; nematodes, such as Caenorhabditis elegans; insects, such as Drosophila melanogaster, mosquitoes, fruit flies, or honey bees; arachnids, such as spiders; fish, such as zebrafish; reptiles; amphibians, such as frogs or Xenopus laevis; Dictyostelium discoideum; fungi, such as Pneumocystis carinii; carinii), Takifugu rubripes, yeast, Saccharamoyces cerevisiae or Schizosaccharomyces pombe; or Plasmodium falciparum.

[0223] (vi) Genome

[0224] "Genome" generally refers to genomic information from a subject, for example, at least part or all of the genetic information encoded by the subject's genes. A genome can include coding regions (e.g., regions encoding proteins) as well as non-coding regions. A genome can include the sequences of some or all of the subject's chromosomes. For example, the human genome typically has a total of 46 chromosomes. The sequences of some or all of these chromosomes can constitute a genome.

[0225] (vii) Adapters, adapters, and tags

[0226] "Adapter," "adapter," and "tag" are terms used interchangeably in this disclosure and refer to substances that can be coupled to a polynucleotide sequence using any of a number of different techniques, including but not limited to ligation, hybridization, and tagmentation (in a process known as "tagging"). An adapter can also be a nucleic acid sequence that adds functionality, such as a spacer sequence, a primer sequence / site, a barcode sequence, or a unique molecular identifier sequence.

[0227] (viii) Hybridization, hybridization, annealing, and chain recombination

[0228] The terms "hybridization," "annealing," and "strand recombination" are used interchangeably in this disclosure and refer to the pairing of substantially complementary or fully complementary nucleic acid sequences within two different molecules. Pairing can be achieved by any process in which a nucleic acid sequence is joined by base pairing with a substantially complementary or fully complementary sequence to form a hybridization complex. For the purposes of hybridization, two nucleic acid sequences are "substantially complementary" if at least 60% (e.g., at least 70%, at least 80%, or at least 90%) of the individual bases in the two nucleic acid sequences are complementary to each other.

[0229] (ix) Primers

[0230] " Primer " is a single-stranded nucleic acid sequence with a 3 'end that can be used as a chemical substrate for nucleic acid polymerase in a nucleic acid extension reaction. RNA primers are formed by RNA nucleotides and are used for RNA synthesis, while DNA primers are composed of DNA nucleotides and are used for DNA synthesis. Primers can also include RNA nucleotides and DNA nucleotides (for example, in a random or designed pattern). Primers can also include other natural or synthetic nucleotides with other functionalities as described herein. In some examples, a DNA primer can be used to initiate RNA synthesis, and vice versa (for example, an RNA primer can be used to initiate DNA synthesis). The length of the primer can be different. For example, a primer can be about 6 bases to about 120 bases. For example, a primer can include up to about 25 bases.

[0231] (x) Primer extension

[0232] "Primer extension" refers to any method in which two nucleic acid sequences (e.g., constant regions from each of two different capture probes) are linked (e.g., hybridized) by overlapping complementary nucleic acid sequences at their respective ends (e.g., 3' ends). Following such ligation, nucleic acid extension (e.g., enzymatic extension) can be performed at one or both ends using another nucleic acid sequence as an extension template. Enzymatic extension can be performed by enzymes including, but not limited to, polymerases and / or reverse transcriptases.

[0233] (xi) Proximity Connection

[0234] "Proximity ligation" is a method of joining two (or more) nucleic acid sequences that are adjacent to each other by enzymatic means (e.g., ligase). In some embodiments, proximity ligation may include a "gap filling" step, which involves the inclusion of one or more nucleic acids spanning the distance between two nucleic acid molecules of interest by a polymerase based on the nucleic acid sequence of the template nucleic acid molecule (see, e.g., U.S. Patent No. 7,264,929, the entire contents of which are incorporated herein by reference).

[0235] A variety of methods can be used for proximity joining of nucleic acid molecules, including, but not limited to, "sticky-end" and "blunt-end" ligation. In addition, single-stranded ligation can be used to proximity join single-stranded nucleic acid molecules. Sticky-end proximity ligation involves hybridization of complementary single-stranded sequences between the two nucleic acid molecules to be joined prior to the ligation event. Blunt-end proximity ligation generally does not involve hybridization of complementary regions from each nucleic acid molecule because both nucleic acid molecules lack single-stranded overhangs at the site of ligation.

[0236] (xii) Nucleic acid extension

[0237] "Nucleic acid extension" generally involves incorporating one or more nucleic acids (e.g., A, G, C, T, U, nucleotide analogs, or derivatives thereof) into a molecule (e.g., but not limited to a nucleic acid sequence) in a template-dependent manner such that the contiguous nucleic acid is incorporated by an enzyme (e.g., a polymerase or reverse transcriptase) to generate a newly synthesized nucleic acid molecule. For example, by using a complementary nucleic acid sequence as a template for nucleic acid synthesis, a primer that hybridizes to the complementary nucleic acid sequence can be used to synthesize a new nucleic acid molecule. Similarly, the 3' polyadenylated tail of an mRNA transcript that hybridizes to a poly (dT) sequence (e.g., a capture domain) can be used as a template for single-stranded synthesis of a corresponding cDNA molecule.

[0238] (xiii) PCR amplification

[0239] "PCR amplification" refers to the use of polymerase chain reaction (PCR) to generate copies of genetic material, including DNA and RNA sequences. For example, suitable reagents and conditions for implementing PCR are described in U.S. Patent Nos. 4,683,202, 4,683,195, 4,800,159, 4,965,188, and 5,512,462, the entire contents of which are incorporated herein by reference. In a typical PCR amplification, the reaction mixture includes the genetic material to be amplified, an enzyme, one or more primers for a primer extension reaction, and reagents for the reaction. The oligonucleotide primers are of sufficient length to hybridize with the complementary genetic material under annealing conditions. The length of the primer generally depends on the length of the amplification domain, but is generally at least 4 bases, at least 5 bases, at least 6 bases, at least 8 bases, at least 9 bases, at least 10 base pairs (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, and can be up to 40 bp or longer, with the length of the primer generally being between 18 and 50 bp. The genetic material can be contacted with a single primer or a set of two primers (forward and reverse primers), depending on whether primer extension, linear or exponential amplification of the genetic material is desired.

[0240] In some embodiments, the PCR amplification process uses a DNA polymerase. The DNA polymerase activity can be provided by one or more different DNA polymerases. In certain embodiments, the DNA polymerase is from bacteria, for example, the DNA polymerase is a bacterial DNA polymerase. For example, the DNA polymerase can be from bacteria of the genus Escherichia coli, Bacillus, Thermobacterium, or Thermococcus.

[0241] Suitable examples of DNA polymerases that can be used include, but are not limited to, E. coli DNA polymerase I, Bsu DNA polymerase, Bst DNA polymerase, Taq DNA polymerase, VENT TM DNA polymerase, DEEPVENT TM DNA polymerase, Taq DNA polymerase, Hot Start Taq DNA Polymerase, Crimson Taq DNA polymerase, Crimson Taq DNA polymerase, DNA polymerase, Quick DNA polymerase, Hemo DNA polymerase, DNA polymerase, DNA polymerase, High-fidelity DNA polymerases, 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.

[0242] The term "DNA polymerase" includes not only naturally occurring enzymes, but also all modified derivatives thereof, and also derivatives of naturally occurring DNA polymerases. For example, in some embodiments, the DNA polymerase can be modified to remove 5'-3' exonuclease activity. Sequence-modified derivatives or mutants of DNA polymerases that can be used include, but are not limited to, mutants that retain at least some of their functions, such as the DNA polymerase activity of the wild-type sequence. Under different reaction conditions, such as temperature, template concentration, primer concentration, etc., mutations can affect the activity profile of the enzyme, such as increasing or decreasing the polymerization rate. Mutations or sequence modifications may also affect the exonuclease activity and / or thermal stability of the enzyme.

[0243] In some embodiments, PCR amplification can include reactions such as, but not limited to, strand displacement amplification, rolling circle amplification, ligase chain reaction, transcription-mediated amplification, isothermal amplification, and / or loop-mediated amplification.

[0244] In some embodiments, PCR amplification uses a single primer that is complementary to the 3' tag of the target DNA fragment. In some embodiments, PCR amplification uses a first and a second primer, wherein at least the 3' terminal portion 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' terminal portion of the second primer displays at least a portion of the sequence of the 5' tag of the target nucleic acid fragment. In some embodiments, the 5' terminal portion of the first primer is not complementary to the 3' tag of the target nucleic acid fragment, and the 5' terminal portion of the second primer does not display at least a portion of the sequence of the 5' tag 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.

[0245] In some embodiments (e.g., when PCR amplification amplifies the captured DNA), DNA ligase can be used to connect the PCR amplification product to other sequences. DNA ligase activity can be provided by one or more different DNA ligases. In some embodiments, the DNA ligase is from bacteria, for example, the DNA ligase is a bacterial DNA ligase. In some embodiments, the DNA ligase is from a virus (e.g., a bacteriophage). For example, the DNA ligase can be T4 DNA ligase. Other enzymes suitable for the ligation step include, but are not limited to, Tth DNA ligase, Taq DNA ligase, Thermococcus (strain 9oN) DNA ligase (9oN™ DNA ligase, available from New England Biolabs, Ipswich, Massachusetts) and Ampligase TM (Available from Epicentre Biotechnologies, Madison, Wisconsin.) Derivatives, such as sequence-modified derivatives and / or mutants thereof, may also be used.

[0246] 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, suitable examples of which include but are not limited to: M-MLV, MuLV, AMV, HIV, ArrayScript TM 、MultiScribe TM 、ThermoScript TM ,and I, II, III and IV enzymes. "Reverse transcriptase" includes not only the naturally occurring enzyme but also all such modified derivatives thereof, and also includes derivatives of the naturally occurring reverse transcriptase.

[0247] In addition, sequence-modified derivatives or mutants of M-MLV, MuLV, AMV, and HIV reverse transcriptases can be used for reverse transcription, including mutants that retain at least some functional (e.g., reverse transcriptase) activity of the wild-type sequence. The reverse transcriptase can be provided as part of a composition comprising other components, such as a stabilizing component that enhances or improves the activity of the reverse transcriptase, such as an RNase inhibitor, an inhibitor of DNA-dependent DNA synthesis, such as actinomycin D. Many sequence-modified derivatives or mutants of reverse transcriptases, such as M-MLV, and compositions comprising unmodified and modified enzymes, such as ArrayScript TM 、MultiScribe TM 、ThermoScript TM ,and I, II, III and IV enzymes are commercially available.

[0248] Certain reverse transcriptases (e.g., 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) as templates to synthesize complementary DNA strands. Therefore, in some embodiments, the reverse transcription reaction can use an enzyme (reverse transcriptase) that can use both RNA and ssDNA as templates for the extension reaction, such as AMV or MMLV reverse transcriptase.

[0249] 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 TM "or or on a capillary (" Capillary". In some embodiments, quantification of genetic material is determined by light absorbance and real-time PCR. In some embodiments, quantification of genetic material is determined by digital PCR. In some embodiments, the analyzed gene can be compared to a reference nucleic acid extract (DNA and RNA) for both expression (mRNA) and quantity (DNA) to compare expression levels of the target nucleic acid.

[0250] (xiv) Antibodies

[0251] An "antibody" is a polypeptide molecule that recognizes and binds to a complementary target antigen. Antibodies typically have a molecular structural shape that resembles a Y-shape. Naturally occurring antibodies, known as immunoglobulins, belong to one of the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE. Antibodies can also be synthesized artificially. For example, recombinant antibodies known as monoclonal antibodies can be synthesized using synthetic genes by recovering the antibody gene from a source cell, amplifying 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 technology can be used to generate antibodies and antibody fragments, including non-endogenous substances.

[0252] Synthetic antibodies can be obtained from non-immunoglobulin sources. For example, antibodies can be produced from nucleic acids (e.g., aptamers) and non-immunoglobulin scaffolds (e.g., peptide aptamers), into which hypervariable loops are inserted to form antigen-binding sites. Synthetic antibodies based on nucleic acid or peptide structures can be smaller than immunoglobulin-derived antibodies, resulting in greater tissue permeabilization.

[0253] Antibodies can also include affimer proteins, which are affinity reagents typically having a molecular weight of about 12-14 kDa. Affimer proteins typically bind to targets (e.g., target proteins) with high affinity and specificity. Examples of such targets include, but are not limited to, ubiquitin chains, immunoglobulins, and C-reactive proteins. In some embodiments, affimer proteins are derived from cysteine ​​protease inhibitors and include a peptide loop and a variable N-terminal sequence that provides a binding site.

[0254] Antibodies may also include single domain antibodies (V H H domain and VNAR domain), scFv and Fab fragments.

[0255] (xv) Affinity group

[0256] An "affinity group" is a molecule or portion of a molecule that has a high affinity or preference for associating or binding to another specific or particular molecule or portion. The association or binding to another specific or particular molecule or portion can be through non-covalent interactions, such as hydrogen bonds, ionic forces, and van der Waals interactions. For example, an affinity group can be biotin, which has a high affinity or preference for associating or binding to the proteins avidin or streptavidin. For example, an affinity group can also refer to avidin or streptavidin that has affinity for biotin. Other examples of affinity groups and specific or particular molecules or portions that they bind or associate with include, but are not limited to, antibodies or antibody fragments and their respective antigens, such as digoxin and anti-digoxin antibodies, lectins, and carbohydrates (e.g., sugars, monosaccharides, disaccharides, or polysaccharides), as well as receptors and receptor ligands.

[0257] Any pair of an affinity group and the specific or particular molecule or moiety to which it is bound or associated can have its effects reversed, for example, between a first molecule and a second molecule, where in the first example the first molecule is characterized as an affinity group for the second molecule and in the second example the second molecule is characterized as an affinity group for the first molecule.

[0258] (xvi) Labels, detectable labels and optical labels

[0259] The terms "detectable marker," "optical marker," and "label" are used interchangeably herein to refer to a directly or indirectly detectable portion that is associated (e.g., coupled) with a molecule to be detected (e.g., a capture probe or analyte). Detectable labels can be directly detectable by themselves (e.g., radioisotope labels or fluorescent labels) or, in the case of enzyme labels, can be indirectly detectable, e.g., by catalyzing the chemical alteration of a chemical substrate compound or composition that is directly detectable. Detectable labels can be suitable for small-scale detection and / or for high-throughput screening. Therefore, suitable detectable labels include, but are not limited to, radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes.

[0260] Detectable markers can be detected qualitatively (e.g., optical or spectral), or can be quantitative. Qualitative detection generally includes a detection method that confirms the presence or occurrence of a detectable marker, while quantitative detection generally includes a detection method with a quantifiable (e.g., digitally reportable) value, such as intensity, duration, polarization and / or other properties. In some embodiments, detectable markers are bound to a feature or a capture probe associated with a feature. For example, a feature that is detectably labeled can include fluorescent, colorimetric or chemiluminescent markers attached to a bead (see, e.g., Rajeswari et al., J.Microbiol Methods 139: 22-28, 2017, and Forcucci et al., J. Biomed Opt. 10: 105010, 2015, the entire contents of which are incorporated herein by reference).

[0261] In some embodiments, multiple detectable labels can be attached to a feature, capture probe, or composition to be detected. For example, a detectable label can be incorporated during nucleic acid polymerization or amplification (e.g., -labeled nucleotides, e.g. -dCTP). Any suitable detectable label can be used. In some embodiments, the detectable label is a fluorophore. For example, the fluorophore can be from the following group: 7-AAD (7-aminoactinomycin D), acridine orange (+DNA), acridine orange (+RNA), Alexa- 350、Alexa- 430、Alexa- 488、Alexa- 532、Alexa- 546、Alexa- 555、Alexa- 568、Alexa- 594、Alexa- 633、Alexa- 647, Alexa 660、Alexa 680、Alexa 700、Alexa 750, allophycocyanin (APC), AMCA / AMCA-X, 7-aminoactinomycin D (7-AAD), 7-amino-4-methylcoumarin, 6-aminoquinoline, aniline blue, ANS, APC-Cy7, ATTO-TAG TM CBQCA, ATTO-TAG TM FQ, Auramine O-Feulgen, BCECF (high pH), BFP (blue fluorescent protein), BFP / GFP FRET, BOBO TM -1 / BO-PRO TM -1. BOBO TM -3 / BO-PRO TM -3. FL, TMR, TR-X, 530 / 550, 558 / 568, 564 / 570, 581 / 591, 630 / 650-X, 650-665-X, BTC, Calcein, Calcein Blue, Calcium Crimson TM , Calcium Green-1 TM CalciumOrange TM , White, 5-carboxyfluorescein (5-FAM), 5-carboxynaphthylfluorescein, 6-carboxyrhodamine 6G, 5-carboxytetramethylrhodamine (5-TAMRA), carboxy-X-rhodamine (5-ROX), Cascade Cascade Yellow TM , CCF2 (GeneBLAzer TM), CFP (cyan fluorescent protein), CFP / YFP FRET, chromomycin A3, Cl-NERF (low pH), CPM, 6-CR 6G, CTC-formazan 、 Cychrome (PE-Cy5), dansylamide, dansylcadaverine, dansyl chloride, DAPI, dapoxyl, DCFH, DHR, DiA (4-Di-16-ASP), DiD (DilC18 (5)), DIDS, Dil (DilC18 (3)), DiO (DiOC18 (3)), DiR (DilC18 (7)), Di-4ANEPPS, Di-8ANEPPS, DM-NERF (4.5-6.5 pH), DsRed (red fluorescent protein), EBFP, ECFP, EGFP, -97 alcohol, eosin, erythromycin, ethidium bromide, ethidium homodimer-1 (EthD-1), europium (III) chloride, 5-FAM (5-carboxyfluorescein), fast blue, fluorescein dT phosphoamidite, FITC, Fluo-3, Fluo-4, Fluoro-G0ld TM (High pH), Fluoro-Gold TM (low pH), Fluoro-Jade, 1-43, Fura-2 (high calcium), Fura-2 / BCECF, Fura Red TM (High Calcium), FuraRed TM / Fluo-3, GeneBLAzer TM (CCF2), red-shifted GFP (rsGFP), wild-type GFP, GFP / BFP-FRET, GFP / DsRed-FRET, Hoechst 33342 & 33258, 7-hydroxy-4-methylcoumarin (pH 9), 1,5-indoleacetic acid, Indo-1 (high calcium), Indo-1 (low calcium), indole dicarbocyanine, indole tricarbocyanine, JC-1, 6-JOE, JOJO TM -1 / JO-PRO TM -1, LDS751(+DNA), LDS751(+RNA), LOLO TM -1 / LO-PRO TM -1, Lucifer Yellow, LysoSensor TM Blue (pH 5), LysoSensor TM Green (pH 5), LysoSensor TM Yellow / blue (pH 4.2), green, red, Yellow, Mag-Fura-2, Mag-Indo-1, Magnesium Green TM , Marina 4-Methylumbelliferone, Mitramycin, green, orange, Red, NBD(amine), Nile Red, Oregon 488, Oregon 500, Oregon 514, Pacific Blue, PBF1, PE (R-phycoerythrin), PE-Cy5, PE-Cy7, PE-Texas Red, PerCP (Peridinyl Chlorophyll Protein), PerCP-Cy5.5 (TruRed), PharRed (APC-Cy7), C-phycocyanin, R-phycocyanin, R-phycoerythrin (PE), PI (propidium iodide), PKH26, PKH67, POPO TM -1 / PO-PRO TM -1, POPO TM -3 / PO-PRO TM -3, propidium iodide (PI), PyMPO, pyrene, pyrroline Y, Quantum Red (PE-Cy5), quinacridone mustard, R670 (PE-Cy5), Red 613 (PE-Texas Red), red fluorescent protein (DsRed), resorufin, RH 414, Rhod-2, rhodamine B, Rhodamine Green TM , Rhodamine Red TM , Rhodamine phalloidin, Rhodamine 110, Rhodamine 123, 5-ROX (carboxy-X-rhodamine), S65A, S65C, S65L, S65T, SBFI, SITS, -1 (high pH), -2, -1 (high pH), -1 (low pH), Sodium Green TM , #1, #2, 11, 13, 17, 45, blue, green, Orange, 5-TAMRA (5-carboxytetramethylrhodamine), tetramethylrhodamine (TRITC), Texas / Texas -X,Texas -X(NHS ester), thiadicarbocyanine, thiazole orange, -1 / TO- -1. -3 / TO- -3, TO- -5, three-color (PE-Cy5), TRITC (tetramethylrhodamine), TruRed (PerCP-Cy5.5), WW 781, X-rhodamine (XRITC), Y66F, Y66H, Y66W, YFP (yellow fluorescent protein), -1 / YO- -1. -3 / YO- -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, 700, 800, 800CW (NHS ester), WellRED D4 dye, WellRED D3 dye, WellRED D2 dye, 640 (NHS ester) and Dy 750 (NHS ester).

[0262] As described above, in some embodiments, the detectable label is or includes a luminescent or chemiluminescent moiety. Common luminescent / chemiluminescent moieties include, but are not limited to, peroxidases, such as horseradish peroxidase (HRP), soybean peroxidase (SP), alkaline phosphatase, and luciferase. These protein moieties can catalyze a chemiluminescent reaction given an appropriate chemical substrate (e.g., an oxidizing agent plus a chemiluminescent compound). Many families of compounds are known to provide chemiluminescence under various conditions. Non-limiting examples of chemiluminescent compound families include 2,3-dihydro-1,4-phthalazinedione luminal, 5-amino-6,7,8-trimethoxy- and dimethylamino[ca]benzene analogs. These compounds can luminesce in the presence of alkaline hydrogen peroxide or calcium hypochlorite and a base. Other examples of chemiluminescent compound families include, for example, 2,4,5-triphenylimidazole, p-dimethylamino and -methoxy substituents, oxalates (e.g., oxalyl active esters), p-nitrophenyl, N-alkyl acridinium esters, fluorescein, lucigenin, or acridinium esters.

[0263] (xvii) Template switching oligonucleotides

[0264] A "template switching oligonucleotide" is an oligonucleotide that hybridizes to non-templated nucleotides added by a reverse transcriptase (e.g., an enzyme with terminal transferase activity) during reverse transcription. In some embodiments, the template switching oligonucleotide hybridizes to non-templated poly(C) nucleotides added by the reverse transcriptase. In some embodiments, the template switching oligonucleotide adds a common 5' sequence to the full-length cDNA used for cDNA amplification.

[0265] In some embodiments, a template-switching oligonucleotide adds a common sequence to the 5' end of the RNA being reverse transcribed. For example, the template-switching oligonucleotide can hybridize to an untemplated poly(C) nucleotide added to the end of a cDNA molecule and provide a template for the reverse transcriptase to continue replication to the 5' end of the template-switching oligonucleotide, thereby generating a full-length cDNA capable of further amplification. In some embodiments, once the full-length cDNA molecule is generated, the template-switching oligonucleotide can be used as a primer in a cDNA amplification reaction.

[0266] In some embodiments, a 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 a capture probe. In certain embodiments, a sample analysis method using a template-switching oligonucleotide can involve generating a nucleic acid product from an analyte in a tissue sample, followed by further processing of the nucleic acid product using a template-switching oligonucleotide.

[0267] The template switching oligonucleotide may include a hybridization region and a template region. The hybridization region may include any sequence capable of hybridizing with a target. In some embodiments, the hybridization region may, for example, include continuous G bases to complement the overhanging C bases at the 3' end of the cDNA molecule. Continuous 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. The template sequence may include any sequence to be incorporated into the cDNA. In other embodiments, the hybridization region may include at least one base in addition to at least one G base. In other embodiments, hybridization may include bases other than G bases. In some embodiments, the template region includes at least one (e.g., at least 2, 3, 4, 5 or more) tag sequence and / or functional sequence. In some embodiments, the template region and the hybridization region are separated by a spacer.

[0268] In some embodiments, the template region includes a barcode sequence. The barcode sequence can function as a spatial barcode and / or as a unique molecular identifier. The template switching oligonucleotide can include deoxyribonucleic acid; RNA; modified nucleic acid, including 2-aminopurine, 2,6-diaminopurine (2-amino-dA), reverse dT, 5-methyl dC, 2′-deoxyinosine, super T (5-hydroxybutyl-2′-deoxyuridine), super G (8-aza-7-deazaguanosine), locked nucleic acid (LNA), unlocked nucleic acid (UNA, e.g., UNA-A, UNA-U, UNA-C, UNA-g), Iso-dG, Iso-dC, 2′ fluoro bases (e.g., fluoro C, fluoro U, fluoro A, and fluoro G), or any combination thereof.

[0269] In some embodiments, the template-switching oligonucleotide can be at least about 1, 2, 10, 20, 50, 75, 100, 150, 200, or 250 nucleotides in length or longer. In some embodiments, the template-switching oligonucleotide can be at most about 2, 10, 20, 50, 100, 150, 200, or 250 nucleotides in length or longer.

[0270] (xviii) Splint oligonucleotides

[0271] A "splint oligonucleotide" is an oligonucleotide that, when hybridized with other polynucleotides, acts as a "splint," bringing the polynucleotides adjacent to each other so that they can be linked together. In some embodiments, the splint oligonucleotide is DNA or RNA. The splint oligonucleotide may include a nucleotide sequence that is partially complementary to a nucleotide sequence from two or more different oligonucleotides. In some embodiments, the splint oligonucleotide assists in joining a "donor" oligonucleotide and an "acceptor" oligonucleotide. Generally, RNA ligase, DNA ligase, or other types of ligases are used to join the two nucleotide sequences together.

[0272] In some embodiments, the splint oligonucleotide is between 10 and 50 oligonucleotides in length, for example, between 10 and 45, 10 and 40, 10 and 35, 10 and 30, 10 and 25, or 10 and 20 oligonucleotides in length. In some embodiments, the splint oligonucleotide is 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.

[0273] (c) Analyte

[0274] 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 the present invention, an "analyte" can include any biological substance, structure, part, or component to be analyzed. The term "target" can similarly refer to an analyte of interest.

[0275] Analytes can be broadly divided into two categories: 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 phosphorylation or acetylation variants of proteins, amidation variants of proteins, hydroxylation variants of proteins, methylation variants of proteins, ubiquitination variants of proteins, sulfated variants of proteins, viral coat proteins, extracellular and intracellular proteins, antibodies, and antigen-binding fragments. In some embodiments, the analyte can be an organelle (e.g., a nucleus or mitochondria).

[0276] Cell surface features corresponding to the analyte may include, but are not limited to, receptors, antigens, surface proteins, transmembrane proteins, clusters of differentiation proteins, protein channels, protein pumps, carrier proteins, phospholipids, glycoproteins, glycolipids, cell-cell interaction protein complexes, antigen presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, or lipidation) status of cell surface proteins, gap junctions, and adherens junctions.

[0277] Analytes can be derived from specific cell types and / or specific subcellular compartments. For example, analytes can be derived from the cytoplasm, nucleus, mitochondria, microsomes, or more generally, any other compartment, organelle, or part of a cell. Permeabilizing agents specifically targeted to certain cellular compartments and organelles can be used to selectively release analytes from cells for analysis.

[0278] 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.

[0279] Examples of nucleic acid analytes also include RNA analytes, such as various types of coding and non-coding RNA. Different types of RNA analytes include messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small RNA (miRNA) and viral RNA. RNA can be a transcript (e.g., present in a tissue section). RNA can be small (e.g., less than 200 nucleic acid bases in length) or large (e.g., RNA with a length greater than 200 nucleic acid bases). Small RNA mainly includes 5.8S ribosomal RNA (rRNA), 5S rRNA, transfer RNA (tRNA), microRNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snRNA), Piwi interacting RNA (piRNA), tRNA-derived small RNA (tsRNA) and small rDNA-derived RNA (srRNA). RNA can be double-stranded RNA or single-stranded RNA. RNA can be circular RNA. RNA can be bacterial rRNA (e.g., 16srRNA or 23s rRNA).

[0280] Other examples of analytes include mRNA and cell surface features (e.g., using markers 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 micrococcal enzyme-seq), mRNA and metabolites (e.g., using markers described herein), barcoded markers (e.g., oligonucleotide-tagged antibodies described herein) and V(D)J sequences of immune cell receptors (e.g., T cell receptors), mRNA and perturbants (e.g., CRISPR crRNA / sgRNA, TALEN, zinc finger nucleases, and / or antisense oligonucleotides as described herein). In some embodiments, the perturbant can be a small molecule, an antibody, a drug, an aptamer, miRNA, a physical environment (e.g., temperature change), or any other known perturbant.

[0281] 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 (e.g., a TCR or BCR). In some embodiments, the nucleic acid molecule is first generated from reverse transcription of the corresponding mRNA using a poly(T)-containing primer. The generated cDNA can then be barcoded 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 the poly(C) tail added to the 3′ end of the cDNA by a reverse transcriptase. The original mRNA template and the template-switching oligonucleotide can be denatured from the cDNA, and the barcoded capture probe can then hybridize to the cDNA and generate a complement of the cDNA. 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 compositions and barcoding methods comprising template-switching oligonucleotides are described in PCT patent application PCT / US2017 / 057269, filed on October 18, 2017, and U.S. patent application serial number 15 / 825,740, filed on November 29, 2017, both of which are incorporated herein by reference in their entirety. V(D)J analysis can also be accomplished by using one or more markers that bind to specific surface features of immune cells and are associated with a barcode sequence. The one or more markers can include MHC or MHC multimers.

[0282] As described above, the analyte may include a nucleic acid capable of functioning as a component of a gene editing reaction, such as, for example, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing. Thus, the capture probe may include a nucleic acid sequence complementary to the analyte (e.g., a sequence hybridizable to a CRISPR RNA (crRNA), a single guide RNA (sgRNA), or an adapter sequence engineered into a crRNA or sgRNA).

[0283] In certain embodiments, analytes can be extracted from living cells. Processing conditions can be adjusted to ensure that the biological sample remains viable during analysis and that the analyte is extracted (or released) from the living cells of the sample. Analytes derived from living cells can be obtained only once from the sample, or at intervals while the sample remains viable.

[0284] In general, the systems, devices, methods, and compositions can be used to analyze any number of analytes. For example, the number of analytes analyzed can 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 1000, at least about 10,000, at least about 100,000, or more different analytes present in a sample region or within an individual feature of a substrate. Methods for performing multiplex analysis to analyze two or more different analytes will be discussed in subsequent sections of the present invention.

[0285] (d) Biological samples

[0286] (i) Type of biological sample

[0287] 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 capture microscopy (LCM), and typically includes cells and / or other biological material from the subject. In addition to the above-mentioned subjects, biological samples can 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 also be obtained from prokaryotes, such as bacteria, such as Escherichia coli, Staphylococci, or Mycoplasma pneumoniae; Archaea; viruses, such as hepatitis C virus or human immunodeficiency virus; or viroids. Biological samples can be obtained from eukaryotic organisms, such as patient-derived organoids (PDOs) or patient-derived xenografts (PDXs). Biological samples can include organoids, miniaturized and simplified versions of organs produced in vitro that can display realistic microanatomical structures in three dimensions. Organoids can produce one or more cells of self-organization, embryonic stem cells and / or induced pluripotent stem cells, which can self-organize in three-dimensional culture due to their self-renewal and differentiation capabilities. In some embodiments, the organoid is a brain organoid, intestinal organoid, stomach organoid, tongue organoid, thyroid organoid, thymus organoid, testicular organoid, liver organoid, pancreatic organoid, epithelial organoid, lung organoid, kidney organoid, gastrula, heart organoid or retinal organoid. The object from which a biological sample can be obtained can be a healthy or asymptomatic individual, an individual suffering from or suspected of having a disease (e.g., cancer) or an individual with a pre-treatment for the disease, and / or an individual in need of treatment or suspected of needing treatment.

[0288] A biological sample may be from a homogenous culture or population of a subject or organism as described herein, or may alternatively be from a collection of several different organisms, for example, in a community or ecosystem.

[0289] A biological sample may include one or more diseased cells. Diseased cells may exhibit 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 may be derived from solid tumors, hematologic malignancies, cell lines, or obtained as circulating tumor cells.

[0290] Biological samples can also include fetal cells. For example, a procedure such as amniocentesis can be performed to obtain a fetal cell sample from the maternal circulation. Fetal cell sequencing can be used to identify any of a number of genetic diseases, including, for example, aneuploidy, such as Down syndrome, Edwards syndrome, and Patau syndrome. In addition, cell surface characteristics of fetal cells can be used to identify any of a variety of diseases or conditions.

[0291] Biological samples can also include immune cells. Sequence analysis of the immune function of such cells, including genomics, proteomics, and cell surface characteristics, can provide rich information to help understand the state and function of the immune system. For example, determining the status (e.g., negative or positive) of minimal residual disease (MRD) in multiple myeloma (MM) patients after autologous stem cell transplantation is considered to be a predictor of MRD in MM patients (see, e.g., U.S. Patent Application Publication No. 2018 / 0156784, which is incorporated herein by reference in its entirety).

[0292] Examples of immune cells in a biological sample include, but are not limited to, B cells, T cells (e.g., cytotoxic T cells, natural killer T cells, regulatory T cells, and helper T cells), natural killer cells, cytokine-induced killer (CIK) cells, myeloid cells, such as granulocytes (basophils, eosinophils, neutrophils / multinuclear neutrophils), monocytes / macrophages, mast cells, platelets / megakaryocytes, and dendritic cells.

[0293] The biological sample can include any number of macromolecules, such as cellular macromolecules and organelles (such as mitochondria and nuclei). The biological sample can be a nucleic acid sample and / or a protein sample. The biological sample can be a carbohydrate sample or a lipid sample. The biological sample can be obtained as a tissue sample, such as a tissue section, a biopsy, a core biopsy, a needle aspiration or a fine needle aspiration. The sample can be a liquid sample, such as a blood sample, a urine sample or a saliva sample. The sample can be a skin sample, a colon sample, a cheek swab, a histological sample, a histopathological sample, a plasma or serum sample, a tumor sample, a living cell, a cultured cell, a clinical sample, such as whole blood or a blood-derived product, a blood cell or a cultured tissue or cell, including a cell suspension.

[0294] Cell-free biological samples may include extracellular polynucleotides. Extracellular polynucleotides can be isolated from body samples such as blood, plasma, serum, urine, saliva, mucosal excretions, sputum, feces, and tears.

[0295] As described above, a biological sample may include a single analyte of interest or multiple analytes of interest. Methods for performing multiplexing to analyze two or more different analytes in a single biological sample are discussed in subsequent sections of the present invention.

[0296] (ii) Preparation of biological samples

[0297] Various steps can be performed to prepare a biological sample for analysis. Unless otherwise indicated, the preparation steps described below can generally be combined in any manner to appropriately prepare a particular sample for analysis.

[0298] (1) Tissue sections

[0299] Biological samples can be obtained from a subject (e.g., by surgical biopsy, whole subject sectioning), grown in vitro as a cell population on a growth substrate or culture dish, or prepared as a tissue slice or tissue section. The grown sample can be thin enough to be analyzed without further processing steps. Alternatively, the grown sample and the sample obtained by biopsy or sectioning can be prepared as thin tissue sections using a mechanical cutting device (e.g., a vibrating blade microtome). As another alternative, in some embodiments, thin tissue sections can be prepared by applying a biological sample tough imprint to a suitable substrate material.

[0300] The thickness of the tissue section can be a fraction of the maximum cross-sectional dimension of the cells (e.g., less than 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1). However, tissue sections having a thickness greater than the maximum cross-sectional cell dimension can also be used. For example, cryostat sections, such as 10-20 micrometer thick, can be used.

[0301] In some embodiments, the thickness of tissue section is 1-100 micron, 1-50 micron, 1-30 micron, 1-25 micron, 1-20 micron, 1-15 micron, 1-10 micron, 2-8 micron, 3-7 micron or 4-6 micron, but as mentioned above, thickness can also be analyzed to be greater than or less than the section of these scopes.

[0302] It is also possible to obtain multiple slices from a single biological sample. For example, multiple tissue slices can be obtained from a surgical biopsy sample by serially sectioning the biopsy sample using a microtome blade. In this way, spatial information between serial slices can be preserved, and the slices can be analyzed sequentially to obtain three-dimensional information about the biological sample.

[0303] (2) Freezing

[0304] In some embodiments, biological sample (for example, tissue section as described above) can be prepared by deep freezing at a temperature suitable for maintaining or keeping the integrity (for example, physical properties) of tissue structure. For example, the temperature can be lower than -20 ° C, or lower than -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 sample can be cut (for example, thin slice) onto the substrate surface using any number of suitable methods. For example, a freezing microtome (for example, cryostat) can be used to prepare a tissue sample, and the freezing microtome is arranged on a temperature suitable for maintaining the structural integrity of the tissue sample and the chemical properties of nucleic acid in the sample. For example, this temperature can be lower than -15 ° C, lower than -20 ° C or lower than -25 ° C. Samples can be snap-frozen in isopentane and liquid nitrogen. Frozen samples can be stored in airtight containers prior to embedding.

[0305] (3) Formaldehyde fixation and paraffin embedding

[0306] In some embodiments, biological samples can be prepared using formaldehyde fixation and paraffin embedding (FFPE), which is an established method. In some embodiments, cell suspensions and other non-tissue samples can be prepared using formaldehyde fixation and paraffin embedding. After the sample is fixed and embedded in paraffin or resin blocks, the sample can be sectioned as described above. Prior to analysis, paraffin-embedded material can be removed from the tissue sections (e.g., deparaffinized) by incubating the tissue sections in an appropriate solvent (e.g., xylene) and then rinsing (e.g., 99.5% ethanol for 2 minutes, 96% ethanol for 2 minutes, and 70% ethanol for 2 minutes).

[0307] (4) Fixed

[0308] As an alternative to formaldehyde fixation described above, biological samples can be fixed in any of a variety of other fixatives to preserve the biological structure of the sample prior to analysis. For example, the sample can be fixed by immersion in ethanol, methanol, acetone, formaldehyde (e.g., 2% formaldehyde), paraformaldehyde-triton, glutaraldehyde, or a combination thereof.

[0309] In some embodiments, acetone fixation is used together with fresh frozen samples, and fresh frozen samples may include but are not limited to cortical tissue, mouse olfactory bulb, human brain tumor, human postmortem brain and breast cancer samples. In some embodiments, based on the workflow selection and / or optimization compatible fixation method of expectation. For example, formaldehyde fixation can be selected to be compatible with the workflow using IHC / IF scheme to carry out protein visualization. As another example, methanol can be selected to fix for the workflow that attaches importance to RNA / DNA library quality. Acetone fixation can be selected to penetrate tissue in some applications. When carrying out acetone fixation, pre-permeabilization step (as described below) can not be carried out. Or, acetone fixation can be carried out in conjunction with permeabilization step.

[0310] (5) Embedding

[0311] As an alternative to paraffin embedding, biological samples can be embedded in any of a variety of other embedding materials to provide a substrate for the sample prior to sectioning and other processing steps. Generally, the embedding material is removed prior to analyzing the tissue sections obtained from the sample. Suitable embedding materials include, but are not limited to, waxes, resins (e.g., methacrylates), epoxies, and agar.

[0312] (6) Dyeing

[0313] For ease of visualization, biological samples can be stained using a variety of stains and staining techniques. In some embodiments, any number of biological stains can be used to stain the sample, including but not limited to acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hodgkin's stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine, or safranine.

[0314] The sample can be stained using known staining techniques, including Can-Grunwald, Giemsa, hematoxylin and eosin (H&E), Jenner's, Leishman, Masson's trichrome, Papanicolaou, Romanowsky, silver, Sudan, Wright's and / or PAS staining techniques. PAS staining is typically performed after formaldehyde or acetone fixation.

[0315] In some embodiments, a biological sample can be stained using a detectable marker as described elsewhere herein (e.g., radioisotopes, fluorophores, chemiluminescent compounds, bioluminescent compounds, and dyes). In some embodiments, only one type of staining agent or a technique is used to stain the biological sample. In some embodiments, staining includes biological staining techniques, such as H&E staining. In some embodiments, staining includes using fluorescently coupled antibodies to identify analytes. In some embodiments, two or more different types of staining agents or two or more different staining techniques are used to stain the biological sample. For example, a biological sample can be prepared by staining and imaging using a technique (e.g., H&E staining and bright field imaging) and then staining and imaging the same biological sample using another technique (e.g., IHC / IF staining and fluorescence microscopy).

[0316] In some embodiments, the biological sample can be decolorized. Decontamination or decolorization methods for biological samples are known in the art and generally depend on the properties of the staining agent applied to the sample. For example, H&E staining can be decolorized by washing the sample in HCl. In some embodiments, decolorization can be included in 1, 2, 3 or more washes in HCl. In some embodiments, decolorization can include adding HCl to a downstream solution (e.g., a permeabilization solution). As another example, in some embodiments, one or more immunofluorescent stains are applied to the sample by antibody coupling. These stains can be removed using techniques such as cleavage of disulfide bonds processed by reducing agents and detergent washes, hygroscopic salt treatment, antigen retrieval solution treatment, and acidic glycine buffer treatment. For example, methods for multiple staining and destaining are described in Bolognesi et al., J. Histochem. Cytochem. 2017; 65(8): 431-444, Lin et al., Nat Commun. 2015; 6: 8390, Pirici et al., J. Histochem. Cytochem. 2009; 57: 567-75, and Glass et al., J. Histochem. Cytochem. 2009; 57: 899-905, the entire contents of which are incorporated herein by reference.

[0317] (7) Hydrogel embedding

[0318] In some embodiments, hydrogel formation occurs within a biological sample. In some embodiments, a biological sample (e.g., a tissue slice) is embedded in a hydrogel. In some embodiments, hydrogel subunits are injected into the biological sample, and polymerization of the hydrogel is initiated 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" may be a hydrophilic monomer, molecular precursor, or polymer that can be polymerized (e.g., cross-linked) to form a three-dimensional (3D) hydrogel network.

[0319] The hydrogel can swell in the presence of water. In some embodiments, the hydrogel comprises a natural material. In some embodiments, the hydrogel comprises a synthetic material. In some embodiments, the hydrogel comprises a hybrid material, for example, the hydrogel material comprises a component having both a synthetic polymer and a natural polymer. Any material used in a hydrogel or a hydrogel comprising a polypeptide-based material as described herein can be used. Embedding a sample in this manner generally involves contacting a biological sample with a hydrogel so that the biological sample is surrounded by the hydrogel. For example, the sample can be embedded by contacting the sample with a suitable polymer material and activating the polymer material to form a hydrogel. In some embodiments, the hydrogel is formed so that the hydrogel is internalized within the biological sample.

[0320] In some embodiments, the biological sample is fixed in the hydrogel by crosslinking of the polymeric material forming the hydrogel. Crosslinking can be performed chemically and / or photochemically, or by any other hydrogel formation method known in the art. For example, the biological sample can be fixed in the hydrogel by polyacrylamide crosslinking. In addition, the analyte of the biological sample can be fixed in the hydrogel by crosslinking (e.g., polyacrylamide crosslinking).

[0321] The composition of the hydrogel and its application to biological samples generally depends on the nature and preparation of the biological sample (e.g., sliced, non-sliced, fresh frozen tissue, fixed type). The hydrogel can be any suitable hydrogel, wherein after the hydrogel is formed on the biological sample, the biological sample becomes anchored or embedded in the hydrogel. Non-limiting examples of hydrogels are described herein or are known in the art. As an example, where the biological sample is a tissue slice, the hydrogel may include a monomer solution and an ammonium persulfate (APS) initiator / tetramethylethylenediamine (TEMED) promoter solution. As another example, when the biological sample consists of cells (e.g., cultured cells or cells separated from a tissue sample), the cells can be incubated with a monomer solution and an APS / TEMED solution. For cells, the hydrogel is formed in a compartment, including but not limited to a device for culturing, maintaining or transporting cells. For example, the hydrogel can be formed using a monomer solution plus APS / TEMED, the depth of which added to the compartment is between about 0.1 μm and about 5 mm.

[0322] In some embodiments, the hydrogel includes a joint that allows biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel includes a joint that allows biological analyte to be anchored to the hydrogel. In this case, the joint can be added to the hydrogel before, simultaneously or afterwards, the hydrogel is formed. Non-limiting examples of joints for anchoring nucleic acids to the hydrogel can include 6-((acryloyl) amino) hexanoic acid (acryloyl-XSE) (available from ThermoFisher, Waltham, Massachusetts), Label-IT amine (available from MirusBio, Madison, Wisconsin) and Label X).

[0323] In some embodiments, functionalization chemistry can be used. In some embodiments, functionalization chemistry includes hydrogel tissue chemistry (HTC). Any hydrogel tissue skeleton suitable for HTC (e.g., synthetic or natural) can be used to anchor biomacromolecules and regulate functionalization. Non-limiting examples of methods using HTC skeleton variants include CLARITY, PACT, ExM, SWITCH, and ePACT. In some embodiments, the hydrogel formation in the biological sample is permanent. For example, biomacromolecules can be permanently adhered to the hydrogel, thereby allowing multiple rounds of interrogation. In some embodiments, the hydrogel formation in the biological sample is reversible.

[0324] In some embodiments, other reagents are added to the hydrogel subunits before, simultaneously and / or after polymerization. For example, other reagents may include but are not limited to oligonucleotides (e.g., capture probes), endonucleases for fragmented DNA, fragmentation buffers for DNA, DNA polymerases, dNTPs for amplifying nucleic acids and attaching barcodes to the amplified fragments. Other enzymes may be used, including but not limited to RNA polymerases, transposases, ligases, proteinase K and DNA enzymes. Other reagents may also include reverse transcriptases, including enzymes with terminal transferase activity, primers and conversion oligonucleotides. In some embodiments, optical labels are added to the hydrogel subunits before, simultaneously and / or after polymerization.

[0325] In some embodiments, an HTC agent is added to the hydrogel before, simultaneously with, and / or after polymerization. In some embodiments, a cell spiking agent is added to the hydrogel before, simultaneously with, and / or after polymerization. In some embodiments, a cell penetrating agent is added to the hydrogel before, simultaneously with, and / or after polymerization.

[0326] In some embodiments, biological sample is embedded in hydrogel to promote sample transfer to another location (for example, to array).For example, archived biological sample (for example, FFPE tissue slice) can be transferred to spatial array from storage for spatial analysis.In some embodiments, biological sample on substrate can be covered with any prepolymer solution as described herein.In some embodiments, prepolymer solution can be polymerized, thereby forming hydrogel above and / or around biological sample.Hydrogel formation can occur in a manner sufficient to anchor (for example, embed into) biological sample to hydrogel.After hydrogel is formed, biological sample is anchored to (for example, embed into) hydrogel, wherein hydrogel is separated from substrate (for example, slide) and causes biological sample to be separated from substrate together with hydrogel.Then the biological sample contained in hydrogel can be contacted with spatial array, and biological sample is carried out spatial analysis.

[0327] Any of a variety of characteristics can determine the transfer conditions required for a given biological sample. Non-limiting examples of characteristics that may affect transfer conditions include the sample (e.g., thickness, fixation, and cross-linking) and / or the analyte of interest (different conditions are used to preserve and / or transfer different analytes (e.g., DNA, RNA, and protein)).

[0328] In some embodiments, the hydrogel is removed after the biological sample is contacted with the spatial array. For example, the methods described herein may include event-dependent (e.g., light or chemical) depolymerizing hydrogels, wherein upon application of an event (e.g., an external stimulus), the hydrogel depolymerizes. In one example, a biological sample can be anchored to a DTT-sensitive hydrogel, wherein the addition of DTT can cause the hydrogel to depolymerize and release the anchored biological sample.

[0329] The hydrogel embedded in the biological sample can be cleared using any suitable method. For example, electrophoretic tissue clearing methods can be used to remove biomacromolecules from the hydrogel-embedded sample. In some embodiments, the hydrogel-embedded sample is stored in a medium (e.g., a fixing medium, methylcellulose, or other semisolid medium) before or after the hydrogel is cleared.

[0330] In some embodiments, the hydrogel chemistry can be adjusted to specifically bind (e.g., retain) a particular type of analyte (e.g., RNA, DNA, protein, etc.). In some embodiments, the hydrogel includes a joint that allows a biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel includes a joint that allows a biological analyte to be anchored to the hydrogel. In this case, the joint can be added to the hydrogel before, simultaneously with, or after the hydrogel is formed. Non-limiting examples of joints that anchor nucleic acids to hydrogels can include 6-((acryloyl)amino)hexanoic acid (acryloyl-X SE), Label-IT amine, and Label X. Non-limiting examples of characteristics that may affect transfer conditions include samples (e.g., thickness, fixation, and cross-linking) and / or analytes of interest (different conditions for preserving and / or transferring different analytes (e.g., DNA, RNA, and proteins)).

[0331] Other methods and aspects of hydrogel embedding of 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.

[0332] (8) Biological sample transfer

[0333] In some embodiments, a biological sample (e.g., a biological sample prepared by methanol fixation or formalin fixation and paraffin embedding (FFPE)) fixed on a substrate is transferred to a spatial array using a hydrogel. In some embodiments, a hydrogel is formed above the biological sample on a substrate (e.g., a glass slide). For example, hydrogel formation can occur in a manner sufficient to anchor (e.g., embed) the biological sample to the hydrogel. After the hydrogel is formed, the biological sample is anchored to (e.g., embedded into) the hydrogel, wherein the hydrogel is separated from the substrate causing the biological sample to be separated from the substrate together with the hydrogel. The biological sample can then be brought into contact with the spatial array, thereby allowing spatial analysis of the biological sample. In some embodiments, the hydrogel is removed after the biological sample is contacted with the spatial array. For example, the methods described herein may include event-dependent (e.g., light or chemical) depolymerization hydrogels, wherein when an event (e.g., an external stimulus) is applied, the hydrogel depolymerizes. In one example, the biological sample can be anchored on a DTT-sensitive hydrogel, wherein adding DTT can cause the hydrogel to depolymerize and release the anchored biological sample. The hydrogel can be any suitable hydrogel, wherein after forming the hydrogel on the biological sample, the biological sample becomes anchored or embedded in the hydrogel. The non-limiting examples of hydrogels are described in this article or are known in the art. In some embodiments, the hydrogel includes a joint that allows the biological sample to be anchored to the hydrogel. In some embodiments, the hydrogel includes a joint that allows the biological analyte to be anchored to the hydrogel. In this case, the joint can be added to the hydrogel before, simultaneously, or after the hydrogel is formed. The non-limiting examples of the joint that nucleic acids are anchored to the hydrogel can include 6-((acryloyl) amino) hexanoic acid (acryloyl-XSE) (available from ThermoFisher, Waltham, Massachusetts), Label-IT amine (available from MirusBio, Madison, Wisconsin) and Label X). Any kind of characteristic can determine the required transfer conditions for a given biological sample. Non-limiting examples of characteristics that may affect transfer conditions include the sample (e.g., thickness, fixation, and cross-linking) and / or the analyte of interest (different conditions for preserving and / or transferring different analytes (e.g., DNA, RNA, and protein)). In some embodiments, hydrogel formation can occur in a manner sufficient to anchor (e.g., embed) the analyte in the biological sample to the hydrogel. In some embodiments, the hydrogel can invaginate (e.g., shrink) along with the anchored analyte present in the biological sample (e.g., embedded in the hydrogel). In some embodiments, the hydrogel can expand (e.g., expand isometrically) along with the anchored analyte present in the biological sample (e.g., embedded in the hydrogel). In some embodiments, the hydrogel can be invaginated (e.g., shrink) and then expand with the anchored analyte present in the biological sample (e.g., embedded in the hydrogel).

[0334] (9) Isometric Expansion

[0335] In some embodiments, the biological sample embedded in the hydrogel can be expanded isometrically. Available isometric expansion methods include hydration, which is a preparative step in expansion microscopy, as described in the following literature, Chen et al., Science 347 (6221): 543-548, 2015; Asano et al. Current Protocols. 2018, 80: 1, doi: 10.1002 / cpcb.56 and Gao et al. BMC Biology. 2017, 15: 50, doi: 10.1186 / s12915-017-0393-3, Wassie, AT et al., Expansion microscopy: principles and uses in biological research, Nature Methods, 16 (1): 33-41 (2018), each of which is incorporated herein by reference in its entirety.

[0336] In general, the steps used to perform isometric spreading 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 for anchoring RNA, DNA, and proteins to gels).

[0337] Isometric expansion can be achieved by anchoring one or more components of the biological sample to the gel, followed by gel formation, protein hydrolysis, and swelling. Isometric expansion of the biological sample can occur before the biological sample is fixed on the substrate, or after the biological sample is fixed on the substrate. In some embodiments, the isometrically expanded biological sample can be removed from the substrate before the extended biological sample is contacted with a spatial barcode array (e.g., a capture probe of spatial barcoding on a substrate).

[0338] In some embodiments, proteins in a biological sample are anchored to a swellable gel (e.g., a polyelectrolyte gel). Antibodies can be directed to the protein before, after, or in conjunction with anchoring to the swellable gel. DNA and / or RNA in a biological sample can also be anchored to the swellable gel via a suitable linker. Examples of such linkers include, but are not limited to, 6-((acryloyl)amino)hexanoic acid (acryloyl-X-SE) (available from ThermoFisher, Waltham, Massachusetts), Label IT amine (available from MirusBio, Madison, Wisconsin), and Label X (described, for example, in Chen et al., Nat. Methods 13: 679-684, 2016, the entire contents of which are incorporated herein by reference).

[0339] Isometric spreading of a sample can improve the spatial resolution of subsequent analysis of the sample. For example, isometric spreading of a biological sample can result in increased resolution of spatial analysis (e.g., single cell analysis). The increased resolution in a spatial cross-section can be determined by comparing an isometrically spread sample with a non-isometrically spread sample.

[0340] In some embodiments, the biological sample is isometrically expanded to at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non-expanded amount. In some embodiments, the sample is isometrically expanded to at least 2 times and less than 20 times its non-expanded amount.

[0341] In some embodiments, a biological sample embedded in a hydrogel isometrically expanded to at least 2x, 2.1x, 2.2x, 2.3x, 2.4x, 2.5x, 2.6x, 2.7x, 2.8x, 2.9x, 3x, 3.1x, 3.2x, 3.3x, 3.4x, 3.5x, 3.6x, 3.7x, 3.8x, 3.9x, 4x, 4.1x, 4.2x, 4.3x, 4.4x, 4.5x, 4.6x, 4.7x, 4.8x, or 4.9x its non-expanded amount. In some embodiments, a biological sample embedded in a hydrogel isometrically expanded to at least 2x and less than 20x its non-expanded amount.

[0342] (10) Substrate attachment

[0343] In some embodiments, a biological sample can be attached to a hydrogel substrate. Examples of substrates suitable for this purpose are described in detail below. Attachment of the biological sample can be irreversible or reversible, depending on the nature of the sample and the subsequent steps in the analytical method.

[0344] In certain embodiments, a sample can be reversibly attached to a substrate by applying a suitable polymer coating to the substrate and contacting the sample 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.

[0345] More generally, in some embodiments, the substrate can be coated or functionalized with one or more substances to facilitate sample attachment 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.

[0346] (11) Unaggregated cells

[0347] In some embodiments, the biological sample corresponds to a cell (e.g., from a cell culture or tissue sample). In a cell sample having a plurality of cells, the individual cells may not naturally aggregate. For example, the cells may be from a cell suspension and / or from isolated or disaggregated cells of a tissue or tissue section.

[0348] Alternatively, the cells in the sample can aggregate and can be depolymerized into a plurality of individual cells using, for example, enzymes or mechanical techniques. Examples of enzymes for enzymatic depolymerization include, but are not limited to, dispase, collagenase, trypsin, or a combination thereof. For example, a tissue homogenizer can be used to perform mechanical depolymerization.

[0349] In some embodiments of the cell of non-aggregated or disaggregated cells, cells are distributed on substrate so that at least one cell occupies different spatial features on substrate.Cell can be fixed on substrate (for example, to prevent the lateral diffusion of cell).In some embodiments, cell fixative can be used for fixing non-aggregated or disaggregated samples on spatial bar coding array before analyte capture. " cell fixative " can refer to the antibody attached to substrate, which can be bound to cell surface markers.In some embodiments, the distribution of multiple cells on substrate follows Poisson statistics.

[0350] In some embodiments, cells from the plurality of cells are immobilized on a substrate. In some embodiments, cells are immobilized to prevent lateral diffusion, for example, by adding a hydrogel and / or by applying an electric field.

[0351] (12) Suspension and adherent cells

[0352] In some embodiments, the biological sample can be obtained from a cell culture grown in vitro. A sample from a cell culture can include one or more suspended cells that are anchorage-independent within the cell culture. Examples of such cells include, but are not limited to, hematopoietic cells and cell lines derived from the following cell lines: Colo205, CCRF-CEM, HL-60, K562, MOLT-4, RPMI-8226, SR, HOP-92, NCI-H322M, and MALME-3M.

[0353] The sample from the cell culture may include one or more adherent cells grown on the surface of a container containing culture medium. Non-limiting examples of adherent cells include DU145 (prostate cancer) cells, H295R (adrenocortical carcinoma) cells, HeLa (cervical cancer) cells, KBM-7 (chronic myeloid leukemia) cells, LNCaP (prostate cancer) cells, MCF-7 (breast cancer) cells, MDA-MB-468 (breast cancer) cells, PC3 (prostate cancer) cells, SaOS-2 (bone cancer) cells, SH-SY5Y (neuroblastoma, cloned from myeloma) cells, cells, T-47D (breast cancer) cells, THP-1 (acute myeloid leukemia) cells, U87 (glioblastoma) cells, National Cancer Institute Cancer Cell Line Panel 60 (NCI60), Vero (African green monkey kidney epithelial cell line), MC3T3 (embryonic skull) cells, GH3 (pituitary tumor) cells, PC12 (pheochromocytoma) cells, dog MDCK kidney epithelial cells, Xenopus laevis A6 kidney epithelial cells, zebrafish AB9 cells, and Sf9 insect epithelial cells.

[0354] Other examples of adherent cells are shown in Table 1 and are categorized in, for example, “A Catalog of in Vitro Cell Lines, Transplantable Animal and Human Tumors and Yeast,” Division of Cancer Treatment and Diagnostics (DCTD), National Cancer Institute (2013) and Abaan et al., “The exomes of the NCI-60 panel: a genomic resource for cancer biology and systems pharmacology,” Cancer Research 73(14): 4372-82, 2013, the entire contents of which are incorporated herein by reference.

[0355] Table 1: Examples of adherent cells

[0356]

[0357]

[0358]

[0359] In some embodiments, the adherent cells are cells corresponding to one or more of the following cell lines: BT549, HS578T, 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, dog MDCK kidney epithelial, Xenopus A6 kidney epithelial, zebrafish AB9, and Sf9 insect epithelial cell lines.

[0360] (13) Tissue permeabilization

[0361] In some embodiments, the biological sample can be permeabilized to facilitate the transfer of analytes from the sample and / or to facilitate the transfer of substances (e.g., capture probes) into the sample. If the sample is not permeabilized enough, the amount of analyte captured from the sample may be too low to be fully analyzed. Conversely, if the tissue sample is too permeabilized, the relative spatial relationships of the analytes within the tissue sample may be lost. Therefore, it is ideal to fully permeabilize the tissue sample to obtain good signal intensity while still maintaining a balance between the spatial resolution of the analyte distribution in the sample.

[0362] Generally, biological samples can be permeabilized by exposing the sample to one or more permeabilizing agents. Suitable agents for this purpose include, but are not limited to, organic solvents (e.g., acetone, ethanol, and methanol), cross-linking agents (e.g., paraformaldehyde), detergents (e.g., saponin, Triton X-100, TM or Tween-20 TM ), or sodium dodecyl sulfate (SDS), and an enzyme (such as trypsin, a protease (e.g., proteinase K)). In some embodiments, the detergent is an anionic detergent (e.g., SDS or a solution of N-lauroylsarcosine sodium salt). In some embodiments, the biological sample can be permeabilized using any of the methods described herein (e.g., using any detergent described herein, such as SDS and / or a solution of N-lauroylsarcosine sodium salt) before or after enzymatic treatment (e.g., treatment with any enzyme described herein, such as trypsin, a protease (e.g., pepsin and / or proteinase K)).

[0363] In some embodiments, a biological sample can be permeabilized by exposing the sample to 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 permeabilized by exposing the sample 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 12.0 w / v%, about 2.0 w / v% to about 10.0 w / v%, about 4.0 w / v% to about 14.0 w / v%, about 4.0 w / v% to about 12.0 w / v%, or about 45°C to about 50°C) (e.g., at a temperature of about 4°C to about 35°C, about 4°C 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, 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). %, 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 10.0 w / v%, about 8.0 w / v% to about 14.0 w / v%, about 8.0 w / v% to about 12.0 w / v%, about 8.0 w / v% to about 10.0 w / 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%) of SDS and / or N-lauroyl sarcosinate solution and / or proteinase K (e.g., for 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, or about 5 minutes to about 10 minutes).

[0364] In some embodiments, the biological sample can be incubated with a permeabilizing agent to facilitate permeabilization of the sample. For example, other methods of sample permeabilization are described in Jamur et al., Method Mol. Biol. 588: 63-66, 2010, the entire contents of which are incorporated herein by reference.

[0365] Lysis reagent

[0366] In some embodiments, the biological sample can be permeabilized by adding one or more lysing agents to the sample. Examples of suitable lysing agents include, but are not limited to, bioactive agents, such as lysing enzymes for cracking different cell types, such as Gram-positive or -negative bacteria, plants, yeasts, mammals, such as lysozyme, colorless peptidase, lysostaphin, Labiase enzyme, kitalase, lytic enzyme, and a variety of other commercially available lysing enzymes.

[0367] Other lysing agents can be added to the biological sample in addition or alternatively to promote permeabilization. For example, a lysis solution based on a surfactant can be used for lysis sample cells. The lysis solution can include an ionic surfactant, such as sarkosyl and sodium lauryl sulfate (SDS). More generally, chemical lysing agents can include but are not limited to organic solvents, chelating agents, detergents, surfactants and chaotropic agents.

[0368] In some embodiments, the biological sample can be permeabilized by a non-chemical permeabilization method. Non-chemical permeabilization methods are known in the art. For example, usable non-chemical permeabilization methods include but are not limited to physical dissolution techniques, such as electroporation, mechanical permeabilization methods (e.g., bead milling using a homogenizer and grinding balls to mechanically disrupt the sample's tissue structure), acoustic permeabilization (e.g., ultrasound), and thermal decomposition techniques, such as heating to induce thermal permeabilization of the sample.

[0369] Protease

[0370] In some embodiments, culture medium, solution or permeabilization solution can contain one or more proteases.In some embodiments, the biological sample processed with the protease that can degrade histone can cause the generation of fragmented genomic DNA.The same capture domain (for example, the capture domain with poly (T) sequence) for capturing mRNA can be used to capture fragmented genomic DNA.In some embodiments, before spatial profile analysis, biological sample is processed with the protease that can degrade histone and RNA protective agent, to promote the capture of genomic DNA and mRNA.

[0371] In some embodiments, the biological sample is permeabilized by exposing the sample to a protease capable of degrading histones. As used herein, the term "histone" generally refers to a linker histone (e.g., H1) and / or a core histone (e.g., H2A, H2B, H3, and H4). In some embodiments, a protease can degrade a linker histone, a core histone, or both. Any suitable protease capable of degrading histones in a biological sample can be used. Non-limiting examples of proteases capable of degrading histones include proteases inhibited by leupeptin and TLCK (tosyl-L-lysyl-chloromethane hydrochloride), proteases encoded by the EUO gene from Chlamydia trachomatis serovar A, granzyme A, serine proteases (e.g., trypsin or trypsin-like proteases, neutral serine proteases, elastase, cathepsin G), aspartyl proteases (e.g., cathepsin D), peptidase family C1 enzymes (e.g., cathepsin L), pepsin, proteinase K, proteases inhibited by the diazomethane inhibitor Z-Phe-Phe-CHN (2) or the epoxide inhibitor E-64, lysosomal proteases or azurase (e.g., cathepsin G, elastase, proteinase 3, neutral serine proteases).In some embodiments, the serine protease is trypsin, a trypsin-like enzyme, or a functional variant or derivative thereof (e.g., P00761; COHK48; Q8IYP2; Q8BW11; Q6IE06; P35035; P00760; P06871; Q90627; P16049; P07477; P00762; P35031; P19799; P350 36; Q29463; P06872; Q90628; P07478; P07146; P00763; P35032; P70059; P29786; P3503 7; Q90629; P35030; P08426; P35033; P35038; P12788; P29787; P35039; P35040; Q8NHM4; P35041;P35043;P35044;P54624;P04814;P35045;P32821;P54625;P35004;P35046;P 32822;P35047;C0HKA5;C0HKA2;P54627;P35005;C0HKA6;C0HKA3;P52905;P83348;P00 In some embodiments, the protease capable of degrading one or more histones comprises an amino acid sequence having at least 80% sequence identity to P00761, P00760, or Q29463. In some embodiments, the protease capable of degrading one or more histones comprises an amino acid sequence that is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to P00761, P00760, or Q29463. A protease is considered a functional variant if it has at least 50%, e.g., at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity of the protease under optimal conditions for the enzyme.In some embodiments, enzymatic treatment with pepsin or a pepsin-like enzyme may include: P03954 / PEPA1_MACFU; P28712 / PEPA1_RABIT; P27677 / PEPA2_MACFU; P27821 / PEPA2_RABIT; P0DJD8 / PEPA3_HUMAN; P27822 / PEPA3_RABIT; P0DJD7 / PEPA4_HUMAN; P27678 / PEPA4_MACFU; P28713 / PEPA4_RABIT; P0DJD9 / PEPA5_HUMAN; Q9D106 / PEPA5_MOUSE;P27823 / PEPAF_RABIT;P00792 / PEPA_BOVIN;Q9N2D4 / PEPA_CALJA;Q9GMY6 / PEPA_CANLF;P00793 / PEPA_CHICK;P11489 / PEPA_MACMU;P00791 / PEPA_PIG;Q9GMY7 / PEPA_RHIFE;Q9GMY8 / PEPA_SORUN;P81497 / PEPA_SUNMU;P13636 / PEPA_URSTH and functional variants and derivatives thereof, or combinations thereof. In some embodiments, pepsin may include: P00791 / PEPA_PIG;P00792 / PEPA_BOVIN, functional variants, derivatives or combinations thereof.

[0372] In addition, the protease may be included in a reaction mixture (solution) that also includes other components (e.g., buffers, salts, chelating agents (e.g., EDTA) and / or detergents (e.g., SDS, N-lauroylsarcosine sodium salt solution). The reaction mixture may be buffered and have a pH of about 6.5-8.5, for example, about 7.0-8.0. In addition, the reaction mixture may be used at any suitable temperature, for example, about 10 to 50°C, for example, about 10 to 44°C, 11 to 43°C, 12 to 42°C, 13 to 41°C, 14 to 40°C, 15 to 39°C, 16 to 38°C, 17 to 37°C, for example, about 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 28°C, 30°C, 33°C, 35°C or 37°C, preferably about 35 to 45°C, for example, about 37°C.

[0373] Other reagents

[0374] In some embodiments, the permeabilization solution can include other reagents or can be used to treat the biological sample with other reagents to optimize the permeabilization of the biological sample. In some embodiments, other reagents are RNA protective agents. As used herein, the term "RNA protective agent" generally refers to a reagent that protects RNA from RNA nucleases (e.g., RNase). Any suitable RNA protective agent that protects RNA from degradation can be used. The non-limiting example of an RNA protective agent includes an organic solvent (e.g., at least 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% v / v organic solvent), including but not limited to ethanol, methanol, propan-2-ol, acetone, trichloroacetic acid, propanol, polyethylene glycol, acetic acid or a combination thereof. In some embodiments, the RNA protective agent includes ethanol, methanol and / or propan-2-ol, or a combination thereof. In some embodiments, the RNA protective agent includes RNAlater ICE (Thermo Fisher Scientific). In some embodiments, the RNA protective agent includes at least about 60% ethanol. In some embodiments, the RNA protectant comprises about 60-95% ethanol, about 0-35% methanol, and about 0-35% propan-2-ol, wherein the total amount of organic solvents in the culture medium does not exceed about 95%. In some embodiments, the RNA protectant comprises about 60-95% ethanol, about 5-20% methanol, and about 5-20% propan-2-ol, wherein the total amount of organic solvents in the culture medium does not exceed about 95%.

[0375] In some embodiments, RNA protective agent includes salt. The salt may include ammonium sulfate, ammonium bisulfate, ammonium chloride, ammonium acetate, cesium sulfate, cadmium sulfate, cesium iron sulfate (II), chromium sulfate (III), cobalt sulfate (II), copper sulfate (II), 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 and zinc sulfate. In some embodiments, salt is sulfate, such as ammonium sulfate, ammonium bisulfate, cesium sulfate, cadmium sulfate, cesium iron sulfate (II), chromium sulfate (III), cobalt sulfate (II), copper sulfate (II), lithium sulfate, magnesium sulfate, manganese sulfate, potassium sulfate, sodium sulfate, or zinc sulfate. In some embodiments, 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, such as about 4 g, 3 g, 2 g or 1 g / 100 mL.

[0376] Additionally, the RNA protectant can be included in a culture medium that also contains a chelating agent (e.g., EDTA), a buffer (e.g., sodium citrate, sodium acetate, potassium citrate, or potassium acetate, preferably sodium acetate), and / or is buffered to a pH between about 4-8 (e.g., about 5).

[0377] In some embodiments, before, simultaneously or after permeabilization, one or more RNA protective agents are used to treat the biological sample. For example, before being treated with one or more permeabilization reagents (e.g., one or more proteases), the biological sample is treated with one or more RNA protective agents. In another example, the biological sample is treated with a solution comprising one or more RNA protective agents and one or more permeabilization reagents (e.g., one or more proteases). In another example, after the biological sample has been treated with one or more permeabilization reagents (e.g., one or more proteases), the biological sample is treated with one or more RNA protective agents. In some embodiments, the biological sample is treated with one or more RNA protective agents before fixation.

[0378] In some embodiments, the position of the analyte captured in the identification biological sample includes a nucleic acid extension reaction. In some embodiments in which the capture probe captures fragmented genomic DNA molecules, the nucleic acid extension reaction includes a DNA polymerase. For example, the nucleic acid extension reaction includes using the captured analyte (e.g., fragmented genomic DNA) as a template, utilizing a DNA polymerase to extend the capture probe hybridized with the captured analyte (e.g., fragmented genomic DNA). The product of the extension reaction includes a spatial barcoded analyte (e.g., spatially barcoded fragmented genomic DNA). Spatial barcoded analytes (e.g., spatially barcoded fragmented genomic DNA) can be used to identify the spatial position of the analyte in the biological sample. Any DNA polymerase that can use the captured analyte as a template to extend the capture probe can be used for the method described herein. Non-limiting examples of DNA polymerases include T7 DNA polymerase; Bsu DNA polymerase; and Escherichia coli DNA polymerase pol I.

[0379] Diffusion-resistant media

[0380] In some embodiments, the anti-diffusion medium typically used to limit the diffusion of analytes may include at least one permeabilization agent. For example, the anti-diffusion medium (e.g., a hydrogel) may include pores (e.g., micropores, nanopores, or picopores or pores) containing a permeabilization buffer or reagent. In some embodiments, the anti-diffusion medium (e.g., a hydrogel) is soaked in a permeabilization buffer before contacting the hydrogel with the sample. In some embodiments, when the anti-diffusion medium is applied to a biological sample, the hydrogel or other anti-diffusion medium may include a dry reagent or monomer to deliver the permeabilization agent. In some embodiments, the anti-diffusion medium (e.g., a hydrogel) is covalently attached to a solid substrate (e.g., an acrylic glass slide).

[0381] In some embodiments, the hydrogel can be modified to deliver permeabilization reagents and include captured objects. For example, the hydrogel film can be modified to include spatial barcoding capture probes. Then, before the spatial barcoding hydrogel film is contacted with the sample, the spatial barcoding hydrogel film is immersed in a permeabilization buffer. In another example, the hydrogel can be modified to include spatial barcoding capture probes and is designed to be used as a porous membrane (for example, permeable hydrogel) when exposed to permeabilization buffer or any other biological sample preparation reagent. The permeabilization reagent diffuses through the spatial barcoding permeable hydrogel and permeates the biological sample on the other side of the hydrogel. After being exposed to the permeabilization reagent, the analyte is then diffused into the spatial barcoding hydrogel. In this case, the spatial barcoding hydrogel (for example, porous membrane) promotes that the biological analyte in the biological sample diffuses into the hydrogel. In some embodiments, the biological analyte first diffuses into the hydrogel and is then exposed to the permeabilization reagent (for example, when the secreted analyte is present outside the biological sample, or when the biological sample is dissolved or permeated by other means before the permeabilization reagent is added). In some embodiments, the permeabilization reagent flows through the hydrogel with a variable flow rate (for example, any flow rate that promotes the diffusion of the permeabilization reagent through the spatial barcoded hydrogel). In some embodiments, the permeabilization reagent flows through the microfluidic chamber or channel on the spatial barcoded hydrogel. In some embodiments, after the permeabilization reagent is introduced into the biological sample by flow, the biological sample preparation reagent can be made to flow through the hydrogel to further promote the diffusion of biological analytes into the spatial barcoded hydrogel. Therefore, the spatial barcoded hydrogel membrane transports the permeabilization reagent to the sample surface in contact with the spatial barcoded hydrogel, enhancing analyte migration and capture. In some embodiments, the spatial barcoded hydrogel is applied to the sample and placed in a large amount of permeabilization solution. In some embodiments, the hydrogel membrane soaked in the permeabilization reagent is sandwiched between the sample and the spatial barcoded array. In some embodiments, the target analyte can diffuse through the hydrogel soaked with the permeabilization reagent and hybridize or bind to the capture probe on the other side of the hydrogel. In some embodiments, the thickness of the hydrogel is proportional to the resolution loss. In some embodiments, the hole (e.g., micropore, nanopore or picometer hole) may include a spatial barcoding capture probe and a permeabilization reagent and / or a buffer. In some embodiments, the spatial barcoding capture probe and the permeabilization reagent are maintained between the spacers. In some embodiments, the sample is punched, cut or transferred into the hole, wherein the target analyte diffuses to the spatial barcoding capture probe by the permeabilization reagent / buffer. In some embodiments, the resolution loss may be proportional to the gap thickness (e.g., the amount of permeabilization buffer between the sample and the capture probe). In some embodiments, the thickness of the anti-diffusion medium (e.g., hydrogel) is between about 50-500 microns, including a thickness of 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 microns, or any thickness between 50 and 500 microns.

[0382] In some embodiments, the biological sample is exposed to a porous membrane (e.g., a permeable hydrogel) to help permeabilize and limit the loss of diffusion analytes while allowing the permeabilizing agent to reach the sample. The membrane chemistry and pore volume can be controlled to minimize analyte loss. In some embodiments, the porous membrane can be made of glass, silicon, paper, hydrogel, polymer monolith or other materials. In some embodiments, the material can be naturally porous. In some embodiments, the material can have holes or holes etched into the solid material. In some embodiments, the permeabilizing agent flows through the microfluidic chamber or channel on the porous membrane. In some embodiments, the flow controls the accessibility of the sample to the permeabilizing agent. In some embodiments, the porous membrane is a permeable hydrogel. For example, when the permeabilizing agent and / or biological sample preparation reagent can utilize diffusion to pass through the hydrogel, the hydrogel is permeable. Any suitable permeabilizing agent and / or biological sample preparation reagent described herein can be used under conditions sufficient to release analytes (e.g., nucleic acids, proteins, metabolites, lipids, etc.) from the biological sample. In some embodiments, the hydrogel is exposed to the biological sample on one side and to the permeabilizing agent on the other side. The permeabilization agent diffuses through the permeable hydrogel and permeabilizes the biological sample on the other side of the hydrogel. In some embodiments, the permeabilization agent is flowed through the hydrogel with a variable flow rate (e.g., any flow rate that promotes the diffusion of the permeabilization agent through the hydrogel). In some embodiments, the permeabilization agent flows through the microfluidic chamber or channel above the hydrogel. The operation of flowing the permeabilization agent through the hydrogel allows the concentration of the agent to be controlled. In some embodiments, the hydrogel chemistry and pore volume can be adjusted to enhance permeabilization and limit the loss of diffused analytes.

[0383] In some embodiments, a porous membrane is sandwiched between a spatial barcoding array and a sample, wherein a permeabilization solution is applied to the porous membrane. Permeabilization reagent diffuses into the biological sample through the membrane pores. In some embodiments, the biological sample can be placed on a substrate (e.g., a glass slide). The biological analyte then diffuses through the porous membrane and enters the space containing the capture probe. In some embodiments, the porous membrane is modified to include a capture probe. For example, the capture probe can be connected to the surface of the porous membrane using any method described herein. In another example, the capture probe 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 on the biological sample in a structure that allows the capture probe on the porous membrane to interact with the biological analyte from the biological sample. For example, the capture probe is located on one side of the porous membrane 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 position containing the biological sample and the capture probe to promote the permeabilization of the biological sample (e.g., also promoting the capture probe to capture the biological analyte). In some embodiments, the porous membrane is located between the sample and the capture probe. In some embodiments, the permeabilization reagent is flowed through a microfluidic chamber or channel on a porous membrane.

[0384] Selective permeabilization / selective lysis

[0385] In some embodiments, a biological sample can be processed according to established methods to selectively release an analyte from a subcellular region of a cell. In some embodiments, the methods provided herein can include detecting at least one biological analyte present in a subcellular region of a cell in a biological sample. As used herein, "subcellular region" can refer to any subcellular region. For example, a subcellular region can refer to the cytoplasm, mitochondria, nucleus, nucleolus, endoplasmic reticulum, lysosome, vesicle, Golgi apparatus, plastid, vacuole, ribosome, cytoskeleton, or a combination thereof. In some embodiments, the subcellular region comprises at least one of the 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 the mitochondria. In some embodiments, the subcellular region is the microsome.

[0386] For example, a biological analyte can be selectively released from a subcellular region of a cell by selective permeabilization or selective lysis. In some embodiments, "selective permeabilization" can refer to a permeabilization method that can permeabilize the membrane of a subcellular region while keeping a different subcellular region substantially intact (e.g., the biological analyte is not released from the subcellular region due to the permeabilization method applied). Non-limiting examples of selective permeabilization methods include the use of electrophoresis and / or the application of a permeabilizing agent. In some embodiments, "selective lysis" can refer to a lysis method that can lyse the membrane of a subcellular region while keeping a different subcellular region substantially intact (e.g., the biological analyte is not released from the subcellular region due to the lysis method applied). Several methods for selective permeabilization or lysis are known to those skilled in the art, including those described in: Lu et al. Lab Chip. 2005 Jan;5(1):23-9; Niklas et al. Anal Biochem. 2011 Sep 15;416(2):218-27; Cox and Emili. Nat Protoc. 2006;1(4):1872-8; Chiang et al. J Biochem. Biophys. Methods. 2000 Nov 20;46(1-2):53-68; and Yamauchi and Herr et al. Microsyst. Nanoeng. 2017;3.pii:16079; each of which is incorporated herein by reference in its entirety.

[0387] In some embodiments, "selective permeabilization" or "selective lysis" refers to the selective permeabilization or selective lysis of a particular cell type. For example, "selective permeabilization" or "selective lysis" can refer to the lysis of one cell type while keeping a different cell type substantially intact (e.g., a biological analyte is not released from the cell due to the permeabilization or lysis method used). A cell that is "a different cell type" from another cell can refer to cells from different classification circles, prokaryotic cells versus eukaryotic cells, cells from different tissue types, etc. Many methods for selectively permeabilizing or lysing different cell types are known to those skilled in the art. Non-limiting examples include the use of permeabilizing agents, electroporation, and / or ultrasonic treatment. See, e.g., International Application No. WO 2012 / 168003; Han et al., Microsyst Nanoeng. 2019 Jun 17;5:30; Gould et al. Oncotarget. 2018 Mar 20;9(21):15606-15615; Oren and Shai. Biochemistry. 1997 Feb 18;36(7):1826-35; Algayer et al. Molecules. 2019 May 31;24(11).pii:E2079; Hipp et al. Leukemia. 2017 Oct;31(10):2278; International Application No. WO 2012 / 168003; and, U.S. Patent No. 7,785,869; all of which are incorporated herein by reference in their entireties.

[0388] In some embodiments, applying a selective permeabilizing or lysing agent comprises contacting the biological sample with a hydrogel comprising the permeabilizing or lysing agent.

[0389] In some embodiments, a biological sample is contacted with two or more arrays (e.g., flexible arrays as described herein). For example, after a subcellular region is permeabilized and biological analytes from the subcellular region are captured by a first array, the first array can be removed and biological analytes from a different subcellular region can be captured on a second array.

[0390] (14) Selective enrichment of RNA substances

[0391] In some embodiments where RNA is the analyte, one or more RNA analyte species of interest can be selectively enriched (e.g., Adiconis et al., Comparative analysis of RNA sequencing methods for degraded and low-input samples, Nature, Vol. 10, July 2013, pp. 623-632, incorporated herein 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 other oligonucleotides are sequences used to initiate a reaction by a polymerase. For example, one or more primer sequences having sequence complementarity to one or more RNAs of interest can be used to amplify the one or more RNAs of interest, thereby selectively enriching these RNAs. In some embodiments, oligonucleotides having sequence complementarity to the complementary strand of the captured RNA (e.g., cDNA) can bind to the cDNA. For example, biotinylated oligonucleotides having a sequence complementary to one or more cDNAs of interest can bind to the cDNA and can be selected using biotinylation-streptavidin affinity using any of a variety of methods known in the art (e.g., streptavidin beads).

[0392] Alternatively, any of a variety of methods can be used to down-select (e.g., remove, deplete) one or more RNAs (e.g., ribosomal and / or mitochondrial RNAs). Non-limiting examples of hybridization and capture methods for ribosomal RNA depletion include RiboMinus TM 、RiboCop TM and Ribo-Zero TM Another non-limiting RNA depletion method involves hybridizing complementary DNA oligonucleotides to the unwanted RNA and then degrading the RNA / DNA hybrid using RNase H. Non-limiting examples of hybridization and degradation methods include rRNA depletion, NuGEN AnyDeplete, TruSeq TM Another non-restrictive ribosomal RNA depletion method includes ZapR TMDigestion, such as SMARTer. In the SMARTer method, random nucleic acid conjugates and RNA hybridization are used for carrying out first chain synthesis and tailing by reverse transcriptase, and then template switching and extension are carried out by reverse transcriptase. In addition, the first round of PCR amplification adds full-length Illumina sequencing conjugates (sequencing adapter) (for example, Illumina index). Ribosomal RNA is cracked by ZapRv2 and R probe v2. Carry out second round PCR, amplify non-rRNA molecules (for example, cDNA). The part or step of these ribosome depletion schemes / test kits can be further combined with the method described herein to optimize the scheme for specific biological samples.

[0393] In the depletion scheme, the probe can be given to a sample that selectively hybridizes with ribosomal RNA (rRNA), thereby reducing the pool and concentration of rRNA in the sample. The probe can be applied to a biological sample that selectively hybridizes with mitochondrial RNA (mtRNA), thereby reducing the pool and concentration of mtRNA in the sample. In some embodiments, a probe complementary to mitochondrial RNA can be added during cDNA synthesis, or a probe complementary to both ribosomal and mitochondrial RNA can be added during cDNA synthesis. Subsequently, the capture probe is applied to the sample and may result in an improvement in the capture of other types of RNA due to the reduction of non-specific RNA (e.g., downwardly selected RNA) present in the sample. Alternatively, double-stranded specific nuclease (DSN) treatment can remove rRNA (see, e.g., Archer et al., Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage at the cDNA stage, BMC Genomics, 15401, (2014), the entire contents of which are incorporated herein by reference). Additionally, hydroxyapatite chromatography can remove abundant species (e.g., rRNA) (see, e.g., Vandernoot, VA, cDNA normalization by hydroxyapatite chromatography to enrich transcriptome diversity in RNA-seq applications, Biotechniques, 53(6)373-80, (2012), the entire contents of which are incorporated herein by reference).

[0394] (15) Other reagents

[0395] Before analyzing the sample, other reagents can be added to the biological sample to perform various functions. In some embodiments, nuclease inhibitors such as DNA enzyme and RNA enzyme inactivators or proteinase inhibitors and / or chelating agents such as EDTA can be added to the biological sample. In other embodiments, nucleases such as DNA enzyme or RNA enzyme, or proteases such as pepsin or proteinase K are added to the sample. In some embodiments, other reagents can be dissolved in solution or applied to the sample as a medium. In some embodiments, other reagents (such as pepsin) can be dissolved in HCl before being applied to the sample.

[0396] In some embodiments, the biological sample can be treated with one or more enzymes. For example, one or more endonucleases for fragmenting DNA, DNA polymerases, and dNTPs for amplifying nucleic acids can be added. Other enzymes that can also be added to the biological sample include, but are not limited to, polymerases, transposases, ligases, DNA enzymes, and RNA enzymes.

[0397] In some embodiments, a reverse transcriptase can be added to a sample, comprising an enzyme with terminal transferase activity, primers, and a template switching oligonucleotide (TSO). Template switching can be used to increase the length of a cDNA, for example, by appending a predefined nucleic acid sequence to the cDNA. In some embodiments, the appended nucleic acid sequence comprises one or more ribonucleotides.

[0398] In some embodiments, other reagents can be added to improve the recovery rate of one or more target molecules (e.g., 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 the input or target molecule is low compared to the remaining molecules. Typically, a single target molecule cannot form a precipitate, and adding a carrier molecule helps to form a precipitate. Some target molecule recovery schemes use carrier RNA to prevent the small amount of target nucleic acid present in the sample from irreversibly binding. In some embodiments, carrier RNA can be added immediately before the second chain synthesis step. In some embodiments, carrier RNA can be added immediately before the synthesis of the second chain cDNA on the oligonucleotides released from the array. In some embodiments, carrier RNA can be added immediately before the in vitro transcription cleanup step. In some embodiments, carrier RNA can be added before the amplified RNA is purified and quantified. In some embodiments, carrier RNA can be added before RNA quantification. In some embodiments, carrier RNA can be added immediately before the second chain cDNA synthesis and in vitro transcription cleanup step.

[0399] (16) Pretreatment for capture probe interaction

[0400] In some embodiments, the analyte in the biological sample can be pre-treated before interacting with the capture probe. For example, before interacting with the capture probe, a polymerization reaction catalyzed by a polymerase (such as DNA polymerase or reverse transcriptase) is carried out in the biological sample. In some embodiments, the primer for polymerization reaction includes a functional group that enhances hybridization with the capture probe. The capture probe can include an appropriate capture domain to capture the biological analyte of interest (for example, a poly (dT) sequence to capture poly (A) mRNA).

[0401] In some embodiments, the biological analyte is pre-processed to generate a library by next generation sequencing. For example, the analyte can be pre-processed by adding a modifier (e.g., attaching a sequence that interacts with the capture probe). In some embodiments, the analyte (e.g., DNA or RNA) is fragmented using a fragmentation technique (e.g., using a transposase and / or a fragmentation buffer).

[0402] After fragmentation, the analyte can be modified. For example, the modification can be added by connecting an adapter sequence that allows hybridization with the capture probe. In some embodiments, where the analyte of interest is RNA, poly (A) tailing is performed. Adding a poly (A) tail to an RNA that does not contain a poly (A) tail can promote hybridization with a capture probe that contains a capture domain with a functional amount of a poly (dT) sequence.

[0403] In some embodiments, before interacting with the capture probe, a ligation reaction catalyzed by a ligase is carried out in a biological sample. In some embodiments, connection can be carried out by chemical connection. In some embodiments, click chemistry can be used to connect, as described below. In some embodiments, the capture domain includes a DNA sequence with complementarity to an RNA molecule, wherein the RNA molecule has complementarity with the second DNA sequence, and wherein the RNA-DNA sequence complementarity is used to connect the second DNA sequence to the DNA sequence in the capture domain. In these embodiments, the RNA molecule can be directly detected.

[0404] In some embodiments, a target-specific reaction is performed in a biological sample prior to interacting with a capture probe. Examples of target-specific reactions include, but are not limited to, connection of target-specific adapters, probes, and / or other oligonucleotides, target-specific amplification using one or more analyte-specific primers, and target-specific detection using in situ hybridization, DNA microscopy, and / or antibody detection. In some embodiments, a capture probe includes a capture domain (e.g., amplification or connection) that is targeted to a target-specific product.

[0405] II. General spatial array-based analysis methods

[0406] Provided herein are methods, devices, systems, and compositions for spatial array-based analysis of biological samples.

[0407] (a) Spatial analysis methods

[0408] Array-based spatial analysis methods involve transferring one or more analytes from a biological sample to an array of features on a substrate, where each feature is associated with a unique spatial location on the array. Subsequent analysis of the transferred analytes involves determining the identity of the analytes and the spatial location of each analyte in the biological sample. The spatial location of each analyte in the biological sample is determined based on the features bound to each analyte in the array and the relative spatial locations of the features on the array.

[0409] There are at least two general approaches to associating a spatial barcode with one or more adjacent cells such that the spatial barcode identifies the one or more cells and / or the contents of the one or more cells as being associated with a particular spatial location. One general approach is to facilitate the analyte out of the cell and toward a spatially barcoded array. Figure 1 An exemplary embodiment of this general method is depicted. Figure 1 In the present invention, a spatially barcoded array having a collection of capture probes (as further described herein) is contacted with a biological sample 101, and the biological sample is permeabilized to allow analytes to migrate from the sample to the array. The analytes interact with the capture probes on the spatially barcoded array 102. Once the analytes hybridize / bind to the capture probes, the sample is optionally removed from the array and the capture probes are analyzed to obtain spatially resolved analyte information 103.

[0410] Another general approach is to cleave the spatially barcoded capture probes from the array and facilitate the spatially barcoded capture probes toward and / or into or onto the biological sample. Figure 2 An exemplary embodiment of the general method is depicted, whereby a spatially barcoded array aggregated with capture probes (as further described herein) can be contacted with a sample 201. The spatially barcoded capture probes are cleaved and then interact with cells within a provided biological sample 202. This interaction can be a covalent or non-covalent cell surface interaction. The interaction can be an intracellular interaction promoted by a delivery system or a cell penetrating peptide. Once the spatially barcoded capture probes are associated with specific cells, the sample can be optionally removed for analysis. The sample can be selectively separated before analysis. Once the labeled cells are associated with the spatially barcoded capture probes, the capture probes can be analyzed to obtain spatially resolved information about the labeled cells 203.

[0411] Figure 3Shown is an exemplary workflow for preparing a biological sample on a spatial barcoding array 301. Sample preparation can include placing the sample on a slide, fixing the sample and / or staining the biological sample for imaging. Bright field (for imaging of sample hematoxylin and eosin staining) and fluorescence (for imaging of features) modes can then be used to image the stained sample on array 302. Alternatively, the sample can be decolorized before permeabilization. In some embodiments, the analyte is then released from the sample, and the capture probes forming the spatial barcoding array are hybridized or combined with the released analyte 303. The sample is then removed from the array 304, and the capture probes are cracked from the array 305. The biological sample and array are then optionally imaged for a second time in two modes 305B, while the analyte is reversely transcribed into cDNA, and an amplicon library 306 is prepared and sequenced 307. The two sets of images are then spatially overlapped to associate the biological sample information 308 identified in space. When the sample and array do not have a second imaging 305B, a point coordinate file is provided instead. The spot coordinate file replaces the second imaging step 305B. Additionally, amplicon library preparation 306 can be performed using unique PCR adapters and sequencing 307.

[0412] Figure 4 Another exemplary workflow is shown, which utilizes a spatial barcoding array on a substrate, wherein spatial barcoding capture probes are gathered in areas called features. The spatial barcoding capture probe can include a cleavage domain, one or more functional domains, a spatial barcode, a unique molecular identifier, and a capture domain. The spatial barcoding capture probe can also include a 5' end modification for reversibly attaching to the substrate. The spatial barcoding array is contacted with a biological sample 401, and the sample is permeabilized by applying a permeabilization reagent 402. The permeabilization reagent can be given by placing the array / sample assembly in a large amount of solution. Alternatively, the permeabilization reagent can be given to the sample via an anti-diffusion medium and / or a physical barrier (e.g., a cover), wherein the sample is sandwiched between the anti-diffusion medium and / or barrier and the substrate comprising the array. Analytes are migrated to the spatial barcoding capture array using any number of techniques disclosed herein. For example, analyte migration can be performed using an anti-diffusion medium cover and passive migration. As another example, for example, using an electrophoretic transfer system, analyte migration can be active migration. Once the analyte approaches the spatial barcoding capture probe, the capture probe can hybridize or otherwise bind to the target analyte 403. The biological sample can optionally be removed from the array 404 .

[0413] The capture probe can be optionally cleaved from the array 405, and the captured analyte can be spatially barcoded by performing a reverse transcriptase first-chain cDNA reaction. The first-chain cDNA reaction can optionally be performed using a template conversion oligonucleotide. For example, the template conversion oligonucleotide can hybridize with a poly (C) tail added to the 3′ end of the cDNA by reverse transcriptase in a template-independent manner. The original mRNA template and template conversion oligonucleotide can be denatured from the cDNA, and then the spatial barcoded capture probe can hybridize with the cDNA and can generate a complement of the cDNA. The first-chain cDNA can then be purified and collected for downstream amplification steps. The first-chain cDNA can be amplified using PCR 406, in which the forward and reverse primers flank the spatial barcode and analyte regions of interest to produce a library associated with a specific spatial barcode. In some embodiments, the cDNA comprises a synthetic sequencing (SBS) primer sequence. The library amplicons are sequenced and analyzed to decode spatial information 407.

[0414] Using the methods, compositions, systems, kits and devices described herein, RNA transcripts present in biological samples (e.g., tissue samples) can be used for spatial transcriptome analysis. In particular, in some cases, barcoded oligonucleotides can be configured to initiate, replicate and thereby produce barcoded extension products from an RNA template or a derivative thereof. For example, in some cases, the barcoded oligonucleotides can include an mRNA-specific priming sequence, such as a poly-T primer segment or other targeted priming sequence that allows priming and replication of mRNA in a reverse transcription reaction. Alternatively or in addition, random RNA priming can be performed using a random N-polymer (N-mer) primer segment of the barcoded oligonucleotide. Reverse transcriptase (RT) can use an RNA template and a primer complementary to the 3′ end of the RNA template to guide the synthesis of a first-strand complementary DNA (cDNA). Many RTs can be used 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 thereof. Some recombinant M-MuLV reverse transcriptases, such as II reverse transcriptases, compared to their wild-type counterparts, may have reduced RNase H activity and increased thermostability, and provide higher specificity, higher cDNA yields, and more complete cDNA products up to 12 kilobases (kb) in length. In some embodiments, the reverse transcriptase is a mutant reverse transcriptase, such as, but not limited to, a mutant MMLV reverse transcriptase. In another embodiment, the reverse transcriptase is a mutant MMLV reverse transcriptase, such as, but not limited to, one or more variants described in U.S. Patent Publication No. 20180312822, which is incorporated herein by reference in its entirety.

[0415] Figure 5An exemplary workflow is described in which a biological sample is removed from a spatial barcoded array and spatial barcoded capture probes are removed from the array for barcoded analyte amplification and library preparation. Another embodiment includes using template switching oligonucleotides to perform first-strand synthesis on a spatial barcoded array without cutting the capture probes. In this embodiment, sample preparation 501 and permeabilization 502 are performed as described elsewhere herein. Once the capture probe captures the analyte, the first-strand cDNA produced by template switching and reverse transcriptase 503 is subsequently denatured, and the second strand is subsequently extended 504. The second-strand cDNA is then denatured, neutralized, and transferred to a tube 505 from the first-strand cDNA. cDNA quantification and amplification can be performed using standard techniques discussed herein. The cDNA can then undergo library preparation 506 and indexing 507, including fragmentation, end repair, A-tailing, and indexing PCR steps.

[0416] In the non-limiting example of the above workflow, biological samples (e.g., tissue sections) can be fixed with methanol, stained with hematoxylin and eosin, and imaged. Alternatively, the sample can be decolorized before permeabilization. These images can be used to map spatial gene expression patterns back to the biological sample. Permeabilization enzymes can be used to permeabilize biological samples directly on a glass slide. Analytes (e.g., polyadenylated mRNA) released from the overlying cells of the biological sample can be captured by capture probes in the capture area on the substrate. Reverse transcription (RT) reagents can be added to the permeabilized biological sample. Incubation with the RT reagent can produce spatially barcoded full-length cDNA from the captured analyte (e.g., polyadenylated mRNA). Second chain reagents (e.g., second chain primers, enzymes) can be added to the biological sample on the glass slide to initiate second chain synthesis. The resulting cDNA can be denatured and transferred (e.g., to a clean tube) for amplification and / or library construction from the capture probe template. The full-length cDNA of spatial barcoding can be amplified by PCR before library construction. The cDNA can then be enzymatically fragmented and size-selected to optimize the size of the cDNA amplicon. P5, P7, i7, and i5 can serve as sample indices, and TruSeq Read 2 can be added through methods such as end repair, A-tailing, adapter extension, and PCR. The cDNA fragments can then be sequenced using paired-end sequencing, using TruSeq Read 1 and TruSeq Read 2 as sequencing primer sites.

[0417] In some embodiments, correlation analysis of data generated by this workflow and other workflows described herein can produce a 95% or greater correlation (e.g., 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater) of genes expressed across two capture regions. When the workflow is performed using single-cell RNA sequencing of nuclei, in some embodiments, correlation analysis of the data can produce a correlation of greater than 90% (e.g., greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of genes expressed across two capture regions.

[0418] In some embodiments, after cDNA is produced (e.g., by reverse transcription), the cDNA can be amplified directly on the substrate surface. Multiple copies of the cDNA (e.g., synthesized from captured analytes) generated by amplification directly on the substrate surface can increase the complexity of the final sequencing library. Therefore, in some embodiments, 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.

[0419] In some embodiments, isothermal amplification can be faster than standard PCR reaction. In some embodiments, isothermal amplification can be linear amplification (for example, asymmetric with a single primer) or exponential amplification (for example, with two primers). In some embodiments, isothermal nucleic acid amplification can be carried out by template switching oligonucleotide primers. In some embodiments, template switching oligonucleotide adds a common sequence to the 5 ' end of the RNA being reverse transcribed. For example, after the capture probe interacts with the analyte (for example, mRNA) and performs reverse transcription, other nucleotides are added to the end of the cDNA to produce 3 ' overhangs as described herein. In some embodiments, the non-templated poly (C) nucleotide hybridization that template switching oligonucleotide adds to reverse transcriptase continues to replicate to the 5 ' end of the template switching oligonucleotide, thereby generating a full-length cDNA prepared for further amplification. In some embodiments, template switching oligonucleotide adds a common 5 ' sequence (for example, the reverse complement of template switching oligonucleotide) to the full-length cDNA for cDNA amplification.

[0420] In some embodiments, once a full-length cDNA molecule is generated, the template-switching oligonucleotide can be used as a primer in a cDNA amplification reaction (e.g., using a DNA polymerase). In some embodiments, a double-stranded cDNA (e.g., a first-strand cDNA and a second-strand reverse-complement cDNA) can be isothermally amplified using a helicase or recombinase followed by a strand-displacing DNA polymerase. The strand-displacing DNA polymerase can produce a displaced second strand, thereby producing an amplified product.

[0421] In any isothermal amplification method described herein, barcode exchange (e.g., spatial barcode) can occur after the first amplification cycle, wherein unused capture probes are present on the substrate surface. In some embodiments, the free 3'OH end of the unused capture probe can be blocked by any suitable 3'OH blocking method. In some embodiments, the 3'OH can be blocked by hairpin ligation.

[0422] Isothermal nucleic acid amplification can be used as a supplement or alternative to standard PCR reactions (e.g., PCR reactions that require heating to about 95°C to denature double-stranded DNA). Isothermal nucleic acid amplification generally does not require the use of a thermal cycler, however, in some embodiments, isothermal amplification can be performed in a thermal cycler. In some embodiments, isothermal amplification can be performed at about 35°C to 75°C. In some embodiments, isothermal amplification can 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 any temperature therebetween, depending on the polymerase and auxiliary enzymes used.

[0423] 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, loop-mediated DNA isothermal amplification (LAMP), isothermal multiple displacement amplification, recombinase polymerase amplification, helicase-dependent amplification, single primer isothermal amplification, and circular helicase-dependent amplification (see, e.g., Gill and Ghaemi, Nucleic acid isothermal amplification technologies: a review, Nucleosides, Nucleotides, & Nucleic Acids, 27(3), 224-43, doi: 10.1080 / 15257770701845204 (2008), which is incorporated herein by reference in its entirety.

[0424] In some embodiments, isothermal nucleic acid amplification is helicase-dependent nucleic acid amplification. Helicase-dependent isothermal nucleic acid amplification is described in Vincent et al., Helicase-dependent isothermal DNA amplification, EMBO Rep., 795-800 (2004) and U.S. Patent No. 7,282,328, both of which are incorporated herein by reference in their entirety. Helicase-dependent nucleic acid amplification on a substrate (e.g., on a chip) is described in Andresen et al., Helicase-dependent amplification: use in OnChip amplification and potential for 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, isothermal nucleic acid amplification is recombinase polymerase nucleic acid amplification. Recombinase polymerase nucleic acid amplification is described in Piepenburg et al., DNA Detection Using Recombinant Proteins, PLoS Biol., 4, 7e204 (2006) and Li et al., Review: a comprehensive summary of adecade development of the recombinase polymerase amplification, Analyst, 144, 31-67, doi: 10.1039 / C8AN01621F (2019), both of which are incorporated herein by reference in their entirety.

[0425] Typically, isothermal amplification techniques use standard PCR reagents known in the art (e.g., buffer, dNTPs, etc.). Some isothermal amplification techniques may require other reagents. For example, helicase-dependent nucleic acid amplification uses single-stranded binding proteins and auxiliary proteins. In another example, recombinase polymerase nucleic acid amplification uses recombinase (e.g., T4UvsX), recombinase loading factor (e.g., TF UvsY), single-stranded binding protein (e.g., T4gp32), crowding agent (e.g., PEG-35K) and ATP.

[0426] After isothermal nucleic acid amplification of the full-length cDNA described by any of the methods herein, the isothermal amplified cDNA (e.g., single-stranded or double-stranded) can be recovered from the substrate and optionally subsequently amplified using typical cDNA PCR in a microcentrifuge tube. The sample can then be used together with any of the spatial methods described herein.

[0427] (i) Immunohistochemistry and immunofluorescence

[0428] In some embodiments, immunofluorescence or immunohistochemistry protocols (direct and indirect staining techniques) can be performed as part or additional parts of the exemplary spatial workflow presented herein. For example, tissue sections can be fixed according to the methods described herein. Biological samples can be transferred to arrays (for example, capture probe arrays), wherein analytes (for example, proteins) are detected using immunofluorescence protocols. For example, samples can be rehydrated, sealed and permeabilized (3XSSC, 2% BSA, 0.1% Triton X, 1U / μl RNAse inhibitors, 4°C for 10 minutes), followed by staining with a fluorescent primary antibody (1: 100 in 3XSSC, 2% BSA, 0.1% Triton X, 1U / μl RNAse inhibitors, 4°C for 30 minutes). Biological samples can be washed, covered with a glass slide (in glycerol+1U / μl RNAse inhibitor), imaged (for example, using a confocal microscope or other device capable of fluorescence detection), washed and processed (according to analyte capture or spatial workflow as described herein).

[0429] As used herein, "antigen retrieval buffer" can improve antibody capture in IF / IHC protocols. An exemplary protocol for antigen retrieval can be preheating the antigen retrieval buffer (e.g., 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 washing the biological sample.

[0430] In some embodiments, optimizing permeabilization may be useful for identifying intracellular analytes. Permeabilization optimization can include selecting a permeabilizing agent, the concentration of the permeabilizing agent, and the duration of permeabilization. Tissue permeabilization is discussed elsewhere herein.

[0431] In some embodiments, blocking arrays and / or biological samples during preparation of labeling biological samples can reduce the non-specific binding of antibodies to arrays and / or biological samples (reduce background). Some embodiments provide blocking buffer / blocking solution that can be applied before and / or during the application of a marker, wherein the blocking buffer can include a blocking agent and an optional surfactant and / or saline solution. In some embodiments, the blocking agent can be bovine serum albumin (BSA), serum, gelatin (e.g., fish gelatin), milk (e.g., skim milk powder), casein, polyethylene glycol (PEG), polyvinyl alcohol (PVA), or polyvinyl pyrrolidone (PVP), biotin blocking agent, peroxidase blocking agent, levamisole, Carnoy's solution, glycine, lysine, sodium borohydride, pontamine sky blue, Sudan black, trypan blue, FITC blocking agent and / or acetic acid. The blocking buffer / blocking solution can be applied to the array and / or the biological sample prior to and / or during labeling of the biological sample (eg, using an antibody conjugated to a fluorophore).

[0432] In some embodiments, additional steps or optimizations can be included in performing IF / IHC protocols in conjunction with spatial arrays.Additional steps or optimizations can be included in performing the spatially spiked analyte capture agent workflows discussed herein.

[0433] In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting an analyte, such as a biological analyte) from a biological sample (e.g., an analyte present in a biological sample, such as a tissue section), the methods comprising: (a) providing a biological sample on a substrate; (b) staining the biological sample on the substrate, imaging the stained biological sample, and selecting the biological sample or a subsection of the biological sample (e.g., a region of interest) for analysis; (c) providing an array comprising one or more sets of multiple capture probes on the substrate; (d) contacting the biological sample with the array, thereby allowing the capture probes in one or more sets of multiple capture probes to capture the analyte of interest; and (e) analyzing the captured analyte, thereby spatially analyzing the analyte of interest. Any type of staining and imaging techniques described herein or known in the art can be used according to the methods described herein. In some embodiments, staining includes optical labels as described herein, including but not limited to fluorescent labels, radioactive labels, chemiluminescent labels, calorimetric or colorimetric detectable labels. In some embodiments, staining includes fluorescent antibodies to target analytes (e.g., cell surface or intracellular proteins) in the biological sample. In some embodiments, staining includes immunohistochemical staining for target analytes (e.g., cell surface or intracellular proteins) in biological samples. In some embodiments, staining includes chemical stains, such as hematoxylin and eosin (H&E) or periodic acid Schiff (PAS). In some embodiments, a considerable period of time (e.g., days, months, or years) can be separated between staining and / or imaging of biological samples and analysis. In some embodiments, before, simultaneously, or after the array contacts the biological sample, a reagent for analysis is added to 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 barcoding features (e.g., substrates with capture probes, beads with capture probes) are attached to a flexible substrate. In some embodiments, before the array contacts the biological sample, measures are taken to slow down the reaction (e.g., reducing 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). In some embodiments, step (e) is performed without disengaging the biological sample from 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 spiked with an analyte capture agent before, simultaneously with, or after staining and / or imaging of the biological sample. In such cases, a considerable amount of time (e.g., days, months, or years) may elapse between staining and / or imaging and analysis. In some embodiments, the array is adapted to facilitate migration of biological analytes from the stained and / or imaged biological sample to the array (e.g., using any of the materials or methods described herein).In some embodiments, the biological sample is permeabilized before contacting with the array. In some embodiments, the permeabilization rate is slowed down before the biological sample contacts with the array (for example, to limit the diffusion of analytes from their original position in the biological sample). In some embodiments, regulating the permeabilization rate (for example, regulating the activity of permeabilization reagents) can be achieved by regulating the conditions to which the biological sample is exposed (for example, regulating temperature, pH and / or light). In some embodiments, regulating the permeabilization rate includes using external stimuli (for example, small molecules, enzymes and / or activating agents) to regulate the permeabilization rate. For example, a permeabilization reagent can be provided to the biological sample before contacting with the array, and the permeabilization reagent is inactive before conditions (for example, temperature, pH and / or light) change or external stimuli (for example, small molecules, enzymes and / or activating agents) are provided.

[0434] In some embodiments, provided herein are methods for spatially detecting an analyte (e.g., detecting an analyte, such as a biological analyte) from a biological sample (e.g., an analyte present in a biological sample, such as a tissue section), the methods comprising: (a) providing a biological sample on a substrate; (b) staining the biological sample on the substrate, imaging the stained biological sample, and selecting the biological sample or a subsection of the biological sample (e.g., a region of interest) for spatial transcriptomic analysis; (c) providing an array comprising one or more sets of multiple capture probes on the substrate; (d) contacting the biological sample with the array, thereby allowing a capture probe from the one or more sets of multiple capture probes to capture the biological analyte of interest; and (e) analyzing the captured biological analytes, thereby performing spatial analysis of the biological analytes of interest.

[0435] (b) Capture probe

[0436] "Capture probe" refers to any molecule capable of capturing (directly or indirectly) and / or labeling an analyte (e.g., an analyte of interest) in a biological sample. In some embodiments, the capture probe is a nucleic acid or a polypeptide. In some embodiments, the capture probe is a conjugate (e.g., an oligonucleotide-antibody conjugate). In some embodiments, the capture probe comprises a barcode (e.g., a spatial barcode and / or a unique molecular identifier (UMI)) and a capture domain.

[0437] Figure 6It is a schematic diagram illustrating an example of a capture probe as described herein. As shown in the figure, capture probe 602 is optionally coupled to feature 601 by a cleavage domain 603 such as a disulfide bond joint. The capture probe may include a functional sequence useful to subsequent processing, such as a functional sequence 604, which may include a sequencer-specific flow cell attachment sequence, such as a P5 sequence, and a functional sequence 606, which may include a sequencing primer sequence, such as an R1 primer binding site. In some embodiments, sequence 604 is a P7 sequence, and sequence 606 is an R2 primer binding site. Spatial barcode 605 may be included in the capture probe for barcoding target analytes. Typically, functional sequences may be selected to be compatible with any of a variety of sequencing systems, such as 454 sequencing, ion torrent proton (Ion Torrent Proton) or PGM, Illumina X10, PacBio, nanopore (Nanopore) etc. and requirements thereof. In some embodiments, functional sequences may be selected to be compatible with non-commercial sequencing systems. Examples of such sequencing systems and technologies for which suitable functional sequences can be used include, but are not limited to, Roche 454 sequencing, Ion Torrent proton or PGM sequencing, Illumina X10 sequencing, PacBio SMRT sequencing, and Oxford nanopore sequencing. In addition, in some embodiments, functional sequences can be selected for compatibility with other sequencing systems, including non-commercial sequencing systems.

[0438] In some embodiments, the spatial barcode 605, functional sequence 604 (e.g., flow cell attachment sequence) and 606 (e.g., sequencing primer sequence) can be common to all probes attached to a given feature. The spatial barcode can also include a capture domain 607 to facilitate capture of target analytes.

[0439] (i) Capture domain

[0440] As described 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, which specifically binds to a desired analyte. In some embodiments, a capture domain can be used to capture or detect a desired analyte.

[0441] 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 (e.g., RNA molecules and DNA molecules). In some embodiments, the functional nucleic acid sequence may include an N-polymer sequence (e.g., a random N-polymer sequence), wherein the N-polymer sequence is configured to interact with multiple DNA molecules. In some embodiments, the functional sequence may include a poly (T) sequence, which is configured to interact with a messenger RNA (mRNA) molecule via a poly (A) tail of an mRNA transcript. In some embodiments, the functional nucleic acid sequence is a binding target of a protein (e.g., a transcription factor, a DNA binding protein, or an RNA binding protein), wherein the analyte of interest is a protein.

[0442] The capture probe can include ribonucleotides and / or deoxyribonucleotides and synthetic nucleotide residues that can participate in Watson-Crick type or similar base pair interactions. In some embodiments, the capture domain can trigger a reverse transcription reaction to produce cDNA complementary to the captured RNA molecule. In some embodiments, the capture domain of the capture probe can trigger a DNA extension (polymerase) reaction to produce DNA complementary to the captured DNA molecule. In some embodiments, the capture domain can serve as a template for the ligation reaction between the captured DNA molecule and the surface probe directly or indirectly fixed on the substrate. In some embodiments, the capture domain can be connected to one chain of the captured DNA molecule. For example, SplintR ligase together with RNA or DNA sequence (e.g., degenerate RNA) can be used to connect single-stranded DNA or RNA to the capture domain. In some embodiments, a ligase with RNA templated ligase activity (e.g., SplintR ligase, T4 RNA ligase 2 or KOD ligase) can be used to connect single-stranded DNA or RNA to the capture domain. In some embodiments, the capture domain includes a splint oligonucleotide. In some embodiments, the capture domain captures the splint oligonucleotide.

[0443] In some embodiments, the capture domain is located at the 3' end of the capture probe and includes a free 3' end, which 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 with nucleic acids (e.g., RNA or other analytes) present in cells of a biological sample in contact with the array. In some embodiments, the capture domain can be selected or designed to selectively or specifically bind to the target nucleic acid. For example, the capture domain can be selected or designed to capture mRNA by hybridizing with the mRNA poly (A) tail. Therefore, in some embodiments, the capture domain includes a poly (T) DNA oligonucleotide, for example, a series of consecutive deoxythymidine residues connected by a phosphodiester bond, which can hybridize with the poly (a) tail of the mRNA. In some embodiments, the capture domain may include nucleotides that are functionally or structurally similar to the poly (T) tail. For example, a poly (U) oligonucleotide or an oligonucleotide comprising a deoxythymidine analog. 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.

[0444] In some embodiments, the capture probe includes a capture domain having a sequence capable of binding to mRNA and / or genomic DNA. For example, the capture probe may include a capture domain comprising a nucleic acid sequence capable of binding to a poly (A) tail of an mRNA and / or a poly (A) homopolymeric sequence present in genomic DNA (e.g., a poly (T) sequence). In some embodiments, a homopolymeric sequence is added to an mRNA molecule or a genomic DNA molecule using a terminal transferase to produce an analyte having a poly (A) or poly (T) sequence. For example, a poly (A) sequence can be added to an analyte (e.g., a fragment of genomic DNA) so that the analyte can be captured by the poly (T) capture domain.

[0445] In some embodiments, a random sequence (e.g., a random hexamer or similar sequence) can be used to form all or part of a capture domain. For example, a random sequence can be used together with a poly(T) (or poly(T) analog) sequence. Thus, where the capture domain includes a poly(T) (or "poly(T)-like") oligonucleotide, it can also include a random oligonucleotide sequence (e.g., a "poly(T)-random sequence" probe). For example, this can be located 5' or 3' of the poly(T) sequence, such as at the 3' end of the capture domain. The poly(T)-random sequence probe can facilitate the capture of the mRNA poly(A) tail. In some embodiments, the capture domain can be a completely random sequence. In some embodiments, a degenerate capture domain can be used.

[0446] In some embodiments, the collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly (T) sequence, and the capture domain of one or more capture probes includes a random sequence. In some embodiments, the collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly (T) sample sequence, and the capture domain of one or more capture probes includes a random sequence. In some embodiments, the collection of two or more capture probes forms a mixture, wherein the capture domain of one or more capture probes includes a poly (T) random sequence, and the capture domain of one or more capture probes includes a random sequence. In some embodiments, probes with degenerate capture domains can be added to any of the aforementioned combinations listed herein. In some embodiments, probes with degenerate capture domains can replace one of the probes in each pair described herein.

[0447] The capture domain can be based on the specific gene sequence or specific motif sequence or common / conserved sequence (i.e., sequence-specific capture domain) that it is designed to capture. Thus, in some embodiments, the capture domain can selectively bind to a desired nucleic acid subtype or subset, such as a specific type of RNA, such as mRNA, rRNA, tRNA, SRP RNA, tRNA, snRNA, snRNA, SmY RNA, sARNA, 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 can selectively bind to a desired subset of ribonucleic acids, such as microbiome RNA, such as 16SrRNA.

[0448] In some embodiments, the capture domain includes an "anchor" or "anchoring sequence," which is a nucleotide sequence designed to ensure that the capture domain hybridizes with the intended biological analyte. In some embodiments, the anchoring sequence includes a nucleotide sequence, including 1-mers, 2-mers, 3-mers, or longer sequences. In some embodiments, the short sequence is random. For example, a capture domain including a poly (T) sequence can be designed to capture mRNA. In such embodiments, the anchoring sequence can include a random 3-mer (e.g., GGG) that helps ensure that the poly (T) capture domain hybridizes with the mRNA. In some embodiments, the anchoring sequence can be VN, N, or NN. Alternatively, a specific nucleotide sequence can be used to design the sequence. In some embodiments, the anchoring sequence is located at the 3' end of the capture domain. In some embodiments, the anchoring sequence is located at the 5' end of the capture domain.

[0449] In some embodiments, the capture domain of the capture probe is blocked before the biological sample is contacted with the array, and a blocking probe is used when the nucleic acid in the biological sample is modified before it is captured on the array. In some embodiments, the blocking probe is used to block or modify the free 3' end of the capture domain. In some embodiments, the blocking probe can hybridize with the capture probe to mask the free 3' end of the capture domain, such as a hairpin probe, a partially double-stranded probe, or a complementary sequence. In some embodiments, the free 3' end of the capture domain can be blocked by chemical modification, for example, adding an azidomethyl group as a chemically reversible capping portion so that the capture probe does not include a free 3' end. Before the biological sample is contacted with the array, the capture probe is blocked or modified, particularly at the free 3' end of the capture domain, to prevent modification of the capture probe, for example, to prevent the addition of a poly (A) tail to the free 3' end of the capture probe.

[0450] 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, nucleic acids in a biological sample can be modified so that they can be captured by a capture domain. For example, an adapter sequence (including a binding domain capable of binding to the capture domain of a capture probe) can be added to the end of a nucleic acid (e.g., fragmented genomic DNA). In some embodiments, this is achieved by connecting adapter sequences or extending nucleic acids. In some embodiments, enzymes are used to incorporate additional nucleotides, such as poly (A) tails, at the ends of the nucleic acid sequence. In some embodiments, the capture probe can be reversibly shielded 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 made inaccessible so that the capture domain is not susceptible to the process (e.g., connection or extension) used to modify the nucleic acid of the biological sample.

[0451] In some embodiments, the capture domain of the capture probe is modified to remove any modifications of the capture probe that occurred during modification of the nucleic acid molecules of the biological sample. In some embodiments, the capture probe can include additional sequences downstream of the capture domain, such as 3' to the capture domain, i.e., a blocking domain.

[0452] 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 only nucleic acid-based include, but are not limited to, proteins, peptides, aptamers, antigens, antibodies, and molecular analogs that mimic the function of any capture domain described herein.

[0453] (ii) Cleavage domain

[0454] Each capture probe can optionally include at least one cleavage domain. A cleavage domain represents the portion of the probe that is used to reversibly attach the probe to an array feature, as described herein. In addition, one or more segments or regions of the capture probe can optionally be released from the array feature by cleavage of the cleavage domain. For example, a spatial barcode and / or a universal molecular identifier (UMI) can be released by cleavage of the cleavage domain.

[0455] Figure 7 Figure 7 is a schematic diagram illustrating a cleavable capture probe, wherein the cleaved capture probe can enter non-permeabilized cells and bind to the analyte in the sample. Capture probe 701 comprises a cleavage domain 702, a cell-penetrating peptide 703, a reporter molecule 704, and a disulfide bond (-SS-). 705 represents all other parts of the capture probe, such as the spatial barcode and capture domain.

[0456] In some embodiments, the cleavage domain that connects the capture probe to the feature is a disulfide bond. A reducing agent can be added to break the disulfide bond, thereby releasing the capture probe from the feature. As another example, heating can also cause degradation of the cleavage domain and release of the attached capture probe from the array feature. In some embodiments, laser radiation is used to heat and degrade the cleavage domain of the capture probe at a specific location. In some embodiments, the cleavage domain is a photosensitive chemical bond (e.g., a chemical bond that dissociates when exposed to light such as ultraviolet light).

[0457] Oligonucleotides with light-sensitive chemical bonds (e.g., photocleavable linkers) have a variety of advantages. They can be efficiently and rapidly cleaved (e.g., in nanoseconds and milliseconds). In some cases, a photomask can be used so that only specific areas of the array are exposed to a cleavable stimulus (e.g., exposure to ultraviolet light, exposure to light, exposure to laser-induced heat). When using a photocleavable linker, the cleavage reaction is triggered by light and is highly selective for the linker, resulting in a biorthogonal reaction. Typically, the wavelength absorption of the photocleavable linker is in the near-ultraviolet range of the spectrum. In some embodiments, the λ of the photocleavable linker is less than 100 nm. 最大 is about 300 nm to about 400 nm, or about 310 nm to about 365 nm. In some embodiments, the λ of the photocleavable linker 最大 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.

[0458] Non-limiting examples of photosensitive chemical bonds that can be used in cleavage domains include those described in Leriche et al. Bioorg Med Chem. 2012 Jan 15; 20(2): 571-82 and U.S. Publication No. 2017 / 0275669, both of which are incorporated herein by reference in their entirety. For example, linkers comprising photosensitive chemical bonds include: 3-amino-3-(2-nitrophenyl)propionic acid (ANP), benzoyl ester derivatives, 8-quinolinebenzenesulfonate, dicoumarol, 6-bromo-7-alkylcoumarin-4-ylmethoxycarbonyl, bimane-based linkers, and biarylhydrazone-based linkers. In some embodiments, the photosensitive bond is part of a cleavable linker, such as the following o-nitrobenzyl (ONB) linker:

[0459]

[0460] in:

[0461] X is selected from O and NH;

[0462] R 1 Selected from H and C 1-3 alkyl;

[0463] R 2 Selected from H and C 1-3 alkoxy;

[0464] n is 1, 2, or 3; and

[0465] a and b represent the connection point between the linker and the substrate, or the connection point between the linker and the capture probe, respectively.

[0466] In some embodiments, at least one spacer is included between the substrate and the o-nitrobenzyl (ONB) linker, and at least one spacer is included between the o-nitrobenzyl (ONB) linker and the capture probe. In some aspects of these embodiments, the spacer comprises at least one group 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-, -SS-, 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, R 1 is H. In some embodiments, R 1 C 1-3 In some embodiments, R 1 In some embodiments, R 2 is H. In some embodiments, R 2 It is C 1-3 In some embodiments, R2 In some embodiments, R 1 H and R 2 is H. In some embodiments, R 1 H and R 2 In some embodiments, R 1 is methyl and R 2 is H. In some embodiments, R 1 is methyl and R 2 It is a methoxy group.

[0467] In some embodiments, the photocleavable linker has the formula:

[0468]

[0469] In some embodiments, the photocleavable linker has the formula:

[0470]

[0471] In some embodiments, the photocleavable linker has the formula:

[0472]

[0473] In some embodiments, the photocleavable linker has the formula:

[0474]

[0475] In some embodiments, the photocleavable linker has the formula:

[0476]

[0477] Without being bound by any particular theory, it is believed that excitation of the o-nitrobenzyl (ONB) linker leads to a Norrish-type hydrogen abstraction at the γ position, followed by the formation of an azine acid that is highly reactive and rearranges to a nitroso compound, resulting in complete cleavage of the linker, as shown below:

[0478]

[0479] In some embodiments, the photocleavable linker is a 3-amino-3-(2-nitrophenyl)propionic acid (ANP) linker:

[0480]

[0481] Among them, X, R 2 , n, a, and b are as described herein for the o-nitrobenzyl (ONB) linker.

[0482] In some embodiments, the photocleavable linker has the formula:

[0483]

[0484] In some embodiments, the photocleavable linker is a benzoyl ester linker:

[0485]

[0486] wherein a and b are as described herein for the o-nitrobenzyl (ONB) linker.

[0487] Other examples of photosensitive chemical bonds that can be used in the cleavage domain include halogenated nucleosides, such as bromodeoxyuridine (BrdU). BrdU is an analog of thymidine that can be readily incorporated into oligonucleotides (e.g., in the cleavage domain of a capture probe) and is sensitive to UVB light (280-320 nm range). Upon exposure to UVB light, a photocleavage reaction occurs (e.g., at the nucleoside immediately 5' to the site of BrdU incorporation (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.

[0488] Other examples of cleavage domains include labile chemical bonds, such as, but not limited to, ester bonds (e.g., cleavable with acid, base, or hydroxylamine), vicinal diol bonds (e.g., cleavable by sodium periodate), Diels-Alder bonds (e.g., cleavable by heat), sulfone bonds (e.g., cleavable by base), silyl ether bonds (e.g., cleavable by acid), glycosidic bonds (e.g., cleavable by amylases), peptide bonds (e.g., cleavable by proteases), abasic or apurinic / apyrimidinic (AP) sites (e.g., cleavable by base or AP endonucleases), or phosphodiester bonds (e.g., cleavable by nucleases (e.g., DNase)).

[0489] In some embodiments, the cleavage domain includes a sequence recognized by one or more enzymes capable of cleaving nucleic acid molecules, for example, capable of destroying the phosphodiester bond between two or more nucleotides. The bond can be targeted by other nucleic acid molecules, such as restriction enzymes (such as restriction endonucleases) for cleavage. For example, the cleavage domain can include a restriction endonuclease (restriction enzyme) recognition sequence. Restriction endonucleases cut double-stranded or single-stranded DNA on a specific recognition nucleotide sequence known as a restriction site. In some embodiments, rare-cut restriction enzymes are used, for example, enzymes with long recognition sites (at least 8 base pairs in length) to reduce the possibility of cleavage at other positions in the capture probe.

[0490] In some embodiments, the cleavage domain includes a poly (U) sequence that can be cleaved by uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII (commercially known as USER TM Once released, the releasable capture probe can be used for reaction. Thus, for example, the activatable capture probe can be activated by releasing the capture probe from the feature.

[0491] In some embodiments, when the capture probe is, for example, indirectly attached to the substrate via a surface probe, the cleavage domain includes one or more mismatched nucleotides so that the complementary portion of the surface probe and the capture probe is not 100% complementary (for example, the number of mismatched base pairs can be one, two or three base pairs). For example, this mismatch is recognized by MutY and T7 endonuclease I enzymes, which result in the cracking of the nucleic acid molecules at the mismatched position. As described herein, a "surface probe" can be any portion that is present on the surface of the substrate and can be connected to a reagent (for example, a capture probe). In some embodiments, the surface probe is an oligonucleotide. In some embodiments, the surface probe is a part of the capture probe.

[0492] In some embodiments in which the capture probe is attached (e.g., fixed) to a feature indirectly (e.g., via a surface probe), the cleavage domain includes a nickase recognition site or sequence. A nickase is an endonuclease that can only cut a single strand of a DNA duplex. Thus, the cleavage domain can 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 destabilizes the duplex between the surface probe and the capture probe, thereby releasing the capture probe from the feature.

[0493] Nickase can also be used for capturing probe and directly attaching (for example, fixing) in some embodiments of feature.For example, substrate can be contacted with the nucleic acid molecule that hybridizes to the cleavage domain of capture probe to provide or reorganize nickase recognition site, for example cracking auxiliary probe.Therefore, contacting with nickase will cause the cracking of cleavage domain, thereby discharges capture probe from feature.This cracking auxiliary probe can also be used for providing or reorganizing cracking recognition site for other cleavage enzymes (for example restriction endonucleases).

[0494] Some nickases, by binding and recognizing specific nucleotide recognition sequences, introduce single-stranded nicks only at specific sites of the DNA molecule. Many natural nickases have been discovered, and the sequence recognition characteristics of at least four nickases have been determined. Nickases are described in U.S. Patent No. 6,867,028, which is incorporated herein by reference in its entirety. In general, any suitable nickase can be used to bind to the complementary nickase recognition site of the cleavage domain. After use, the nickase can be removed from the analysis, or inactivated after releasing the capture probe to prevent unnecessary cleavage of the capture probe.

[0495] Examples of suitable capture domains that are not solely nucleic acid based 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.

[0496] In some embodiments, the cleavage domain is absent from the capture probe. Examples of substrates with attached capture probes lacking a cleavage domain are described, for example, in Macosko et al., (2015) Cell 161, 1202-1214, the entire contents of which are incorporated herein by reference.

[0497] In some embodiments, the capture probe region corresponding to the cleavage domain can be used for certain other functions. For example, other regions for nucleic acid extension or amplification can be included in the position where the cleavage domain would normally be located. In such embodiments, this region can supplement the functional domain or even exist as another functional domain. In some embodiments, there is a cleavage domain, but its use is optional.

[0498] (iii) Functional domain

[0499] Each capture probe can optionally include at least one functional domain. Each functional domain typically includes a functional nucleotide sequence for use in a downstream analysis step in the overall analysis procedure.

[0500] In some embodiments, the capture probe may include a functional domain for attachment to a sequencing flow cell, e.g., In some embodiments, the capture probe or its derivative may include another functional domain, such as for attachment to a The functional domain can be selected to be compatible with any of a variety of different sequencing systems, such as 454 sequencing, Ion Torrent Proton or PGM, Illumina X10, etc. and their requirements.

[0501] In some embodiments, the functional domain comprises a primer. The primer may comprise a primer for R1 primer sequence for sequencing, and in some embodiments, includes Examples of such capture probes and their uses are described in U.S. Patent Publication Nos. 2014 / 0378345 and 2015 / 0376609, the entire contents of which are incorporated herein by reference.

[0502] (iv) Spatial barcode

[0503] As described above, the capture probe may include one or more spatial barcodes (e.g., two or more, three or more, four or more, five or more) spatial barcodes. A "spatial barcode" is a continuous nucleic acid segment or two or more non-continuous nucleic acid segments that serve as a label or identifier that transmits or is capable of transmitting spatial information. In some embodiments, the capture probe includes a spatial barcode with a spatial aspect, wherein the barcode is associated with a specific position within the array or a specific position on the substrate.

[0504] The spatial barcode can be part of the analyte or independent of the analyte (e.g., part of a capture probe). The spatial barcode can be a label attached to the analyte (e.g., a nucleic acid molecule) or a combination of labels in addition to the endogenous characteristics of the analyte (e.g., the size or terminal sequence of the analyte). The spatial barcode can be unique. In some embodiments where the spatial barcode is unique, the spatial barcode acts both as a spatial barcode and as a unique molecular identifier (UMI) associated with a specific capture probe.

[0505] Spatial barcodes can have a variety of different formats. For example, spatial barcodes can 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, the spatial barcode is added to a fragment of, for example, a DNA or RNA sample before, during, and / or after sample sequencing. In some embodiments, the spatial barcode enables identification and / or quantification of individual sequence readings. In some embodiments, the spatial barcode is used as a fluorescent barcode, wherein a fluorescently labeled oligonucleotide probe hybridizes to the spatial barcode.

[0506] 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 nucleic acid sequence over a substantial portion (e.g., 80% or more) of the nucleic acid molecules in the biological sample.

[0507] The spatial barcode sequence may include about 6 to about 20 or more nucleotides within the capture probe sequence. In some embodiments, the length of the spatial barcode sequence may be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer. In some embodiments, the length of the spatial barcode sequence may 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 length of the spatial barcode sequence may be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or shorter.

[0508] These nucleotides can be completely continuous, such as in a single sequence segment of adjacent nucleotides, or they can be divided into two or more separate subsequences separated by 1 or more nucleotides. The length of the separated spatial barcode subsequences is about 4 to 16 nucleotides. In some embodiments, the spatial barcode subsequences can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the spatial barcode subsequences 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 spatial barcode subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0509] For multiple capture probes attached to a common array feature, the one or more spatial barcode sequences of the multiple capture probes can include a sequence that is the same for all capture probes coupled to the feature and / or a sequence that differs between all capture probes coupled to the feature.

[0510] Figure 8 is a schematic diagram of an exemplary multiple spatial barcoding feature. Figure 8, feature 801 can be coupled to spatially barcoded capture probes, wherein the spatial barcoded probes of a particular feature can have the same spatial barcode, but have different capture domains designed to associate the spatial barcode of the feature with multiple target analytes. For example, a feature can be coupled to four different types of spatially barcoded capture probes, each type of spatially barcoded capture probe having a spatial barcode 802. One type of capture probe associated with the feature comprises a combination of a spatial barcode 802 and a poly (T) capture domain 803, which is designed to capture mRNA target analytes. A second type of capture probe associated with the feature comprises a combination of a spatial barcode 802 and a random N-mer capture domain 804 for gDNA analysis. A third type of capture probe associated with the feature comprises a combination of a spatial barcode 802 and a capture domain that is complementary to a capture domain on an analyte capture agent capture agent barcode domain 805. The fourth type of capture probe associated with this feature includes a spatial barcode 802 in combination with a capture probe that can specifically bind to a nucleic acid molecule 806 that can function in a CRISPR assay (e.g., CRISPR / Cas9). Figure 8 Only four different capture probe barcoding constructs are shown, but capture probe barcoding constructs can be customized for analysis of any given analyte associated with nucleic acids and can be combined with such constructs. For example, Figure 8 The scheme shown in can also be used for the simultaneous analysis of other analytes disclosed herein, including but not limited to: (a) mRNA, lineage tracing constructs, cell surface or intracellular proteins and metabolites, and gDNA; (b) mRNA, accessible chromatin (e.g., ATAC-seq, DNA enzyme-seq, and / or MNA enzyme-seq) cell surface or intracellular proteins and metabolites, and perturbations (e.g., CRISPR-crRNA / sgRNA, TALEN, zinc finger nucleases and / or antisense oligonucleotides as described herein); (c) mRNA, cell surface or intracellular proteins and / or metabolites, barcoding markers (e.g., MHC multimers described herein) and V(D)J sequences of immune cell receptors (e.g., T cell receptors). In some embodiments, the perturbation agent can be a small molecule, an antibody, a drug, an aptamer, miRNA, a physical environment (e.g., temperature change), or any other known perturbation agent.

[0511] The capture probes attached to a single array feature can include the same (or common) spatial barcode sequence, different spatial barcode sequences, or a combination of the two. The capture probes attached to a feature can include multiple groups of capture probes. The capture probes of a given group can include the same spatial barcode sequence. The same spatial barcode sequence can be different from the spatial barcode sequence of another group of capture probes.

[0512] A plurality of capture probes may include a spatial barcode sequence (e.g., a nucleic acid barcode sequence) associated with a specific position 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 common to the capture probes within the first region, and a second plurality of capture probes may be associated with a second region based on a spatial barcode sequence common to the capture probes within the second region. The second region may or may not be associated with the first region. Other multiple capture probes may be associated with a spatial barcode sequence shared by capture probes within other regions. In some embodiments, the spatial barcode sequence may be the same between multiple capture probe molecules.

[0513] In some embodiments, multiple different spatial barcodes are incorporated into a single array capture probe. For example, a mixed but known set of spatial barcode sequences can provide stronger addressing or attributes of the spatial barcode to a given point or position by providing repeated or independent confirmation of positional characteristics. In some embodiments, multiple spatial barcodes represent increased specificity of the position of a particular array point.

[0514] (v) Unique molecular identifier

[0515] The capture probe can include one or more (e.g., 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 segment or two or more non-contiguous nucleic acid segments that serves as a tag or identifier for a specific analyte or a capture probe that binds a specific analyte (e.g., via a capture domain).

[0516] A UMI can be unique. A UMI can include one or more specific polynucleotide sequences, one or more random nucleic acid and / or amino acid sequences, and / or one or more synthetic nucleic acid and / or amino acid sequences.

[0517] In some embodiments, a UMI is a nucleic acid sequence that does not substantially hybridize to an analyte nucleic acid molecule in a biological sample. In some embodiments, a UMI has less than 80% sequence identity (e.g., less than 70%, 60%, 50%, or less than 40% sequence identity) to a nucleic acid sequence over a substantial portion (e.g., 80% or more) of the nucleic acid molecules in a biological sample.

[0518] A UMI can include about 6 to about 20 or more nucleotides within a capture probe sequence. In some embodiments, a UMI can be about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or longer. In some embodiments, a UMI can be at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or longer. In some embodiments, a UMI can be at most about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides in length or shorter.

[0519] These nucleotides can be completely contiguous, i.e., in a single sequence stretch of adjacent nucleotides, or they can be separated into two or more separate subsequences separated by one or more nucleotides. The length of the isolated UMI subsequences is about 4 to 16 nucleotides. In some embodiments, the UMI subsequences can be about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or longer. In some embodiments, the UMI subsequences 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 subsequences can be at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 nucleotides or shorter.

[0520] In some embodiments, a UMI is attached to an analyte in a reversible or irreversible manner. In some embodiments, a UMI is added to fragments of, for example, a DNA or RNA sample before, during, and / or after sequencing of the analyte. In some embodiments, a UMI enables identification and / or quantification of individual sequencing reads. In some embodiments, a UMI is used as a fluorescent barcode, where a fluorescently labeled oligonucleotide probe hybridizes to the UMI.

[0521] (vi) Other aspects of capture probes

[0522] For the capture probes attached to array features, individual array features can include one or more capture probes. In some embodiments, individual array features include hundreds or thousands of capture probes. In some embodiments, the capture probes are associated with specific individual features, wherein the individual features include capture probes that include a spatial barcode unique to a region or position defined on the array.

[0523] In some embodiments, a specific feature may include capture probes that include more than one spatial barcode (e.g., one capture probe at a specific feature may include a spatial barcode that is different from the spatial barcode included in another capture probe at the same specific feature, while both capture probes include a second common spatial barcode), wherein each spatial barcode corresponds to a specific defined area or position on the array. For example, multiple spatial barcode sequences associated with a specific feature on the array can provide a stronger address or attribute to a given position by providing repetition or independent confirmation of the position. In some embodiments, multiple spatial barcodes represent increased specificity of the position of a specific array point. In a non-limiting example, a specific array point can be encoded using two different spatial barcodes, wherein each spatial barcode identifies a specific defined area within the array, and an array point having two spatial barcodes identifies a sub-area where the two defined areas overlap, for example, such as the overlapping portion of a Venn diagram.

[0524] In another non-limiting example, a particular array spot can be coded with three different spatial barcodes, where a first spatial barcode identifies a first region within the array, a second spatial barcode identifies a second region, where the second region is a sub-region entirely within the first region, and a third spatial barcode identifies a third region, where the third region is a sub-region entirely within the first and second sub-regions.

[0525] In some embodiments, the capture probe attached to the array features is released from the array features to perform sequencing. Or, in some embodiments, the capture probe remains attached to the array features, and the probe is sequenced while remaining attached to the array features (e.g., by in situ sequencing). Other aspects of the sequencing of the capture probe are described in subsequent parts of the present invention.

[0526] In some embodiments, array features can include different types of capture probes attached to features. For example, an array feature can include a first class capture probe and a second class capture probe, wherein the first class capture probe has a capture domain designed to be bound to a class of analytes, and the second class capture probe has a capture domain designed to be bound to a second class of analytes. Typically, an array feature can include one or more (e.g., 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) different types of capture probes attached to a single array feature.

[0527] In some embodiments, the capture probe is a nucleic acid. In some embodiments, the capture probe is attached to the array feature via its 5′ end. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a barcode (e.g., spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs) and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end to the 3′ end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), a second functional domain and a capture domain. In some embodiments, the capture probe comprises, from the 5′ end 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 does not include a spatial barcode. In some embodiments, the capture probe does not include a UMI. In some embodiments, the capture probe includes a sequence for initiating a sequencing reaction.

[0528] In some embodiments, the capture probe is fixed to the feature via its 3' end. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: one or more barcodes (e.g., spatial barcodes and / or UMIs) and one or more capture domains. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: a barcode (e.g., spatial barcode or UMI) and a capture domain. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: a cleavage domain, a functional domain, one or more barcodes (e.g., spatial barcodes and / or UMIs), and a capture domain. In some embodiments, the capture probe comprises, from the 3' end to the 5' end: a cleavage domain, a functional domain, a spatial barcode, a UMI, and a capture domain.

[0529] In some embodiments, the capture probe includes an oligonucleotide synthesized in situ. The oligonucleotide synthesized in situ can be attached to a substrate, or attached to a feature on a substrate. In some embodiments, the oligonucleotide synthesized in situ includes one or more constant sequences, one or more of which are used as initiator sequences (e.g., primers for amplifying target nucleic acids). The oligonucleotide synthesized in situ can, for example, include a constant sequence at the 3' end, which is attached to the substrate, or attached to a feature on the substrate. In addition or alternatively, the oligonucleotide synthesized in situ can include a constant sequence at a free 5' end. In some embodiments, one or more constant sequences can be cleavable sequences. In some embodiments, the oligonucleotide synthesized in situ includes a barcode sequence, such as a variable barcode sequence. The barcode can be any barcode as described herein. The length of the barcode can be approximately 8 to 16 nucleotides (e.g., 8, 9, 10, 11, 12, 13, 14, 15, or 16 nucleotides). The length of the oligonucleotide synthesized in situ can be less than 100 nucleotides (e.g., less than 90, 80, 75, 70, 60, 50, 45, 40, 35, 30, 25 or 20 nucleotides). In some cases, the length of the oligonucleotide synthesized in situ is about 20 to about 40 nucleotides. Exemplary in situ synthesized oligonucleotides are produced by Affymetrix. In some embodiments, the oligonucleotide synthesized in situ is attached to the features of the array.

[0530] In some embodiments, the capture probe is a probe that is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ. In some embodiments, the capture probe is a collection of oligonucleotides that are synthesized in situ.

[0531] In some embodiments, a capture probe can be prepared using a splint oligonucleotide (e.g., any splint oligonucleotide described herein). Two or more oligonucleotides can be ligated together using a splint oligonucleotide and any type of ligase known in the art or described herein (e.g., a splint ligase).

[0532] One of the oligonucleotides can include, for example, a constant sequence (e.g., a sequence that is complementary to a portion of the splint oligonucleotide), a degenerate sequence, and / or a capture domain (e.g., as described herein). One of the oligonucleotides can also include a sequence that is compatible with connection or hybridization to an analyte of interest in a biological sample. The analyte of interest (e.g., mRNA) can also be used as a splint oligonucleotide to connect more oligonucleotides to the capture probe. In some embodiments, the capture probe is produced by causing an enzyme to add a polynucleotide to the end of the oligonucleotide sequence. The capture probe can include a degenerate sequence that can be used as a unique molecular identifier.

[0533] A degenerate sequence is a sequence in which certain positions of a nucleotide sequence contain many possible bases. A degenerate sequence can 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, a 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, a degenerate sequence is used as a UMI.

[0534] In some embodiments, the capture probe includes a restriction endonuclease recognition sequence or a nucleotide sequence that can be cut by a specific enzymatic activity. For example, uracil DNA glycosylase (UDG) or a uracil specific excision reagent (USER) can be used to enzymatically cut a uracil sequence from a nucleotide sequence. As another example, other modified bases (e.g., methylated) can be recognized and cut by specific endonucleases. The capture probe can be subjected to enzyme cleavage, which removes the blocking domain and any other nucleotides added to the 3' end of the capture probe during the modification process. The removal of the blocking domain will expose and / or restore the free 3' end of the capture domain of the capture probe. In some embodiments, other nucleotides can be removed to display and / or restore the 3' end of the capture domain of the capture probe.

[0535] In some embodiments, the blocking domain can be incorporated into the capture probe during or after its synthesis. The terminal nucleotide of the capture domain is a reversible terminator nucleotide (e.g., a 3′-O-blocked reversible terminator and a 3′-unblocked reversible terminator) and can be included in the capture probe during or after probe synthesis.

[0536] (vii) Extended capture probe

[0537] An "extended capture probe" is a capture probe having an expanded nucleic acid sequence. For example, where the capture probe comprises a nucleic acid, an extended "3' end" means that additional nucleotides are added to the very terminal 3' nucleotide of the capture probe to extend the length of the capture probe, e.g., by extending the nucleic acid molecule via a standard polymerization reaction, including template polymerization catalyzed by a polymerase (e.g., DNA polymerase or reverse transcriptase).

[0538] In some embodiments, extending the capture probe includes generating cDNA from the captured (hybridized) RNA. This process involves synthesizing a complementary strand of a hybrid nucleic acid, for example, generating cDNA based on a captured RNA template (RNA hybridized to the capture domain of the capture probe). Thus, in the initial step of extending the capture probe (e.g., cDNA generation), the captured (hybridized) nucleic acid (e.g., RNA) serves as a template for extension (e.g., a reverse transcription step).

[0539] In some embodiments, the capture probe is extended using reverse transcription. For example, reverse transcription includes using reverse transcriptase to synthesize cDNA (complementary or copy DNA) from RNA (such as messenger RNA). In some embodiments, reverse transcription is carried out when the tissue is still in situ, to produce an analyte library, wherein the analyte library includes a spatial barcode from a neighboring capture probe. In some embodiments, the capture probe is extended using one or more DNA polymerases.

[0540] In some embodiments, the capture domain of the capture probe includes a primer for generating a complementary strand of nucleic acid that hybridizes to the capture probe, for example, a primer for DNA polymerase and / or reverse transcription. The nucleic acid (e.g., DNA and / or cDNA) molecule produced by the extension reaction comprises the sequence of the capture probe. The extension of the capture probe, such as DNA polymerase and / or reverse transcription reaction, can be performed using various suitable enzymes and protocols.

[0541] In some embodiments, full-length DNA (e.g., cDNA) molecules are produced. In some embodiments, a "full-length" DNA molecule refers to the entire captured nucleic acid molecule. However, if the nucleic acid (e.g., RNA) is partially degraded in the tissue sample, the captured nucleic acid molecule will be different from the initial RNA length in the tissue sample. In some embodiments, the 3' end of the extended probe (e.g., the first-chain cDNA molecule) is modified. For example, a linker or adapter can be connected to the 3' end of the extended probe. This can be accomplished by using a single-stranded ligase such as T4 RNA ligase or CircleGase TM(available from Epicentre Biotechnologies, Madison, Wisconsin). In some embodiments, a template switching oligonucleotide is used to extend the cDNA to produce a full-length cDNA (or as close to a full-length cDNA as possible). In some embodiments, a second-strand synthesis auxiliary probe (a partially double-stranded DNA molecule capable of hybridizing to the 3′ end of the extended capture probe) can be ligated to the 3′ end of the extended probe, e.g., a first-strand cDNA molecule, using a double-stranded ligase (e.g., 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 (strain 9°N) DNA ligase (9°N TM DNA ligase, New England Biolabs), Ampligase TM (available from Epicentre Biotechnologies, Madison, Wisconsin) and SplintR (available from New England Biolabs, Ipswich, Massachusetts). 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 enzymatically incorporated using terminal transferase activity.

[0542] In some embodiments, the double-stranded extended capture probe is treated to remove any unextended capture probe prior to amplification and / or analysis (e.g., sequence analysis). This can be achieved by a variety of methods, for example, using an enzyme to degrade the unextended probe, such as an exonuclease or a purification column.

[0543] In some embodiments, the extended capture probe is amplified to produce a quantity sufficient for analysis, such as by DNA sequencing. In some embodiments, the first strand (e.g., a DNA and / or cDNA molecule) of the extended capture probe is used as a template for an amplification reaction (e.g., a polymerase chain reaction).

[0544] In some embodiments, the amplification reaction uses a primer comprising an affinity group to incorporate the affinity group onto an extended capture probe (e.g., RNA-cDNA hybrid). In some embodiments, the primer comprises an affinity group and the extended capture probe comprises an affinity group. The affinity group can correspond to any affinity group described above.

[0545] In some embodiments, the capture probe comprising an extension of an affinity group can be coupled to an array feature specific for the affinity group. In some embodiments, the substrate can 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 array feature includes maltose, and the affinity group includes maltose binding protein. In some embodiments, the array feature includes maltose binding protein, and the affinity group includes maltose. In some embodiments, as long as a copy of the extended probe is not attached to the array feature, the capture probe that has been amplified can serve to release the amplified probe from the array feature.

[0546] In some embodiments, the capture probe or its complement or amplicon that extends is released from the array characteristics.The step of releasing the capture probe or its complement or amplicon that extends from the array characteristics can be achieved in many ways.In some embodiments, the capture probe or its complement that extends is released from the feature by nucleic acid cleavage and / or denaturation (for example, by heating to denature double-stranded molecules).

[0547] In some embodiments, the extended capture probe or its complement or amplicon is released from the array feature by physical means. For example, the method of inducing physical release includes denaturing double-stranded nucleic acid molecules. Another method of releasing the extended capture probe is to use a solution that interferes with the hydrogen bonds of the double-stranded molecules. In some embodiments, the extended capture probe is released by applying heated water (e.g., water or buffer at least 85°C, such as water at least 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C or 99°C). In some embodiments, a solution comprising salts, surfactants, etc. is added to further destabilize the interactions between nucleic acid molecules to release the extended capture probe from the array feature. In some embodiments, formamide solution can be used to destabilize the interactions between nucleic acid molecules to release the extended capture probe from the array feature.

[0548] (viii) Analyte capture agent

[0549] The present invention also provides methods and materials for spatial profiling of biological analytes (e.g., mRNA, genomic DNA, accessible chromatin and cell surface or intracellular proteins and / or metabolites) using analyte capture agents. As used herein, an "analyte capture agent" (sometimes also previously referred to as a "cell spike agent") refers to a substance that interacts with an analyte (e.g., an analyte in a sample) and a capture probe (e.g., a capture probe attached to a substrate) to recognize the analyte. In some embodiments, the analyte capture agent comprises an analyte binding portion and a capture agent barcode domain.

[0550] Figure 9Schematic diagram of an exemplary analyte capture agent 902 consisting of an analyte binding portion 904 and a capture agent barcode domain 908. The analyte binding portion 904 is a molecule capable of binding to an analyte 906 and interacting with a spatially barcoded capture probe. The analyte binding portion can bind to the analyte 906 with high affinity and / or high specificity. The analyte capture agent may include a capture agent barcode domain 908, a nucleotide sequence (e.g., an oligonucleotide) that can hybridize with at least a portion or all of the capture domain of the capture probe. The analyte binding portion 904 may include a polypeptide and / or an aptamer (e.g., an oligonucleotide or peptide molecule that binds to a specific target analyte). The analyte binding portion 904 may include an antibody or antibody fragment (e.g., an antigen binding fragment).

[0551] As used herein, the term "analyte binding moiety" refers to a molecule or portion that is capable of binding to a macromolecular component (e.g., an analyte, e.g., a biological analyte). In some embodiments of any of the spatial profiling methods described herein, the analyte binding moiety of the analyte capture agent that binds to a biological analyte may include, but is not limited to, an antibody or epitope binding fragment thereof, a cell surface receptor binding molecule, a receptor ligand, a small molecule, a bispecific antibody, a bispecific T cell engager, a T cell receptor engager, a B cell receptor engager, a precursor, an aptamer, a monomer, an adhesin, a DARPin, and a protein scaffold, or any combination thereof. The analyte binding moiety can bind to a macromolecular component (e.g., an analyte) with high affinity and / or high specificity. The analyte binding moiety may include a nucleotide sequence (e.g., an oligonucleotide) that may correspond to at least part or all of the analyte binding moiety. The analyte binding moiety may include a polypeptide and / or an aptamer (e.g., a polypeptide and / or an aptamer that binds to a specific target molecule, e.g., an analyte). The analyte binding moiety may include an antibody or antibody fragment (e.g., an antigen binding fragment) that binds to a specific analyte (e.g., a polypeptide).

[0552] In some embodiments, the analyte-binding portion of an analyte-capture agent comprises one or more antibodies or antigen-binding fragments thereof. The antibody or antigen-binding fragment comprising the analyte-binding portion can specifically bind to the target analyte. In some embodiments, the analyte is a protein (e.g., a protein on the surface of a biological sample (e.g., a cell) or an intracellular protein). In some embodiments, multiple analyte-capture agents comprising multiple analyte-binding portions bind to multiple analytes present in a biological sample. In some embodiments, the multiple analytes comprise a single type of analyte (e.g., a single type of polypeptide). In some embodiments where the multiple analytes comprise a single type of analyte, the analyte-binding portions of the multiple analyte-capture agents are identical. In some embodiments where the multiple analytes comprise a single type of analyte, the analyte-binding portions of the multiple analyte-capture agents are different (e.g., members of the multiple analyte-capture agents can have two or more analyte-binding portions, wherein each of the two or more analyte-binding portions binds to a single type of analyte (e.g., at different binding sites)). In some embodiments, the multiple analytes comprise multiple different types of analytes (e.g., multiple different types of polypeptides).

[0553] The analyte capture agent may include an analyte binding moiety. The analyte binding moiety may be an antibody. Exemplary non-limiting antibodies that can be used as analyte binding moieties in analyte capture agents or for IHC / IF applications disclosed herein include any of the following, including variants thereof: A-ACT, A-AT, ACTH, actin-muscle specific, 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-1 / CD20, 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, calmodulin binding protein, calmopin, calreticulin, 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, CD 23, 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-5 / 6, 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, glycophorin 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, K-ISH, KERPAN, Ki-67, λ-IHC, λ-ISH, LH, lipase, lysozyme (MURA), mammaglobin, MART-1, MBP, M-cell tryptase, MEL-5, Melan-A, , Melan-A / Ki-67, mesothelin, MiTF, MLH-1, MOC-31, MPO, MSH-2, MSH-6, MUC1, MUC2, MUC4, MUC5AC, MUM-1, MYO D1, myogenin, myoglobin, myoin heavy chain, new aspartic proteinase A, NB84a, NEW-N, NF, NK1-C3, NPM, NSE, OCT-2, OCT-3 / 4, OSCAR, p16, p21, p27 / Kip1, p53, p57, p63, p120, P504S, pan-melanoma, PANC.POLY, parvovirus B19, PAX-2, PAX-5, PAX-5 / CD43, PAX5 / 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), synaptobrevin, synuclein, TAU, TCL-1, TCRβ, TdT, thrombomodulin, thyroglobulin, TIA-1, TOXO, TRAP, TriView, TM Chest, TriView TM Prostate, trypsin, TS, TSH, TTF-1, tyrosinase, ubiquitin, urinary protein, VEGF, villin, vimentin (VIM), VIP, VZV, WT1 (M) N-terminus, WT1 (P) C-terminus, ZAP-70.

[0554] In addition, exemplary non-limiting antibodies that can be used as analyte-binding moieties in analyte capture agents or in the 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 (e.g., 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 (e.g., ASK1, MAPK, MEK1, MEK2, MEK3MEK4, MEK5, PAK1, PAK2, PAK4, PAK6), CMGC kinase family (e.g., Cdk2, Cdk4, Cdk5, Cdk6, Cdk7, Cdk9, Erk1, GSK3, Jnk / MAPK8, Jnk2 / MAPK9, JNK3 / MAPK10, p38 / MAPK), and TKL kinase family (e.g., 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, FOXO1, GATA), growth factor receptors, tumor suppressors (e.g., anti-p53, anti-BLM, anti-Cdk2, anti-Chk2, anti-BRCA-1, anti-NBS1, anti-BRCA-2, anti-WRN, anti-PTEN, anti-WT1, anti-p38).

[0555] In some embodiments, the analyte capture agent can bind to an analyte present in the cell. In some embodiments, the analyte capture agent can bind to a cell surface analyte, 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, cell-cell interaction protein complexes, antigen presenting complexes, major histocompatibility complexes, engineered T cell receptors, T cell receptors, B cell receptors, chimeric antigen receptors, extracellular matrix proteins, post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation or lipidation) state, gap junctions and adherens junctions. In some embodiments, the analyte capture agent can be bound to a post-translationally modified cell surface analyte. In such embodiments, the analyte capture agent can be specific to a cell surface analyte based on a given state of post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation or lipidation) such that a cell surface analyte profile may include post-translational modification information of one or more analytes.

[0556] In some embodiments, the analyte c...

Claims

1. A method for generating a spatial array, comprising: (a) immobilizing a first plurality of primers and a second plurality of primers on a substrate, wherein: (i) a first primer of the first plurality of primers comprises one or more functional domains and a sequence complementary to the hybridization domain, wherein the one or more functional domains of the first primer comprises a sequencing specific site or a primer binding site; and (ii) a second primer of the second plurality of primers comprises a sequence complementary to the capture domain; (b) providing a template oligonucleotide comprising a hybridization domain capable of hybridizing to a sequence complementary to the hybridization domain of the first primer, a complement of the spatial barcode, and a complement of the capture domain; (c) hybridizing the hybridization domain of the template oligonucleotide to a sequence complementary to the hybridization domain of the first primer; (d) extending the first primer using the template oligonucleotide as a template to generate a capture probe comprising the first primer, the spatial barcode and the capture domain; and (e) releasing the template oligonucleotide from the capture probes, thereby generating the spatial array. 2 . The method of claim 1 , further comprising amplifying the capture probes to generate clonal clusters of capture probes.

3. The method of claim 2, wherein the amplifying comprises: (a) hybridizing the capture domain of the capture probe to a sequence complementary to the capture domain of the second primer; (b) extending the second primer using the capture probe as a template to generate a copy of the template oligonucleotide immobilized on the substrate; (c) releasing a copy of the template oligonucleotide from the capture probe; (d) hybridizing a copy of the template oligonucleotide to the unextended first primer; and (e) extending the unextended first primer using the copy of the template oligonucleotide as a template to produce a second capture probe immobilized on the substrate, thereby generating clonal clusters of capture probes.

4. The method of claim 3, further comprising extending the template oligonucleotide using the first primer as a template.

5. The method of claim 1, wherein the template oligonucleotide further comprises a unique molecular identifier (UMI) and one or more functional domains, wherein the one or more functional domains of the template oligonucleotide comprise a sequencing specific site or a primer binding site.

6. The method of claim 5, wherein the UMI is disposed between a complement of the spatial barcode and a complement of the capture domain. The method of claim 1 , wherein the first primer and the second primer are fixed to the substrate at their 5′ ends. The method of claim 1 , wherein the ratio of the first primer to the second primer is 1:1.5 to 1.5:

1.

9. The method of claim 1, wherein the first primer and the second primer are separated by less than 5 μm.

10. The method of claim 1, wherein the sequence complementary to the capture domain of the second primer comprises a poly(A) sequence.

11. The method of claim 3, wherein the second primer further comprises a cleavage domain.

12. The method of claim 11, wherein releasing the copy of the template oligonucleotide comprises cleaving the cleavage domain of the second primer and denaturing the copy of the template oligonucleotide from the capture probe.

13. The method of claim 1, wherein the template oligonucleotide is attached to a bead.

14. The method of claim 13, wherein the beads are gel beads.

15. The method of claim 13, wherein the beads have a diameter of 2 microns or less.

16. The method of claim 15, wherein the beads have a diameter of 1 micron or less.

17. The method of claim 13, wherein the beads are removed after the step of extending the first primer.

18. The method of claim 13, wherein the beads are removed between steps (c) and (d).

19. The method of claim 13, wherein the beads are comprised of a plurality of beads dispersed on the substrate such that a monolayer of beads is disposed on the substrate.

20. The method of claim 1, wherein the capture domain comprises a poly(T) sequence.

21. The method of claim 20, wherein the poly(T) sequence hybridizes to an analyte.

22. The method of claim 21, wherein the analyte is mRNA.

23. The method of claim 22, further comprising extending the capture probe using the mRNA as a template to produce an extended capture probe.

24. The method of claim 1, wherein the substrate comprises a glass slide.

25. The method of claim 24, wherein the glass slide is a treated glass slide.

26. The method of claim 1, further comprising determining the sequence of the spatial barcode of the generated capture probe.

27. The method of claim 26, wherein determining the sequence of the spatial barcode of the generated capture probe is performed after release of the template oligonucleotide.

28. The method of claim 27, wherein determining the sequence of the spatial barcode of the generated capture probe comprises sequencing.

29. The method of claim 28, wherein the sequencing comprises sequencing by synthesis (SBS), sequencing by hybridization (SBH), or sequencing by ligation (SBL).

30. A method of generating a spatial array, comprising: (a) providing an array comprising a plurality of oligonucleotides, wherein the 3′ ends of the oligonucleotides in the plurality of oligonucleotides are attached to a substrate; (b) providing a plurality of primers, wherein a primer in the plurality of primers is substantially complementary to a portion of the oligonucleotide; (c) extending the primer using the oligonucleotide as a template, thereby generating a first oligonucleotide having a free 3' end; (d) extending the first oligonucleotide to generate a 3′ overhang; (e) providing a splint oligonucleotide hybridized to the 3' end of the first oligonucleotide; and (f) ligating a second oligonucleotide to the 3' end of the first oligonucleotide, wherein the second oligonucleotide comprises a sequence that is substantially complementary to a portion of the splint oligonucleotide, and wherein the second oligonucleotide comprises a capture domain, thereby generating a spatial array.

31. The method of claim 30, wherein an oligonucleotide in the plurality of oligonucleotides comprises a spatial barcode.

32. The method of claim 30 or 31, wherein an oligonucleotide in the plurality of oligonucleotides comprises a first constant sequence.

33. The method of any one of claims 30-32, wherein an oligonucleotide in the plurality of oligonucleotides further comprises a second constant sequence.

34. The method of any one of claims 30-33, wherein an oligonucleotide in the plurality of oligonucleotides is 20 to 60 nucleotides in length.

35. The method of any one of claims 30-35, wherein the second oligonucleotide further comprises a constant sequence, and wherein the constant sequence is substantially complementary to a portion of the splint oligonucleotide.

36. The method of any one of claims 30-35, wherein the second oligonucleotide further comprises a unique molecular identifier.

37. The method of claim 30, wherein the second oligonucleotide further comprises a spatial barcode.

38. The method of any one of claims 30-38, wherein the capture domain comprises a poly-T sequence.

39. The method of any one of claims 30-38, wherein the capture domain comprises a gene-specific sequence.

40. The method of any one of claims 30-39, wherein the 3'-end overhang of step (d) is generated enzymatically.

41. The method of claim 40, wherein the enzyme is a template-independent polymerase.

42. The method of claim 41, wherein the template-independent polymerase is terminal deoxynucleotidyl transferase (TdT) or poly (A) polymerase.

43. A method for determining the location of an analyte in a biological sample, comprising: (a) contacting a biological sample with a spatial array produced by the method of any one of claims 30-42; (b) permeabilizing the biological sample to allow analytes in the biological sample to specifically bind to the capture domains on the spatial array; and (c) Identifying the analyte that specifically binds to the capture domain, thereby determining the spatial location of the analyte in the biological sample.

44. A method as claimed in claim 43, wherein a second analyte in the biological sample interacts with a second capture domain on the spatial array, and step (c) includes identifying the second analyte specifically bound to the second capture domain, thereby determining the spatial position of the second analyte in the biological sample.

45. A method for preparing a spatial array, comprising: (a) contacting an array comprising one or more sets of first oligonucleotides with a hydrogel comprising one or more sets of second oligonucleotides, wherein a first oligonucleotide in one or more sets of first oligonucleotides comprises a capture domain, a second oligonucleotide in one or more sets of second oligonucleotides comprises a capture domain, or both; (b) attaching the first oligonucleotide to the second oligonucleotide to generate a capture probe comprising a capture domain; (c) Reducing the size of the hydrogel containing the capture probes by removing water to form a shrunk hydrogel containing the capture probes, thereby creating a spatial array.

46. ​​The method of claim 45, wherein one or more sets of second oligonucleotides are immobilized in a hydrogel.

47. The method of claim 46, wherein the immobilized second oligonucleotides are partitioned such that a first population of the immobilized second oligonucleotides is separated from another population of immobilized second oligonucleotides.

48. The method of claim 47, wherein the immobilized second oligonucleotides are partitioned into a plurality of second oligonucleotide partitions such that different populations of immobilized second oligonucleotides are separated from each other.

49. The method of claim 46 or 47, wherein a first immobilized second oligonucleotide of a partitioned population of immobilized second oligonucleotides is different from a second immobilized second oligonucleotide of another partitioned population of immobilized second oligonucleotides.

50. The method of any one of claims 45-49, wherein the first immobilized second oligonucleotide, the second immobilized second oligonucleotide, or both comprise a spatial barcode.

51. The method of claim 50, wherein a first immobilized second oligonucleotide of a partitioned population of immobilized second oligonucleotides comprises a spatial barcode that is different from a spatial barcode of a second immobilized second oligonucleotide of another partitioned population of immobilized second oligonucleotides.

52. A method as described in claim 50 or 51, wherein one or more second oligonucleotide partitions contain a population of fixed capture probes, and the spatial barcode contained in the population of fixed capture probes is different from the spatial barcode of the population of fixed capture probes of another second oligonucleotide partition.

53. The method of any one of claims 45-52, wherein the one or more sets of first oligonucleotides are partitioned.

54. The method of claim 53, wherein the one or more groups of first oligonucleotides are partitioned into first oligonucleotide partitions.

55. The method of claim 53 or 54, wherein one or more first oligonucleotide partitions comprise a population of first oligonucleotides that is different from first oligonucleotides in another first oligonucleotide partition.

56. The method of any one of claims 45-55, wherein a first oligonucleotide of a population of first oligonucleotides in one or more first oligonucleotide partitions comprises a spatial barcode.

57. The method of claim 56, wherein the first oligonucleotides of a population of first oligonucleotides in one or more first oligonucleotide partitions comprise a spatial barcode that is different from the spatial barcode of the first oligonucleotides of a population of first oligonucleotides in another first oligonucleotide partition.

58. A method as claimed in claim 56 or 57, wherein the spatial barcode of the first oligonucleotides of the population of first oligonucleotides in one or more first oligonucleotide partitions is different from the spatial barcode of the second oligonucleotides of the population of second oligonucleotides in one or more second oligonucleotide partitions.

59. The method of any one of claims 56-58, wherein a first oligonucleotide partition comprises a first population of first oligonucleotides comprising a spatial barcode that is different from a spatial barcode of a second population of first oligonucleotides in another first oligonucleotide partition.

60. The method of any one of claims 45-59, wherein the reduction in size of the hydrogel in step (c) is equidistant.

61. The method of any one of claims 45-60, wherein the removal of water in step (c) comprises a dehydrating solvent.

62. The method of claim 61, wherein the solvent comprises at least one of an alcohol, a ketone, an aldehyde, acetonitrile, or a combination thereof.

63. The method of claim 61 or 62, wherein the solvent comprises at least one of glycerol, ethanol, acetonitrile, acetone, 2-propanol, and combinations thereof.

64. The method of any one of claims 45-60, wherein the removal of water in step (c) comprises the use of salt.

65. The method of claim 64, wherein the salt comprises an inorganic salt.

66. The method of any one of claims 45-60, wherein the removal of water in step (c) comprises an acid.

67. The method of any one of claims 45-66, wherein the removal of water in step (c) comprises a dehydration process.

68. The method of any one of claims 45-67, wherein the linear dimension of the shrunken hydrogel is about 2 to about 10 times smaller than the size of the hydrogel prior to shrinking in step (c).

69. The method of any one of claims 45-68, wherein the linear dimension of the shrunken hydrogel is at least 2 times smaller than the pre-shrunken hydrogel.

70. The method of any one of claims 45-69, wherein the linear dimension of the shrunken hydrogel is at least 4 times smaller than the pre-shrunken hydrogel.

71. The method of any one of claims 45-70, wherein the linear dimension of the shrunken hydrogel is at least 8 times smaller than the pre-shrunken hydrogel.

72. The method of any one of claims 45-71, wherein the attaching comprises polymerase chain reaction.

73. The method of claim 72, wherein the hydrogel comprises a DNA polymerase, a plurality of primers, and a plurality of nucleotides.

74. The method of any one of claims 45-73, wherein members of the plurality of first oligonucleotides are cross-linked to the hydrogel.

75. The method of any one of claims 45-74, wherein the capture probe is amplified.

76. The method of claim 75, wherein the capture probes are amplified prior to reducing the size of the hydrogel.

77. The method of any one of claims 45-76, wherein the hydrogel is prepared from hydrogel subunits comprising at least one of acrylamide, bisacrylamide, poly(ethylene glycol), polyaliphatic polyurethanes, polyether polyurethanes, polyester polyurethanes, polyethylene copolymers, polyamides, polyvinyl alcohol, polypropylene glycol, polytetrahydrofuran, polyvinyl pyrrolidone, polyacrylamide, poly(hydroxyethyl acrylate), poly(hydroxyethyl methacrylate), collagen, hyaluronic acid, chitosan, dextran, agarose, gelatin, alginate, protein polymers, methylcellulose, and combinations thereof.

78. The method of any one of claims 45-77, wherein a first oligonucleotide in the one or more sets of first oligonucleotides comprises a spatial barcode.

79. The method of any one of claims 45-78, wherein a first oligonucleotide in the one or more sets of first oligonucleotides comprises a unique molecular identifier.

80. The method of any one of claims 45-79, wherein a first oligonucleotide in the one or more sets of first oligonucleotides comprises a spatial barcode and a capture domain.

81. A method for determining the location of an analyte in a biological sample, comprising: (a) providing a spatial array as described in any one of claims 45 to 80; (b) contacting a biological sample with the spatial array as described in any one of claims 1 to 44 under conditions that allow specific binding of the analyte to the capture probes of the spatial array; and (c) determining the identity of the analyte that specifically binds to the capture probe, thereby determining the location of the analyte in the biological sample.

82. The method of claim 81, wherein the biological sample is a tissue sample.

83. The method of claim 82, wherein the tissue sample is a fresh frozen tissue sample.

84. The method of claim 82, wherein the tissue sample comprises tumor cells.

85. The method of claim 82, wherein the tissue sample comprises a tissue section.

86. The method of any one of claims 82-85, wherein the method further comprises migrating an analyte in the biological sample to the capture probe.

87. The method of claim 86, wherein the migration comprises passive migration.

88. The method of claim 86, wherein the migration comprises active migration.

89. The method of any one of claims 81-88, wherein the analyte comprises at least one of RNA, DNA, protein, lipid, peptide, metabolite, small molecule, and analyte capture agent.

90. A method for generating an array, comprising: (a) providing a substrate comprising a plurality of features immobilized on the substrate, wherein a feature in the plurality of features comprises a first oligonucleotide, and wherein the first oligonucleotide comprises a first spatial barcode; (b) delivering a second oligonucleotide to the first oligonucleotide, wherein the second oligonucleotide comprises a second spatial barcode and a capture domain; and (c) Attaching the first oligonucleotide and the second oligonucleotide, thereby generating a capture probe comprising the first spatial barcode, the second spatial barcode, and the capture domain.

91. The method of claim 90, wherein the first oligonucleotide is associated with one of a plurality of different first spatial barcodes.

92. The method of claim 90 or 91, wherein the second oligonucleotide is associated with one of a plurality of different second spatial barcodes.

93. The method of any one of claims 90-92, wherein step (c) comprises ligating the first oligonucleotide and the second oligonucleotide.

94. The method of claim 93, wherein said ligating comprises using a ligase.

95. The method of any one of claims 90-93, wherein the first oligonucleotide and the second oligonucleotide are attached to each other by a chemical coupling reaction.

96. The method of any one of claims 90-95, wherein the first oligonucleotide comprises a 3' end that is masked by a protecting group.

97. The method of any one of claims 90-96, wherein the protecting group is removed prior to delivering the second oligonucleotide to the first oligonucleotide.

98. The method of claim 97, wherein the protecting group is a photocleavable protecting group.

99. The method of claim 98, wherein the photocleavable protecting group is removed by exposing the first oligonucleotide to light.

100. The method of claim 98, wherein the photocleavable protecting group coupled to the first oligonucleotide is protected by applying a photomask.

101. The method of any one of claims 90-100, wherein the first feature is separated from other features of the plurality by one or more physical barriers, wherein the one or more physical barriers form one or more chambers, and wherein the first oligonucleotide is delivered to a chamber in the one or more chambers.

102. The method of claim 101, wherein the one or more physical barriers comprise a hydrophobic surface.

103. The method of claim 101 or 102, wherein the one or more physical barriers comprise one or more chamber walls having a tapered end in contact with the substrate.

104. The method of any one of claims 90-103, wherein the first oligonucleotide comprises a unique molecular identifier.

105. A method for generating an array, comprising: (a) providing a substrate comprising two or more partitions, wherein a first partition of the two or more partitions comprises a feature immobilized on the substrate, wherein the feature comprises a first oligonucleotide, and wherein the first oligonucleotide comprises a first spatial barcode; (b) delivering a second oligonucleotide to the first partition, wherein the second oligonucleotide comprises a second spatial barcode and a capture domain; and (c) attaching a first oligonucleotide and a second oligonucleotide in the first partition, thereby generating a capture probe comprising a first spatial barcode, a second spatial barcode, and a capture domain in the first partition.

106. The method of claim 105, further comprising: (d) delivering a third oligonucleotide to a second partition of the two or more partitions, wherein the third oligonucleotide comprises a third spatial barcode and a second capture domain, wherein the second partition comprises a second feature, wherein the second feature comprises a fourth oligonucleotide comprising the first spatial barcode; and (e) Attaching a third oligonucleotide and a fourth oligonucleotide, thereby generating a second capture probe comprising the first spatial barcode, the third spatial barcode, and the second capture domain in the second partition.

107. The method of claim 105 or 106, wherein the two or more partitions are separated by one or more physical barriers.

108. The method of claim 107, wherein the one or more physical barriers are printed on the substrate.

109. The method of claim 107 or 108, wherein the one or more physical barriers form one or more chambers comprising one or more chamber walls.

110. The method of any one of claims 107-109, wherein the one or more chamber walls comprise a hydrophobic surface.

111. The method of any one of claims 107-110, wherein the one or more chamber walls include a tapered end that contacts the substrate.

112. The method of any one of claims 105-111, wherein the second oligonucleotide is delivered using a microfluidic device.

113. A method for enriching a population of capture probes, comprising: (a) providing a plurality of capture probes, wherein: (i) the plurality of capture probes comprises a plurality of different spatial barcodes, and (ii) the target capture probe of the population of capture probes to be enriched comprises a target spatial barcode; and (b) Enrichment of a population of target capture probes containing a target spatial barcode from a plurality of capture probes.

114. A method for determining the location of an analyte in a biological sample, comprising: (a) providing a substrate comprising a plurality of capture probes, wherein a subset of the plurality of capture probes is attached to one location on the substrate, and wherein two or more capture probes of the subset of capture probes comprise a first common spatial barcode; (b) contacting the biological sample with a substrate, such that capture probes of a subset of capture probes specifically bind to analytes from the biological sample, wherein the analytes specifically bound to the capture probes comprise the first common spatial barcode or its complement; (c) identifying a region of interest within the biological sample, wherein the region of interest comprises an analyte that specifically binds to the capture probe; and (d) Enriching the analyte that specifically binds to the capture probe to determine the location of the analyte in the biological sample.

115. The method of claim 114, wherein the substrate further comprises a second subset of capture probes attached to a second location on the substrate, wherein two or more capture probes of the second subset of capture probes comprise a second common spatial barcode that is different from the first common spatial barcode.

116. A method as described in claim 114 or 115, wherein the substrate further comprises a third subset of capture probes attached at a third position on the substrate, wherein two or more capture probes of the third subset of capture probes comprise a third common spatial barcode that is different from the first common spatial barcode and the second common spatial barcode.

117. The method of claims 114-116, wherein the capture probe comprises a capture domain.

118. The method of claims 114-117, wherein the analyte comprises at least one of RNA, DNA, and a cell spike.

119. The method of claim 118, wherein the analyte capture agent comprises an antigen binding molecule.

120. The method of claim 118 or 119, wherein the analyte-capture agent comprises a region configured to couple to or specifically bind to the capture domain of the capture probe.

121. The method of claims 114-120, wherein a region of interest is identified by shallowly sequencing at least a subset of a plurality of analytes that specifically bind to capture probes, and identifying a first common spatial barcode associated with the region of interest.

122. The method of claims 114-121, wherein the region of interest is identified by at least one of immunostaining, immunohistochemistry, microscopy, visual inspection, and combinations thereof.

123. A method as described in claims 114-122, wherein step (d) comprises performing a nucleic acid extension reaction using (i) an analyte that specifically binds to a capture probe and (ii) a primer comprising a sequence specific to a first common spatial barcode or its complement to produce an enriched plurality of analytes that specifically bind to the capture probe, wherein the capture probe comprises the first common barcode or its complement.

124. The method of any one of claims 114-123, wherein the analyte is amplified by performing a polymerase chain reaction (PCR).

125. The method of any one of claims 114-124, further comprising enriching the population of capture probes present in the sub-region of interest: (a) wherein the sub-region of interest is located within the region of interest, wherein the sub-region of interest comprises a subset of capture probes, and wherein: (i) two or more capture probes in the subset of capture probes comprise a first common subspatial barcode; and (ii) a first common subspatial barcode sequence is associated with the subregion of interest; and (b) Enrichment of a population of capture probes comprising a first common subspatial barcode.

126. The method of claim 125, further comprising enriching the population of capture probes present in the sub-subregion of interest: (a) wherein the sub-subregion of interest is located within the sub-region of interest, wherein the sub-subregion of interest comprises a sub-multiple capture probes, and wherein: (i) two or more capture probes in the sub-plurality of capture probes comprise a first common sub-subspatial barcode; and (ii) a first common sub-subspace barcode is associated with the sub-subregion of interest; and (b) Enrichment of a population of capture probes containing the first sub-subspace barcode.

127. The method of any one of claims 114-126, wherein the first common spatial barcode is associated with a single cell of the biological sample.

128. The method of any one of claims 114-127, wherein the analyte comprises mRNA.

129. The method of any one of claims 114-127, wherein the analyte comprises a protein.

130. The method of any one of claims 114-127, wherein the analyte comprises an antibody.

131. The method of any one of claims 114-127, wherein the analyte comprises DNA.

132. The method of claim 131, wherein the DNA encodes a T cell receptor.

133. The method of claim 131, wherein the DNA comprises one or more chromatin regions in a biological sample.

134. A method for generating a spatial array, comprising: (a) providing a substrate comprising a plurality of first oligonucleotides, wherein a first oligonucleotide of the plurality of first oligonucleotides is attached to a region of the substrate via its 3′-end or 5′-end; (b) attaching a second oligonucleotide to the unattached end of the first oligonucleotide, wherein the second oligonucleotide comprises a spatial barcode.

135. The method of claim 134, wherein the method further comprises, after (b): (c) Attaching a third oligonucleotide to the unattached end of the second oligonucleotide.

136. The method of claim 134 or claim 135, wherein the first oligonucleotide is attached to the substrate via its 5'-end and the second oligonucleotide is produced on the 3'-end of the first oligonucleotide using spatially directed in situ synthesis.

137. The method of claim 134 or claim 135, wherein the first oligonucleotide is attached to the substrate via its 3'-end and the second oligonucleotide is produced on the 5'-end of the first oligonucleotide using spatially directed in situ synthesis.

138. The method of any one of claims 134-137, wherein the phosphodiester and exocyclic amine portions of the first oligonucleotide are protected.

139. The method of any one of claims 134-138, wherein the length of the second oligonucleotide is selected from 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides or 30 nucleotides.

140. The method of any one of claims 136-139, wherein the in situ synthesis is photodirected synthesis.

141. The method of any one of claims 134-140, wherein the area of ​​the substrate is less than about 2500 μm 2 , less than about 1600μm 2 , less than about 400μm 2 , less than about 100μm 2 , less than about 50μm 2 , less than about 25μm 2 , less than about 15μm 2 , less than about 10μm 2 or less than about 5 μm 2 .

142. The method of any one of claims 134-141, wherein two or more second oligonucleotides are attached to the two or more first oligonucleotides, wherein a member of the two or more second oligonucleotides is different from another member of the two or more second oligonucleotides.

143. The method of any one of claims 134-142, wherein the first oligonucleotide comprises protected phosphodiester and exocyclic amine moieties.

144. The method of any one of claims 134-143, wherein the substrate further comprises a substantially complementary oligonucleotide that hybridizes to the first oligonucleotide.

145. The method of any one of claims 134-144, wherein the first oligonucleotide is at least about 2 nucleotides, 5 nucleotides, 8 nucleotides, 10 nucleotides, 15 nucleotides, 18 nucleotides, 20 nucleotides, 22 nucleotides, 25 nucleotides, 27 nucleotides, 30 nucleotides, 33 nucleotides, or 35 nucleotides in length.

146. The method of any one of claims 134-145, wherein the first oligonucleotide is about 2 to 45 nucleotides, 5 to 40 nucleotides, 15 to 40 nucleotides, 25 to 40 nucleotides, 25 to 35 nucleotides, or 27 to 33 nucleotides in length.

147. The method of any one of claims 134-146, wherein the first oligonucleotide is prepared by synthesis outside the substrate and the first oligonucleotide is subsequently attached to the substrate.

148. The method of claim 147, wherein the phosphodiester and exocyclic amine portions of the first oligonucleotide are protected prior to attachment of the first oligonucleotide to a substrate.

149. The method of claim 147 or 148, wherein the phosphodiester and exocyclic amine portions of the first oligonucleotide are protected after the first oligonucleotide is attached to the substrate.

150. The method of any one of claims 134-146, wherein the first oligonucleotide is prepared by in situ synthesis on the substrate.

151. The method of claim 150, wherein the in situ synthesis is not photo-directed synthesis.

152. The method of claim 150, wherein the in situ synthesis is not a spatially directed synthesis.

153. The method of any one of claims 134-152, wherein the first oligonucleotide comprises a sequence comprising a cleavage site.

154. The method of any one of claims 134-153, wherein the first oligonucleotide is attached to the substrate via a linker.

155. The method of claim 154, wherein the linker comprises a chemical group comprising a silane, an amine, a peptide, polyethylene glycol, biotin, desthiobiotin, streptavidin, a thiol, an acrylate, an azide, digoxigenin, a hexynyl, or a combination thereof.

156. The method of claim 155, wherein the linker is a branched linker or a dendritic linker.

157. The method of any one of claims 135-156, wherein the third oligonucleotide is attached to the second oligonucleotide by ligation.

158. The method of claim 157, wherein any protected portions of the first and second oligonucleotides are removed prior to ligating the third oligonucleotide to the second oligonucleotide.

159. The method of any one of claims 135-158, wherein the plurality of third oligonucleotides is at least about 5 nucleotides, 7 nucleotides, 9 nucleotides, 13 nucleotides, 20 nucleotides, 25 nucleotides, 27 nucleotides, 30 nucleotides, 33 nucleotides, or 35 nucleotides in length.

160. The method of any one of claims 135-159, wherein the plurality of third oligonucleotides is about 5 to 40 nucleotides, 5 to 20 nucleotides, 5 to 10 nucleotides, 10 to 35 nucleotides, 15 to 30 nucleotides, 25 to 35 nucleotides, or 27 to 33 nucleotides in length.

161. The method of any one of claims 135-160, wherein the third oligonucleotide comprises a capture domain at the 3'-end that is at least 6 nucleotides in length.

162. The method of claim 161, wherein the capture domain comprises a poly-T sequence.

163. The method of any one of claims 135-162, wherein the third oligonucleotide comprises a random sequence of 6 to 20 nucleotides in length.

164. A composition comprising a plurality of oligonucleotides attached to a plurality of regions on a substrate, wherein an oligonucleotide in the plurality of oligonucleotides comprises: a first oligonucleotide having a length of 2 to 50 nucleotides attached to the substrate via its 3' end or 5' end; and A second oligonucleotide of 8 to 30 nucleotides in length is attached to the unattached end of the first oligonucleotide, wherein the second oligonucleotide comprises a spatial barcode.

165. The composition of claim 164, wherein the first oligonucleotide and the second oligonucleotide comprise protected phosphodiester and exocyclic amine moieties.

166. The composition of any one of claims 164-165, wherein a first oligonucleotide is attached to the substrate via its 5' end and a second oligonucleotide is attached to the 3' end of the first oligonucleotide.

167. The composition of any one of claims 164-166, wherein a first oligonucleotide is attached to the substrate via its 3' end and a second oligonucleotide is attached to the 5' end of the first oligonucleotide.

168. The composition of any one of claims 164-167, wherein the oligonucleotide further comprises: A third oligonucleotide of 5 to 40 nucleotides in length is attached to the unattached end of the second oligonucleotide, wherein the third oligonucleotide comprises a capture domain.

169. The composition of any one of claims 164-168, wherein the length of the second oligonucleotide is selected from 8 nucleotides, 9 nucleotides, 10 nucleotides, 11 nucleotides, 12 nucleotides, 13 nucleotides, 14 nucleotides, 15 nucleotides, 16 nucleotides, 17 nucleotides, 18 nucleotides, 19 nucleotides, 20 nucleotides, 21 nucleotides, 22 nucleotides, 23 nucleotides, 24 nucleotides, 25 nucleotides, 26 nucleotides, 27 nucleotides, 28 nucleotides, 29 nucleotides, or 30 nucleotides.

170. The composition of any one of claims 164-169, wherein the area is less than about 2500 μm 2 , less than about 1600μm 2 , less than about 400μm 2 , less than about 100μm 2 , less than about 50μm 2 , less than about 25μm 2 , less than about 15μm 2 , less than about 10μm 2 , less than about 5μm 2 or less than about 1 μm 2 .

171. The composition of any one of claims 164-170, wherein the oligonucleotides in each region are at least 15 nucleotides, at least 20 nucleotides, at least 40 nucleotides, at least 50 nucleotides, or at least 60 nucleotides in length.

172. A composition as described in any of claims 164-171, wherein the length of the oligonucleotides in each region is 15 to 100 nucleotides, 20 to 100 nucleotides, 40 to 100 nucleotides, 50 to 90 nucleotides or 60 to 80 nucleotides.

173. A spatial array produced by the method of any one of claims 1-29.

174. A spatial array comprising: (a) substrate; and (b) a plurality of sets of primers immobilized on a substrate, wherein a first set of the plurality of sets of primers comprises a first primer and a second primer; and The first primer comprises a sequence complementary to the capture probe.

175. The spatial array of claim 174, further comprising a template oligonucleotide comprising a reverse complement sequence of a unique molecular identifier (UMI), and wherein the capture probe comprises the UMI.

176. The spatial array of claim 174 or 175, wherein the first primer is attached to the substrate at the 5' end of the first primer.

177. A spatial array as described in any of claims 174-176, wherein the first primer comprises a sequence complementary to the template oligonucleotide.

178. A spatial array as described in any of claims 174-177, wherein the first primer comprises a poly (A) domain.

179. A spatial array as described in any of claims 174-178, wherein the first primer comprises a cleavage domain.

180. The spatial array of any one of claims 174-179, wherein the second primer is attached to the substrate at the 5' end of the second primer.

181. A spatial array as described in any of claims 174-180, wherein the second primer comprises a cleavage domain.

182. A spatial array as described in any of claims 174-181, wherein the template oligonucleotide is attached to a bead.

183. A spatial array as described in claim 182, wherein the beads are gel beads.

184. A spatial array as described in claim 182 or 183, wherein the beads have a diameter of 2 microns or less.

185. The spatial array of claim 182 or 183, wherein the beads have a diameter of 1 micron or less.

186. The spatial array of any one of claims 174-185, wherein the substrate comprises a glass slide.

187. The spatial array of claim 186, wherein the slide is a treated glass slide.

188. The spatial array of any one of claims 174-187, wherein the ratio of the first primer to the second primer is 1:1.5 to 1.5:

1.

189. The method of any one of claims 174-188, wherein the first primer and the second primer are separated by less than 5 μm.

190. A method of producing a capture probe bound to a substrate, comprising: dispensing a second solution onto the portion of the surface of the substrate coated with the first solution, wherein after dispensing, molecules in the first solution and the second solution do not substantially mix with each other, Wherein the second solution comprises capture probes, and dispensing results in attachment of the capture probes to the substrate surface.

191. The method of claim 190, further comprising, after step (a): curing the second solution; and The first solution is removed from the substrate, wherein the solidified second solution remains on the surface of the substrate.

192. The method of claim 190 or 191, wherein the area of ​​the substrate surface to which the capture probes are attached is smaller than the corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution.

193. The method of claim 190 or 191, wherein the area of ​​the substrate surface to which the capture probes are attached is approximately two times smaller than the corresponding area produced by dispensing the second solution onto the substrate surface in the absence of the first solution.

194. The method of claim 190, further comprising: The first solution and the second solution are cured on the substrate, thereby producing a matrix.

195. The method of claim 194, wherein the first and second solutions are solidified by chemical methods.

196. The method of claim 194, wherein the first and second solutions are cured by photoreaction.

197. The method of any one of claims 194-196, further comprising: The substrate is shrunk to produce a shrunk substrate having regions with attached capture probes, wherein the area in the shrunk substrate is reduced compared to a corresponding area in the substrate.

198. The method of claim 190, wherein the method further comprises, prior to the step of dispensing the second solution: The first solution is solidified to form a first solution matrix, wherein the first solution matrix is ​​reversibly swellable along one or more axes thereof.

199. The method of claim 198, wherein the step of dispensing the second solution onto a portion of the substrate comprises dispensing the second solution onto a surface of the first solution matrix in a swollen state.

200. The method of claim 199, wherein the method further comprises, after the dispensing step: The first solution matrix is ​​contracted along one or more axes of the substrate surface.

201. The method of any one of claims 190-200, wherein the viscosity of the first solution is approximately 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, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or 10 times greater than the viscosity of the second solution.

202. The method of any one of claims 190-201, wherein the first solution has a lower nucleic acid diffusion rate than the second solution.

203. The method of any one of claims 190-202, wherein the capture probe comprises a spatial barcode.

204. The method of any one of claims 190-203, wherein the capture probe comprises a unique molecular identifier.

205. The method of any one of claims 190-204, wherein the capture probe comprises a functional domain.

206. The method of any one of claims 190-205, wherein the capture probe comprises a cleavage domain, a spatial barcode, a unique molecular identifier, a functional domain, and a capture domain.

207. A method of producing a capture probe array bound to a substrate, comprising: dispensing droplets of the second solution onto portions of the surface of the substrate coated with the first solution, wherein after dispensing, molecules in the droplets of the first solution and the second solution do not substantially mix with each other, Wherein the second solution comprises capture probes, and dispensing results in attachment of the capture probes to the substrate surface.

208. The method of claim 207, further comprising: curing the second solution; and The first solution is removed from the substrate, wherein the solidified second solution remains on the surface of the substrate.

209. The method of claim 207 or 208, wherein the area of ​​the substrate surface to which the capture probes are attached is smaller than the corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution.

210. The method of claim 209, wherein the area of ​​the substrate surface to which the capture probes are attached is about two times smaller than the corresponding area created by dispensing the second solution onto the substrate surface in the absence of the first solution.

211. The method of any one of claims 207-210, further comprising: The first solution and the second solution are cured on the substrate, thereby producing a matrix.

212. The method of claim 211, wherein the first solution and the second solution are solidified by chemical methods.

213. The method of claim 211, wherein the first and second solutions are cured by photoreaction.

214. The method of any one of claims 211 to 213, further comprising: The substrate is shrunk to produce a shrunk substrate having regions with attached capture probes, wherein the area in the shrunk substrate is reduced compared to a corresponding area in the substrate.

215. The method of any one of claims 207-214, wherein the method further comprises, before the step of dispensing the second solution: The first solution is solidified to form a first solution matrix, wherein the first solution matrix is ​​reversibly swellable along one or more axes thereof.

216. The method of claim 215, wherein the step of dispensing the second solution onto a portion of the substrate comprises dispensing the second solution onto a surface of the first solution matrix in a swollen state.

217. The method of claim 216, wherein the method further comprises, after the dispensing step: The first solution matrix is ​​contracted along one or more axes of the substrate surface.

218. The method of any one of claims 207-217, wherein the viscosity of the first solution is approximately 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, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.5 times, 3.0 times, 4.0 times, 5.0 times, 6.0 times, 7.0 times, 8.0 times, 9.0 times, or 10 times greater than the viscosity of the second solution.

219. The method of any one of claims 207-218, wherein the first solution has a lower nucleic acid diffusion rate than the second solution.

220. The method of any one of claims 207-217, wherein the capture probe comprises a spatial barcode.

221. The method of any one of claims 207-220, wherein at least one of the capture probes comprises a unique molecular identifier.

222. The method of any one of claims 207-221, wherein the capture probe comprises a functional domain.

223. The method of any one of claims 207-222, wherein the capture probe comprises a cleavage domain, a spatial barcode, a unique molecular identifier, a functional domain, and a capture domain.

224. A method for spatial profiling of an analyte in a biological sample, comprising: (a) dispensing a droplet of a second solution onto a portion of a surface of a substrate coated with a first solution, wherein after dispensing, molecules in the first solution and in the droplet of the second solution do not substantially mix with each other, wherein the second solution comprises a capture probe, and the dispensing results in the capture probe being attached to the surface of the substrate; (b) contacting the biological sample with the spatial array, such that analytes present in the biological sample are captured by the capture probes; The spatial distribution of the captured analyte is thereby determined.

225. The method of claim 224, wherein at least one of the capture probes comprises a cleavage domain, a spatial barcode, a unique molecular identifier, a functional domain, and a capture domain.

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