Sorting method using barcoding chamber for single cell workflow

Abstract

The content disclosed herein includes systems, methods, compositions, and kits for methods for distributing sequencing data of single cells to cells. In some embodiments, methods are provided for correlating sequencing data and phenotypic data for single cells. The contents disclosed herein include solid supports, each comprising more than one oligonucleotide barcode, the oligonucleotide barcode comprising a cell marker sequence. Each cell marker sequence may comprise a predetermined chamber index subsequence. The oligonucleotide barcodes located within the same chamber may comprise the same chamber index sub-sequence, and the oligonucleotide barcodes located within different chambers may comprise different chamber index sub-sequences.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Application No. 63 / 387,997, filed on December 19, 2022; and U.S. Provisional Application No. 63 / 607,516, filed on December 7, 2023. The entire contents of these applications are hereby expressly incorporated by reference in their entirety.

[0003] Background

[0004] Field

[0005] The present disclosure generally relates to the field of molecular biology, such as determining gene expression using molecular barcoding.

[0006] Description of related technologies

[0007] Current techniques allow for the measurement of single-cell gene expression in a massively parallel manner (e.g., >10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells when each cell is co-localized with barcoded reagent beads in a compartment. Phenotypic information about single cells can be derived from experiments performed prior to measuring gene expression and / or protein expression via sequencing. There is a need for compositions, systems, and methods for correlating sequencing data and phenotypic data of single cells. To correlate image- or fluorescence-based sorting with single-cell sequencing data, there is a need for compositions, systems, and methods for sorting cells into compartments such that information about which cell populations are sorted into which compartments is retained. Generally, there is a need for compositions, systems, and methods for assigning sequencing data to the compartments of a microwell array.

[0008] Overview

[0009] The present disclosure includes compositions. In some embodiments, the compositions comprise two or more pluralities of solid supports. In some embodiments, each solid support comprises more than one oligonucleotide barcode, and each of the more than one oligonucleotide barcodes comprises a cell marker sequence, wherein each cell marker sequence comprises a predetermined compartment index subsequence. In some embodiments, the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences.

[0010] In some embodiments, two or more than one solid support includes more than one first solid support and more than one second solid support, wherein the oligonucleotide barcode associated with the more than one first solid support has a first predetermined chamber index subsequence, wherein the oligonucleotide barcode associated with the more than one second solid support has a second predetermined chamber index subsequence, and wherein the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

[0011] In some embodiments, two or more than one solid support includes more than one first solid support and more than one second solid support, wherein the first predetermined chamber index subsequence of the more than one first solid support is selected from a first set of chamber index subsequences, wherein the second predetermined chamber index subsequence of the more than one second solid support is selected from a second set of chamber index subsequences, and wherein each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences. In some embodiments, based on the predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with more than one first solid support or more than one second solid support.

[0012] Two or more than one solid supports can include: more than one third solid support, optionally: (a) the oligonucleotide barcode associated with the more than one third solid support has a third chamber index subsequence and / or (b) the third chamber index subsequence of the more than one third solid support is selected from a third set of chamber index subsequences; more than one fourth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one fourth solid support has a fourth chamber index subsequence and / or (b) the fourth chamber index subsequence of the more than one fourth solid support is selected from a fourth set of chamber index subsequences; more than one fifth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one fifth solid support has a fifth chamber index subsequence and / or (b) the fifth chamber index subsequence of the more than one fifth solid support is selected from a fifth set of chamber index subsequences; more than one sixth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one sixth solid support has a sixth chamber index subsequence and / or (b) the sixth chamber index subsequence of the more than one sixth solid support is selected from a sixth set of chamber index subsequences; more than one seventh solid support, optionally: (a) the oligonucleotide barcode associated with the more than one seventh solid support has a seventh chamber index subsequence and / or (b) the seventh chamber index subsequence of the more than one seventh solid support is selected from a seventh set of chamber index subsequences; and / or more than one eighth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one eighth solid support has an eighth chamber index subsequence and / or (b) the eighth chamber index subsequence of the more than one eighth solid support is selected from an eighth set of chamber index subsequences. In some embodiments, the first chamber index subsequence, the second chamber index subsequence, the third chamber index subsequence, the fourth chamber index subsequence, the fifth chamber index subsequence, the sixth chamber index subsequence, the seventh chamber index subsequence, and / or the eighth chamber index subsequence do not share sequences with each other. In some embodiments, based on a predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with the first solid support, the second solid support, the third solid support, the fourth solid support, the fifth solid support, the sixth solid support, the seventh solid support, or the eighth solid support.

[0013] In some embodiments, the cell label comprises more than one cell label portion and one or more linkers. In some embodiments, the cell label comprises a first cell label portion, a first linker, and a second cell label portion, optionally the cell label comprises a second linker and a third cell label portion, and further optionally the cell label comprises a third linker and a fourth cell label portion. In some embodiments, the predetermined chamber index subsequence comprises the first cell label portion, the second cell label portion, the third cell label portion, the fourth cell label portion, or any combination thereof. In some embodiments, the first set of chamber index subsequences, the second set of chamber index subsequences, the third set of chamber index subsequences, the fourth set of chamber index subsequences, the fifth set of chamber index subsequences, the sixth set of chamber index subsequences, the seventh set of chamber index subsequences, and / or the eighth set of chamber index subsequences comprises a set of fewer than about 960, about 864, about 768, about 672, about 576, about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences that are different from the chamber index subsequences of other sets of chamber index subsequences. In some embodiments, the first cell label portion, the second cell label portion, the third cell label portion, the fourth cell label portion, or any combination thereof is selected from a set of fewer than about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences. In some embodiments, based on the sequences of the first cell label portion, the second cell label portion, the third cell label portion, the fourth cell label portion, or any combination thereof from sequencing reads derived from an oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with a first solid support, a second solid support, a third solid support, a fourth solid support, a fifth solid support, a sixth solid support, a seventh solid support, or an eighth solid support.

[0014] The present disclosure includes methods. In some embodiments, the method includes: distributing more than one precursor first solid support and a first population of first oligonucleotides into more than one first partitions, wherein the co-localized solid support and the first oligonucleotides become associated; distributing more than one precursor second solid support and a second population of first oligonucleotides into more than one second partitions, wherein the co-localized solid support and the first oligonucleotides become associated; pooling more than one precursor first solid supports associated with the first oligonucleotides; pooling more than one precursor second solid supports associated with the first oligonucleotides; distributing more than one precursor first solid supports associated with the first oligonucleotides and a first population of second oligonucleotides into more than one first partitions of a second set, wherein the co-localized solid support and the second oligonucleotides become associated; distributing more than one precursor second solid supports associated with the first oligonucleotides and a second population of second oligonucleotides into more than one second partitions of a second set, wherein the co-localized solid support and the second oligonucleotides become associated; pooling more than one precursor first solid supports associated with the first and second oligonucleotides; pooling more than one precursor second solid supports associated with the first and second oligonucleotides; distributing more than one precursor first solid supports associated with the first and second oligonucleotides and a first population of third oligonucleotides into more than one first partitions of a third set, wherein the co-localized solid support and the third oligonucleotides become associated; and distributing more than one precursor second solid supports associated with the first and second oligonucleotides and a second population of third oligonucleotides into more than one second partitions of a third set, wherein the co-localized solid support and the third oligonucleotides become associated. In some embodiments, the method includes: pooling more than one precursor first solid supports associated with the first, second, and third oligonucleotides to produce more than one first solid supports; and pooling more than one precursor second solid supports associated with the first, second, and third oligonucleotides to produce more than one second solid supports.

[0015] In some embodiments, (i) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different; (ii) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are the same; (iii) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are different; (iv) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are different; (v) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are the same; (vi) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different; or (vii) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are the same.

[0016] The first and second populations of the first oligonucleotide can each comprise about 192 first cell marker portions having different sequences. In some embodiments, the first and second populations of the second oligonucleotide each comprise about 192 second cell marker portions having different sequences. In some embodiments, the first and second populations of the third oligonucleotide each comprise about 192 third cell marker portions having different sequences.

[0017] In some embodiments, the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different. In some embodiments, the first and second populations of the third oligonucleotide comprise a non-overlapping subset of the cell marker portion sequences of the first and second populations of the first oligonucleotide and / or the first and second populations of the second oligonucleotide.

[0018] The first and second populations of the first oligonucleotides can each comprise about 384 first cell marker moieties having different sequences. In some embodiments, the first and second populations of the second oligonucleotides each comprise about 384 second cell marker moieties having different sequences. In some embodiments, the first and second populations of the third oligonucleotides each comprise about 48 third cell marker moieties having different sequences.

[0019] In some embodiments, the method comprises: distributing more than one precursor first solid support associated with the first, second, and third oligonucleotides and a fourth oligonucleotide to more than one first partitions, wherein the co-localized solid support and the fourth oligonucleotide become associated; distributing more than one precursor second solid support associated with the first, second, and third oligonucleotides and a fifth oligonucleotide to more than one second partitions, wherein the co-localized solid support and the fifth oligonucleotide become associated; pooling more than one precursor first solid support associated with the first, second, third, and fourth oligonucleotides to produce more than one first solid support; and pooling more than one precursor second solid support associated with the first, second, third, and fifth oligonucleotides to produce more than one second solid support.

[0020] In some embodiments, the first and second populations of the first oligonucleotides are the same; the first and second populations of the second oligonucleotides are the same; the first and second populations of the third oligonucleotides are the same; and the fifth and sixth oligonucleotides are different.

[0021] In some embodiments, the first oligonucleotide comprises a first cell marker moiety and a first linker or its complement. In some embodiments, the second oligonucleotide comprises a first linker, a second cell marker moiety, and a second linker or its complement. In some embodiments, the third oligonucleotide comprises a second linker and a third cell marker moiety or its complement, optionally the second oligonucleotide further comprises a third linker or its complement. In some embodiments, the fourth oligonucleotide comprises a third linker and a fourth cell marker moiety or its complement.

[0022] In some embodiments, the first oligonucleotide and the second oligonucleotide are configured to be joined via a first linker. In some embodiments, the second oligonucleotide and the third oligonucleotide are configured to be joined via a second linker. In some embodiments, the third oligonucleotide and the fourth oligonucleotide are configured to be joined via a third linker. In some embodiments, more than one first solid support and / or more than one second solid support comprises at least about 1000, about 10000, about 100000, about 1000000, about 7000000, about 10000000, about 56000000 unique cell marker sequences. In some embodiments, the sequences of the first cell marker portion, the second cell marker portion, and / or the third cell marker portion are the same. In some embodiments, the sequences of the first cell marker portion, the second cell marker portion, and / or the third cell marker portion are different. In some embodiments, the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, and / or the fourth oligonucleotide are single-stranded, double-stranded, and / or comprise one or two single-stranded overhangs. In some embodiments, the first solid support and the second solid support each comprise more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence.

[0023] In some embodiments, the oligonucleotide barcode associated with more than one first solid support has a first predetermined chamber index subsequence, the oligonucleotide barcode associated with more than one second solid support has a second predetermined chamber index subsequence, and the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

[0024] The first predetermined chamber index subsequence of more than one first solid support can be selected from a first set of chamber index subsequences, the second predetermined chamber index subsequence of more than one second solid support is selected from a second set of chamber index subsequences, and each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences. The methods provided herein can produce the compositions provided herein.

[0025] In some embodiments, the method comprises: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning more than one first solid support and more than one second solid support into partitions of an identified first chamber and an identified second chamber, respectively, in more than one chamber. In some embodiments, distributing the oligonucleotide into more than one partition comprises providing more than one partition comprising the oligonucleotide. In some embodiments, the more than one partition comprises a 384-well plate, a 288-well plate, a 192-well plate, a 96-well plate, or a 48-well plate.

[0026] In some embodiments, the solid support comprises synthetic particles. In some embodiments, at least one of the more than one oligonucleotide barcodes is immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, partially encapsulated in the synthetic particles, or a combination thereof. In some embodiments, the synthetic particles are destructible. In some embodiments, the synthetic particles comprise beads. In some embodiments, the beads include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microspheres, anti-fluorescent dye microspheres, or any combination thereof. In some embodiments, the synthetic particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. In some embodiments, the synthetic particles comprise destructible hydrogel particles.

[0027] The disclosure herein includes methods of assigning sequencing data to chambers. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into a partition of an identified chamber of the more than one chamber, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein oligonucleotide barcodes located within the same chamber comprise the same chamber index subsequence, and wherein oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into a partition of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, wherein the single cells comprise copies of nucleic acid targets, wherein single cells of the same population are located within the same chamber, and wherein single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0028] The disclosure herein includes methods for assigning sequencing data to chambers. In some embodiments, the method includes: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each containing a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single cell populations into the partitions of the identified chambers in more than one chamber, wherein each of the two or more single cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located in the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of more than one single cell from at least one single cell population using more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber in more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0029] The disclosure herein includes methods for assigning sequencing data to single cell populations. In some embodiments, the method includes: providing a microwell array including more than one chamber, wherein each chamber includes more than one partition; partitioning each of two or more than one solid supports into a partition of an identified chamber of the more than one chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each contain a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single cell populations into a partition of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a population of the two or more single cell populations based on the chamber assigned to the sequencing read.

[0030] The disclosure herein includes methods of assigning sequencing data to single cell populations. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each contain a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single cell populations into the partitions of the identified chambers of more than one chamber, wherein each of the two or more single cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a population of the two or more single cell populations based on the chamber to which the sequencing read is assigned.

[0031] The disclosure herein includes methods for correlating sequencing data and phenotypic data of a population of single cells. In some embodiments, the methods include: obtaining phenotypic data for each of two or more populations of single cells; providing a microwell array including more than one chamber, wherein each chamber includes more than one partition; partitioning each of two or more than one solid supports into partitions of an identified chamber of the more than one chamber, wherein each solid support includes more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each including a cell marker sequence, wherein each cell marker sequence includes a predetermined chamber index subsequence, wherein oligonucleotide barcodes associated with the same solid support include the same cell marker sequence, wherein oligonucleotide barcodes associated with different solid supports include different cell marker sequences, wherein oligonucleotide barcodes located within the same chamber include the same chamber index subsequence, and wherein oligonucleotide barcodes located in different chambers include different chamber index subsequences; partitioning each of two or more populations of single cells into partitions of an identified chamber of the more than one chamber, wherein each of the two or more populations of single cells includes more than one single cell, wherein the single cells include copies of nucleic acid targets, wherein single cells of the same population are located within the same chamber, and wherein single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one population of single cells using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read including the more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and correlating the sequencing data and the phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0032] The disclosure herein includes methods for correlating sequencing data and phenotypic data of a population of single cells. In some embodiments, the methods include: obtaining phenotypic data for each of two or more populations of single cells; providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes contains a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of the two or more populations of single cells into the partitions of an identified chamber in the more than one chamber, wherein each of the two or more populations of single cells contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein single cells of the same population are located within the same chamber, and wherein single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one single cell in the more than one single cell from at least one population of single cells using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and correlating the sequencing data and phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0033] The method can include: assigning each of the more than one sequencing reads to a population of two or more populations of single cells based on the chamber assigned to the sequencing read. The method can include: obtaining phenotypic data for each of two or more populations of single cells; and correlating the sequencing data and phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0034] In some embodiments, two or more than one solid supports are two or more than one solid supports of the compositions provided herein and / or are generated by the methods provided herein. In some embodiments, two or more than one solid supports include more than one first solid support and more than one second solid support. In some embodiments, the oligonucleotide barcode associated with more than one first solid support has a first predetermined chamber index subsequence. In some embodiments, the oligonucleotide barcode associated with more than one second solid support has a second predetermined chamber index subsequence. In some embodiments, the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

[0035] In some embodiments, two or more than one solid supports include more than one first solid support and more than one second solid support. In some embodiments, the first predetermined chamber index subsequence of more than one first solid support is selected from a first set of chamber index subsequences. In some embodiments, the second predetermined chamber index subsequence of more than one second solid support is selected from a second set of chamber index subsequences. In some embodiments, each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences. In some embodiments, based on the predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with more than one first solid support or more than one second solid support.

[0036] Partitioning each of two or more than one solid supports into an identified chamber among more than one chamber can include partitioning a predetermined more than one solid support selected from two or more than one solid supports into an identified chamber among more than one chamber. In some embodiments, barcoding copies of a nucleic acid target includes: contacting more than one oligonucleotide barcode with copies of the nucleic acid target for hybridization; and extending more than one oligonucleotide barcode hybridized to copies of the nucleic acid target to produce more than one barcoded nucleic acid target.

[0037] The partitioning can be one having a size in the range from about 1,000 μm 3 to about 786,000 μm 3micropores of volumes within the range. In some embodiments, each oligonucleotide barcode contains a molecular marker sequence. In some embodiments, the oligonucleotide barcodes of more than one identical solid support contain the same chamber index subsequence, and wherein the oligonucleotide barcodes of more than one different solid support contain different chamber index subsequences. In some embodiments, each cell label of more than one type of oligonucleotide barcode contains at least 6 nucleotides. In some embodiments, the cell label contains more than one part and one or more linkers. In some embodiments, the cell label contains a first cell label part, a first linker, and a second cell label part, optionally the cell label contains a second linker and a third cell label part, and further optionally the cell label contains a third linker and a fourth cell label part. In some embodiments, the first cell label part contains the chamber index subsequence. The length of the chamber index subsequence can be 2 - 15 nucleotides.

[0038] In some embodiments, the single cell populations are different samples. In some embodiments, each of two or more single cell populations is a biological replicate sample, a technical replicate sample, a control sample, an experimental sample, or a combination thereof. In some embodiments, two or more single cell populations are derived from one or more samples separated based on phenotypic data, optionally more than one single cell includes T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof. In some embodiments, more than one chamber is at least about 4 chambers, about 10 chambers, about 20 chambers, about 40 chambers, about 60 chambers, about 80 chambers, about 100 chambers, about 200 chambers, about 300 chambers, about 400 chambers, about 500 chambers, about 750 chambers, or about 1000 chambers. In some embodiments, more than one partition is at least about 100 partitions, about 500 partitions, about 1000 partitions, about 5000 partitions, about 10000 partitions, about 25000 partitions, about 50000 partitions, about 75000 partitions, or about 100000 partitions. In some embodiments, sequencing of the cell label identifies the starting chamber of each sequenced barcoded nucleic acid target or its product within the micropore array.

[0039] In some embodiments, the partitions in more than one partition contain single cells in more than one single cell and single solid supports in more than one solid support. In some embodiments, more than one barcoded nucleic acid target each contains a sequence complementary to at least a portion of the nucleic acid target and a molecular tag. In some embodiments, each of the more than one sequencing reads contains (1) a cell tag sequence and (2) a molecular tag sequence. In some embodiments, each oligonucleotide barcode contains a first universal sequence. In some embodiments, the oligonucleotide barcode contains a target binding region containing a capture sequence. In some embodiments, the target binding region contains a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof. In some embodiments, partitioning more than one solid support into each of more than one chamber includes depositing each of the more than one solid supports into an identified chamber of a microwell array by flow cytometry. In some embodiments, partitioning each of two or more single cell populations into an identified chamber of more than one chamber includes depositing each single cell population into an identified chamber of a microwell array by flow cytometry. The method can include aligning the sorting component of a flow cytometer with the microwell array.

[0040] Phenotypic data can include event data. Event data can include quantitative biological event data derived from a sorting device. In some embodiments, the event data includes side scatter signals, forward scatter signals, one or more fluorescence signals, or any combination thereof. The method can include performing a correlation analysis on the phenotypic data and the sequencing data of the single cells. In some embodiments, the correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of a therapeutic agent, and / or cellular targets of a candidate therapeutic agent. The method can include lysing one or more single cells. The viability of the single cells can be maintained for a period of time after partitioning and before lysis. The period of time can be at least about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 250 min, 500 min, 750 min, 1000 min, 2500 min, 5000 min, 7500 min, or 10000 min.

[0041] The method can include determining the copy number of a nucleic acid target in one or more of more than one single cell of at least one single cell population. In some embodiments, determining the copy number of a nucleic acid target in one or more of more than one single cell includes determining the copy number of the nucleic acid target in more than one single cell based on the number of molecular tags, their complements, or combinations thereof having different sequences associated with more than one barcoded nucleic acid target or its products.

[0042] The method can include: contacting random primers with more than one barcoded nucleic acid target, wherein each of the random primers comprises a second universal sequence or its complement; and extending the random primers hybridized to the more than one barcoded nucleic acid target to produce more than one extension product. The method can include: amplifying the more than one extension product using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the second universal sequence or its complement, thereby producing a first more than one barcoded amplicon. In some embodiments, amplifying the more than one extension product includes adding a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a sequence of a portion thereof to the more than one extension product. The method can include: determining the copy number of a nucleic acid target in one or more of more than one single cell based on the number of molecular markers having different sequences associated with the first more than one barcoded amplicon or its product. In some embodiments, determining the copy number of a nucleic acid target in one or more of more than one single cell includes determining the number of each of more than one nucleic acid targets in one or more of more than one single cell based on the number of molecular markers having different sequences associated with a barcoded amplicon in the first more than one barcoded amplicon, the first more than one barcoded amplicon including the sequence of each of more than one nucleic acid targets. In some embodiments, the sequence of each of more than one nucleic acid targets includes a subsequence of each of more than one nucleic acid targets. In some embodiments, the sequence of the nucleic acid target in the first more than one barcoded amplicon includes a subsequence of the nucleic acid target. The method can include: amplifying the first more than one barcoded amplicon using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the second universal sequence or its complement, thereby producing a second more than one barcoded amplicon. In some embodiments, amplifying the first more than one barcoded amplicon includes adding a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a sequence of a portion thereof to the first more than one barcoded amplicon. The method can include: determining the copy number of a nucleic acid target in one or more of more than one single cell based on the number of molecular markers having different sequences associated with the second more than one barcoded amplicon or its product. In some embodiments, the first more than one barcoded amplicon and / or the second more than one barcoded amplicon comprises a whole transcriptome amplification (WTA) product.

[0043] The method can include: synthesizing a third plurality of barcoded amplicons using more than one barcoded nucleic acid target as a template to generate a third plurality of barcoded amplicons. In some embodiments, synthesizing the third plurality of barcoded amplicons includes polymerase chain reaction (PCR) amplification of more than one barcoded nucleic acid target. In some embodiments, synthesizing the third plurality of barcoded amplicons includes PCR amplification using primers capable of hybridizing to a first universal sequence or its complement and target-specific primers. The method can include: obtaining sequence data of the third plurality of barcoded amplicons or their products, and optionally obtaining sequence information including attaching sequencing adapters to the third plurality of barcoded amplicons or their products. The method can include: determining the copy number of a nucleic acid target in one or more of more than one single cell based on the number of molecular markers with different sequences associated with the third plurality of barcoded amplicons or their products.

[0044] In some embodiments, the nucleic acid target comprises a nucleic acid molecule. In some embodiments, the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof. In some embodiments, the nucleic acid target comprises a sample index oligonucleotide, and optionally the sample index oligonucleotide comprises a sample index sequence, and the sample index sequences of at least two of more than one sample index compositions comprise different sequences. In some embodiments, the nucleic acid target comprises a cell component binding reagent specific oligonucleotide. In some embodiments, the cell component binding reagent specific oligonucleotide comprises a unique identifier sequence for the cell component binding reagent. In some embodiments, extending more than one oligonucleotide barcode comprises using a reverse transcriptase and / or a DNA polymerase lacking at least one of 5' to 3' exonuclease activity and 3' to 5' exonuclease activity to extend more than one oligonucleotide barcode. In some embodiments, the DNA polymerase comprises the Klenow fragment. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase, optionally wherein the viral reverse transcriptase is murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase. In some embodiments, the first universal sequence and the second universal sequence are the same. In some embodiments, the first universal sequence and the second universal sequence are different. In some embodiments, the first universal sequence and / or the second universal sequence comprises a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a portion thereof. In some embodiments, the sequencing adapter comprises a P5 sequence, a P7 sequence, its complementary sequence, and / or a portion thereof. In some embodiments, the sequencing primer comprises a read 1 sequencing primer, a read 2 sequencing primer, its complementary sequence, and / or a portion thereof. In some embodiments, at least 10 of more than one oligonucleotide barcode comprise different molecular marker sequences. In some embodiments, each molecular marker of more than one oligonucleotide barcode comprises at least 6 nucleotides.

[0045] In some embodiments, the solid support comprises synthetic particles. In some embodiments, the solid support comprises a flat surface. In some embodiments, at least one of more than one oligonucleotide barcode is immobilized on, partially immobilized on, encapsulated in, or partially encapsulated in the synthetic particles. In some embodiments, the synthetic particles are destructible. In some embodiments, the synthetic particles comprise beads. In some embodiments, the beads comprise Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, antibiotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. In some embodiments, the synthetic particles comprise a material selected from: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and combinations thereof. The synthetic particles can be destructible hydrogel particles.

[0046] The present disclosure includes compositions. In some embodiments, the composition comprises: a micro-well array, wherein the micro-well array comprises more than one chamber, wherein each chamber comprises more than one partition, wherein each partition is a micro-well having a volume in the range from about 1,000 μm 3 to about 786,000 μm 3 The composition can comprise: a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

[0047] The present disclosure includes compositions. In some embodiments, the composition comprises: a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, wherein the cartridge comprises a micro-well array, wherein the micro-well array comprises more than one chamber, wherein each chamber comprises more than one partition, wherein each partition is a micro-well having a volume in the range from about 1,000 μm 3 to about 786,000 μm 3 The composition can comprise: a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, wherein the cartridge comprises a micro-well array, wherein the micro-well array comprises more than one chamber, wherein each chamber comprises more than one partition, wherein each partition is a micro-well having a volume in the range from about 1,000 μm

[0048] The disclosure herein includes compositions. In some embodiments, the composition comprises: two or more than one solid support, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcode each comprises a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes of the same more than one solid support comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes of different more than one solid supports comprise different chamber index subsequences. In some embodiments, the two or more than one solid support are the two or more than one solid support of the compositions disclosed herein and / or produced by the methods disclosed herein.

[0049] In some embodiments, a partition in more than one partition comprises a single solid support in more than one solid support, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcode each comprises a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences.

[0050] The composition may include: a chamber index subsequence lookup table. The chamber index subsequence lookup table may identify, for example, the chamber index subsequence associated with each solid support distributed in each microwell of the array.

[0051] In some embodiments, the cartridge is configured to maintain the viability of single cells partitioned within the microwells, optionally for a period of time of at least about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 250 min, 500 min, 750 min, 1000 min, 2500 min, 5000 min, 7500 min, or 10000 min. In some embodiments, the cartridge includes a transparent window for optical imaging of the microwells. The composition can include: an imaging system that is configured to capture and process images of all or a portion of the microwells, wherein the imaging system further includes an illumination subsystem, an imaging subsystem, and a processor. In some embodiments, the imaging system is configured to perform brightfield, darkfield, fluorescence, or quantitative phase imaging. The composition can include: a buffer. The composition can include: one or more reagents for reverse transcription reactions, one or more reagents for amplification reactions, or both.

[0052] In some embodiments, the oligonucleotide barcodes each include a molecular marker sequence. In some embodiments, each cell label of more than one oligonucleotide barcode includes at least 6 nucleotides. In some embodiments, the cell label includes more than one portion and one or more linkers. In some embodiments, the cell label includes a first cell label portion, a first linker, and a second cell label portion, optionally the cell label includes a second linker and a third cell label portion, and further optionally the cell label includes a third linker and a fourth cell label portion. In some embodiments, the first cell label portion includes a chamber index subsequence. In some embodiments, the length of the chamber index subsequence is 2-15 nucleotides. In some embodiments, more than one chamber is at least about 4 chambers, about 10 chambers, about 20 chambers, about 40 chambers, about 60 chambers, about 80 chambers, about 100 chambers, about 200 chambers, about 300 chambers, about 400 chambers, about 500 chambers, about 750 chambers, or about 1000 chambers. In some embodiments, more than one partition is at least about 100 partitions, about 500 partitions, about 1000 partitions, about 5000 partitions, about 10000 partitions, about 25000 partitions, about 50000 partitions, about 75000 partitions, or about 100000 partitions.

[0053] The composition can include: more than one solid support, each of the more than one solid support containing more than one oligonucleotide barcode. In some embodiments, each oligonucleotide barcode contains a molecular marker and a cell marker. In some embodiments, the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, and wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences. In some embodiments, each oligonucleotide barcode contains a first universal sequence. In some embodiments, the oligonucleotide barcode contains a target binding region containing a capture sequence. In some embodiments, the target binding region contains a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof. In some embodiments, each cell marker of more than one oligonucleotide barcode contains at least 6 nucleotides.

[0054] In some embodiments, the solid support includes a flat surface. In some embodiments, the solid support includes synthetic particles. In some embodiments, at least one of the more than one oligonucleotide barcodes is immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, partially encapsulated in the synthetic particles, or a combination thereof. In some embodiments, the synthetic particles are destructible. In some embodiments, the synthetic particles include beads. In some embodiments, the beads include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. In some embodiments, the synthetic particles comprise a material selected from: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and combinations thereof. The synthetic particles can be destructible hydrogel particles. In some embodiments, the composition further comprises instructions for use. Brief Description of the Drawings

[0056] Figure 1 Illustrates non-limiting exemplary barcodes.

[0057] Figure 2 Shows a non-limiting exemplary workflow for barcoding and digital counting.

[0058] Figure 3 Is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of 3'-end barcoded targets from more than one target.

[0059] Figures 4A - 4Bdepicts a non-limiting exemplary schematic diagram of the microwell array ( Figure 4A ) and chamber ( Figure 4B ) provided herein.

[0060] Figure 5 depicts a non-limiting exemplary method for manufacturing an oligonucleotide barcode comprising a chamber index subsequence provided herein.

[0061] Figure 6 depicts a non-limiting exemplary method for manufacturing an oligonucleotide barcode comprising a chamber index subsequence provided herein.

[0062] Figure 7 depicts a non-limiting exemplary method for manufacturing an oligonucleotide barcode comprising a chamber index subsequence provided herein.

[0063] DETAILED DESCRIPTION

[0064] Reference is made in the following detailed description to the accompanying drawings which form a part hereof. In the drawings, like symbols generally identify like components unless the context indicates otherwise. The illustrative embodiments described in the detailed description, drawings, and claims are not meant to be limiting. Other embodiments may be utilized and other changes may be made without departing from the spirit or scope of the subject matter presented herein. It will be readily understood that the aspects of the present disclosure as generally described herein and illustrated in the figures can be arranged, substituted, combined, separated, and designed in a variety of different configurations, all of which are explicitly contemplated herein and form a part of the present disclosure.

[0065] All patents, published patent applications, other publications, and sequences from GenBank and other databases mentioned herein are incorporated by reference in their entirety for relevant art.

[0066] Quantifying small amounts of nucleic acids (e.g., messenger ribonucleic acid (mRNA) molecules) is clinically important for determining genes expressed in cells, for example, at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can also be very challenging, especially when the number of molecules is very small. One method for determining the absolute number of molecules in a sample is digital polymerase chain reaction (PCR). Ideally, PCR produces the same copy of a molecule in each cycle. However, PCR can have drawbacks such that each molecule replicates with a random probability, and this probability varies according to the PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Random barcodes with unique molecular labels (also known as molecular indexes, MI) can be used to count the number of molecules and correct for amplification bias. Such as PreciseTM Assays (Cellular Research, Inc. (Palo Alto, CA)) and Rhapsody TM Random barcoding of assays (Becton, Dickinson and Company (Franklin Lakes, NJ)) can correct biases caused by PCR and library preparation steps by labeling mRNA with molecular labels (ML) during reverse transcription (RT).

[0067] Precise TM Assays can utilize a non-depleting pool of random barcodes with a large number (e.g., 6561 to 65536) of unique molecular label sequences on poly(T) oligonucleotides to hybridize with all poly(A)-mRNA in a sample during the RT step. The random barcodes can contain universal PCR primer sites. During RT, target gene molecules randomly react with the random barcodes. Each target molecule can hybridize with a random barcode, resulting in the generation of randomly barcoded complementary ribonucleic acid (cDNA) molecules. After labeling, the randomly barcoded cDNA molecules from the wells of a microplate can be pooled into a single tube for PCR amplification and sequencing. The raw sequencing data can be analyzed to yield the number of reads, the number of random barcodes with unique molecular label sequences, and the number of mRNA molecules.

[0068] The present disclosure includes compositions. In some embodiments, the composition comprises two or more than one solid support. In some embodiments, each solid support comprises more than one oligonucleotide barcode, and the more than one oligonucleotide barcode each comprises a cell label sequence, wherein each cell label sequence comprises a predetermined chamber index subsequence. In some embodiments, the oligonucleotide barcodes associated with the same solid support comprise the same cell label sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell label sequences.

[0069] The present disclosure includes methods. In some embodiments, the method includes: distributing more than one precursor first solid support and a first population of first oligonucleotides to more than one first partitions, wherein the co-localized solid support and the first oligonucleotides become associated; distributing more than one precursor second solid support and a second population of first oligonucleotides to more than one second partitions, wherein the co-localized solid support and the first oligonucleotides become associated; pooling more than one precursor first solid support associated with the first oligonucleotides; pooling more than one precursor second solid support associated with the first oligonucleotides; distributing more than one precursor first solid support associated with the first oligonucleotides and a first population of second oligonucleotides to more than one first partitions of a second set, wherein the co-localized solid support and the second oligonucleotides become associated; distributing more than one precursor second solid support associated with the first oligonucleotides and a second population of second oligonucleotides to more than one second partitions of a second set, wherein the co-localized solid support and the second oligonucleotides become associated; pooling more than one precursor first solid support associated with the first and second oligonucleotides; pooling more than one precursor second solid support associated with the first and second oligonucleotides; distributing more than one precursor first solid support associated with the first and second oligonucleotides and a first population of third oligonucleotides to more than one first partitions of a third set, wherein the co-localized solid support and the third oligonucleotides become associated; and distributing more than one precursor second solid support associated with the first and second oligonucleotides and a second population of third oligonucleotides to more than one second partitions of a third set, wherein the co-localized solid support and the third oligonucleotides become associated.

[0070] The disclosure herein includes methods of assigning sequencing data to chambers. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into a partition of an identified chamber of the more than one chamber, wherein the solid supports each comprise more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprise a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into a partition of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, wherein the single cells comprise copies of nucleic acid targets, wherein the single cells of the same population are located in the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0071] The disclosure herein includes methods for assigning sequencing data to chambers. In some embodiments, the method includes: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each contain a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single cell populations into the partitions of the identified chambers of more than one chamber, wherein each of the two or more single cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber of more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0072] The disclosure herein includes methods for assigning sequencing data to single cell populations. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into partitions of an identified chamber of the more than one chamber, wherein the solid supports each comprise more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprise a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into partitions of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, wherein the single cells comprise copies of nucleic acid targets, wherein single cells of the same population are located in the same chamber, and wherein single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a population of the two or more single cell populations based on the chamber assigned to the sequencing read.

[0073] The disclosure herein includes methods for assigning sequencing data to single cell populations. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each containing a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single cell populations into the identified partitions of the chambers of more than one chamber, wherein each of the two or more single cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a population of the two or more single cell populations based on the chamber to which the sequencing read is assigned.

[0074] The disclosure herein includes methods for correlating sequencing data and phenotypic data of a population of single cells. In some embodiments, the methods include: obtaining phenotypic data for each of two or more populations of single cells; providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into partitions of an identified chamber of the more than one chamber, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein oligonucleotide barcodes located within the same chamber comprise the same chamber index subsequence, and wherein oligonucleotide barcodes located within different chambers comprise different chamber index subsequences; partitioning each of two or more populations of single cells into partitions of an identified chamber of the more than one chamber, wherein each of the two or more populations of single cells comprises more than one single cell, wherein each single cell comprises a copy of a nucleic acid target, wherein single cells of the same population are located within the same chamber, and wherein single cells of different populations are located within different chambers; barcoding copies of nucleic acid targets of at least one single cell of the more than one single cell from at least one population of single cells using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or a product thereof; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and correlating the sequencing data and the phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0075] The disclosure herein includes methods for correlating sequencing data and phenotypic data of a population of single cells. In some embodiments, the methods include: obtaining phenotypic data for each of two or more populations of single cells; providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, wherein each of the more than one oligonucleotide barcodes contains a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of the two or more populations of single cells into the partitions of the identified chambers in more than one chamber, wherein each of the two or more populations of single cells contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one single cell of more than one single cell from at least one population of single cells using more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and correlating the sequencing data and phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0076] The methods can include: assigning each of the more than one sequencing reads to a population of two or more populations of single cells based on the chamber assigned to the sequencing read. The methods can include: obtaining phenotypic data for each of two or more populations of single cells; and correlating the sequencing data and phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0077] The disclosure herein includes compositions. In some embodiments, the composition comprises: a microwell array, wherein the microwell array comprises more than one chamber, wherein each chamber comprises more than one partition, wherein each partition is having a size in the range from about 1,000 μm 3 to about 786,000 μm 3Micropores having a volume within a range. The composition may include: a cartridge, where the cartridge includes at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

[0078] The present disclosure includes a composition. In some embodiments, the composition includes: a cartridge, where the cartridge includes at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, where the cartridge includes a micropore array, where the micropore array includes more than one chamber, where each chamber includes more than one partition, where each partition is a micropore having a volume in a range from about 1,000 μm 3 to about 786,000 μm 3 within the range.

[0079] The present disclosure includes a composition. In some embodiments, the composition includes: two or more than one solid supports, where each solid support includes more than one oligonucleotide barcode, where each of the more than one oligonucleotide barcodes includes a cell marker sequence, where each cell marker sequence includes a predetermined chamber index subsequence, where the oligonucleotide barcodes associated with the same solid support include the same cell marker sequence, where the oligonucleotide barcodes associated with different solid supports include different cell marker sequences, where the oligonucleotide barcodes of the same more than one solid supports include the same chamber index subsequence, and where the oligonucleotide barcodes of different more than one solid supports include different chamber index subsequences.

[0080] Definitions

[0081] Unless otherwise defined, technical terms and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. See, e.g., Singleton et al., Dictionary of Microbiology and Molecular Biology, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

[0082] As used herein, the term "adapter" can refer to a sequence that facilitates the amplification or sequencing of associated nucleic acids. The associated nucleic acids can include target nucleic acids. The associated nucleic acids can include one or more of a spatial marker, a target marker, a sample marker, an index marker, or a barcode sequence (e.g., a molecular marker). The adapter can be linear. The adapter can be a pre-adenylated adapter. The adapter can be double-stranded or single-stranded. One or more adapters can be located at the 5' end or the 3' end of the nucleic acid. When the adapter contains known sequences at the 5' end and the 3' end, the known sequences can be the same or different sequences. An adapter located at the 5' end and / or 3' end of a polynucleotide can be capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, the adapter can contain a universal sequence. The universal sequence can be a region of a nucleotide sequence that is common to two or more nucleic acid molecules. Two or more nucleic acid molecules can also have regions of different sequences. Thus, for example, the 5' adapter can contain the same and / or a universal nucleic acid sequence, and the 3' adapter can contain the same and / or a universal sequence. A universal sequence that can be present in different members of more than one nucleic acid molecule can allow the replication or amplification of more than one different sequence using a single universal primer that is complementary to the universal sequence. Similarly, at least one, two (e.g., a pair), or more universal sequences that can be present in different members of a collection of nucleic acid molecules can allow the replication or amplification of more than one different sequence using at least one, two (e.g., a pair), or more single universal primers that are complementary to the universal sequences. Thus, the universal primer contains a sequence that can hybridize to such a universal sequence. A molecule having a target nucleic acid sequence can be modified to attach a universal adapter (e.g., a non-target nucleic acid sequence) to one or both ends of different target nucleic acid sequences. One or more universal primers attached to the target nucleic acid can provide a site for universal primer hybridization. One or more universal primers attached to the target nucleic acid can be the same or different from each other.

[0083] As used herein, the term "associated" or "associated with" can mean that two or more substances can be identified as co-localized at a particular point in time. Association can mean that two or more substances are or were within a similar container. Association can be an informatics association. For example, digital information about two or more substances can be stored and can be used to determine that one or more substances are co-localized at a particular point in time. Association can also be a physical association. In some embodiments, two or more associated substances are "tethered", "attached", or "immobilized" to each other or "tethered", "attached", or "immobilized" to a common solid or semi-solid surface. Association can refer to a covalent or non-covalent manner for attaching a marker to a solid or semi-solid support (such as a bead). Association can be a covalent bond between a target and a marker. Association can include hybridization between two molecules (such as a target molecule and a marker).

[0084] As used herein, the term "complementary" can refer to the ability of two nucleotides to pair precisely. For example, if the nucleotides of a nucleic acid at a given position are capable of forming hydrogen bonds with the nucleotides of another nucleic acid, the two nucleic acids are considered to be complementary to each other at that position. Complementarity between two single-stranded nucleic acid molecules can be "partial", where only some nucleotides bind, or it can be complete when there is full complementarity between the single-stranded molecules. If a first nucleotide sequence is complementary to a second nucleotide sequence, the first nucleotide sequence can be referred to as the "complement" of the second sequence. If a first nucleotide sequence is complementary to a sequence that is opposite to the second sequence (i.e., the nucleotide order is reversed), the first nucleotide sequence can be referred to as the "reverse complement" of the second sequence. As used herein, a "complementary" sequence can refer to the "complement" or "reverse complement" of a sequence. It is understood from this disclosure that if one molecule can hybridize with another molecule, it can be complementary or partially complementary to the molecule with which it hybridizes.

[0085] As used herein, the term "digital counting" can refer to a method for estimating the number of target molecules in a sample. Digital counting can include the step of determining the number of unique labels that have been associated with the target in the sample. This method, which can be random in nature, transforms the problem of counting molecules from one of localizing and identifying identical molecules into a series of yes / no digital questions regarding the detection of a predefined set of labels.

[0086] As used herein, the term "a label" or "more than one label" can refer to a nucleic acid code associated with a target in a sample. The label can be, for example, a nucleic acid label. The label can be a fully or partially amplifiable label. The label can be a fully or partially sequenceable label. The label can be part of a naturally occurring nucleic acid that can be identified as distinct. The label can be a known sequence. The label can include a junction of nucleic acid sequences, such as a junction of natural and non-natural sequences. As used herein, the term "label" can be used interchangeably with the terms "index", "tag", or "label-tag". The label can convey information. For example, in various embodiments, a label can be used to determine the identity of a sample, the source of a sample, the identity of a cell, and / or a target.

[0087] As used herein, the term "non-depleting reservoir" can refer to a pool of barcodes (e.g., random barcodes) consisting of many different labels. A non-depleting reservoir can include a large number of different barcodes such that when the non-depleting reservoir is associated with a target pool, each target may be associated with a unique barcode. The uniqueness of each labeled target molecule can be determined by the statistics of random selection and depends on the copy number of the same target molecule in the set compared to the diversity of the labels. The size of the resulting set of labeled target molecules can be determined by the random nature of the barcoding process, and then analysis of the number of detected barcodes allows calculation of the number of target molecules present in the original set or sample. When the ratio of the copy number of the target molecules present to the number of unique barcodes is low, the labeled target molecules are highly unique (i.e., the probability that more than one target molecule is labeled with a given label is very low).

[0088] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. Nucleic acids can include nucleotides. Nucleic acids can be exogenous or endogenous to a cell. Nucleic acids can be present in a cell-free environment. Nucleic acids can be genes or fragments thereof. Nucleic acids can be DNA. Nucleic acids can be RNA. Nucleic acids can include one or more analogs (e.g., altered backbone, sugar, or nucleobase). Some non-limiting examples of analogs include: 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholinos, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to a sugar), thiol-containing nucleotides, biotinylated nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine. The terms "nucleic acid", "polynucleotide", "target polynucleotide", and "target nucleic acid" can be used interchangeably.

[0089] Nucleic acids can include one or more modifications (e.g., base modifications, backbone modifications) to provide the nucleic acid with new or enhanced characteristics (e.g., improved stability). Nucleic acids can contain nucleic acid affinity tags. A nucleoside can be a base-sugar combination. The base portion of a nucleoside can be a heterocyclic base. Two of the most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide can be a nucleoside that further includes a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include ribofuranose, the phosphate group can be linked to the 2′, 3′, or 5′ hydroxyl moiety of the sugar. In forming a nucleic acid, the phosphate groups can covalently link adjacent nucleosides to one another to form a linear polymeric compound. Subsequently, the ends of this linear polymeric compound can be further joined to form a cyclic compound; however, linear compounds are generally suitable. Additionally, the linear compound can have internal nucleobase complementarity and can thus fold in a manner that produces a fully or partially double-stranded compound. In a nucleic acid, the phosphate groups are generally referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone can be a 3′ to 5′ phosphodiester linkage.

[0090] Nucleic acids can include modified backbones and / or modified internucleoside linkages. Modified backbones can include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. Suitable modified nucleic acid backbones that contain a phosphorus atom can include, for example, phosphorothioates, chiral phosphorothioates, dithiophosphates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates such as 3′-alkylene phosphonates, 5′-alkylene phosphonates, chiral phosphonates, phosphonates, phosphoramidates (including 3′-aminophosphoramidates and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates), thioalkyl phosphates, thioalkyl phosphotriesters, selenophosphates, and borophosphates, analogs with normal 3′-5′ linkages, 2′-5′ linkages, and analogs with reverse polarity (where one or more internucleotide linkages are 3′ to 3′, 5′ to 5′, or 2′ to 2′ linkages).

[0091] The nucleic acid can include a polynucleotide backbone formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms, and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatomic or heterocyclic internucleoside linkages. These can include those having morpholino linkages (partially formed from the sugar moiety of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; riboacetyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having a mixed N, O, S, and CH2 component moiety.

[0092] The nucleic acid can include nucleic acid mimics. The term "mimic" can be intended to include polynucleotides in which only the furanose ring or both the furanose ring and the internucleotide linkage are replaced by non-furanose groups, and replacement of only the furanose ring can also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety can be maintained to hybridize with the appropriate target nucleic acid. One such nucleic acid can be peptide nucleic acid (PNA). In PNA, the sugar backbone of the polynucleotide can be replaced by an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides can be retained and directly or indirectly bound to the azanitrogen atom of the amide moiety of the backbone. The backbone in a PNA compound can include two or more linked aminoethylglycine units, which gives PNA an amide-containing backbone. The heterocyclic base moiety can be directly or indirectly bound to the azanitrogen atom of the amide moiety of the backbone.

[0093] The nucleic acid can include a morpholino backbone structure. For example, the nucleic acid can contain a 6-membered morpholino ring replacing the ribose ring. In some of these embodiments, phosphorodiamidate or other non-phosphodiester internucleoside linkages can replace the phosphodiester linkage.

[0094] The nucleic acid can include linked morpholino units having a heterocyclic base attached to the morpholino ring (e.g., morpholino nucleic acid). A linking group can link the morpholino monomer units in the morpholino nucleic acid. Nonionic morpholino-based oligomeric compounds can have fewer undesirable interactions with cellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be linked using different linking groups. Another class of polynucleotide mimics can be called cyclohexenyl nucleic acid (CeNA). The furanose ring commonly present in nucleic acid molecules can be replaced by a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared using phosphoramidite chemistry and used in oligomeric compound synthesis. Incorporating CeNA monomers into nucleic acid strands can increase the stability of DNA / RNA hybrids. CeNA oligoadenylates can form complexes with nucleic acid complements with stability similar to that of native complexes. Additional modifications can include locked nucleic acid (LNA), in which the 2'-hydroxy group is linked to the 4'-carbon atom of the sugar ring, thereby forming a 2'-C,4'-C-oxymethylene linkage, thereby forming a bicyclic sugar moiety. The linkage can be methylene (-CH2-) n , a group bridging the 2'-oxygen atom and the 4'-carbon atom, where n is 1 or 2. LNAs and LNA analogs can exhibit very high duplex thermal stability (Tm = +3 °C to +10 °C) with complementary nucleic acids, stability to 3'-exonuclease degradation, and good solubility.

[0095] The nucleic acid may also include modified or substituted nucleobases (commonly referred to simply as "bases"). As used herein, "unmodified" or "natural" nucleobases may include purine bases (e.g., adenine (A) and guanine (G)), and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases may include other synthetic as well as natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl derivatives and other alkyl derivatives of adenine and guanine, 2-propyl derivatives and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (-C≡C-CH3) uracil and cytosine, and other alkynyl derivatives of pyrimidine bases, 6-azauracil, cytosine, and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thio, 8-thioalkyl, 8-hydroxy, and other 8-substituted adenines and guanines, 5-halo especially 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaadenine, and 8-azaguanine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine. Modified nucleobases may include tricyclic pyrimidines, such as phenoxazine cytidine (1H-pyrimido(5,4-b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido(5,4-b)(1,4)benzothiazin-2(3H)-one), G-clamps such as substituted phenoxazine cytidine (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido(4,5-b)indol-2-one), pyridoindole cytidine (H-pyrido(3’,2’:4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0096] As used herein, the term "sample" may refer to a composition containing a target. Suitable samples for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms.

[0097] As used herein, the term "sampling device" or "device" can refer to a device that can take a sample of a slice and / or place the slice on a substrate. A sampling device can refer to, for example, a fluorescence-activated cell sorting (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or an ultramicrotome.

[0098] As used herein, the term "solid support" can refer to a discrete solid or semi-solid surface to which more than one barcode (e.g., a random barcode) can be attached. A solid support can include any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar configuration made of plastic, ceramic, metal, or polymeric material (e.g., hydrogel) to which nucleic acids can be immobilized (e.g., covalently or non-covalently). A solid support can include discrete particles that can be spherical (e.g., microspheres) or have a non-spherical or irregular shape, such as cubic, rectangular, conical, cylindrical, conical, oval, or disc-shaped, etc. The shape of the beads can be non-spherical. More than one solid support spaced apart in an array may not include a substrate. A solid support can be used interchangeably with the term "bead".

[0099] As used herein, the term "random barcode" can refer to a polynucleotide sequence of the present disclosure that contains a label. A random barcode can be a polynucleotide sequence that can be used for random barcoding. A random barcode can be used to quantify a target in a sample. A random barcode can be used to control errors that may occur after a label is associated with a target. For example, a random barcode can be used to evaluate amplification or sequencing errors. A random barcode associated with a target can be referred to as a random barcode-target or a random barcode-label-target.

[0100] As used herein, the term "gene-specific random barcode" can refer to a polynucleotide sequence that contains a label and a gene-specific target-binding region. A random barcode can be a polynucleotide sequence that can be used for random barcoding. A random barcode can be used to quantify a target in a sample. A random barcode can be used to control errors that may occur after a label is associated with a target. For example, a random barcode can be used to evaluate amplification or sequencing errors. A random barcode associated with a target can be referred to as a random barcode-target or a random barcode-label-target.

[0101] As used herein, the term "random barcoding" can refer to the random labeling (e.g., barcoding) of nucleic acids. Random barcoding can utilize a recursive Poisson strategy to associate and quantify labels associated with a target. As used herein, the term "random barcoding" can be used interchangeably with "randomly labeling".

[0102] As used herein, the term "target" can refer to a composition that can be associated with a barcode (e.g., a random barcode). Exemplary suitable targets for analysis by the disclosed methods, devices, and systems include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, etc. The target can be single-stranded or double-stranded. In some embodiments, the target can be a protein, peptide, or polypeptide. In some embodiments, the target is a lipid. As used herein, "target" can be used interchangeably with "species".

[0103] As used herein, the term "reverse transcriptase" can refer to a group of enzymes having reverse transcriptase activity (i.e., catalyzing the synthesis of DNA from an RNA template). Generally, such enzymes include, but are not limited to, retroviral reverse transcriptases, retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retroposon reverse transcriptases, bacterial reverse transcriptases, group II intron-derived reverse transcriptases, and mutants, variants, or derivatives thereof. Non-retroviral reverse transcriptases include non-LTR retrotransposon reverse transcriptases, retroplasmid reverse transcriptases, retroposon reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include Lactococcus lactis LI.LtrB intron reverse transcriptase, Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases can include many types of non-retroviral reverse transcriptases (i.e., especially retroposons, group II introns, and diversity-generating retroelements).

[0104] The terms "universal adapter primer", "universal primer adapter", or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can be used to hybridize to a barcode (e.g., a random barcode) to generate a gene-specific barcode. The universal adapter sequence can be, for example, a known sequence that is common to all barcodes used in the methods of the present disclosure. For example, when more than one target is labeled using the methods disclosed herein, each target-specific sequence can be ligated to the same universal adapter sequence. In some embodiments, more than one universal adapter sequence can be used in the methods disclosed herein. For example, when more than one target is labeled using the methods disclosed herein, at least two target-specific sequences are ligated to different universal adapter sequences. The universal adapter primer and its complement can be included in two oligonucleotides, one of which contains the target-specific sequence and the other of which contains the barcode. For example, the universal adapter sequence can be part of the oligonucleotide containing the target-specific sequence to generate a nucleotide sequence complementary to the target nucleic acid. A second oligonucleotide containing the complementary sequence of the barcode and the universal adapter sequence can hybridize to the nucleotide sequence and generate a target-specific barcode (e.g., a target-specific random barcode). In some embodiments, the universal adapter primer has a different sequence from the universal PCR primers used in the methods of the present disclosure.

[0105] Barcode

[0106] Barcoding, such as random barcoding, has been described in, for example, Fu et al., Proc Natl Acad Sci U.S.A., May 31, 2011, 108(22):9026 - 31; US2011 / 0160078; Fan et al., Science, Feb 6, 2015, 347(6222):1258367; US2015 / 0299784; and WO2015 / 031691; the content of each of these, including any supporting or supplementary information or materials, is incorporated herein by reference in its entirety. In some embodiments, the barcodes disclosed herein can be random barcodes, which can be polynucleotide sequences that can be used to randomly label (e.g., barcode, tag) targets. If the ratio of the number of different barcode sequences of the random barcode to the number of occurrences of any target to be labeled can be the following or can be about the following: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values, then the barcode can be referred to as a random barcode. The target can be an mRNA population that includes mRNA molecules having the same or nearly the same sequence. If the ratio of the number of different barcode sequences of the random barcode to the number of occurrences of any target to be labeled is at least the following or is at most the following: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1, then the barcode can be referred to as a random barcode. The barcode sequence of the random barcode can be referred to as a molecular tag.

[0107] Barcodes (e.g., random barcodes) can include one or more labels. Exemplary labels can include universal labels, cell labels, barcode sequences (e.g., molecular tags), sample labels, plate labels, spatial labels, and / or pre - spatial labels. Figure 1Illustrated is an exemplary barcode 104 with spatial labels. The barcode 104 can include a 5' amine that can connect the barcode to a solid support 105. The barcode can include universal labels, dimensional labels, spatial labels, cellular labels, and / or molecular labels. The order of different labels in the barcode (including but not limited to universal labels, dimensional labels, spatial labels, cellular labels, and molecular labels) can vary. For example, as shown in Figure 1 , the universal label can be the 5'-most label, and the molecular label can be the 3'-most label. The spatial labels, dimensional labels, and cellular labels can be in any order. In some embodiments, the universal label, spatial label, dimensional label, cellular label, and molecular label are in any order. The barcode can include a target binding region. The target binding region can interact with a target in a sample (e.g., a target nucleic acid, RNA, mRNA, DNA). For example, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. In some cases, the labels of the barcode (e.g., universal labels, dimensional labels, spatial labels, cellular labels, and barcode sequences) can be separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides.

[0108] Labels (e.g., cellular labels) can include a set of unique nucleic acid subsequences of defined length, e.g., seven nucleotides each (equivalent to the number of bits used in some Hamming error correction codes), which can be designed to provide error correction capabilities. A set of error correction subsequences containing seven-nucleotide sequences can be designed such that any pairwise combination of sequences in the set exhibits a defined "genetic distance" (or number of mismatched bases), e.g., a set of error correction subsequences can be designed to exhibit a genetic distance of three nucleotides. In this case, examination of the error correction sequences in a sequence data set of the target nucleic acid molecules of the label (described in more detail below) can allow one to detect or correct amplification errors or sequencing errors. In some embodiments, the length of the nucleic acid subsequences used to generate the error correction code can vary, e.g., their length can be the following or can be about the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides or a number or range of nucleotides between any two of these values. In some embodiments, nucleic acid subsequences of other lengths can be used to generate the error correction code.

[0109] The barcode can include a target binding region. The target binding region can interact with a target in a sample. The target can be or include the following: ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNAs each containing a poly(A) tail, or any combination thereof. In some embodiments, more than one target can include deoxyribonucleic acid (DNA).

[0110] In some embodiments, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more labels of the barcode (e.g., universal labels, dimensional labels, spatial labels, cell labels, and barcode sequences (e.g., molecular labels)) can be separated from the other one or two remaining labels of the barcode by a spacer. The spacer can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotides. In some embodiments, none of the labels of the barcode are separated by a spacer.

[0111] Universal label

[0112] Barcodes can include one or more universal tags. In some embodiments, one or more universal tags can be the same for all barcodes in a set of barcodes attached to a given solid support. In some embodiments, one or more universal tags can be the same for all barcodes attached to more than one bead. In some embodiments, the universal tag can include a nucleic acid sequence capable of hybridizing to a sequencing primer. The sequencing primer can be used to sequence the barcode including the universal tag. The sequencing primer (e.g., a universal sequencing primer) can include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal tag can include a nucleic acid sequence capable of hybridizing to a PCR primer. In some embodiments, the universal tag can include a nucleic acid sequence capable of hybridizing to both a sequencing primer and a PCR primer. The nucleic acid sequence of the universal tag capable of hybridizing to a sequencing primer or a PCR primer can be referred to as a primer binding site. The universal tag can include a sequence that can be used to initiate transcription of the barcode. The universal tag can include a sequence that can be used to extend the barcode or a region within the barcode. The length of the universal tag can be the following or can be about the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or a number or range between any two of these values of nucleotides. For example, the universal tag can include at least about 10 nucleotides. The length of the universal tag can be at least the following or can be at most the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. In some embodiments, a cleavable linker or modified nucleotide can be part of the universal tag sequence to enable the barcode to be cleaved from the support.

[0113] Dimension label

[0114] Barcodes can include one or more dimensional tags. In some embodiments, the dimensional tags can include nucleic acid sequences that provide information about the dimension in which the tagging (e.g., random tagging) occurs. For example, the dimensional tags can provide information about the time at which the target is barcoded. The dimensional tags can be associated with the time of barcoding (e.g., random barcoding) in the sample. The dimensional tags can be activated at the time of tagging. Different dimensional tags can be activated at different times. The dimensional tags provide information about the order in which the targets, groups of targets, and / or samples are barcoded. For example, a population of cells can be barcoded in the G0 phase of the cell cycle. In the G1 phase of the cell cycle, the cells can be pulsed again with barcodes (e.g., random barcodes). In the S phase of the cell cycle, the cells can be pulsed again with barcodes, and so on. The barcodes at each pulse (e.g., each phase of the cell cycle) can include different dimensional tags. In this way, the dimensional tags provide information about which targets are tagged at which phase of the cell cycle. The dimensional tags can interrogate many different biological times. Exemplary biological times can include, but are not limited to, the cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., cells, a population of cells) can be tagged before and / or after treatment with a drug and / or therapy. Changes in the copy number of different targets can indicate the response of the sample to the drug and / or therapy.

[0115] The dimensional tags can be activatable. The activatable dimensional tags can be activated at a specific time point. The activatable tags can be activated, for example, constitutively (e.g., not turned off). The activatable dimensional tags can be activated, for example, reversibly (e.g., the activatable dimensional tags can be turned on and off). The dimensional tags can be reversibly activated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. The dimensional tags can be reversibly activated, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more. In some embodiments, the dimensional tags can be activated with fluorescence, light, chemical events (e.g., cleavage, ligation of another molecule, addition of a modification (e.g., PEGylation, sumoylation, acetylation, methylation, deacetylation, demethylation), photochemical events (e.g., photocaging)), and introduction of unnatural nucleotides.

[0116] In some embodiments, the dimensional label can be the same for all barcodes (e.g., random barcodes) attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, at least 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% of the barcodes on the same solid support can contain the same dimensional label. In some embodiments, at least 60% of the barcodes on the same solid support can contain the same dimensional label. In some embodiments, at least 95% of the barcodes on the same solid support can contain the same dimensional label.

[0117] Up to 10 6 unique dimensional label sequences can be present in more than one solid support (e.g., bead). The length of the dimensional label can be the following or can be about the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The length of the dimensional label can be at least the following or can be at most the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. The dimensional label can contain between about 5 and about 200 nucleotides. The dimensional label can contain between about 10 and about 150 nucleotides. The dimensional label can contain nucleotides with a length between about 20 and about 125.

[0118] Spatial label

[0119] The barcode can contain one or more spatial labels. In some embodiments, the spatial label can contain a nucleic acid sequence that provides information about the spatial orientation of the target molecule associated with the barcode. The spatial label can be associated with coordinates in the sample. The coordinates can be fixed coordinates. For example, the coordinates can be fixed relative to a substrate. The spatial label can refer to a two-dimensional or three-dimensional grid. The coordinates can be fixed relative to a landmark. The landmark can be identified in space. The landmark can be a structure that can be imaged. The landmark can be a biological structure, such as an anatomical landmark. The landmark can be a cellular landmark, such as an organelle. The landmark can be an unnatural landmark, such as a structure with an identifiable identifier (such as a color code, barcode, magnetic property, fluorescence, radioactivity, or unique size or shape). The spatial label can be associated with a physical partition (e.g., a well, a container, or a droplet). In some embodiments, more than one spatial label is used together to encode one or more positions in space.

[0120] The spatial barcode can be the same for all barcodes attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes on the same solid support that contain the same spatial barcode can be below or about below: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. In some embodiments, the percentage of barcodes on the same solid support that contain the same spatial barcode can be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, at least 60% of the barcodes on the same solid support can contain the same spatial barcode. In some embodiments, at least 95% of the barcodes on the same solid support can contain the same spatial barcode.

[0121] Up to 10 6 or more unique spatial barcode sequences can be present in more than one solid support (e.g., bead). The length of the spatial barcode can be below or about below: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range between any two of these values of nucleotides. The length of the spatial barcode can be at least below or at most below: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides. The spatial barcode can contain between about 5 and about 200 nucleotides. The spatial barcode can contain between about 10 and about 150 nucleotides. The spatial barcode can contain nucleotides with a length between about 20 and about 125.

[0122] Cell label

[0123] Barcodes (e.g., random barcodes) can contain one or more cell markers. In some embodiments, the cell markers can contain nucleic acid sequences that provide information for determining which target nucleic acid is derived from which cell. In some embodiments, the cell markers are the same for all barcodes attached to a given solid support (e.g., bead), but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support can be below or can be about below: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100% or a number or range between any two of these values. In some embodiments, the percentage of barcodes containing the same cell marker on the same solid support can be below or can be about below: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. For example, at least 60% of the barcodes on the same solid support can contain the same cell marker. As another example, at least 95% of the barcodes on the same solid support can contain the same cell marker.

[0124] Up to 10 6 unique cell marker sequences can be present in more than one solid support (e.g., bead). The length of the cell marker can be below or can be about below: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or a number or range between any two of these values of nucleotides. The length of the cell marker can be at least below or can be at most below: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides. For example, the cell marker can contain between about 5 and about 200 nucleotides. As another example, the cell marker can contain between about 10 and about 150 nucleotides. As yet another example, the cell marker can contain nucleotides with a length between about 20 and about 125.

[0125] Barcode sequence

[0126] Barcodes can contain one or more barcode sequences. In some embodiments, the barcode sequences can contain nucleic acid sequences that provide identification information for a specific type of target nucleic acid material that hybridizes to the barcode. The barcode sequences can contain nucleic acid sequences that provide a counter (e.g., provide a rough estimate) for a specific occurrence of a target nucleic acid material that hybridizes to the barcode (e.g., target binding region).

[0127] In some embodiments, a set of diverse barcode sequences is attached to a given solid support (e.g., a bead). In some embodiments, there may be or may be about the following number of unique molecular marker sequences: 10 2 species, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, 10 9 species or a number or range between any two of these values. For example, more than one barcode may include about 6561 barcode sequences with different sequences. As another example, more than one barcode may include about 65536 barcode sequences with different sequences. In some embodiments, there may be at least or at most the following number of unique barcode sequences: 10 2 species, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species or 10 9 species. The unique molecular marker sequences can be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular marker sequences are partially or fully contained within particles (e.g., hydrogel beads).

[0128] In different embodiments, the length of the barcode can be different. For example, the length of the barcode can be the following or can be about the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides or a number or range between any two of these values of nucleotides. As another example, the length of the barcode can be at least or at most the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides.

[0129] Molecular label

[0130] Barcodes (e.g., random barcodes) can contain one or more molecular markers. The molecular marker can contain a barcode sequence. In some embodiments, the molecular marker can contain a nucleic acid sequence that provides identification information for a particular type of target nucleic acid material that hybridizes to the barcode. The molecular marker can contain a nucleic acid sequence that provides a counter for the specific occurrence of a target nucleic acid material that hybridizes to the barcode (e.g., a target binding region).

[0131] In some embodiments, a set of distinct molecular tags are attached to a given solid support (e.g., bead). In some embodiments, there may be or may be about the following number of unique molecular tag sequences: 10 2 species, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, 10 9 species, or a number or range between any two of these values. For example, more than one barcode may include about 6561 molecular tags with different sequences. As another example, more than one barcode may include about 65536 molecular tags with different sequences. In some embodiments, there may be at least or at most the following number of unique molecular tag sequences: 10 2 species, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, or 10 9 species. Barcodes with unique molecular tag sequences can be attached to a given solid support (e.g., bead).

[0132] For barcoding using more than one random barcode (e.g., random barcoding), the ratio of the number of different molecular tag sequences to the number of occurrences of any target can be the following or can be about the following: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, or a number or range between any two of these values. The target can be an mRNA species comprising mRNA molecules having the same or nearly the same sequence. In some embodiments, the ratio of the number of different molecular tag sequences to the number of occurrences of any target is at least or at most the following: 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, or 100:1.

[0133] The length of the molecular marker can be the following or can be about the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or a number or range of nucleotides between any two of these values. The length of the molecular marker can be at least the following or can be at most the following: 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200 or 300 nucleotides.

[0134] Target binding region

[0135] The barcode can comprise one or more target binding regions, such as capture probes. In some embodiments, the target binding region can hybridize to a target of interest. In some embodiments, the target binding region can comprise a nucleic acid sequence that specifically hybridizes (e.g., specifically hybridizes to a particular gene sequence) to a target (e.g., a target nucleic acid, a target molecule, such as a cellular nucleic acid to be analyzed). In some embodiments, the target binding region can comprise a nucleic acid sequence that can attach (e.g., hybridize) to a specific location of a particular target nucleic acid. In some embodiments, the target binding region can comprise a nucleic acid sequence that is capable of specifically hybridizing to a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be ligated to any nucleic acid molecule comprising a sequence complementary to the restriction site overhang.

[0136] In some embodiments, the target binding region can comprise a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence can refer to a sequence that can bind more than one target nucleic acid independently of the specific sequence of the target nucleic acid. For example, the target binding region can comprise a random polymer sequence, a poly(dA) sequence, a poly(dT) sequence, a poly(dG) sequence, a poly(dC) sequence, or a combination thereof. For example, the target binding region can be an oligo(dT) sequence that hybridizes to the poly(A) tail on an mRNA molecule. A random polymer sequence can be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or higher polymer sequence of any length. In some embodiments, the target binding region is the same for all barcodes attached to a given bead. In some embodiments, for more than one barcode attached to a given bead, the target binding region can comprise two or more different target binding sequences. The length of the target binding region can be the following or can be about the following: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides, or a number or range of nucleotides between any two of these values. The length of the target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase such as Moloney murine leukemia virus (MMLV) reverse transcriptase to produce a cDNA molecule having a poly(dC) tail. The barcode can comprise a target binding region having a poly(dG) tail. After base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues to replicate towards the 5' end of the barcode. By doing so, the resulting cDNA molecule contains the barcode sequence (such as a molecular tag) at the 3' end of the cDNA molecule.

[0137] In some embodiments, the target binding region can comprise oligo(dT), which can hybridize to mRNA containing a polyadenylated terminus. The target binding region can be gene-specific. For example, the target binding region can be configured to hybridize to a specific region of the target. The length of the target binding region can be the following or can be about the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides or a number or range of nucleotides between any two of these values. The length of the target binding region can be at least the following or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The length of the target binding region can be about 5 - 30 nucleotides. When the barcode contains a gene-specific target binding region, the barcode can be referred to herein as a gene-specific barcode.

[0138] Orientation Property

[0139] Random barcodes (e.g., random barcodes) can comprise one or more orienting properties that can be used to orient (e.g., align) the barcodes. The barcode can comprise a portion for isoelectric focusing. Different barcodes can have different isoelectric focusing points. When these barcodes are introduced into a sample, the sample can be subjected to isoelectric focusing to facilitate orienting the barcodes in a known manner. In this way, the orienting properties can be used to develop a known mapping of the barcodes in the sample. Exemplary orienting properties can include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, barcodes with the orienting property of self-assembly can self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0140] Affinity Property

[0141] Barcodes (e.g., random barcodes) can include one or more affinity features. For example, spatial tags can include affinity features. Affinity features can include chemical and / or biological moieties that can facilitate binding of the barcode to another entity (e.g., a cell receptor). For example, an affinity feature can include an antibody, e.g., an antibody specific for a particular moiety (e.g., a receptor) on a sample. In some embodiments, the antibody can direct the barcode to a specific cell type or molecule. A target at and / or near a specific cell type or molecule can be labeled (e.g., randomly labeled). In some embodiments, the affinity feature can provide spatial information in addition to the nucleotide sequence of the spatial tag, as the antibody can direct the barcode to a specific location. The antibody can be a therapeutic antibody, e.g., a monoclonal or polyclonal antibody. The antibody can be humanized or chimeric. The antibody can be a naked antibody or a fusion antibody.

[0142] An antibody can be a full-length (i.e., naturally occurring or formed by recombinant processes of normal immunoglobulin gene segments) immunoglobulin molecule (e.g., an IgG antibody) or an immunologically active (i.e., specifically binding) portion of an immunoglobulin molecule (such as an antibody fragment).

[0143] Antibody fragments can be, for example, a portion of an antibody, such as F(ab’)2, Fab’, Fab, Fv, sFv, etc. In some embodiments, the antibody fragment can bind to the same antigen recognized by the full-length antibody. Antibody fragments can include isolated fragments consisting of the variable regions of the antibody, such as an “Fv” fragment consisting of the variable regions of the heavy and light chains and a recombinant single-chain polypeptide molecule (an “scFv protein”) in which the variable regions of the light and heavy chains are joined by a peptide linker. Exemplary antibodies can include, but are not limited to, cancer cell antibodies, virus antibodies, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0144] Universal adapter primer

[0145] Barcodes can contain one or more universal adapter primers. For example, gene-specific barcodes (such as gene-specific random barcodes) can contain universal adapter primers. Universal adapter primers can refer to universal nucleotide sequences across all barcodes. Universal adapter primers can be used to construct gene-specific barcodes. The length of the universal adapter primer can be the following or can be about the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or a number or range of nucleotides between any two of these values. The length of the universal adapter primer can be at least the following or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. The length of the universal adapter primer can be 5 - 30 nucleotides.

[0146] Adapter

[0147] When a barcode contains more than one type of marker (e.g., more than one cell marker or more than one barcode sequence, such as a molecular marker), linker marker sequences can be interspersed between the markers. The length of the linker marker sequence can be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. The length of the linker marker sequence can be at most about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides. In some cases, the length of the linker marker sequence is 12 nucleotides. Linker marker sequences can be used to facilitate barcode synthesis. Linker markers can include error correction (e.g., Hamming) codes.

[0148] Solid support

[0149] In some embodiments, the barcodes disclosed herein (such as random barcodes) can be associated with a solid support. The solid support can be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences of more than one barcode (e.g., a first more than one barcode) on the solid support (such as the molecular markers of a random barcode (e.g., a first barcode sequence)) differ by at least one nucleotide. The cellular markers of the barcodes on the same solid support can be the same. The cellular markers of the barcodes on different solid supports can differ by at least one nucleotide. For example, the first cellular markers of the first more than one barcodes on a first solid support can have the same sequence, and the second cellular markers of the second more than one barcodes on a second solid support can have the same sequence. The first cellular markers of the first more than one barcodes on the first solid support and the second cellular markers of the second more than one barcodes on the second solid support can differ by at least one nucleotide. The cellular marker can be, for example, about 5-20 nucleotides in length. The barcode sequence can be, for example, about 5-20 nucleotides in length. The synthetic particle can be, for example, a bead.

[0150] The bead can be, for example, a silica bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a Sephadex / Sepharose bead, a cellulose bead, a polystyrene bead, or any combination thereof. The bead can comprise materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substances, ceramics, plastics, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof.

[0151] In some embodiments, the bead can be a polymer bead (such as a deformable bead or a gel bead) functionalized with a barcode or a random barcode (such as a gel bead from 10X Genomics (San Francisco, CA)). In some embodiments, the gel bead can comprise a polymer-based gel. The gel bead can be produced, for example, by encapsulating one or more polymer precursors into droplets. The gel bead can be produced after exposing the polymer precursor to a promoter (e.g., tetramethylethylenediamine (TEMED)).

[0152] The particle can be destructible (e.g., soluble, degradable). For example, the polymer bead can dissolve, melt, or degrade under desired conditions. The desired conditions can include environmental conditions. The desired conditions can cause the polymer bead to dissolve, melt, or degrade in a controlled manner. The gel bead can dissolve, melt, or degrade due to chemical stimulation, physical stimulation, biological stimulation, thermal stimulation, magnetic stimulation, electrical stimulation, light stimulation, or any combination thereof.

[0153] For example, an analyte and / or a reagent (such as an oligonucleotide barcode) can be coupled / fixed to the inner surface of a gel bead (e.g., the interior accessible via diffusion of the oligonucleotide barcode and / or the material used to generate the oligonucleotide barcode) and / or the outer surface of the gel bead or any other microcapsule described herein. The coupling / fixing can be via any form of chemical bonding (e.g., covalent bond, ionic bond) or physical phenomenon (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the coupling / fixing of the reagents described herein to the gel bead or any other microcapsule can be reversible, such as, for example, via a labile moiety (e.g., via a chemical crosslinker, including the chemical crosslinkers described herein). After application of a stimulus, the labile moiety can be cleaved and release the immobilized reagent. In some embodiments, the labile moiety is a disulfide bond. For example, in the case of immobilizing an oligonucleotide barcode to a gel bead via a disulfide bond, exposing the disulfide bond to a reducing agent can cleave the disulfide bond and release the oligonucleotide barcode from the bead. The labile moiety can be included as part of the gel bead or microcapsule, as part of a chemical linker that connects the reagent or analyte to the gel bead or microcapsule, and / or as part of the reagent or analyte. In some embodiments, at least one barcode of more than one barcode can be immobilized on the particle, partially immobilized on the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof.

[0154] In some embodiments, the gel beads can comprise a wide range of different polymers, including but not limited to: polymers, thermosensitive polymers, photosensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. The polymers can include but are not limited to the following materials: such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethyl-ammonium chloride) (PDADMAC), poly(pyrrole) (PPy), poly(vinylpyrrolidone) (PVPON), poly(vinylpyridine) (PVP), poly(methacrylic acid) (PMAA), poly(methyl methacrylate) (PMMA), polystyrene (PS), poly(tetrahydrofuran) (PTHF), poly(phthalaldehyde) (PPA), poly(hexyl viologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), poly(lactic-co-glycolic acid) (PLGA).

[0155] Many chemical stimuli can be used to trigger the disruption, dissolution, or degradation of the beads. Examples of such chemical alterations can include, but are not limited to, pH-mediated changes to the bead wall, disintegration of the bead wall via chemical cleavage of crosslink bonds, triggered depolymerization of the bead wall, and bead wall conversion reactions. Bulk changes can also be used to trigger bead disruption.

[0156] Bulk or physical alterations of the microcapsules by various stimuli also offer many advantages in designing capsules for reagent release. The bulk or physical changes occur on a macroscopic scale, where bead rupture is the result of mechanical-physical forces induced by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, melting of the bead wall, or alteration of the porosity of the bead wall.

[0157] Biological stimuli can also be used to trigger the disruption, dissolution, or degradation of the beads. Generally, biological triggers are similar to chemical triggers, but many examples use biomolecules or molecules common in living systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, etc. For example, the beads can contain a polymer crosslinked with a peptide that is sensitive to cleavage by a specific protease. More particularly, one example can include microcapsules crosslinked with the GFLGK peptide. After the addition of a biological trigger, such as the protease cathepsin B, the peptide crosslinks in the shell wall are cleaved and the contents of the beads are released. In other cases, the protease can be heat-activated. In another example, the beads include a shell wall containing cellulose. The addition of chitosan hydrolase serves as a biological trigger for cellulose bond cleavage, shell wall depolymerization, and release of its internal contents.

[0158] It is also possible to induce the beads to release their contents after application of a heat stimulus. Alteration of the temperature can cause various changes to the beads. A change in heat can cause the beads to melt, such that the bead wall disintegrates. In other cases, heat can increase the internal pressure of the internal components of the beads, such that the beads rupture or explode. In still other cases, heat can cause the beads to transform into a shrunk and dehydrated state. Heat can also act on thermosensitive polymers within the bead wall, thereby causing bead disruption.

[0159] Including magnetic nanoparticles within the bead wall of the microcapsules can permit triggered rupture of the beads as well as guiding the beads into an array. The devices of the present disclosure can include magnetic beads for either purpose. In one example, incorporation of Fe3O4 nanoparticles into polyelectrolyte-containing beads triggers rupture in the presence of an oscillating magnetic field stimulus.

[0160] The beads can also be disrupted, dissolved, or degraded as a result of an electrical stimulus. Similar to the magnetic particles described in the previous section, electro-sensitive beads can permit triggered rupture of the beads as well as other functions, such as alignment in an electric field, conductivity, or redox reactions. In one example, beads containing electro-sensitive materials align in an electric field, such that the release of internal reagents can be controlled. In other examples, the electric field can cause a redox reaction within the bead wall itself, which can increase the porosity.

[0161] It is also possible to use light stimulation to disrupt the beads. Many photo triggers are possible and can include systems that use various molecules such as nanoparticles and chromophores that are capable of absorbing photons in a specific wavelength range. For example, a metal oxide coating can be used as a capsule trigger. UV irradiation of a polyelectrolyte capsule coated with SiO2 can cause the disintegration of the bead wall. In yet another example, a photoswitchable material such as an azobenzene group can be incorporated into the bead wall. After application of UV or visible light, chemicals such as these undergo reversible cis-to-trans isomerization upon absorption of photons. In this regard, incorporation of a photon switch results in a bead wall that can disintegrate or become more porous upon application of a photo trigger.

[0162] For example, in Figure 2 a non-limiting example of barcoding (e.g., random barcoding) as illustrated in, after introducing cells (such as single cells) onto more than one micropore of a micropore array at block 208, beads can be introduced onto more than one micropore of the micropore array at block 212. Each micropore can contain one bead. The beads can contain more than one barcode. The barcode can contain an attached 5'-amine region. The barcode can contain a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0163] The barcodes disclosed herein can be associated (e.g., attached) with a solid support (e.g., a bead). The barcodes associated with the solid support can each contain a barcode sequence selected from a group that includes at least 100 or 1000 barcode sequences having unique sequences. In some embodiments, different barcodes associated with the solid support can contain barcode sequences having different sequences. In some embodiments, a certain percentage of the barcodes associated with the solid support contain the same cell label. For example, the percentage can be the following or can be about the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values. As another example, the percentage can be at least the following or can be at most the following: 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or 100%. In some embodiments, the barcodes associated with the solid support can have the same cell label. The barcodes associated with different solid supports can have different cell labels selected from a group that includes at least 100 or 1000 cell labels having unique sequences.

[0164] The barcodes disclosed herein can be associated (e.g., attached) with a solid support (e.g., a bead). In some embodiments, a solid support comprising more than one synthetic particle associated with more than one barcode can be used to barcode more than one target in a sample. In some embodiments, the solid support can comprise more than one synthetic particle associated with more than one barcode. The spatial labeling of more than one barcode on different solid supports can differ by at least one nucleotide. The solid support can comprise more than one barcode, e.g., in two or three dimensions. The synthetic particle can be a bead. The bead can be a silica bead, a controlled pore glass bead, a magnetic bead, a Dynabead, a Sephadex / Sepharose bead, a cellulose bead, a polystyrene bead, or any combination thereof. The solid support can comprise a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support can be free-floating. In some embodiments, the solid support can be embedded in a semi-solid or solid array. The barcode can be unassociated with the solid support. The barcode can be a single nucleotide. The barcode can be associated with a substrate.

[0165] As used herein, the terms “tethered,” “attached,” and “fixed” can be used interchangeably and can refer to covalent or non-covalent means for attaching a barcode to a solid support. Any of a variety of different solid supports can be used as the solid support for attaching a pre-synthesized barcode or for in situ solid-phase synthesis of a barcode.

[0166] In some embodiments, the solid support is a bead. The bead can comprise one or more types of solid, porous, or hollow spheres, balls, sockets, cylinders, or other similar configurations that can immobilize nucleic acids (e.g., covalently or non-covalently). The bead can be composed of, e.g., plastic, ceramic, metal, polymeric materials, or any combination thereof. The bead can be or include spherical (e.g., microspheres) or discrete particles having a non-spherical or irregular shape, such as cubic, rectangular, conical, cylindrical, frustoconical, oval, or disk-shaped, etc. In some embodiments, the shape of the bead can be non-spherical.

[0167] The bead can comprise a variety of materials, including but not limited to paramagnetic materials (e.g., magnesium, molybdenum, lithium, and tantalum), superparamagnetic materials (e.g., ferrite (Fe3O4; magnetite) nanoparticles), ferromagnetic materials (e.g., iron, nickel, cobalt, some of their alloys, and some rare earth metal compounds), ceramics, plastics, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof.

[0168] In some embodiments, the beads (e.g., beads to which a label is attached) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0169] Some embodiments disclosed herein include one or more particles (e.g., beads). Each particle may contain more than one oligonucleotide (e.g., barcode). Each of the more than one oligonucleotides may contain a barcode sequence (e.g., molecular marker sequence), a cell marker, and a target binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random polymer, or a combination thereof). The cell marker sequences of each of the more than one oligonucleotides may be the same. The cell marker sequences of the oligonucleotides on different particles may be different such that the oligonucleotides on different particles can be identified. In different embodiments, the number of different cell marker sequences may be different. In some embodiments, the number of cell marker sequences may be the following or may be about the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 、10 7 、10 8 、10 9 、a number or range between any two of these values, or more. In some embodiments, the number of cell marker sequences may be at least the following or may be at most the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 10 6 、10 7 、10 8 、or 10 9In some embodiments, no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more particles in more than one particle include oligonucleotides with the same cell sequence. In some embodiments, more than one particle including oligonucleotides with the same cell sequence can be at most 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more. In some embodiments, all of the more than one particles do not have the same cell marker sequence.

[0170] More than one oligonucleotide on each particle can comprise a different barcode sequence (e.g., a molecular marker). In some embodiments, the number of barcode sequences can be or can be about the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 , 10 9 , or a number or range between any two of these values. In some embodiments, the number of barcode sequences can be at least the following or can be at most the following: 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000, 100000 6 , 10 7 , 10 8 or 10 9For example, at least 100 of the more than one oligonucleotides contain different barcode sequences. As another example, in a single particle, at least 100, 500, 1000, 5000, 10000, 15000, 20000, 50000, a number or range between any two of these values, or more of the more than one oligonucleotides contain different barcode sequences. Some embodiments provide more than one particle containing a barcode. In some embodiments, the ratio of the target to be labeled to the occurrence (or copy or number) of different barcode sequences can be at least 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, or higher. In some embodiments, each of the more than one oligonucleotides further contains a sample label, a universal label, or both. The particle can be, for example, a nanoparticle or a microparticle.

[0171] The beads can be of different sizes. For example, the diameter of the beads can range from 0.1 micrometers to 50 micrometers. In some embodiments, the diameter of the beads can be the following or can be about the following: 0.1 micrometer, 0.5 micrometer, 1 micrometer, 2 micrometers, 3 micrometers, 4 micrometers, 5 micrometers, 6 micrometers, 7 micrometers, 8 micrometers, 9 micrometers, 10 micrometers, 20 micrometers, 30 micrometers, 40 micrometers, 50 micrometers, or a number or range between any two of these values.

[0172] The diameter of the bead can be related to the diameter of the pore of the substrate. In some embodiments, the diameter of the bead can be longer or shorter than or about the following of the diameter of the pore: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values. The diameter of the bead can be related to the diameter of a cell (e.g., a single cell captured by the pore of the substrate). In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the pore by at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. The diameter of the bead can be related to the diameter of a cell (e.g., a single cell captured by the pore of the substrate). In some embodiments, the diameter of the bead can be longer or shorter than or about the following of the diameter of the cell: 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, or a number or range between any two of these values. In some embodiments, the diameter of the bead can be longer or shorter than the diameter of the cell by at least or at most 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300%.

[0173] The bead can be attached to the substrate and / or embedded in the substrate. The bead can be attached to a gel, hydrogel, polymer, and / or matrix and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial position of the bead in the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using the spatial markers present on the barcode on the bead, and the spatial marker can be used as a location address.

[0174] Examples of beads can include but are not limited to streptavidin beads, agarose beads, magnetic beads, microbeads, antibody-conjugated beads (e.g., anti-immunoglobulin microbeads), protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, antibiotin microbeads, anti-fluorescent dye microbeads, and BcMag TM carboxyl-terminated magnetic beads.

[0175] Beads can be associated with quantum dots or fluorescent dyes (e.g., impregnated with quantum dots or fluorescent dyes) to make them fluoresce in one or more fluorescent optical channels. Beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. Beads can be identifiable. For example, a camera can be used to image the beads. Beads can have a detectable code associated with them. For example, beads can contain barcodes. Beads can change size, e.g., due to swelling in an organic or inorganic solution. Beads can be hydrophobic. Beads can be hydrophilic. Beads can be biocompatible.

[0176] Solid supports (e.g., beads) can be visualized. Solid supports can contain visualization tags (e.g., fluorescent dyes). Solid supports (e.g., beads) can be etched with identifiers (e.g., numbers). The identifiers can be visualized by imaging the beads.

[0177] Solid supports can include soluble, semi-soluble, or insoluble materials. When a solid support includes a linker, scaffold, building block, or other reactive moiety attached thereto, the solid support can be referred to as "functionalized", while when the solid support lacks such an attached reactive moiety, the solid support can be referred to as "non-functionalized". Solid supports can be free in solution, such as in the form in a microtiter well; in a flow-through form, such as in a column; or used as a dipstick.

[0178] Solid supports can include membranes, paper, plastics, coated surfaces, flat surfaces, glass, slides, chips, or any combination thereof. Solid supports can take the form of resins, gels, microspheres, or other geometric configurations. Solid supports can include silica chips, micron particles, nano particles, plates, arrays, capillaries, flat supports such as glass fiber filters, glass surfaces, metal surfaces (steel, gold, silver, aluminum, silicon, and copper), glass supports, plastic supports, silicon supports, chips, filters, membranes, microtiter plates, slides, plastic materials including porous plates or membranes (e.g., formed from polyethylene, polypropylene, polyamide, polyvinylidene fluoride), and / or wafers, combs, needles, or needle arrays (e.g., needle arrays suitable for combinatorial synthesis or analysis) or beads, depressions or nanoliter pore arrays on flat surfaces such as wafers (e.g., silicon wafers), wafers with depressions (with or without a filter bottom).

[0179] Solid supports can include polymer matrices (e.g., gels, hydrogels). The polymer matrix can be able to penetrate the intracellular space (e.g., around organelles). The polymer matrix can be able to be pumped through the entire circulatory system.

[0180] Substrate and microwell array

[0181] As used herein, a substrate can refer to a type of solid support. A substrate can refer to a solid support that can contain the barcodes or random barcodes of the present disclosure. The substrate can include, for example, more than one micropore. The substrate can be, for example, a pore array including two or more micropores. In some embodiments, the micropores can include small reaction chambers defining a volume. In some embodiments, the micropores can capture one or more cells. In some embodiments, the micropores can capture only one cell. In some embodiments, the micropores can capture one or more solid supports. In some embodiments, the micropores can capture only one solid support. In some embodiments, the micropores capture single cells and single solid supports (e.g., beads). The micropores can contain the barcode reagents of the present disclosure.

[0182] Barcoding method

[0183] The present disclosure provides methods for estimating the number of different targets at different positions in a body sample (e.g., tissue, organ, tumor, cell). The method can include placing a barcode (e.g., a random barcode) in close proximity to the sample, lysing the sample, associating different targets with the barcode, amplifying the targets and / or digitally counting the targets. The method can also include analyzing the information obtained from the spatial markers on the barcode and / or visualizing the information. In some embodiments, the method includes visualizing more than one target in the sample. Mapping more than one target onto a map of the sample can include generating a two-dimensional map or a three-dimensional map of the sample. The two-dimensional map and the three-dimensional map of the sample can be generated before or after barcoding (e.g., random barcoding) more than one target in the sample. Visualizing more than one target in the sample can include mapping more than one target onto a map of the sample. Mapping more than one target onto a map of the sample can include generating a two-dimensional map or a three-dimensional map of the sample. The two-dimensional map and the three-dimensional map of the sample can be generated before or after barcoding more than one target in the sample. In some embodiments, the two-dimensional map and the three-dimensional map of the sample can be generated before or after lysing the sample. Lysing the sample before or after generating the two-dimensional map or the three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0184] In some embodiments, barcoding more than one target includes hybridizing more than one barcode with more than one target to produce barcoded targets (e.g., randomly barcoded targets). Barcoding more than one target can include generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets can be performed using a solid support containing more than one barcode (e.g., a random barcode).

[0185] Contact the sample with the barcode

[0186] The present disclosure provides methods for contacting a sample (e.g., a cell) with a substrate of the present disclosure. A sample including, for example, a cell, a thin section of an organ or tissue can be contacted with a barcode (e.g., a random barcode). The cell can be contacted, for example, by gravity flow, where the cell can be allowed to sediment and a monolayer can be produced. The sample can be a thin section of tissue. The thin section can be placed on the substrate. The sample can be one-dimensional (e.g., forming a flat surface). The sample (e.g., a cell) can be dispersed over the substrate, for example, by growing / culturing the cell on the substrate.

[0187] When the barcode is in close proximity to the target, the target can hybridize with the barcode. The barcodes can be contacted in non-exhaustible ratios such that each different target can be associated with a different barcode of the present disclosure. To ensure an effective association between the target and the barcode, the target and the barcode can be crosslinked.

[0188] Cell lysis

[0189] After the dispensing of the cells and the barcodes, the cells can be lysed to release the target molecules. The cell lysis can be accomplished by any of a variety of means, such as by chemical or biochemical means, by osmotic shock, or by means of thermal lysis, mechanical lysis, or optical lysis. The cells can be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, lithium dodecyl sulfate, Triton X-100, Tween-20, or NP-40), an organic solvent (e.g., methanol or acetone), or a digestive enzyme (e.g., proteinase K, pepsin, or trypsin) or any combination thereof. To increase the association between the target and the barcode, the diffusion rate of the target molecules can be altered, for example, by lowering the temperature of the lysate and / or increasing the viscosity of the lysate.

[0190] In some embodiments, a filter paper can be used to lyse the sample. The filter paper can be soaked with a lysis buffer on top. Pressure can be applied to the sample with the filter paper, which can facilitate the lysis of the sample and the hybridization of the target of the sample with the substrate.

[0191] In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include using digestive enzymes such as proteinase K, pepsin, and trypsin. Lysis can be performed by adding a lysis buffer to the substrate. The lysis buffer can contain Tris HCl. The lysis buffer can contain at least about 0.01 M, 0.05 M, 0.1 M, 0.5 M, or 1 M or more of Tris HCl. The lysis buffer can contain at most about 0.01 M, 0.05 M, 0.1 M, 0.5 M, or 1 M or more of Tris HCl. The lysis buffer can contain about 0.1 M Tris HCl. The pH of the lysis buffer can be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher. The pH of the lysis buffer can be at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or higher. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer can contain salts (e.g., LiCl). The salt concentration in the lysis buffer can be at least about 0.1 M, 0.5 M, or 1 M or higher. The salt concentration in the lysis buffer can be at most about 0.1 M, 0.5 M, or 1 M or higher. In some embodiments, the concentration of the salt in the lysis buffer is about 0.5 M. The lysis buffer can contain detergents (e.g., SDS, lithium dodecyl sulfate, triton X, tween, NP-40). The detergent concentration in the lysis buffer can be at least about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or higher. The detergent concentration in the lysis buffer can be at most about 0.0001%, 0.0005%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, or 7% or higher. In some embodiments, the detergent concentration in the lysis buffer is about 1% lithium dodecyl sulfate. The time used in the lysis method can depend on the amount of detergent used. In some embodiments, the more detergent used, the less time required for lysis. The lysis buffer can contain chelating agents (e.g., EDTA, EGTA). The chelating agent concentration in the lysis buffer can be at least about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM or higher. The chelating agent concentration in the lysis buffer can be at most about 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM or higher. In some embodiments, the chelating agent concentration in the lysis buffer is about 10 mM. The lysis buffer can contain reducing agents (e.g., β-mercaptoethanol, DTT). The reducing agent concentration in the lysis buffer can be at least about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or higher.The concentration of the reducing agent in the lysis buffer can be at most about 1 mM, 5 mM, 10 mM, 15 mM, or 20 mM or higher. In some embodiments, the concentration of the reducing agent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer can comprise about 0.1 M Tris HCl, about pH 7.5, about 0.5 M LiCl, about 1% lithium dodecyl sulfate, about 10 mM EDTA, and about 5 mM DTT.

[0192] Lysis can be carried out at a temperature of about 4 °C, 10 °C, 15 °C, 20 °C, 25 °C, or 30 °C. Lysis can be carried out for about 1 minute, 5 minutes, 10 minutes, 15 minutes, or 20 minutes or more. The lysed cells can contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules. The lysed cells can contain at most about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, or 700,000 or more target nucleic acid molecules.

[0193] Attach the barcode to the target nucleic acid molecule

[0194] After cell lysis and release of nucleic acid molecules from the cells, the nucleic acid molecules can be randomly associated with the barcodes of the co-localized solid support. The association can include hybridizing the target recognition region of the barcode with the complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode can interact with the poly(A) tail of the target). The assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) for hybridization can be selected to promote the formation of specific stable hybrids. In some embodiments, the nucleic acid molecules released from the lysed cells can be associated with more than one probe on the substrate (e.g., hybridized with the probe on the substrate). When the probe contains oligo(dT), the mRNA molecules can be hybridized with the probe and reverse transcribed. The oligo(dT) portion of the oligonucleotide can serve as a primer for the first-strand synthesis of cDNA molecules. For example, in Figure 2 the non-limiting example of barcoding illustrated, at box 216, the mRNA molecules can be hybridized with the barcodes on the beads. For example, single-stranded nucleotide fragments can be hybridized with the target binding region of the barcode.

[0195] Attachment can also include linking the target recognition region of the barcode to a portion of the target nucleic acid molecule. For example, the target binding region can contain a nucleic acid sequence that can specifically hybridize to a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure can also include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to generate a restriction site overhang. The barcode can then be linked to any nucleic acid molecule containing a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to join the two fragments.

[0196] For example, in Figure 2 the non-limiting example of barcoding illustrated in, at block 220, labeled targets (e.g., target-barcode molecules) from more than one cell (or more than one sample) can subsequently be pooled, e.g., into a tube. The labeled targets can be pooled by, e.g., retrieving the barcode and / or beads to which the target-barcode molecules are attached.

[0197] Recovery of the solid support-based collection of attached target-barcode molecules can be achieved by using magnetic beads and an externally applied magnetic field. After pooling the target-barcode molecules, all further processing can be performed in a single reaction vessel. Further processing can include, e.g., a reverse transcription reaction, an amplification reaction, a lysis reaction, a dissociation reaction, and / or a nucleic acid extension reaction. The further processing reactions can be performed within a micro-well, i.e., without first pooling labeled target nucleic acid molecules from more than one cell.

[0198] Reverse transcription or nucleic acid extension

[0199] The present disclosure provides methods of using reverse transcription (e.g., at block 224 of Figure 2 ) or nucleic acid extension to generate target-barcode conjugates. The target-barcode conjugates can contain the barcode and a complementary sequence of all or a portion of the target nucleic acid (i.e., a barcoded cDNA molecule, such as a randomly barcoded cDNA molecule). Reverse transcription of the associated RNA molecule can occur by adding a reverse transcription primer along with a reverse transcriptase. The reverse transcription primer can be an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. The length of the oligo(dT) primer can be 12 - 18 nucleotides or can be about 12 - 18 nucleotides and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexanucleotide primer can bind to the mRNA at various complementary sites. The target-specific oligonucleotide primer typically selectively primes the mRNA of interest.

[0200] In some embodiments, reverse transcription of the mRNA molecule into a labeled RNA molecule can occur by adding a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexamer primer, or a target-specific oligonucleotide primer. Typically, the oligo(dT) primer is 12 - 18 nucleotides in length and binds to the endogenous poly(A) tail at the 3' end of mammalian mRNA. The random hexamer primer can bind to the mRNA at various complementary sites. The target-specific oligonucleotide primer typically selectively primes the mRNA of interest.

[0201] In some embodiments, the target is a cDNA molecule. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase such as Moloney murine leukemia virus (MMLV) reverse transcriptase to produce a cDNA molecule with a poly(dC) tail. The barcode can include a target-binding region with a poly(dG) tail. After base pairing between the poly(dG) tail of the barcode and the poly(dC) tail of the cDNA molecule, the reverse transcriptase switches the template strand from the cellular RNA molecule to the barcode and continues to replicate towards the 5' end of the barcode. By doing so, the resulting cDNA molecule contains a barcode sequence (such as a molecular tag) at the 3' end of the cDNA molecule.

[0202] Reverse transcription can occur repeatedly to produce more than one labeled cDNA molecule. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 reverse transcription reactions. The method can include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 reverse transcription reactions.

[0203] Amplification

[0204] One or more nucleic acid amplification reactions can be performed (e.g., in Figure 2at the frame 228) to generate more than one copy of the labeled target nucleic acid molecule. The amplification can be carried out in a multiplexed manner, where more than one target nucleic acid sequence is amplified simultaneously. The amplification reaction can be used to add sequencing adapters to the nucleic acid molecule. The amplification reaction can include amplifying at least a portion of the sample label (if present). The amplification reaction can include amplifying at least a portion of the cell label and / or barcode sequence (e.g., molecular label). The amplification reaction can include amplifying at least a portion of the sample tag, cell label, spatial label, barcode sequence (e.g., molecular label), target nucleic acid, or a combination thereof. The amplification reaction can include amplifying 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 100% or a range or number between any two of these values of more than one nucleic acid. The method can also include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of the target-barcode molecule containing the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0205] In some embodiments, the amplification can be carried out using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for in vitro amplification of a specific DNA sequence by simultaneous extension of primers of the complementary strands of DNA. As used herein, PCR can encompass derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplexed PCR, digital PCR, and assembly PCR.

[0206] The amplification of the labeled nucleic acid can include non-PCR-based methods. Examples of non-PCR-based methods include but are not limited to multiple displacement amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, rolling circle amplification, or circle-to-circle amplification. Other non-PCR-based amplification methods include DNA-dependent RNA polymerase-driven RNA transcription amplification or more than one cycle of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), and Qβ replicase (Qβ) method, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods that hybridize primers to nucleic acid sequences and cleave the resulting duplex before extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and ramification extension amplification (RAM). In some embodiments, the amplification does not produce circular transcripts.

[0207] In some embodiments, the methods disclosed herein further include performing polymerase chain reaction on a labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to produce a labeled amplicon (e.g., randomly labeled amplicon). The labeled amplicon can be a double-stranded molecule. The double-stranded molecule can include a double-stranded RNA molecule, a double-stranded DNA molecule, or an RNA molecule hybridized to a DNA molecule. One or both strands of the double-stranded molecule can comprise a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure can include synthetic or altered nucleic acids.

[0208] Amplification can include using one or more unnatural nucleotides. Unnatural nucleotides can include photo-labile or triggerable nucleotides. Examples of unnatural nucleotides can include, but are not limited to, peptide nucleic acid (PNA), morpholino nucleic acid, and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). The unnatural nucleotides can be added to one or more cycles of the amplification reaction. Adding the unnatural nucleotides can be used to identify the products at a specific cycle or time point in the amplification reaction.

[0209] Performing one or more amplification reactions can include using one or more primers. One or more primers can include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. One or more primers can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more nucleotides. One or more primers can contain fewer than 12-15 nucleotides. One or more primers can anneal to at least a portion of more than one labeled target (e.g., randomly labeled target). One or more primers can anneal to the 3' end or 5' end of more than one labeled target. One or more primers can anneal to an internal region of more than one labeled target. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900 or 1000 nucleotides away from the 3' end of more than one labeled target. One or more primers can include a set of fixed primers. One or more primers can include at least one or more custom primers. One or more primers can include at least one or more control primers. One or more primers can include at least one or more gene-specific primers.

[0210] One or more primers can include universal primers. The universal primers can anneal to universal primer binding sites. One or more custom primers can anneal to a first sample label, a second sample label, a spatial label, a cell label, a barcode sequence (e.g., a molecular label), a target, or any combination thereof. One or more primers can include universal primers and custom primers. The custom primers can be designed to amplify one or more targets. The targets can include a subset of the total nucleic acids in one or more samples. The targets can include a subset of the total labeled targets in one or more samples. One or more primers can include at least 96 or more custom primers. One or more primers can include at least 960 or more custom primers. One or more primers can include at least 9600 or more custom primers. One or more custom primers can anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids can correspond to one or more genes.

[0211] Any amplification protocol can be used in the methods of the present disclosure. For example, in one protocol, a first round of PCR can use gene-specific primers and primers targeting the universal Illumina sequencing primer 1 sequence to amplify molecules attached to beads. A second round of PCR can use nested gene-specific primers flanked by the Illumina sequencing primer 2 sequence and primers targeting the universal Illumina sequencing primer 1 sequence to amplify the first PCR product. A third round of PCR adds P5 and P7 as well as a sample index to turn the PCR product into an Illumina sequencing library. Sequencing using 150bp×2 can reveal the cell label and barcode sequence (e.g., a molecular label) on read 1, the gene on read 2, and the sample index on the index 1 read.

[0212] In some embodiments, nucleic acids can be removed from a substrate using chemical cleavage. For example, chemical groups or modified bases present in the nucleic acids can be used to facilitate removal of the nucleic acids from the solid support. For example, enzymes can be used to remove nucleic acids from a substrate. For example, nucleic acids can be removed from a substrate by restriction endonuclease digestion. For example, treatment of nucleic acids containing dUTP or ddUTP with uracil-d-glycosidase (UDG) can be used to remove nucleic acids from a substrate. For example, enzymes that perform nucleotide excision (such as base excision repair enzymes, such as apurinic / apyrimidinic (AP) endonucleases) can be used to remove nucleic acids from a substrate. In some embodiments, photocleavable groups and light can be used to remove nucleic acids from a substrate. In some embodiments, cleavable linkers can be used to remove nucleic acids from a substrate. For example, cleavable linkers can include at least one of the following: biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig protein A, photo-labile linkers, acid- or base-labile linker groups, or aptamers.

[0213] When the probe is gene-specific, molecules can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, the nucleic acids can be amplified after they have been synthesized (e.g., reverse transcribed). Amplification can be performed in multiplex, where multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acids.

[0214] In some embodiments, amplification can be performed on a substrate, for example, by bridge amplification. cDNA can be homopolymer tailed to generate compatible ends for bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, the primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair of primers covalently attached to the solid particles. When a sample containing the template nucleic acid contacts the particles and undergoes a single thermal cycle, the template molecule can be annealed to the first primer, and the first primer is extended forward by adding nucleotides to form a duplex molecule composed of the template molecule and the newly formed DNA strand complementary to the template. In the heating step of the next cycle, the duplex molecule can denature, releasing the template molecule from the particles and leaving the complementary DNA strand attached to the particles via the first primer. In the annealing phase of the subsequent annealing and extension steps, the complementary strand can hybridize with the second primer, which is complementary to a segment of the complementary strand at the position removed from the first primer. This hybridization can result in the formation of a bridge between the first primer and the second primer by the complementary strand, connecting the first primer by a covalent bond and the second primer by hybridization. In the extension phase, by adding nucleotides in the same reaction mixture, the second primer can be extended in the reverse direction, converting the bridge into a double-stranded bridge. Then the next cycle begins, and the double-stranded bridge can denature to produce two single-stranded nucleic acid molecules, each with one end attached to the particle surface via the first primer and the second primer respectively, and the other end of each single-stranded nucleic acid molecule is unattached. In the annealing and extension steps of this second cycle, each strand can hybridize with additional complementary primers previously unused on the same particle to form new single-stranded bridges. The two previously unused primers that are now hybridized are extended to convert the two new bridges into double-stranded bridges.

[0215] The amplification reaction can include amplifying at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97% or 100% of more than one nucleic acid.

[0216] The amplification of the labeled nucleic acid can include PCR-based methods or non-PCR-based methods. The amplification of the labeled nucleic acid can include exponential amplification of the labeled nucleic acid. The amplification of the labeled nucleic acid can include linear amplification of the labeled nucleic acid. Amplification can be performed by polymerase chain reaction (PCR). PCR can refer to a reaction for in vitro amplification of a specific DNA sequence by simultaneous extension of primers of the complementary strands of DNA. PCR can cover derivative forms of the reaction, including but not limited to, RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, digital PCR, suppression PCR, semi-suppression PCR, and assembly PCR.

[0217] Amplification of the labeled nucleic acid can include non-PCR-based methods, including but not limited to, MDA, TMA, NASBA, SDA, real-time SDA, rolling circle amplification, circle-to-circle amplification, DNA-dependent RNA polymerase-driven RNA transcription amplification, or multiple cycles of RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ) method, use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods that hybridize primers to a nucleic acid sequence and cleave the resulting duplex prior to extension reaction and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5'-exonuclease activity, rolling circle amplification, and / or RAM.

[0218] In some embodiments, the methods disclosed herein further include performing nested polymerase chain reaction on the amplified amplicons (e.g., targets). The amplicons can be double-stranded molecules. The double-stranded molecules can include double-stranded RNA molecules, double-stranded DNA molecules, or RNA molecules hybridized to DNA molecules. One or both strands of the double-stranded molecule can contain a sample tag or molecular identifier label. Alternatively, the amplicon can be a single-stranded molecule. The single-stranded molecule can include DNA, RNA, or a combination thereof. The nucleic acids of the present invention can include synthetic or altered nucleic acids.

[0219] In some embodiments, the method includes repeatedly amplifying the labeled nucleic acid to produce more than one amplicon. The methods disclosed herein can include performing at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amplification reactions. Alternatively, the method includes performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0220] Amplification can also include adding one or more control nucleic acids to one or more samples containing more than one nucleic acid. Amplification can also include adding one or more control nucleic acids to more than one nucleic acid. The control nucleic acid can contain a control label.

[0221] Amplification can include using one or more unnatural nucleotides. Unnatural nucleotides can include photo-labile and / or triggerable nucleotides. Examples of unnatural nucleotides include, but are not limited to, peptide nucleic acid (PNA), morpholino nucleic acid, and locked nucleic acid (LNA), as well as glycol nucleic acid (GNA) and threose nucleic acid (TNA). Unnatural nucleotides can be added to one or more cycles of the amplification reaction. Adding unnatural nucleotides can be used to identify products at specific cycles or time points in the amplification reaction.

[0222] Performing one or more amplification reactions can include using one or more primers. One or more primers can include one or more oligonucleotides. One or more oligonucleotides can comprise at least about 7-9 nucleotides. One or more oligonucleotides can comprise fewer than 12-15 nucleotides. One or more primers can anneal to at least a portion of more than one labeled nucleic acid. One or more primers can anneal to the 3' end and / or 5' end of more than one labeled nucleic acid. One or more primers can anneal to an internal region of more than one labeled nucleic acid. The internal region can be at least about 50, 100, 150, 200, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 650, 700, 750, 800, 850, 900, or 1000 nucleotides away from the 3' end of more than one labeled nucleic acid. One or more primers can include a set of fixed primers. One or more primers can include at least one or more custom primers. One or more primers can include at least one or more control primers. One or more primers can include at least one or more housekeeping gene primers. One or more primers can include universal primers. Universal primers can anneal to universal primer binding sites. One or more custom primers can anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. One or more primers can include universal primers and custom primers. Custom primers can be designed to amplify one or more target nucleic acids. Target nucleic acids can include a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are probes attached to the arrays of the present disclosure.

[0223] In some embodiments, barcoding (e.g., random barcoding) more than one target in a sample further includes generating an indexed library of barcoded targets (e.g., randomly barcoded targets) or barcoded fragments of targets. The barcode sequences of different barcodes (e.g., the molecular tags of different random barcodes) can be different from each other. Generating an indexed library of barcoded targets includes generating more than one indexed polynucleotide from more than one target in the sample. For example, for an indexed library of barcoded targets that includes a first indexed target and a second indexed target, the tagged region of the first indexed polynucleotide can differ by, differ by about, differ by at least, or differ by at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides or a number or range of nucleotides between any two of these values from the tagged region of the second indexed polynucleotide. In some embodiments, generating an indexed library of barcoded targets includes contacting more than one target (e.g., mRNA molecules) with more than one oligonucleotide that includes a poly(T) region and a tagged region; and performing first-strand synthesis using reverse transcriptase to generate single-stranded tagged cDNA molecules (each including a cDNA region and a tagged region), wherein more than one target includes at least two mRNA molecules of different sequences and more than one oligonucleotide includes at least two oligonucleotides of different sequences. Generating an indexed library of barcoded targets can further include amplifying the single-stranded tagged cDNA molecules to generate double-stranded tagged cDNA molecules; and performing nested PCR on the double-stranded tagged cDNA molecules to generate tagged amplicons. In some embodiments, the method can include generating adapter-tagged amplicons.

[0224] Barcoding (e.g., random barcoding) can include using nucleic acid barcodes or tags to label individual nucleic acid (e.g., DNA or RNA) molecules. In some embodiments, it involves adding a DNA barcode or tag to a cDNA molecule when generating the cDNA molecule from mRNA. Nested PCR can be performed to minimize PCR amplification bias. Adapters for sequencing (e.g., next-generation sequencing (NGS)) use can be added. For example, at Figure 2 box 232, sequencing results can be used to determine the sequences of cell tags, molecular tags, and nucleotide fragments of one or more copies of a target.

[0225] Figure 3It is a schematic diagram showing a non-limiting exemplary process of generating an indexed library of barcoded targets (e.g., randomly barcoded targets), such as an indexed library of barcoded mRNAs or fragments thereof. As shown in step 1, the reverse transcription process can encode each mRNA molecule with a unique molecular tag sequence, a cell tag sequence, and a universal PCR site. Specifically, by hybridizing a set of barcodes (e.g., random barcodes) 310 to the poly(A) tail region 308 of the RNA molecule 302 (e.g., randomly hybridizing), the RNA molecule 302 can be reverse transcribed to generate a labeled cDNA molecule 304 (including a cDNA region 306). Each of the barcodes 310 can include a target binding region, such as a poly(dT) region 312, a tag region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0226] In some embodiments, the cell tag sequence can comprise 3 to 20 nucleotides. In some embodiments, the molecular tag sequence can comprise 3 to 20 nucleotides. In some embodiments, each of more than one random barcode further includes one or more of a universal tag and a cell tag, wherein the universal tag is the same for more than one random barcode on a solid support, and the cell tag is the same for more than one random barcode on a solid support. In some embodiments, the universal tag can comprise 3 to 20 nucleotides. In some embodiments, the cell tag comprises 3 to 20 nucleotides.

[0227] In some embodiments, the tag region 314 can include a barcode sequence or molecular tag 318 and a cell tag 320. In some embodiments, the tag region 314 can include one or more of a universal tag, a dimension tag, and a cell tag. The length of the barcode sequence or molecular tag 318 can be the following, can be about the following, can be at least the following, or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides between any two of these values. The length of the cell tag 320 can be the following, can be about the following, can be at least the following, or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides between any two of these values. The length of the universal tag can be the following, can be about the following, can be at least the following, or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides between any two of these values. The universal tag can be the same for more than one random barcode on the solid support, and the cell tag is the same for more than one random barcode on the solid support. The length of the dimension tag can be the following, can be about the following, can be at least the following, or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range of nucleotides between any two of these values.

[0228] In some embodiments, the tagging region 314 can include the following, can include about the following, can include at least the following, or can include at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 different tags, such as barcode sequences or molecular tags 318 and cell tags 320, or a number or range of numbers between any two of these values. The length of each tag can be the following, can be about the following, can be at least the following, or can be at most the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 nucleotides, or a number or range of numbers between any two of these values. A set of barcodes or random barcodes 310 can contain the following, can contain about the following, can contain at least the following, or can contain at most the following: 10, 20, 40, 50, 70, 80, 90, 10 2 species, 10 3 species, 10 4 species, 10 5 species, 10 6 species, 10 7 species, 10 8 species, 10 9 species, 10 10 species, 10 11 species, 10 12 species, 10 13 species, 10 14 species, 10 15 species, 10 20 species, or a number or range of numbers between any two of these values of barcodes or random barcodes 310. And the set of barcodes or random barcodes 310 can, for example, each contain a unique tagging region 314. The tagged cDNA molecules 304 can be purified to remove excess barcodes or random barcodes 310. The purification can include Ampure bead purification.

[0229] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled into 1 tube and PCR amplified with the first PCR primer pool and the first universal PCR primer. Pooling is possible because of the unique tag region 314. In particular, the tagged cDNA molecules 304 can be amplified to generate nested PCR tagged amplicons 322. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification with 96 multiplex primers in a single reaction volume. In some embodiments, in a single reaction volume, the multiplex PCR amplification can utilize, utilize about, utilize at least, or utilize at most 10, 20, 40, 50, 70, 80, 90, 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、10 10 、10 11 、10 12 、10 13 、10 14 、10 15 、10 20 or a number or range between any two of these values of multiplex primers. The amplification can include using the first PCR primer pool 324 including custom primers 326A-C targeting specific genes and the universal primer 328. The custom primer 326 can hybridize to a region within the cDNA portion 306' of the tagged cDNA molecule 304. The universal primer 328 can hybridize to the universal PCR region 316 of the tagged cDNA molecule 304.

[0230] As Figure 3As shown in step 3, the product from the PCR amplification in step 2 can be amplified with a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, the amplicon 322 labeled with nested PCR can be further amplified by nested PCR. Nested PCR can include multiplex PCR performed in a single reaction volume with a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328'. The nested PCR primer pool 328 can contain, can contain approximately, can contain at least, or can contain at most: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, or a number or range between any two of these values of different nested PCR primers 330. The nested PCR primers 332 can contain adapters 334 and hybridize to regions within the cDNA portion 306" of the labeled amplicon 322. The universal primer 328' can contain an adapter 336 and hybridize to the universal PCR region 316 of the labeled amplicon 322. Thereby, step 3 generates adapter-labeled amplicon 338. In some embodiments, the nested PCR primers 332 and the second universal PCR primer 328' may not contain adapters 334 and 336. Instead, adapters 334 and 336 can be ligated to the product of the nested PCR to generate adapter-labeled amplicon 338.

[0231] As shown in step 4, the PCR product from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, one or more additional assays can be performed on the adapter-labeled amplicon 338 using adapters 334 and 336. The adapters 334 and 336 can hybridize to primers 340 and primer 342. One or more of the primers 340 and 342 can be PCR amplification primers. One or more of the primers 340 and 342 can be sequencing primers. One or more of the adapters 334 and 336 can be used for further amplification of the adapter-labeled amplicon 338. One or more of the adapters 334 and 336 can be used for sequencing the adapter-labeled amplicon 338. The primer 342 can contain a plate index 344 such that amplicons generated using the same set of barcodes or random barcodes 310 can be sequenced in a single sequencing reaction using next-generation sequencing (NGS).

[0232] Chambered Single Cell Barcoding

[0233] High-throughput screening can be performed with a sample multiplexing kit, where different cell populations can be stained with antibodies containing sample-barcode oligonucleotides. However, in some embodiments, these additional staining steps are not preferred because they may alter the biological response. Provided herein are compositions, systems, and methods for a compartmentalized Rhapsody with unique barcodes (e.g., well index sub-sequences). Compositions and methods are provided for sorting into barcoded wells for a single-cell workflow on a Rhapsody cartridge. To associate image- or fluorescence-based sorting with single-cell data, it can be important to have a device for sorting cells into wells such that information about which cell population (based on gating) is sorted into which well is retained. Provided herein are compositions and methods that include a compartmentalized Rhapsody cartridge, where each well can be loaded with beads containing a unique set of cell markers.

[0234] In some embodiments, a protocol for the manufacture of solid supports (e.g., beads) is provided. In some embodiments, x unique sets of cell markers are provided, where each set contains y unique cell markers. For example, a 384-well compartmentalized cartridge can contain 384 unique sets of cell markers. There can be thousands of microwells within each of these wells. A cell sorter can directly sort different cell populations into these wells (e.g., as it would sort into a 384-well plate). In some embodiments, a workflow for maintaining cell viability in the cartridge and the remainder of the single-cell workflow provided herein (e.g., Rhapsody) is provided. Some embodiments of the compositions and methods provided herein include automated liquid handling. Figures 4A - 4B A non-limiting exemplary schematic of the microwell array Figure 4A ( Figure 4B ) and well Figure 4A provided herein is depicted. A non-limiting exemplary schematic of a microwell array 400 including more than one well 402 is depicted. Each well can include more than one partition 404 (e.g., microwell).

[0235] In some embodiments, methods and compositions are provided that are capable of differentiating library samples after cell barcoding sequencing, which can simplify the user workflow. The disclosure herein includes methods and compositions for sample and lane identification using cell barcodes. In some embodiments, the method includes assigning specific cell barcodes (e.g., containing a chamber-specific chamber index subsequence) to each chamber of a multi-chamber cartridge (e.g., an 8-lane cartridge of an HT Xpress system), while maintaining high cell barcode diversity by fabricating, for example, 8 different bead batches. By assigning specific cell labels to each lane (e.g., chamber), the user can identify their library samples when performing a multi-lane experiment without having to assign specific forward and reverse primers to each lane and library type. This can enable an efficient workflow for the user to prepare their libraries for sequencing.

[0236] The disclosure herein provides more than one solid support containing a chamber-specific chamber index subsequence (e.g., 8 different barcoded beads for each HT lane), such that the user can differentiate chambers (e.g., lanes) without having to use indexed library primers (e.g., 8 different reverse primers). Current methods require the user to use different forward and reverse primers to differentiate their library samples. By enabling the user to differentiate based on cell barcodes, the methods and compositions provided herein can eliminate the need to use multiple different forward and reverse primers.

[0237] In some embodiments, the cell label diversity is 384 * 384 * 384 = 56623104 different cell barcode combinations, where there are 384 cell labels 1 (first cell label portion; CL1), 384 cell labels 2 (second cell label portion; CL2), and 384 cell labels 3 (third cell label portion; CL3). It has been simulated that up to 192 * 192 * 192 = 7077888 different cell barcode combinations are needed to maintain a cell barcode conflict of less than 1%. In some embodiments, the methods and compositions provided herein use the same 384 cell label portions spanning three different oligonucleotides, but are distributed in such a way that each bead batch has 7077888 different cell barcodes. The methods provided herein can generate 8 different bead batches with specific cell barcodes. Early users can assign specific bead batches to each lane. For example, for 8 lanes, the user can use 8 different bead batches. Currently, users assign different reverse primers to distinguish their lanes. For example, if a user wants to perform whole transcriptome analysis (WTA) on 8 lanes with 8 different samples, instead of tracking 8 different reverse primers, using the provided compositions and methods, as long as they input their beads into their respective lanes, ultimately they will only need to use 1 reverse primer and can resolve their samples bioinformatically. For example, Table 1 illustrates the current method of using library indices on primers to distinguish starting lanes (for targeted or WTA library amplification).

[0238] Table 1: Current library indexing method

[0239]

[0240]

[0241] In addition, as the complexity of the library combination increases, the number of forward and reverse indices tracked using the current method increases. Thus, for example, for 8 lanes used only for WTA, 8 library indices are employed, but for 8 lanes used for WTA + AbSeq, this increases to 16 library indices, and for 8 lanes used for WTA + AbSeq + ST (e.g., sample tagging), this increases to 24 library indices. In contrast, Table 2 illustrates an exemplary setup using the compositions and methods provided herein - in the case of assigning different cell barcodes to each lane, the user may only need one forward primer / reverse primer to track each library type.

[0242] Table 2: Improved method where cell barcodes are assigned to each lane

[0243]

[0244] The present disclosure provides methods and compositions for preparing and using oligonucleotide barcodes (e.g., solid supports comprising chamber-specific chamber index subsequences) provided herein that include chamber index subsequences. Figure 5 Depicted is a non-limiting exemplary method for fabricating oligonucleotide barcodes comprising chamber index subsequences provided herein. Each box represents a 2 x 96 well plate (192 total CLs). The fabrication of eight or more solid supports (which can be used by a user to distinguish 8 lanes / chambers) is depicted. The method can use (192 CL1) x (192 CL2) x (192 CL3) to generate 7,077,888 different combinations. Letters A, B, C, and D represent different oligonucleotide populations having different cell marker portion sequences. Figure 6 Depicted is another non-limiting exemplary method for fabricating oligonucleotide barcodes comprising chamber index subsequences provided herein. For example, a user can combine 384 CL1, 384 CL2, and 48 different CL3 for each lane. The method can simplify bead fabrication and can maintain 7,077,888 cell barcode diversities (384 x 384 x 48). The fabrication method can be similar to current processes, but only pool half of the plate in the oligonucleotide 3 step. Figure 7 Depicted is another non-limiting exemplary method for fabricating oligonucleotide barcodes comprising chamber index subsequences provided herein. The fabrication method adds a large number of ligations at the end of the current process to add a differentiated cell marker with oligonucleotide 4. The cell barcode diversity can be 384 x 384 x 384 x 1 = ~56 million. In some embodiments, oligonucleotide 4 is configured to reduce the increased sequencing read length, which can reduce the library diversity of R1 in some embodiments. The sequence of the cell marker portion can be the same between the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide. For example, in some embodiments, the first oligonucleotide and the second oligonucleotide and / or the third oligonucleotide can each have a first cell marker portion, a second cell marker portion, or a third cell marker portion comprising the AAAGG sequence, respectively.

[0245] The present disclosure provides two or more than one solid supports (e.g., a first solid support, a second solid support, a third solid support, etc.), wherein each of the more than one solid supports has a unique chamber-specific chamber index subsequence. Each of the more than one solid supports can be incorporated into a different chamber (e.g., the first solid support is placed in lane 1 and the second solid support is placed in lane 2). Based on the unique chamber-specific chamber index subsequence of each of the more than one solid supports, it can be determined from the sequencing reads whether it is a product of an oligonucleotide barcode associated with, for example, the first solid support or the second solid support (and thus whether it originated from lane 1 or lane 2). For example, the first solid support among the more than one first solid supports can have a first chamber index subsequence that enables a user to uniquely identify from the sequencing reads originating from the first solid support. In some embodiments, the first chamber index subsequence is a single subsequence of a cell label. For example, referring to Figure 7 , in some embodiments, the first chamber index subsequence is a single cell label portion (such as a fourth cell label portion, as shown in Figure 7 ), and the unique subsequence is the same on all first solid supports (e.g., not selected from a set of chamber index subsequences). In this case, only the fourth cell label portion of the cell label is used to distinguish the solid support (and thus the chamber). In some embodiments, as shown in Figure 6 , a single subsequence of the cell label is used to distinguish the solid support (and thus the chamber), but the chamber index subsequence is selected from a set of chamber index subsequences. For the oligonucleotide barcode of the method derived from Figure 6 , the chamber index subsequence can be a third cell label portion. For example, if 48 different oligonucleotides 3 (including CL3; e.g., the first half of the third oligonucleotide of population A) are used to generate the first solid support, the first chamber index subsequence has one of the sequences of a set of 48 chamber index subsequences. The second solid support can have CL3 derived from the second half of the third oligonucleotide of population A. Thus, the second solid support can have a second chamber index subsequence that has one of the sequences of a non-overlapping set of another 48 chamber index subsequences (the second half of the third oligonucleotide of population A). Since the third cell label portions of the first solid support and the second solid support are derived from non-overlapping subsets of the third cell label portion sequence, the user can determine whether the sequencing reads originated from the first solid support or the second solid support based on the sequence of the third label portion. Thus, the chamber index subsequence is predetermined because for both the first solid support and the second solid support, the user knows what the possible set of sequences of the chamber index subsequence is for each.

[0246] In some embodiments, the predetermined chamber index subsequence can include multiple (or all) components of the cell label. For example, usingFigure 5 The process shown in Figure 5 can produce eight or more than one solid supports (to distinguish different chambers / lanes). Each of the more than one solid supports can be derived from different combinations of populations of a first oligonucleotide, a second oligonucleotide, and a third oligonucleotide. For example, with respect to the first solid support (for chamber / lane 1), these can be derived from a first population of the first oligonucleotide, a first population of the second oligonucleotide, and a first population of the third oligonucleotide, while the second solid support (for chamber / lane 2) can be derived from a second population of the first oligonucleotide, a second population of the second oligonucleotide, and a second population of the third oligonucleotide. In this case, the first and second populations of the first oligonucleotide are the same (A / B), the first and second populations of the second oligonucleotide are the same (A / B), and the first and second populations of the third oligonucleotide are different (A / B versus C / D). Since the CL3 sequences of the first and second populations of the third oligonucleotide are different from each other, the first solid support and the second solid support can be distinguished from each other based on the third cell marker portion (chamber index subsequence). All eight lanes are different from each other in terms of at least one of oligonucleotide 1, oligonucleotide 2, or oligonucleotide 3, and can thus be distinguished based on one, two, or three chamber index subsequences.

[0247] The compositions disclosed herein can comprise two or more than one solid supports. The solid supports can each comprise more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence. In some embodiments, the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences.

[0248] In some embodiments, two or more than one solid supports comprise more than one first solid support and more than one second solid support, wherein the oligonucleotide barcodes associated with the more than one first solid support have a first predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the more than one second solid support have a second predetermined chamber index subsequence, and wherein the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

[0249] In some embodiments, two or more than two solid supports include more than one first solid support and more than one second solid support, wherein a first predetermined chamber index subsequence of the more than one first solid supports is selected from a first set of chamber index subsequences, wherein a second predetermined chamber index subsequence of the more than one second solid supports is selected from a second set of chamber index subsequences, and wherein each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences. In some embodiments, based on a predetermined chamber index subsequence of sequencing reads derived from an oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with more than one first solid support or more than one second solid support.

[0250] Two or more than one solid supports may include: more than one third solid support, optionally: (a) the oligonucleotide barcode associated with the more than one third solid support has a third chamber index subsequence and / or (b) the third chamber index subsequence of the more than one third solid support is selected from a third set of chamber index subsequences; more than one fourth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one fourth solid support has a fourth chamber index subsequence and / or (b) the fourth chamber index subsequence of the more than one fourth solid support is selected from a fourth set of chamber index subsequences; more than one fifth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one fifth solid support has a fifth chamber index subsequence and / or (b) the fifth chamber index subsequence of the more than one fifth solid support is selected from a fifth set of chamber index subsequences; more than one sixth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one sixth solid support has a sixth chamber index subsequence and / or (b) the sixth chamber index subsequence of the more than one sixth solid support is selected from a sixth set of chamber index subsequences; more than one seventh solid support, optionally: (a) the oligonucleotide barcode associated with the more than one seventh solid support has a seventh chamber index subsequence and / or (b) the seventh chamber index subsequence of the more than one seventh solid support is selected from a seventh set of chamber index subsequences; and / or more than one eighth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one eighth solid support has an eighth chamber index subsequence and / or (b) the eighth chamber index subsequence of the more than one eighth solid support is selected from an eighth set of chamber index subsequences. In some embodiments, the first chamber index subsequence, the second chamber index subsequence, the third chamber index subsequence, the fourth chamber index subsequence, the fifth chamber index subsequence, the sixth chamber index subsequence, the seventh chamber index subsequence, and / or the eighth chamber index subsequence do not share sequences with each other. In some embodiments, based on a predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, the user can determine whether the oligonucleotide barcode is associated with the first solid support, the second solid support, the third solid support, the fourth solid support, the fifth solid support, the sixth solid support, the seventh solid support, or the eighth solid support.

[0251] In some embodiments, a cellular tag comprises more than one cellular tag portion and one or more linkers. In some embodiments, a cellular tag comprises a first cellular tag portion, a first linker, and a second cellular tag portion, optionally the cellular tag comprises a second linker and a third cellular tag portion, and further optionally the cellular tag comprises a third linker and a fourth cellular tag portion. In some embodiments, a predetermined chamber index subsequence comprises the first cellular tag portion, the second cellular tag portion, the third cellular tag portion, the fourth cellular tag portion, or any combination thereof. In some embodiments, the first set of chamber index subsequences, the second set of chamber index subsequences, the third set of chamber index subsequences, the fourth set of chamber index subsequences, the fifth set of chamber index subsequences, the sixth set of chamber index subsequences, the seventh set of chamber index subsequences, and / or the eighth set of chamber index subsequences comprise a set of fewer than about 960, about 864, about 768, about 672, about 576, about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences that are distinct from the chamber index subsequences of other sets of chamber index subsequences. In some embodiments, the first cellular tag portion, the second cellular tag portion, the third cellular tag portion, the fourth cellular tag portion, or any combination thereof is selected from a set of fewer than about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences. In some embodiments, based on the sequences of the first cellular tag portion, the second cellular tag portion, the third cellular tag portion, the fourth cellular tag portion, or any combination thereof from sequencing reads derived from an oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with a first solid support, a second solid support, a third solid support, a fourth solid support, a fifth solid support, a sixth solid support, a seventh solid support, or an eighth solid support.

[0252] The present disclosure includes methods. In some embodiments, the method includes: distributing more than one precursor first solid support and a first population of first oligonucleotides to more than one first partitions, wherein the co-localized solid support and the first oligonucleotides become associated; distributing more than one precursor second solid support and a second population of first oligonucleotides to more than one second partitions, wherein the co-localized solid support and the first oligonucleotides become associated; pooling more than one precursor first solid supports associated with the first oligonucleotides; pooling more than one precursor second solid supports associated with the first oligonucleotides; distributing more than one precursor first solid supports associated with the first oligonucleotides and a first population of second oligonucleotides to more than one first partitions of a second set, wherein the co-localized solid support and the second oligonucleotides become associated; distributing more than one precursor second solid supports associated with the first oligonucleotides and a second population of second oligonucleotides to more than one second partitions of a second set, wherein the co-localized solid support and the second oligonucleotides become associated; pooling more than one precursor first solid supports associated with the first and second oligonucleotides; pooling more than one precursor second solid supports associated with the first and second oligonucleotides; distributing more than one precursor first solid supports associated with the first and second oligonucleotides and a first population of third oligonucleotides to more than one first partitions of a third set, wherein the co-localized solid support and the third oligonucleotides become associated; and distributing more than one precursor second solid supports associated with the first and second oligonucleotides and a second population of third oligonucleotides to more than one second partitions of a third set, wherein the co-localized solid support and the third oligonucleotides become associated. In some embodiments, the method includes: pooling more than one precursor first solid supports associated with the first, second, and third oligonucleotides to produce more than one first solid supports; and pooling more than one precursor second solid supports associated with the first, second, and third oligonucleotides to produce more than one second solid supports.

[0253] In some embodiments, (i) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different; (ii) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are the same; (iii) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are different; (iv) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are different; (v) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are the same; (vi) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different; or (vii) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are the same.

[0254] In some embodiments, the first and second populations of the first oligonucleotide each comprise about 192 first cell marker portions having different sequences. In some embodiments, the first and second populations of the second oligonucleotide each comprise about 192 second cell marker portions having different sequences. In some embodiments, the first and second populations of the third oligonucleotide each comprise about 192 third cell marker portions having different sequences.

[0255] In some embodiments, the first and second populations of the first oligonucleotide are the same; the first and second populations of the second oligonucleotide are the same; and the first and second populations of the third oligonucleotide are different. In some embodiments, the first and second populations of the third oligonucleotide comprise a non-overlapping subset of the cell marker portion sequences of the first and second populations of the first oligonucleotide and / or the first and second populations of the second oligonucleotide.

[0256] In some embodiments, the first population and the second population of the first oligonucleotide each comprise about 384 first cell marker portions having different sequences. In some embodiments, the first population and the second population of the second oligonucleotide each comprise about 384 second cell marker portions having different sequences. In some embodiments, the first population and the second population of the third oligonucleotide each comprise about 48 third cell marker portions having different sequences.

[0257] In some embodiments, the method comprises: distributing more than one precursor first solid support associated with the first, second, and third oligonucleotides and a fourth oligonucleotide into more than one first partition, wherein the co-localized solid support and the fourth oligonucleotide become associated; distributing more than one precursor second solid support associated with the first, second, and third oligonucleotides and a fifth oligonucleotide into more than one second partition, wherein the co-localized solid support and the fifth oligonucleotide become associated; pooling more than one precursor first solid support associated with the first, second, third, and fourth oligonucleotides to produce more than one first solid support; and pooling more than one precursor second solid support associated with the first, second, third, and fifth oligonucleotides to produce more than one second solid support.

[0258] In some embodiments, the first population and the second population of the first oligonucleotide are the same; the first population and the second population of the second oligonucleotide are the same; the first population and the second population of the third oligonucleotide are the same; and the fifth oligonucleotide and the sixth oligonucleotide are different.

[0259] In some embodiments, the first oligonucleotide comprises a first cell marker portion and a first linker or its complement. In some embodiments, the second oligonucleotide comprises a first linker, a second cell marker portion, and a second linker or its complement. In some embodiments, the third oligonucleotide comprises a second linker and a third cell marker portion or its complement, optionally the second oligonucleotide further comprises a third linker or its complement. In some embodiments, the fourth oligonucleotide comprises a third linker and a fourth cell marker portion or its complement.

[0260] In some embodiments, the first oligonucleotide and the second oligonucleotide are configured to be joined via a first linker. In some embodiments, the second oligonucleotide and the third oligonucleotide are configured to be joined via a second linker. In some embodiments, the third oligonucleotide and the fourth oligonucleotide are configured to be joined via a third linker. In some embodiments, more than one first solid support and / or more than one second solid support comprise at least about 1000, about 10000, about 100000, about 1000000, about 7000000, about 10000000, about 56000000 unique cell marker sequences. In some embodiments, the sequences of the first cell marker portion, the second cell marker portion, and / or the third cell marker portion are the same. In some embodiments, the sequences of the first cell marker portion, the second cell marker portion, and / or the third cell marker portion are different. In some embodiments, the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, and / or the fourth oligonucleotide are single-stranded, double-stranded, and / or comprise one or two single-stranded overhangs. In some embodiments, the first solid support and the second solid support each comprise more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence.

[0261] In some embodiments, the oligonucleotide barcode associated with more than one first solid support has a first predetermined chamber index subsequence, the oligonucleotide barcode associated with more than one second solid support has a second predetermined chamber index subsequence, and the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

[0262] In some embodiments, the first predetermined chamber index subsequence of more than one first solid support is selected from a first set of chamber index subsequences, the second predetermined chamber index subsequence of more than one second solid support is selected from a second set of chamber index subsequences, and each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences. The methods provided herein can produce the compositions provided herein.

[0263] In some embodiments, the method comprises: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning more than one first solid support and more than one second solid support into partitions of an identified first chamber and an identified second chamber in more than one chamber. In some embodiments, distributing the oligonucleotides into more than one partition comprises providing more than one partition comprising the oligonucleotides. In some embodiments, the more than one partition comprises a 384-well plate, a 288-well plate, a 192-well plate, a 96-well plate, or a 48-well plate.

[0264] In some embodiments, the solid support comprises synthetic particles. In some embodiments, at least one of the more than one oligonucleotide barcodes is immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, partially encapsulated in the synthetic particles, or a combination thereof. In some embodiments, the synthetic particles are destructible. In some embodiments, the synthetic particles comprise beads. In some embodiments, the beads include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. In some embodiments, the synthetic particles comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. In some embodiments, the synthetic particles include destructible hydrogel particles.

[0265] In some embodiments, methods of assigning sequencing data to chambers are provided. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into partitions of an identified chamber of the more than one chamber, wherein the solid supports each comprise more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprise a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into partitions of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, wherein the single cells comprise copies of nucleic acid targets, wherein the single cells of the same population are located in the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0266] In some embodiments, methods of assigning sequencing data to chambers are provided. In some embodiments, the method comprises: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each contain a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single cell populations into the partitions of the identified chambers in more than one chamber, wherein each of the two or more single cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber in more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0267] The present disclosure provides methods for assigning sequencing data to single cell populations. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into a partition of an identified chamber of the more than one chamber, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein oligonucleotide barcodes located within the same chamber comprise the same chamber index subsequence, and wherein oligonucleotide barcodes located within different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into a partition of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, wherein each single cell comprises a copy of a nucleic acid target, wherein single cells of the same population are located within the same chamber, and wherein single cells of different populations are located within different chambers; barcoding copies of nucleic acid targets of at least one single cell of the more than one single cell from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or its products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; assigning each of the more than one sequencing reads to a chamber of the more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a population of the two or more single cell populations based on the chamber assigned to the sequencing read.

[0268] In some embodiments, methods are provided for assigning sequencing data to single-cell populations. In some embodiments, the methods include: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each containing a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of two or more single-cell populations into the partitions of identified chambers in more than one chamber, wherein each of the two or more single-cell populations contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single-cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a chamber in more than one chamber based on the chamber index subsequence of each cell marker sequence in the sequencing data; and assigning each of the more than one sequencing reads to a population of two or more single-cell populations based on the chamber to which the sequencing read is assigned.

[0269] Some embodiments provide methods for correlating sequencing data and phenotypic data of a single cell population. In some embodiments, the methods include: obtaining phenotypic data for each of two or more single cell populations; providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into partitions of an identified chamber of the more than one chamber, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into partitions of an identified chamber of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, wherein the single cells comprise copies of a nucleic acid target, wherein the single cells of the same population are located in the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of the nucleic acid target of at least one single cell of the more than one single cell from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read comprising the more than one barcoded nucleic acid target or its product; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and correlating the sequencing data and the phenotypic data of at least one single cell population based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0270] In some embodiments, methods are provided for correlating sequencing data and phenotypic data of a population of single cells. In some embodiments, the method comprises: obtaining phenotypic data for each of two or more populations of single cells; providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each solid support contains more than one oligonucleotide barcode, the more than one oligonucleotide barcodes each containing a cell marker sequence, wherein each cell marker sequence contains a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers contain different chamber index subsequences; partitioning each of the two or more populations of single cells into the partitions of the identified chambers in more than one chamber, wherein each of the two or more populations of single cells contains more than one single cell, wherein the single cells contain copies of nucleic acid targets, wherein the single cells of the same population are located within the same chamber, and wherein the single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one single cell in more than one single cell from at least one population of single cells using more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; obtaining sequencing data of more than one sequencing read containing more than one barcoded nucleic acid target or a product thereof; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and correlating the sequencing data and the phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0271] The method may comprise: assigning each of the more than one sequencing reads to a population of two or more populations of single cells based on the chamber assigned to the sequencing read. The method may comprise: obtaining phenotypic data for each of two or more populations of single cells; and correlating the sequencing data and the phenotypic data of at least one population of single cells based on the chamber index subsequence of each cell marker sequence in the sequencing data.

[0272] In some embodiments, partitioning of each of two or more than one solid supports into identified chambers of more than one chamber may include partitioning a predetermined more than one solid support selected from the two or more than one solid supports into the identified chambers of the more than one chamber. In some embodiments, barcoding of copies of a nucleic acid target includes: contacting more than one oligonucleotide barcode with copies of the nucleic acid target for hybridization; and extending the more than one oligonucleotide barcode hybridized to the copies of the nucleic acid target to produce more than one barcoded nucleic acid target.

[0273] The partitioning may be a microwell having a volume in the range of from about 1,000 μm 3 to about 786,000 μm 3 The oligonucleotide barcodes may each comprise a molecular marker sequence. In some embodiments, the oligonucleotide barcodes of the same more than one solid support may comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes of different more than one solid supports may comprise different chamber index subsequences. Each cell marker of the more than one oligonucleotide barcode may comprise at least 6 nucleotides. The cell marker may comprise more than one portion and one or more linkers. The cell marker may comprise a first cell marker portion, a first linker, and a second cell marker portion, optionally the cell marker comprises a second linker and a third cell marker portion, and further optionally the cell marker comprises a third linker and a fourth cell marker portion. The first cell marker portion may comprise a chamber index subsequence. In some embodiments, the length of the chamber index subsequence may be 2-15 nucleotides. The two or more than one solid supports may include from about 2 to about 100 different more than one solid supports. The oligonucleotide barcodes of the same more than one solid support may comprise the same chamber index subsequence, and the oligonucleotide barcodes of different more than one solid supports may comprise different chamber index subsequences.

[0274] The chamber index subsequences can be selected from a set of distinct chamber index subsequences. A set of distinct chamber index subsequences can include at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 2,000, at least 5,000, or more different chamber index subsequences. In some embodiments, a set of chamber index subsequences is designed to have minimal sequence homology to the DNA or RNA sequence of the sample to be analyzed. In some embodiments, the sequences of a set of chamber index subsequences differ from each other or from their complements by at least 1 nucleotide, at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 6 nucleotides, at least 7 nucleotides, at least 8 nucleotides, at least 9 nucleotides, at least 10 nucleotides, or more. In some embodiments, the sequences of a set of chamber index subsequences differ from each other or from their complements by at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or more.

[0275] The single cell populations can be different samples. Each of two or more single cell populations can be a biological replicate sample, a technical replicate sample, a control sample, an experimental sample, or a combination thereof. Two or more single cell populations can be derived from one or more samples separated based on phenotypic data. Optionally, more than one single cell can include T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof. More than one chamber can be at least about 4 chambers, about 10 chambers, about 20 chambers, about 40 chambers, about 60 chambers, about 80 chambers, about 100 chambers, about 200 chambers, about 300 chambers, about 400 chambers, about 500 chambers, about 750 chambers, or about 1,000 chambers. More than one partition can be at least about 100 partitions, about 500 partitions, about 1,000 partitions, about 5,000 partitions, about 10,000 partitions, about 25,000 partitions, about 50,000 partitions, about 75,000 partitions, or about 100,000 partitions.

[0276] In some embodiments, sequencing-based identification of cell labels determines the starting chamber of each sequenced barcoded nucleic acid target or its product within the microwell array. A partition in more than one partition can contain single cells in more than one single cell and single solid supports in more than one solid support. Each of more than one barcoded nucleic acid target can contain a sequence complementary to at least a portion of the nucleic acid target and a molecular label. Each of more than one sequencing read can contain (1) a cell label sequence and (2) a molecular label sequence. Each oligonucleotide barcode can contain a first universal sequence. The oligonucleotide barcode can include a target-binding region containing a capture sequence. The target-binding region can contain a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof. In some embodiments, partitioning each of more than one solid support into each of more than one chamber can include depositing each of more than one solid support into an identified chamber of the microwell array by flow cytometry. In some embodiments, partitioning each of two or more single cell populations into an identified chamber in more than one chamber can include depositing each single cell population into an identified chamber of the microwell array by flow cytometry. The method can include aligning a sorting component of a flow cytometer with the microwell array.

[0277] Phenotypic data can include event data. Event data can include quantitative biological event data derived from a sorting device. Event data can include side scatter signals, forward scatter signals, one or more fluorescence signals, or any combination thereof. The method can include performing a correlation analysis on the phenotypic data and the sequencing data of single cells. In some embodiments, the correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of a therapeutic agent, and / or cell targets of a candidate therapeutic agent. The method can include lysing one or more single cells. The viability of the single cells can be maintained for a period of time after partitioning and before lysis, optionally for at least about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 250 min, 500 min, 750 min, 1000 min, 2500 min, 5000 min, 7500 min, or 10000 min.

[0278] The solid support may include synthetic particles. The solid support may include a flat surface. At least one of more than one oligonucleotide barcode may be immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, or partially encapsulated in the synthetic particles. The synthetic particles may be destructible. The synthetic particles may include beads. The beads may include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles may comprise a material selected from: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof. The synthetic particles may include destructible hydrogel particles.

[0279] Method for determining the copy number of a nucleic acid target

[0280] The methods provided herein can include determining the copy number of a nucleic acid target in one or more of more than one single cell of at least one single cell population. Determining the copy number of a nucleic acid target in one or more of more than one single cell can include determining the copy number of the nucleic acid target in more than one single cell based on the number of molecular markers, their complements, or combinations thereof having different sequences associated with more than one barcoded nucleic acid target or its products. The methods can include: contacting random primers with more than one barcoded nucleic acid target, wherein each of the random primers comprises a second universal sequence or its complement; and extending the random primers hybridized to the more than one barcoded nucleic acid target to produce more than one extension product. The methods can include: amplifying the more than one extension product using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a second universal sequence or its complement, thereby producing a first more than one barcoded amplicon. Amplifying the more than one extension product can include adding a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a sequence of a portion thereof to the more than one extension product. The methods can include: determining the copy number of the nucleic acid target in one or more of more than one single cell based on the number of molecular markers having different sequences associated with the first more than one barcoded amplicon or its products. In some embodiments, determining the copy number of a nucleic acid target in one or more of more than one single cell can include determining the number of each of more than one nucleic acid targets in one or more of more than one single cell based on the number of molecular markers having different sequences associated with a barcoded amplicon in the first more than one barcoded amplicon, the first more than one barcoded amplicon comprising the sequence of each of the more than one nucleic acid targets. The sequence of each of the more than one nucleic acid targets can include a subsequence of each of the more than one nucleic acid targets. The sequence of the nucleic acid target in the first more than one barcoded amplicon can include a subsequence of the nucleic acid target. The methods can include: amplifying the first more than one barcoded amplicon using a primer capable of hybridizing to a first universal sequence or its complement and a primer capable of hybridizing to a second universal sequence or its complement, thereby producing a second more than one barcoded amplicon. Amplifying the first more than one barcoded amplicon can include adding a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a sequence of a portion thereof to the first more than one barcoded amplicon. The methods can include: determining the copy number of the nucleic acid target in one or more of more than one single cell based on the number of molecular markers having different sequences associated with the second more than one barcoded amplicon or its products. The first more than one barcoded amplicon and / or the second more than one barcoded amplicon can comprise whole transcriptome amplification (WTA) products.

[0281] The method can include: synthesizing a third more than one barcoded amplicon using more than one barcoded nucleic acid target as a template to produce a third more than one barcoded amplicon. Synthesizing the third more than one barcoded amplicon can include polymerase chain reaction (PCR) amplification of the more than one barcoded nucleic acid target. Synthesizing the third more than one barcoded amplicon can include PCR amplification using primers capable of hybridizing to a first universal sequence or its complement and target-specific primers. The method can include: obtaining sequence data of the third more than one barcoded amplicon or its product, and optionally obtaining sequence information including attaching sequencing adapters to the third more than one barcoded amplicon or its product. The method can include: determining the copy number of a nucleic acid target in one or more of more than one single cell based on the number of molecular markers having different sequences associated with the third more than one barcoded amplicon or its product.

[0282] The nucleic acid target can include a nucleic acid molecule. The nucleic acid molecule can include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNA containing a poly(A) tail, or any combination thereof. The nucleic acid target can include a sample index oligonucleotide, and optionally the sample index oligonucleotide contains a sample index sequence, and the sample index sequences of at least two sample index compositions in more than one sample index composition contain different sequences. The nucleic acid target can include a cell component binding reagent-specific oligonucleotide. The cell component binding reagent-specific oligonucleotide can contain a unique identifier sequence for the cell component binding reagent. In some embodiments of the methods and compositions provided herein, the nucleic acid target is a binding reagent oligonucleotide (e.g., an antibody oligonucleotide (“AbOligo” or “AbO”), a binding reagent oligonucleotide, a cell component binding reagent-specific oligonucleotide, a sample index oligonucleotide). Some embodiments disclosed herein provide more than one composition, each of the more than one composition comprising a cell component binding reagent (such as a protein binding reagent) conjugated to an oligonucleotide (such as a binding reagent oligonucleotide), wherein the oligonucleotide contains a unique identifier for the cell component binding reagent conjugated thereto. Cell component binding reagents (such as barcoded antibodies) and their uses (such as sample indexing of cells) have been described in US2018 / 0088112 and US2018 / 0346970; the content of each of these is incorporated herein by reference in its entirety.

[0283] Extending more than one oligonucleotide barcode can include extending more than one oligonucleotide barcode using a reverse transcriptase and / or a DNA polymerase lacking at least one of 5'-to-3' exonuclease activity and 3'-to-5' exonuclease activity. The DNA polymerase can include the Klenow fragment. The reverse transcriptase can include a viral reverse transcriptase, optionally wherein the viral reverse transcriptase is murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase. The first universal sequence and the second universal sequence can be the same. The first universal sequence and the second universal sequence can be different. The first universal sequence and / or the second universal sequence can comprise a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a portion thereof. The sequencing adapter can include a P5 sequence, a P7 sequence, its complementary sequence, and / or a portion thereof. The sequencing primer can include a read 1 sequencing primer, a read 2 sequencing primer, its complementary sequence, and / or a portion thereof. At least 10 of the more than one oligonucleotide barcodes can comprise different molecular marker sequences. Each molecular marker of the more than one oligonucleotide barcodes can comprise at least 6 nucleotides.

[0284] Particle analyzer

[0285] Phenotypic data can include data derived from a sorting device (e.g., a flow cytometer). The phenotypic data can include event data. The event data can include quantitative biological event data derived from a sorting device. The event data can include side scatter signals, forward scatter signals, one or more fluorescence signals, or any combination thereof. As used herein, the terms "event" or "event data" shall have their ordinary meaning and shall also be interchangeable with each other and shall also refer to data measured from a single particle (such as a cell or a synthetic particle). Generally, data measured from a single particle includes multiple parameters, including one or more light scattering parameters and at least one fluorescence intensity parameter. Thus, each event is represented as a vector of parameter measurements, where each measured parameter corresponds to a dimension of the data space. In some biological applications, the event data can correspond to quantitative biological data indicating the expression of a specific protein or gene. The method can include: performing a correlation analysis on the phenotypic data and the sequencing data of single cells. The correlation analysis can identify one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of a therapeutic agent, and / or cellular targets of a candidate therapeutic agent.

[0286] Particle analyzers, such as flow cytometers and scanning cytometers, are analytical tools capable of characterizing particles (e.g., single cells) based on optical parameters such as light scattering and fluorescence. In a flow cytometer, for example, particles in a fluid suspension (such as molecules, beads conjugated with analytes, or single cells) pass through a detection region where the particles are exposed to excitation light, typically from one or more laser beams, and the light scattering and fluorescence characteristics of the particles are measured. The particles or their components are typically labeled with fluorescent dyes for ease of detection. By using fluorescent dyes with different spectra to label different particles or components, more than one different particle or component can be detected simultaneously. In some embodiments, the analyzer includes more than one photodetector, one photodetector for each scattering parameter to be measured and one photodetector for each different dye to be detected. The data obtained includes signals measured for each light scattering parameter and fluorescence emission.

[0287] Parameters measured using a flow cytometer typically include the excitation light scattered by the particles in the main forward direction (referred to as forward scatter (FSC)), the excitation light scattered by the particles in the main lateral direction (referred to as side scatter (SSC)), and the light emitted from fluorescent molecules in one or more channels (frequency ranges) of the spectrum (referred to as FL1, FL2, etc.), or the light emitted by fluorescent dyes detected primarily in that channel. Since antibodies labeled with dyes label different cellular proteins, different cell types can be identified by the scattering parameters and fluorescence emission.

[0288] Both flow cytometers and scanning cytometers are commercially available, for example, from BD Biosciences (San Jose, California). Flow cytometry is described, for example, in Landy et al. (eds.), Clinical Flow Cytometry, Annals of the New York Academy of Sciences Volume 677 (1993); Bauer et al. (eds.), Clinical Flow Cytometry: Principles and Applications, Williams & Wilkins (1993); Ormerod (ed.), Flow Cytometry: A Practical Approach, Oxford Univ. Press (1994); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); and Practical Shapiro, Flow Cytometry, 4th Edition, Wiley-Liss (2003); all of which are incorporated herein by reference. Fluorescence imaging microscopy is described, for example, in Pawley (ed.), Handbook of Biological Confocal Microscopy, 2nd Edition, Plenum Press (1989), which is incorporated herein by reference.

[0289] Data obtained from the analysis of cells (or other particles) by multicolor flow cytometry are multidimensional, where each cell corresponds to a point in a multidimensional space defined by the measured parameters. A population of cells or particles is identified as a cluster of points in the data space. Identifying the clusters, and thus the populations, can be done manually by drawing gates around the populations shown in one or more two-dimensional plots, called "scatter plots" or "dot plots" of the data. Alternatively, the clusters can be identified and the gates defining the population limits can be automatically determined. Examples of methods for automatic gating have been described, for example, in U.S. Patent Nos. 4,845,653, 5,627,040, 5,739,000, 5,795,727, 5,962,238, 6,014,904, and 6,944,338, and U.S. Patent Publication No. 2012 / 0245889, each of which is incorporated herein by reference.

[0290] Flow cytometry is a valuable method for analyzing and separating biological particles such as cells and their constituent molecules. As such, it has a wide range of diagnostic and therapeutic applications. The method uses a fluid stream to linearly separate the particles so that they can pass through the detection device in a single file. Individual cells can be distinguished based on their position in the fluid stream and the presence of detectable markers. Thus, flow cytometers can be used to generate diagnostic profiles of populations of biological particles.

[0291] Separation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles in a segregated stream that are detected as having one or more desired characteristics are separated individually from the sample stream by mechanical or electrical removal. This flow sorting method has been used to sort different types of cells, separate sperm carrying X and Y chromosomes for animal breeding, sort chromosomes for genetic analysis, and isolate specific organisms from complex biological populations.

[0292] In flow cytometry sorting, using index sorting means that additional information linking individual cell events to their destination positions in a plate or slide holder is available. This information can be used after acquisition for additional analysis of the physical location of where the cells are on the plate device. It also allows the user to see where these cells are located on a bivariate plot.

[0293] Index sorting is a cell sorting in which the sorting device can record the sorting decision for each event (usually a cell or other particle suspended in a flow stream) and the data can be used for post-sorting analysis. Typically, index sorting is performed by detecting characteristics of the particles (such as color) and directing the particles to a collection plate. The plate can include several plate destinations (e.g., well positions). Sorting can include directing the particles to a specific plate location (e.g., well) within the plate. The sorting device can record the destination plate and / or well position in association with an identifier for the event. Thus, each sorted event has all the measurements from the detector (PMT, photodiode) as well as the well position and sorting destination. The user can examine the data for the sorted cells and correlate it with subsequent operations on the plate (e.g., gene expression obtained from sequencing the sorted cells).

[0294] A sorting mode can be specified for a given sample. The sorting mode includes parameters that control which events are sorted. For example, after receiving a sample at a sorting device, the sorting device can receive a sorting mode to control which characteristics are used for sorting and where the detected values of these characteristics should be sorted. The sorting mode can include a purity mode that can configure a cell sorter to ensure the desired cell type and only the desired cell type is in the gate. The sorting mode can include a single cell mode that can configure a cell sorter to ensure that only single cells are within the droplet. Due to the uncertainty of where a cell may be near the boundary of which droplet, even if subsequent droplets can be sorted, they are generally not sorted. For single cell sorting, this can be desirable for some experiments (such as genome-based assays where the user may want to correlate gene expression with measurements from flow cytometry). In such cases, if there is more than one cell in the well, it is unclear which cell the gene sequence came from. The sorting mode can configure the cell sorter by setting a mask that examines the position of events within and around the droplet. Some single cell sorting mode configurations can include a state machine or examining a queue of events that fall within the droplet.

[0295] In some embodiments, a target gate can be used to identify events of interest. The target gate can be provided by selecting a region on a two-dimensional plot. Events that have characteristic values within the two-dimensional selected region are considered to be within the target gate and can be sorted to a specific location. An event may be within the target gate, but in some sorting modes (e.g., purity mode or single cell mode), the event may not be sorted correctly, such as if another event is in the same droplet in the fluid stream as the event. In some embodiments, this can be referred to as entrainment or cohesion.

[0296] As part of recording the sorting decision, the sorting electronics of the sorting device can transmit the sorting destination along with the event raw data. The event raw data can include detected event characteristics (e.g., reflected light values, fluorescence information, light scattering information, the time of the event, the serial number of the event, the sorting device operating characteristics at the time of analyzing the event (e.g., temperature, flow rate, sorting mode, etc.), etc.). For indexed sorting, the coordinates of the position where the cell of a particular event is deposited can also be transmitted for the current tray, plate, microscope slide, or other physical medium with spatially separated wells where the droplets containing the cells can be deposited.

[0297] In some embodiments, the methods disclosed herein can include enriching a sample comprising more than one cell for cells of interest to produce an enriched cell sample comprising more than one single cell for analysis as provided herein. Enriching the sample can include focusing the cells of interest in the sample; separating one or more cells of interest from the enriched cell sample using a flow cytometer; and obtaining sequence information of one or more polynucleotides from each of the one or more separated cells as described herein. A variety of focusing methods and techniques can be used, for example, hydrodynamic focusing, magnetic field focusing, electric field focusing, gravitational field focusing, optical field focusing, and any combination thereof. In some embodiments, enriching the sample includes depleting cells in the sample that are not of interest. In some embodiments, enriching the sample includes both acoustic focusing and depleting cells in the sample that are not of interest. In some embodiments, one or more of the cells in the sample that are not of interest, interfering cells, and debris can be depleted (e.g., using magnetic depletion).

[0298] Compositions and kits

[0299] The present disclosure includes compositions. In some embodiments, the composition comprises: a micro-well array, wherein the micro-well array comprises more than one chamber, wherein each chamber comprises more than one partition, and wherein each partition is a micro-well having a volume in the range from about 1,000 μm 3 to about 786,000 μm 3 The composition can comprise: a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

[0300] The present disclosure includes compositions. In some embodiments, the composition comprises: a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, and wherein the cartridge comprises a micro-well array, wherein the micro-well array comprises more than one chamber, wherein each chamber comprises more than one partition, and wherein each partition is a micro-well having a volume in the range from about 1,000 μm 3 to about 786,000 μm 3 The composition can comprise: a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, and wherein the cartridge comprises a micro-well array, wherein the micro-well array comprises more than one chamber, wherein each chamber comprises more than one partition, and wherein each partition is a micro-well having a volume in the range from about 1,000 μm

[0301] The present disclosure includes compositions. In some embodiments, the composition comprises: two or more than one solid support, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcode each comprises a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes of the same more than one solid support comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes of different more than one solid supports comprise different chamber index subsequences.

[0302] A partition in more than one partition comprises a single solid support in more than one solid support, wherein each solid support comprises more than one oligonucleotide barcode, the more than one oligonucleotide barcode each comprises a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences.

[0303] The composition may include: a chamber index subsequence lookup table. In some embodiments, the chamber index subsequence lookup table identifies the chamber index subsequences associated with each solid support distributed in each microwell of the array.

[0304] The cartridge may be configured to maintain the viability of single cells partitioned within the microwells, optionally for a period of at least about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 250 min, 500 min, 750 min, 1000 min, 2500 min, 5000 min, 7500 min or 10000 min. The cartridge may include a transparent window for optical imaging of the microwells. The composition may comprise: an imaging system configured to capture and process images of all or a portion of the microwells, wherein the imaging system further includes an illumination subsystem, an imaging subsystem, and a processor. The imaging system may be configured to perform brightfield, darkfield, fluorescence, or quantitative phase imaging.

[0305] The composition may comprise: a buffer. The composition may comprise: one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or both. Each oligonucleotide barcode may comprise a molecular marker sequence. Each cell label of more than one oligonucleotide barcode may comprise at least 6 nucleotides. The cell label may comprise more than one part and one or more linkers. The cell label may comprise a first cell label part, a first linker, and a second cell label part, optionally the cell label comprises a second linker and a third cell label part, and further optionally the cell label comprises a third linker and a fourth cell label part. The first cell label part may comprise a chamber index subsequence. The length of the chamber index subsequence may be 2-15 nucleotides. More than one chamber may be at least about 4 chambers, about 10 chambers, about 20 chambers, about 40 chambers, about 60 chambers, about 80 chambers, about 100 chambers, about 200 chambers, about 300 chambers, about 400 chambers, about 500 chambers, about 750 chambers, or about 1000 chambers. More than one partition may be at least about 100 partitions, about 500 partitions, about 1000 partitions, about 5000 partitions, about 10000 partitions, about 25000 partitions, about 50000 partitions, about 75000 partitions, or about 100000 partitions.

[0306] The composition can include: more than one solid support, each of the more than one solid supports containing more than one oligonucleotide barcode. Each oligonucleotide barcode can include a molecular tag and a cell tag. In some embodiments, the oligonucleotide barcodes associated with the same solid support can include the same cell tag sequence, and wherein the oligonucleotide barcodes associated with different solid supports can include different cell tag sequences. Each oligonucleotide barcode can include a first universal sequence. The oligonucleotide barcode can include a target binding region containing a capture sequence. The target binding region can include a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof. Each molecular tag of the more than one oligonucleotide barcodes can include at least 6 nucleotides. The solid support can include a flat surface. The solid support can include synthetic particles. At least one of the more than one oligonucleotide barcodes can be immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, partially encapsulated in the synthetic particles, or a combination thereof. The synthetic particles can be destructible. The synthetic particles can include beads. The beads can include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, antibiotin microbeads, anti-fluorescent dye microbeads, or any combination thereof. The synthetic particles can include a material selected from: polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and combinations thereof. The synthetic particles can include destructible hydrogel particles. In some embodiments, the composition further includes instructions for use.

[0307] Terms

[0308] In at least some of the previously described embodiments, one or more elements used in one embodiment can be used interchangeably in another embodiment, unless such substitution is technically infeasible. Those skilled in the art will understand that various other omissions, additions, and modifications can be made to the above methods and structures without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

[0309] Those skilled in the art will understand that for this and other processes and methods disclosed herein, the functions performed in the processes and methods can be implemented in a different order. In addition, the steps and operations outlined are provided only as examples, and some of the steps and operations can be optional, combined into fewer steps and operations, or expanded into additional steps and operations without departing from the essence of the disclosed embodiments.

[0310] Regarding the use of substantially any plural and / or singular terms herein, the skilled person can convert from plural to singular and / or from singular to plural as appropriate for the context and / or application. For clarity, various singular / plural permutations may be set forth explicitly herein. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Any reference to "or" herein is intended to cover "and / or" unless otherwise stated.

[0311] Those skilled in the art will understand that, generally speaking, the terms used herein, and in particular the terms in the appended claims (e.g., the subject matter of the appended claims), are generally intended to be "open" terms (e.g., the term "including" should be interpreted as "including but not limited to", the term "having" should be interpreted as "having at least", the term "includes" should be interpreted as "includes but is not limited to", etc.). Those skilled in the art will further understand that if a specific number is intended to be introduced in the claim statement, such an intention will be explicitly stated in the claim, and in the absence of such a statement, there is no such intention. For example, for the sake of understanding, the following appended claims may contain the use of introductory phrases "at least one" and "one or more" to introduce claim statements. However, the use of such phrases should not be construed to mean that introducing a claim statement by the indefinite article "a" or "an" will limit any particular claim containing such an introduction to an embodiment containing only one such statement, even when the same claim includes introductory phrases "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be interpreted to mean "at least one" or "one or more"); this also applies to the use of the definite article to introduce a claim statement. In addition, even if a specific number of the introduced claim statement is explicitly stated, those skilled in the art will recognize that such a statement should be interpreted to mean at least the stated number (e.g., merely stating "two statements" without additional modifiers means at least two statements or two or more statements). Further, in those cases where a convention similar to "at least one of A, B, and C, etc." is used, generally such a syntactic construction is intended to be understood by those skilled in the art as the meaning of the convention (e.g., "a system having at least one of A, B, and C" will include but not be limited to a system having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, generally such a syntactic construction is intended to be understood by those skilled in the art as the meaning of the convention (e.g., "a system having at least one of A, B, or C" will include but not be limited to a system having A alone, having B alone, having C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.).Those skilled in the art will further understand that, in fact, whether in the specification, the claims or the drawings, any disjunctive word and / or phrase presenting two or more alternative terms should be understood as contemplating the possibility of including one of the terms, either term or both terms. For example, the phrase "A or B" should be understood as including the possibilities of "A" or "B" or "A and B".

[0312] In addition, when a feature or aspect of the present disclosure is described in terms of a Markush group, those skilled in the art will recognize that the present disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0313] As will be understood by those skilled in the art, for any and all purposes, such as in providing a written description, all ranges disclosed herein also include any and all possible sub-ranges and combinations of sub-ranges of that range. Any recited range can be readily identified as fully describing and enabling the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. By way of non-limiting example, each range discussed herein can be readily divided into lower third, middle third, and upper third, etc. As will also be understood by those skilled in the art, all language such as "up to", "at least", "greater than", "less than", etc. includes the recited number and refers to ranges that can then be divided into sub-ranges as discussed above. Finally, as will be understood by those skilled in the art, a range includes each individual member. Thus, for example, a group having 1 - 3 items refers to a group having 1, 2, or 3 items. Similarly, a group having 1 - 5 items refers to a group having 1, 2, 3, 4, or 5 items, and so on.

[0314] From the foregoing, it should be understood that the various embodiments of the present disclosure have been described herein for purposes of illustration, and various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting, and the true scope and spirit are indicated by the following claims.

Claims

1. A method for allocating sequencing data to chambers, comprising: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; partitioning each of two or more than one solid supports into the partitions of the identified chambers of the more than one chamber, wherein each of the solid supports comprises more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprises a cell marker sequence, and each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, and wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into the partitions of the identified chambers of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, and wherein each single cell comprises a copy of a nucleic acid target, wherein single cells of the same population are located in the same chamber, and wherein single cells of different populations are located in different chambers; barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcodes to produce more than one barcoded nucleic acid targets; obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid targets or their products; identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and allocating each of the more than one sequencing reads to a chamber of the more than one chambers based on the chamber index subsequence of each cell marker sequence in the sequencing data.

2. A method for allocating sequencing data to chambers, comprising: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, each chamber contains a predetermined more than one solid supports selected from two or more than one solid supports, the solid supports are located within the partitions of the chamber, each of the solid supports comprises more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprises a cell marker sequence, and each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, and wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; partitioning each of two or more single cell populations into the partitions of the identified chambers of the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, and wherein each single cell comprises a copy of a nucleic acid target, Single cells of the same population are located in the same chamber, and single cells of different populations are located in different chambers; Barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; Obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; Identifying the chamber index subsequence of each cell marker sequence in the sequencing data; and Based on the chamber index subsequence of each cell marker sequence in the sequencing data, assigning each of the more than one sequencing reads to a chamber in the more than one chamber.

3. A method for assigning sequencing data to single cell populations, comprising: Providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; Partitioning each of two or more than one solid supports into the partitions of the identified chambers in the more than one chamber, wherein each of the solid supports comprises more than one oligonucleotide barcode, and the more than one oligonucleotide barcodes each comprise a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, and wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences; Partitioning each of two or more single cell populations into the partitions of the identified chambers in the more than one chamber, wherein each of the two or more single cell populations comprises more than one single cell, and wherein the single cells comprise copies of nucleic acid targets, wherein single cells of the same population are located in the same chamber, and wherein single cells of different populations are located in different chambers; Barcoding copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; Obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; Identifying the chamber index subsequence of each cell marker sequence in the sequencing data; Based on the chamber index subsequence of each cell marker sequence in the sequencing data, assigning each of the more than one sequencing reads to a chamber in the more than one chamber; and Based on the chamber to which the sequencing read is assigned, assigning each of the more than one sequencing reads to a population of the two or more single cell populations.

4. A method for assigning sequencing data to single cell populations, comprising: Provided is a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition, wherein each chamber contains a predetermined more than one solid support selected from two or more than one solid supports, wherein the solid supports are located within the partitions of the chamber, wherein each of the solid supports contains more than one oligonucleotide barcode, and the more than one oligonucleotide barcodes each contain a cell marker sequence, and each cell marker sequence contains a predetermined chamber index subsequence. Wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, and the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences. Wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and the oligonucleotide barcodes located in different chambers contain different chamber index subsequences. Partition each of two or more single cell populations into the partitions of the identified chambers of the more than one chamber. Wherein each of the two or more single cell populations contains more than one single cell, and the single cells contain copies of nucleic acid targets. Wherein single cells of the same population are located within the same chamber, and single cells of different populations are located in different chambers. Barcode copies of nucleic acid targets of at least one of the more than one single cells from at least one single cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target. Obtain sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its products. Identify the chamber index subsequence of each cell marker sequence in the sequencing data. And Based on the chamber index subsequence of each cell marker sequence in the sequencing data, assign each of the more than one sequencing reads to a chamber of the more than one chamber. And Based on the chamber to which the sequencing read is assigned, assign each of the more than one sequencing reads to a population of the two or more single cell populations.

5. A method for associating sequencing data and phenotypic data of a single cell population, comprising: Obtain phenotypic data of each of two or more single cell populations. Provide a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition. Partition each of two or more than one solid supports into the partitions of the identified chambers of the more than one chamber, wherein each of the solid supports contains more than one oligonucleotide barcode, and the more than one oligonucleotide barcodes each contain a cell marker sequence, and each cell marker sequence contains a predetermined chamber index subsequence. Wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, and the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences. Wherein the oligonucleotide barcodes located within the same chamber contain the same chamber index subsequence, and the oligonucleotide barcodes located in different chambers contain different chamber index subsequences. Partitioning each of two or more single-cell populations into the identified compartments of the more than one compartment, wherein each of the two or more single-cell populations comprises more than one single cell, and wherein the single cell comprises a copy of a nucleic acid target, wherein single cells of the same population are located in the same compartment, and wherein single cells of different populations are located in different compartments; Barcoding copies of the nucleic acid target of at least one of the more than one single cell from at least one single-cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; Obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; Identifying the compartment index subsequence of each cell marker sequence in the sequencing data; and Associating the sequencing data of at least one single-cell population and the phenotypic data based on the compartment index subsequence of each cell marker sequence in the sequencing data.

6. A method for associating sequencing data and phenotypic data of a single-cell population, comprising: Obtaining phenotypic data of each of two or more single-cell populations; Providing a microwell array comprising more than one compartment, wherein each compartment comprises more than one partition, wherein each compartment contains a predetermined more than one solid support selected from two or more more than one solid supports, wherein the solid support is located within the partition of the compartment, wherein each of the solid supports comprises more than one oligonucleotide barcode, and the more than one oligonucleotide barcode each comprises a cell marker sequence, and each cell marker sequence comprises a predetermined compartment index subsequence, wherein the oligonucleotide barcodes associated with the same solid support contain the same cell marker sequence, and wherein the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences, wherein the oligonucleotide barcodes located in the same compartment contain the same compartment index subsequence, and wherein the oligonucleotide barcodes located in different compartments contain different compartment index subsequences; Partitioning each of two or more single-cell populations into the identified compartments of the more than one compartment, wherein each of the two or more single-cell populations comprises more than one single cell, and wherein the single cell comprises a copy of a nucleic acid target, wherein single cells of the same population are located in the same compartment, and wherein single cells of different populations are located in different compartments; Barcoding copies of the nucleic acid target of at least one of the more than one single cell from at least one single-cell population using the more than one oligonucleotide barcode to produce more than one barcoded nucleic acid target; Obtaining sequencing data of more than one sequencing read containing the more than one barcoded nucleic acid target or its product; Identifying the compartment index subsequence of each cell marker sequence in the sequencing data; and Associating the sequencing data of at least one single-cell population and the phenotypic data based on the compartment index subsequence of each cell marker sequence in the sequencing data.

7. The method according to any one of claims 1-6, wherein the two or more than one solid supports comprise more than one first solid support and more than one second solid support, wherein the oligonucleotide barcodes associated with the more than one first solid support have a first predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the more than one second solid support have a second predetermined chamber index subsequence, and wherein the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

8. The method according to any one of claims 1-7, wherein the two or more than one solid supports comprise more than one first solid support and more than one second solid support, wherein the first predetermined chamber index subsequence of the more than one first solid support is selected from a first set of chamber index subsequences, wherein the second predetermined chamber index subsequence of the more than one second solid support is selected from a second set of chamber index subsequences, and wherein each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences.

9. The method according to any one of claims 1-8, wherein based on the predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with the more than one first solid support or the more than one second solid support.

10. The method according to any one of claims 1-2 or 5-6, comprising: assigning each of the more than one sequencing reads to a population of the two or more single cell populations based on the chamber assigned to the sequencing reads.

11. The method according to any one of claims 1-4 or 10, comprising: obtaining phenotypic data for each of the two or more single cell populations; and associating the sequencing data and the phenotypic data of at least one single cell population based on the chamber index subsequence of each cell marker sequence in the sequencing data.

12. The method according to any one of claims 1-11, wherein the partitioning of each of the two or more than one solid supports into the identified chambers of the more than one chamber comprises partitioning a predetermined more than one solid support selected from the two or more than one solid supports into the identified chambers of the more than one chamber.

13. The method according to any one of claims 1-12, wherein barcoding the copies of the nucleic acid target comprises: contacting the more than one oligonucleotide barcode with the copies of the nucleic acid target for hybridization; and extending the more than one oligonucleotide barcode hybridized to the copies of the nucleic acid target to produce more than one barcoded nucleic acid target.

14. The method according to any one of claims 1-13, wherein the partition is a micropore having a volume in the range from about 1,000 μm 3 to about 786,000 μm 3 inclusive.

15. The method according to any one of claims 1-14, wherein each of the oligonucleotide barcodes comprises a molecular marker sequence.

16. The method according to any one of claims 1-15, wherein the oligonucleotide barcodes of more than one identical solid support contain identical chamber index subsequences, and wherein the oligonucleotide barcodes of more than one different solid support contain different chamber index subsequences.

17. The method according to any one of claims 1-16, wherein: each cell label of the more than one oligonucleotide barcode contains at least 6 nucleotides; the cell label contains more than one part and one or more linkers; the cell label contains a first cell label part, a first linker, and a second cell label part, optionally the cell label contains a second linker and a third cell label part, and further optionally the cell label contains a third linker and a fourth cell label part; and / or the first cell label part, the second cell label part, the third cell label part, and / or the fourth cell label part includes the chamber index subsequence; and / or the length of the chamber index subsequence is 2-80 nucleotides.

18. The method according to any one of claims 1-17, wherein the single cell population is different samples.

19. The method according to any one of claims 1-18, wherein each of the two or more single cell populations is a biological replicate sample, a technical replicate sample, a control sample, an experimental sample, or a combination thereof.

20. The method according to any one of claims 1-19, wherein the two or more single cell populations are derived from one or more samples separated based on phenotypic data, optionally, the more than one single cell includes T cells, B cells, tumor cells, myeloid cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof.

21. The method according to any one of claims 1-20, wherein the more than one chamber is at least about 4 chambers, about 10 chambers, about 20 chambers, about 40 chambers, about 60 chambers, about 80 chambers, about 100 chambers, about 200 chambers, about 300 chambers, about 400 chambers, about 500 chambers, about 750 chambers, or about 1000 chambers, optionally the more than one chamber includes 8 lanes.

22. The method according to any one of claims 1-21, wherein the more than one partition is at least about 100 partitions, about 500 partitions, about 1000 partitions, about 5000 partitions, about 10000 partitions, about 25000 partitions, about 50000 partitions, about 75000 partitions, or about 100000 partitions.

23. The method according to any one of claims 1-22, wherein sequencing of the cell label identifies the starting chamber of each sequenced barcoded nucleic acid target or its product within the microwell array.

24. The method according to any one of claims 1-23, wherein the partitions in the more than one partition include the single cells in the more than one single cell and the single solid supports in the more than one solid support.

25. The method according to any one of claims 1-24, wherein each of the more than one barcoded nucleic acid targets comprises a sequence complementary to at least a portion of the nucleic acid target and a molecular tag.

26. The method according to any one of claims 1-25, wherein each of the more than one sequencing reads comprises (1) a cell tag sequence and (2) a molecular tag sequence.

27. The method according to any one of claims 1-26, wherein each oligonucleotide barcode comprises a first universal sequence.

28. The method according to any one of claims 1-27, wherein the oligonucleotide barcode comprises a target binding region, the target binding region comprising a capture sequence.

29. The method according to claim 28, wherein the target binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof.

30. The method according to any one of claims 1-29, wherein partitioning each of the more than one solid supports into each of the more than one chambers comprises depositing each of the more than one solid supports into an identified chamber of the microwell array by flow cytometry.

31. The method according to any one of claims 1-30, wherein partitioning each of two or more single cell populations into an identified chamber of the more than one chambers comprises depositing each single cell population into an identified chamber of the microwell array by flow cytometry.

32. The method according to any one of claims 1-31, the method further comprising aligning a sorting assembly of a flow cytometer with the microwell array.

33. The method according to any one of claims 1-32, wherein the phenotypic data comprises event data.

34. The method according to claim 33, wherein the event data comprises quantitative biological event data derived from a sorting device.

35. The method according to any one of claims 33-34, wherein the event data comprises a side scatter signal, a forward scatter signal, one or more fluorescence signals, or any combination thereof.

36. The method according to any one of claims 1-35, the method comprising performing a correlation analysis on the phenotypic data and the sequencing data of the single cell.

37. The method according to claim 36, wherein the correlation analysis identifies one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of a therapeutic agent, and / or cellular targets of a candidate therapeutic agent.

38. The method according to any one of claims 1-37, the method comprising lysing one or more of the single cells.

39. The method according to any one of claims 1-38, wherein the viability of the single cell is maintained for a period of time after partitioning and before lysis, optionally the period of time is at least about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 250 min, 500 min, 750 min, 1000 min, 2500 min, 5000 min, 7500 min or 10000 min.

40. The method according to any one of claims 1-39, further comprising determining the copy number of the nucleic acid target in one or more of the more than one single cells of at least one single cell population.

41. The method according to claim 40, wherein determining the copy number of the nucleic acid target in one or more of the more than one single cells comprises determining the copy number of the nucleic acid target in the more than one single cells based on the number of molecular markers, their complements or combinations thereof having different sequences associated with the more than one barcoded nucleic acid target or its product.

42. The method according to any one of claims 40-41, comprising: contacting random primers with the more than one barcoded nucleic acid target, wherein each of the random primers comprises a second universal sequence or its complement; and extending the random primers hybridized to the more than one barcoded nucleic acid target to produce more than one extension product.

43. The method according to claim 42, the method comprising amplifying the more than one extension product using primers capable of hybridizing to the first universal sequence or its complement and primers capable of hybridizing to the second universal sequence or its complement to produce a first more than one barcoded amplicon.

44. The method according to claim 43, wherein amplifying the more than one extension product comprises adding a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence and / or a partial sequence thereof to the more than one extension product.

45. The method according to any one of claims 43-44, the method comprising determining the copy number of the nucleic acid target in one or more of the more than one single cells based on the number of molecular markers having different sequences associated with the first more than one barcoded amplicon or its product.

46. The method according to any one of claims 43-45, wherein determining the copy number of the nucleic acid target in one or more of the more than one single cells comprises determining the number of each of the more than one nucleic acid targets in one or more of the more than one single cells based on the number of the molecular markers having different sequences associated with the barcoded amplicon of the first more than one barcoded amplicon, the first more than one barcoded amplicon comprising the sequence of each of the more than one nucleic acid targets.

47. The method according to claim 46, wherein the sequence of each of the more than one nucleic acid targets comprises a subsequence of each of the more than one nucleic acid targets.

48. The method according to any one of claims 43-47, wherein the sequence of the nucleic acid target in the first more than one barcoded amplicon comprises a subsequence of the nucleic acid target.

49. The method according to any one of claims 43-48, the method comprising amplifying the first more than one barcoded amplicon using a primer capable of hybridizing to the first universal sequence or its complement and a primer capable of hybridizing to the second universal sequence or its complement to produce a second more than one barcoded amplicon.

50. The method according to claim 49, wherein amplifying the first more than one barcoded amplicon comprises adding a binding site for a sequencing primer and / or a sequencing adaptor, the sequence of its complement and / or a portion thereof to the first more than one barcoded amplicon.

51. The method according to any one of claims 49-50, the method comprising determining the copy number of the nucleic acid target in one or more of the more than one single cells based on the number of molecular markers having different sequences associated with the second more than one barcoded amplicon or its product.

52. The method according to any one of claims 43-51, wherein the first more than one barcoded amplicon and / or the second more than one barcoded amplicon comprises a whole transcriptome amplification (WTA) product.

53. The method according to any one of claims 1-52, the method comprising using the more than one barcoded nucleic acid target as a template to synthesize a third more than one barcoded amplicon to produce a third more than one barcoded amplicon, optionally: wherein synthesizing the third more than one barcoded amplicon comprises polymerase chain reaction (PCR) amplification of the more than one barcoded nucleic acid target; wherein synthesizing the third more than one barcoded amplicon comprises PCR amplification using a primer capable of hybridizing to the first universal sequence or its complement and a target-specific primer; comprising obtaining sequence data of the third more than one barcoded amplicon or its product, and optionally obtaining the sequence information comprises attaching a sequencing adaptor to the third more than one barcoded amplicon or its product; and / or comprising determining the copy number of the nucleic acid target in one or more of the more than one single cells based on the number of molecular markers having different sequences associated with the third more than one barcoded amplicon or its product.

54. The method according to any one of claims 1-53, wherein: the nucleic acid target comprises a nucleic acid molecule, optionally the nucleic acid molecule comprises ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation product, RNA comprising a poly(A) tail or any combination thereof; the nucleic acid target comprises a sample index oligonucleotide, and optionally the sample index oligonucleotide comprises a sample index sequence, and the sample index sequences of at least two sample index compositions in more than one sample index composition comprise different sequences; and / or The nucleic acid target includes a cell component-binding reagent-specific oligonucleotide, optionally the cell component-binding reagent-specific oligonucleotide contains a unique identifier sequence for the cell component-binding reagent.

55. The method according to any one of claims 1-54, wherein: Extending the more than one oligonucleotide barcode includes using a reverse transcriptase and / or a DNA polymerase lacking at least one of 5'-to-3' exonuclease activity and 3'-to-5' exonuclease activity to extend the more than one oligonucleotide barcode; The DNA polymerase includes the Klenow fragment; The reverse transcriptase includes a viral reverse transcriptase, optionally wherein the viral reverse transcriptase is murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase; The first universal sequence and the second universal sequence are the same; The first universal sequence and the second universal sequence are different; The first universal sequence and / or the second universal sequence contains a binding site for a sequencing primer and / or a sequencing adapter, its complementary sequence, and / or a portion thereof; The sequencing adapter includes a P5 sequence, a P7 sequence, its complementary sequence, and / or a portion thereof; The sequencing primer includes a read 1 sequencing primer, a read 2 sequencing primer, its complementary sequence, and / or a portion thereof; At least 10 of the more than one oligonucleotide barcodes contain different molecular marker sequences; and / or Each molecular marker of the more than one oligonucleotide barcodes contains at least 6 nucleotides.

56. The method according to any one of claims 1-55, wherein the solid support includes a flat surface or synthetic particles, optionally wherein: At least one of the more than one oligonucleotide barcodes is immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, or partially encapsulated in the synthetic particles; The synthetic particles are destructible; The synthetic particles include beads; The beads include Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, antibiotin microbeads, antifluorescent dye microbeads, or any combination thereof; The synthetic particles contain a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof; and / or The synthetic particles include destructible hydrogel particles.

57. A composition comprising: Two or more than one solid supports, wherein each of the solid supports contains more than one oligonucleotide barcode, and each of the more than one oligonucleotide barcodes contains a cell marker sequence, and each cell marker sequence contains a predetermined chamber index subsequence, and The oligonucleotide barcodes associated with the same solid support contain the same cell marker sequences, and the oligonucleotide barcodes associated with different solid supports contain different cell marker sequences.

58. The composition according to claim 57, wherein the two or more than one solid supports include more than one first solid support and more than one second solid support, wherein the oligonucleotide barcodes associated with the more than one first solid support have a first predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the more than one second solid support have a second predetermined chamber index subsequence, and wherein the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

59. The composition according to any one of claims 57-58, wherein the two or more than one solid supports include more than one first solid support and more than one second solid support, wherein the first predetermined chamber index subsequence of the more than one first solid support is selected from a first set of chamber index subsequences, wherein the second predetermined chamber index subsequence of the more than one second solid support is selected from a second set of chamber index subsequences, and wherein each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences.

60. The composition according to any one of claims 57-59, wherein based on the predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with the more than one first solid support or the more than one second solid support.

61. The composition according to any one of claims 57-60, wherein the two or more than one solid supports include: more than one third solid support, optionally: (a) the oligonucleotide barcodes associated with the more than one third solid support have a third chamber index subsequence and / or (b) the third chamber index subsequence of the more than one third solid support is selected from a third set of chamber index subsequences; more than one fourth solid support, optionally: (a) the oligonucleotide barcodes associated with the more than one fourth solid support have a fourth chamber index subsequence and / or (b) the fourth chamber index subsequence of the more than one fourth solid support is selected from a fourth set of chamber index subsequences; more than one fifth solid support, optionally: (a) the oligonucleotide barcodes associated with the more than one fifth solid support have a fifth chamber index subsequence and / or (b) the fifth chamber index subsequence of the more than one fifth solid support is selected from a fifth set of chamber index subsequences; more than one sixth solid support, optionally: (a) the oligonucleotide barcodes associated with the more than one sixth solid support have a sixth chamber index subsequence and / or (b) the sixth chamber index subsequence of the more than one sixth solid support is selected from a sixth set of chamber index subsequences; More than one seventh solid support, optionally: (a) the oligonucleotide barcode associated with the more than one seventh solid support has a seventh chamber index subsequence and / or (b) the seventh chamber index subsequence of the more than one seventh solid support is selected from a seventh set of chamber index subsequences; and / or More than one eighth solid support, optionally: (a) the oligonucleotide barcode associated with the more than one eighth solid support has an eighth chamber index subsequence and / or (b) the eighth chamber index subsequence of the more than one eighth solid support is selected from an eighth set of chamber index subsequences; and wherein the first chamber index subsequence, the second chamber index subsequence, the third chamber index subsequence, the fourth chamber index subsequence, the fifth chamber index subsequence, the sixth chamber index subsequence, the seventh chamber index subsequence, and / or the eighth chamber index subsequence do not share sequences with each other.

62. The composition according to any one of claims 57 - 61, wherein based on the predetermined chamber index subsequence of the sequencing reads derived from the oligonucleotide barcode or its product, a user can determine whether the oligonucleotide barcode is associated with a first solid support, a second solid support, a third solid support, a fourth solid support, a fifth solid support, a sixth solid support, a seventh solid support, or an eighth solid support.

63. The composition according to any one of claims 57 - 62, wherein the cell label comprises more than one cell label portion and one or more linkers.

64. The composition according to any one of claims 57 - 63, wherein the cell label comprises a first cell label portion, a first linker, and a second cell label portion, optionally the cell label comprises a second linker and a third cell label portion, and further optionally the cell label comprises a third linker and a fourth cell label portion.

65. The composition according to any one of claims 57 - 64, wherein the predetermined chamber index subsequence comprises the first cell label portion, the second cell label portion, the third cell label portion, the fourth cell label portion, or any combination thereof.

66. The composition according to any one of claims 57 - 65, wherein the first set of chamber index subsequences, the second set of chamber index subsequences, the third set of chamber index subsequences, the fourth set of chamber index subsequences, the fifth set of chamber index subsequences, the sixth set of chamber index subsequences, the seventh set of chamber index subsequences, and / or the eighth set of chamber index subsequences comprise a set of fewer than about 960, about 864, about 768, about 672, about 576, about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences, the unique sequences being different from the chamber index subsequences of other sets of chamber index subsequences.

67. The composition according to any one of claims 57 - 66, wherein the first cell - marking portion, the second cell - marking portion, the third cell - marking portion, the fourth cell - marking portion, or any combination thereof is selected from a group of fewer than about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences.

68. The composition according to any one of claims 57 - 67, wherein based on the sequences of the first cell - marking portion, the second cell - marking portion, the third cell - marking portion, the fourth cell - marking portion, or any combination thereof from sequencing reads derived from the oligonucleotide barcode or its products, a user can determine whether the oligonucleotide barcode is associated with a first solid support, a second solid support, a third solid support, a fourth solid support, a fifth solid support, a sixth solid support, a seventh solid support, or an eighth solid support.

69. A method, comprising: Distributing a first population of more than one precursor first solid supports and a first population of first oligonucleotides into a first more than one first partitions, wherein the co - located solid supports and the first oligonucleotides become associated; Distributing a first population of more than one precursor second solid supports and a second population of first oligonucleotides into a first more than one second partitions, wherein the co - located solid supports and the first oligonucleotides become associated; Pooling the more than one precursor first solid supports associated with the first oligonucleotide; Pooling the more than one precursor second solid supports associated with the first oligonucleotide; Distributing the more than one precursor first solid supports associated with the first oligonucleotide and a first population of second oligonucleotides into a second more than one first partitions, wherein the co - located solid supports and the second oligonucleotides become associated; Distributing the more than one precursor second solid supports associated with the first oligonucleotide and a second population of second oligonucleotides into a second more than one second partitions, wherein the co - located solid supports and the second oligonucleotides become associated; Pooling the more than one precursor first solid supports associated with the first oligonucleotide and the second oligonucleotide; Pooling the more than one precursor second solid supports associated with the first oligonucleotide and the second oligonucleotide; Distributing the more than one precursor first solid supports associated with the first oligonucleotide and the second oligonucleotide and a first population of third oligonucleotides into a third more than one first partitions, wherein the co - located solid supports and the third oligonucleotides become associated; and Distributing the more than one precursor second solid supports associated with the first oligonucleotide and the second oligonucleotide and a second population of third oligonucleotides into a third more than one second partitions, wherein the co - located solid supports and the third oligonucleotides become associated.

70. The method according to claim 69, comprising: Pooling the more than one precursor first solid supports associated with the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide to produce more than one first solid supports; and Pool the more than one precursor second solid supports associated with the first oligonucleotide, the second oligonucleotide, and the third oligonucleotide to produce more than one second solid support.

71. The method according to any one of claims 69 - 70, wherein: (i) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different; (ii) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are the same; (iii) the first and second populations of the first oligonucleotide are the same, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are different; (iv) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are different; (v) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are different, and the first and second populations of the third oligonucleotide are the same; (vi) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are different; or (vii) the first and second populations of the first oligonucleotide are different, the first and second populations of the second oligonucleotide are the same, and the first and second populations of the third oligonucleotide are the same.

72. The method according to any one of claims 69 - 71, wherein each of the first and second populations of the first oligonucleotide comprises about 192 first cell marker portions having different sequences; wherein each of the first and second populations of the second oligonucleotide comprises about 192 second cell marker portions having different sequences; and wherein each of the first and second populations of the third oligonucleotide comprises about 192 third cell marker portions having different sequences.

73. The method according to any one of claims 69 - 72, wherein: the first and second populations of the first oligonucleotide are the same; the first and second populations of the second oligonucleotide are the same; and the first and second populations of the third oligonucleotide are different, optionally, the first and second populations of the third oligonucleotide comprise non - overlapping subsets of the cell marker portion sequences of the first and second populations of the first oligonucleotide and / or the first and second populations of the second oligonucleotide.

74. The method according to any one of claims 69 - 73, wherein the first and second populations of the first oligonucleotides each comprise about 384 first cell marker moieties having different sequences; wherein the first and second populations of the second oligonucleotides each comprise about 384 second cell marker moieties having different sequences; and wherein the first and second populations of the third oligonucleotides each comprise about 48 third cell marker moieties having different sequences.

75. The method according to any one of claims 69 - 74, comprising: distributing the more than one precursor first solid supports and the fourth oligonucleotide associated with the first, second, and third oligonucleotides into a fourth more than one first partition, wherein the co - localized solid supports and the fourth oligonucleotide become associated; distributing the more than one precursor second solid supports and the fifth oligonucleotide associated with the first, second, and third oligonucleotides into a fourth more than one second partition, wherein the co - localized solid supports and the fifth oligonucleotide become associated; pooling the more than one precursor first solid supports associated with the first, second, third, and fourth oligonucleotides to produce more than one first solid support; and pooling the more than one precursor second solid supports associated with the first, second, third, and fifth oligonucleotides to produce more than one second solid support.

76. The method according to any one of claims 69 - 75, wherein: the first and second populations of the first oligonucleotides are the same; the first and second populations of the second oligonucleotides are the same; the first and second populations of the third oligonucleotides are the same; and the fifth and sixth oligonucleotides are different.

77. The method according to any one of claims 69 - 76, wherein: the first oligonucleotide comprises a first cell marker moiety and a first linker or its complement; the second oligonucleotide comprises a first linker, a second cell marker moiety, and a second linker or its complement; the third oligonucleotide comprises a second linker and a third cell marker moiety or its complement, optionally the second oligonucleotide further comprises a third linker or its complement; and / or the fourth oligonucleotide comprises a third linker and a fourth cell marker moiety or its complement.

78. The method according to any one of claims 69 - 77, wherein: the first oligonucleotide and the second oligonucleotide are configured to be linked via the first linker; the second oligonucleotide and the third oligonucleotide are configured to be linked via the second linker; and / or the third oligonucleotide and the fourth oligonucleotide are configured to be linked via the third linker.

79. The method according to any one of claims 69 - 78, wherein the more than one first solid support and / or the more than one second solid support comprise at least about 1000, about 10000, about 100000, about 1000000, about 7000000, about 10000000, about 56000000 unique cell marker sequences.

80. The method according to any one of claims 69 - 79, wherein the sequences of the first cell marker portion, the second cell marker portion, and / or the third cell marker portion are the same.

81. The method according to any one of claims 69 - 80, wherein the sequences of the first cell marker portion, the second cell marker portion, and / or the third cell marker portion are different.

82. The method according to any one of claims 69 - 81, wherein the first oligonucleotide, the second oligonucleotide, the third oligonucleotide, and / or the fourth oligonucleotide are single - stranded, double - stranded, and / or comprise one or two single - stranded overhangs.

83. The method according to any one of claims 69 - 82, wherein each of the first solid support and the second solid support comprises more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprising a cell marker sequence, wherein each cell marker sequence comprises a predetermined chamber index subsequence.

84. The method according to any one of claims 69 - 83, wherein the oligonucleotide barcode associated with the more than one first solid support has a first predetermined chamber index subsequence, wherein the oligonucleotide barcode associated with the more than one second solid support has a second predetermined chamber index subsequence, and wherein the first predetermined chamber index subsequence and the second predetermined chamber index subsequence are different.

85. The method according to any one of claims 69 - 84, wherein the first predetermined chamber index subsequence of the more than one first solid support is selected from a first set of chamber index subsequences, wherein the second predetermined chamber index subsequence of the more than one second solid support is selected from a second set of chamber index subsequences, and wherein each chamber index subsequence of the first set of chamber index subsequences is different from the chamber index subsequences of the second set of chamber index subsequences.

86. The method according to any one of claims 69 - 85, wherein the method produces a composition according to any one of claims 57 - 68.

87. The method according to any one of claims 69 - 86, further comprising: providing a microwell array comprising more than one chamber, wherein each chamber comprises more than one partition; and partitioning the more than one first solid support and the more than one second solid support into partitions of an identified first chamber and an identified second chamber in the more than one chamber, respectively.

88. The method according to any one of claims 69 - 87, wherein distributing the oligonucleotide to more than one partition comprises providing more than one partition comprising the oligonucleotide.

89. The method according to any one of claims 69 - 88, wherein the more than one partition comprises a 384 - well plate, a 288 - well plate, a 192 - well plate, a 96 - well plate, or a 48 - well plate.

90. The method or composition according to any one of claims 57 - 89, wherein the solid support comprises synthetic particles, optionally wherein: at least one of the more than one oligonucleotide barcodes is immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated in the synthetic particles, partially encapsulated in the synthetic particles, or a combination thereof; the synthetic particles are destructible; the synthetic particles comprise beads, optionally the beads comprise Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A - conjugated beads, protein G - conjugated beads, protein A / G - conjugated beads, protein L - conjugated beads, oligo(dT) - conjugated beads, silica beads, silica - like beads, antibiotin microbeads, anti - fluorescent dye microbeads, or any combination thereof; the synthetic particles contain a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof; and / or the synthetic particles comprise destructible hydrogel particles.

91. The method according to any one of claims 1 - 56, wherein the two or more than one solid supports are the two or more than one solid supports according to any one of claims 57 - 68 and / or are produced by the method according to any one of claims 69 - 89.

92. A composition comprising: a micro - pore array, wherein the microporous array comprises more than one chamber, wherein each chamber comprises more than one partition, and wherein each partition is a micropore having a volume in the range of from about 1,000 μm 3 to about 786,000 μm 3 optionally: further comprising a cartridge, wherein the cartridge comprises at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

93. A composition comprising: a cartridge, wherein the cartridge includes at least one of the following: an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, wherein the cartridge includes a microarray of pores, wherein the microarray of pores includes more than one chamber, wherein each chamber includes more than one partition, and wherein each partition is a micropore having a volume in the range of from about 1,000 μm 3 to about 786,000 μm 3 in volume.

94. A composition comprising: two or more than one solid supports, wherein each solid support comprises more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprises a cell - marking sequence, and each cell - marking sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell - marking sequence, and wherein the oligonucleotide barcodes associated with different solid supports comprise different cell - marking sequences, wherein the oligonucleotide barcodes of the same more than one solid supports comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes of different more than one solid supports comprise different chamber index subsequences.

95. The composition according to any one of claims 92 - 94, wherein a partition in the more than one partition comprises a single solid support among the more than one solid supports, wherein each of the solid supports comprises more than one oligonucleotide barcode, each of the more than one oligonucleotide barcodes comprises a cell marker sequence, and each cell marker sequence comprises a predetermined chamber index subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell marker sequence, and wherein the oligonucleotide barcodes associated with different solid supports comprise different cell marker sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber index subsequence, and wherein the oligonucleotide barcodes located in different chambers comprise different chamber index subsequences.

96. The composition according to any one of claims 92-95, wherein: the cartridge is configured to maintain the viability of single cells partitioned within the microwells, optionally for a period of at least about 10 min, 20 min, 30 min, 40 min, 50 min, 60 min, 100 min, 250 min, 500 min, 750 min, 1000 min, 2500 min, 5000 min, 7500 min, or 10000 min; the cartridge comprises a transparent window for optical imaging of the microwells; each of the oligonucleotide barcodes comprises a molecular marker sequence; each cell marker of the more than one oligonucleotide barcode comprises at least 6 nucleotides; the cell marker comprises more than one portion and one or more linkers; the cell marker comprises a first cell marker portion, a first linker, and a second cell marker portion, optionally the cell marker comprises a second linker and a third cell marker portion, and further optionally the cell marker comprises a third linker and a fourth cell marker portion; the first cell marker portion comprises the chamber index subsequence; the chamber index subsequence has a length of 2-15 nucleotides; the more than one chamber is at least about 4 chambers, about 10 chambers, about 20 chambers, about 40 chambers, about 60 chambers, about 80 chambers, about 100 chambers, about 200 chambers, about 300 chambers, about 400 chambers, about 500 chambers, about 750 chambers, or about 1000 chambers; the more than one partition is at least about 100 partitions, about 500 partitions, about 1000 partitions, about 5000 partitions, about 10000 partitions, about 25000 partitions, about 50000 partitions, about 75000 partitions, or about 100000 partitions; each oligonucleotide barcode comprises a first universal sequence; the oligonucleotide barcode comprises a target binding region, the target binding region comprises a capture sequence, optionally the target binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random polymer, or any combination thereof; and / or each molecular marker of the more than one oligonucleotide barcode comprises at least 6 nucleotides.

97. The composition according to any one of claims 92-96, wherein the solid support comprises a flat surface or synthetic particles, optionally wherein: At least one of the more than one oligonucleotide barcodes is immobilized on the synthetic particle, partially immobilized on the synthetic particle, encapsulated in the synthetic particle, partially encapsulated in the synthetic particle, or a combination thereof; The synthetic particle is destructible; The synthetic particle comprises beads, optionally the beads comprise Sepharose beads, streptavidin beads, agarose beads, magnetic beads, conjugated beads, protein A-conjugated beads, protein G-conjugated beads, protein A / G-conjugated beads, protein L-conjugated beads, oligo(dT)-conjugated beads, silica beads, silica-like beads, antibiotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; The synthetic particle comprises a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic substance, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, Sepharose, cellulose, nylon, silicone, and any combination thereof; and / or The synthetic particle comprises destructible hydrogel particles.

98. The composition according to any one of claims 92-97, wherein the two or more than one solid supports are the two or more than one solid supports according to any one of claims 57-68 and / or are produced by the method according to any one of claims 69-89.

99. The composition according to any one of claims 92-98, the composition further comprising: Instructions for use; A chamber index subsequence lookup table, optionally wherein the chamber index subsequence lookup table identifies the chamber index subsequence associated with each solid support distributed in each microwell of the array; An imaging system configured to capture and process an image of all or a part of the microwell, wherein the imaging system further comprises an illumination subsystem, an imaging subsystem, and a processor, optionally the imaging system is configured to perform bright field, dark field, fluorescence, or quantitative phase imaging; A buffer; and / or One or more reagents for reverse transcription reaction, one or more reagents for amplification reaction, or both.

Images