Sorting method using barcoded chambers for single cell workflow

Abstract

The present disclosure includes systems, methods, compositions, and kits for assigning sequencing data of single cells to chambers. In some embodiments, a method for associating sequencing data of single cells with phenotypic data is provided. The present disclosure includes a solid support comprising a plurality of oligonucleotide barcodes, each of which comprises a cell labeling sequence. Each cell labeling sequence may comprise a predetermined chamber-indexing subsequence. Oligonucleotide barcodes located in the same chamber may comprise the same chamber-indexing subsequence, and oligonucleotide barcodes located in different chambers may comprise different chamber-indexing subsequences.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 387,997, filed December 19, 2022, and U.S. Provisional Patent Application No. 63 / 607,516, filed December 7, 2023, the entire contents of which are expressly incorporated herein by reference in their entirety. The present disclosure relates generally to the field of molecular biology, for example, determining gene expression using molecular barcoding. [Background technology]

[0002] Current technology allows for the measurement of gene expression in single cells in a massively parallel manner (e.g., more than 10,000 cells) by attaching cell-specific oligonucleotide barcodes to poly(A) mRNA molecules from individual cells, with each cell colocalizing with a barcoded reagent bead in a compartment. Phenotypic information for a single call can be derived from experiments performed prior to measuring gene and / or protein expression by sequencing. There is a need for compositions, systems, and methods for correlating single-cell sequencing data with phenotypic data. 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 chambers in a way that preserves information about which cell populations are sorted into which chambers. In general, there is a need for compositions, systems, and methods for assigning sequencing data to chambers of a microwell array. Summary of the Invention

[0003] The present disclosure includes compositions. In some embodiments, the compositions include two or more solid supports. In some embodiments, each solid support includes a plurality of oligonucleotide barcodes, each including a cell labeling sequence, and each cell labeling sequence includes a predetermined chamber-indexing subsequence. In some embodiments, the oligonucleotide barcodes associated with the same solid support include the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports include different cell labeling sequences. In some embodiments, the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports, wherein the oligonucleotide barcodes associated with the plurality of first solid supports have a first predetermined chamber-indexing subsequence and the oligonucleotide barcodes associated with the plurality of second solid supports have a second predetermined chamber-indexing subsequence, and wherein the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different.

[0004] In some embodiments, the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports, wherein the first predetermined chamber-indexing subsequences of the plurality of first solid supports are selected from a first set of chamber-indexing subsequences, and the second predetermined chamber-indexing subsequences of the plurality of second solid supports are selected from a second set of chamber-indexing subsequences, and each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from each chamber-indexing subsequence of the second set of chamber-indexing subsequences. In some embodiments, a user can determine whether an oligonucleotide barcode is associated with a plurality of first solid supports or a plurality of second solid supports based on the predetermined chamber-indexing subsequences of sequencing reads derived from the oligonucleotide barcode or its product.

[0005] The two or more plurality of solid supports may include a plurality of third solid supports, where (a) the oligonucleotide barcodes associated with the plurality of third solid supports may have a third chamber-indexing subsequence, and / or (b) the third chamber-indexing subsequence of the plurality of third solid supports is selected from a third set of chamber-indexing subsequences; a plurality of fourth solid supports, where (a) the oligonucleotide barcodes associated with the plurality of fourth solid supports may have a fourth chamber-indexing subsequence, and / or (b) the fourth chamber-indexing subsequence of the plurality of fourth solid supports is selected from a fourth set of chamber-indexing subsequences; and a plurality of fifth solid supports, where (a) the oligonucleotide barcodes associated with the plurality of fifth solid supports have a fifth chamber-indexing subsequence. a plurality of fifth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of sixth solid supports may have a sixth chamber indexing subsequence, and / or (b) the sixth chamber indexing subsequence of the plurality of sixth solid supports is selected from the sixth set of chamber indexing subsequences; a plurality of seventh solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of seventh solid supports may have a seventh chamber indexing subsequence, and / or (b) the seventh chamber indexing subsequence of the plurality of seventh solid supports is selected from the seventh set of chamber indexing subsequences;and / or a plurality of eighth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of eighth solid supports may have an eighth chamber-indexing subsequence, and / or (b) the plurality of eighth solid supports, the eighth chamber-indexing subsequence of the plurality of eighth solid supports, is selected from an eighth set of chamber-indexing subsequences. In some embodiments, the first chamber-indexing subsequence, the second chamber-indexing subsequence, the third chamber-indexing subsequence, the fourth chamber-indexing subsequence, the fifth chamber-indexing subsequence, the sixth chamber-indexing subsequence, the seventh chamber-indexing subsequence, and / or the eighth chamber-indexing subsequence do not share sequences with each other. In some embodiments, a user can determine whether an 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 based on a predetermined chamber-indexed subsequence of a sequencing read derived from the oligonucleotide barcode or its product;

[0006] In some embodiments, the cell label comprises multiple cell-labeling moieties and one or more linkers. In some embodiments, the cell label comprises a first cell-labeling moiety, a first linker, and a second cell-labeling moiety; the cell label may comprise a second linker and a third cell-labeling moiety; and the cell label may further comprise a third linker and a fourth cell-labeling moiety. In some embodiments, the predetermined chamber-indexing moiety sequence comprises a first cell-labeling moiety, a second cell-labeling moiety, a third cell-labeling moiety, a fourth cell-labeling moiety, or any combination thereof. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh, and / or eighth sets of chamber-indexing subsequences comprise a set of about 960, about 864, about 768, about 672, about 576, about 480, about 384, about 288, about 192, about 96, or less than about 48 unique sequences that are distinct from the chamber-indexing subsequences of the other sets of chamber-indexing subsequences. In some embodiments, the first cell-labeling moiety, the second cell-labeling moiety, the third cell-labeling moiety, the fourth cell-labeling moiety, or any combination thereof, is selected from a set of about 480, about 384, about 288, about 192, about 96, or less than about 48 unique sequences. In some embodiments, a user can determine whether an 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 based on the sequence of the first cell labeling portion, the second cell labeling portion, the third cell labeling portion, the fourth cell labeling portion, or any combination thereof, of a sequencing read derived from the oligonucleotide barcode or a product thereof.

[0007] The disclosure herein includes a method. In some embodiments, the method includes the steps of: allocating a first solid support of a precursor type and a first population of a first oligonucleotide to a first plurality of first compartments, wherein the solid supports and the first oligonucleotides are associated with co-localized solid supports; allocating a second solid support of a precursor type and a second population of the first oligonucleotide to a first plurality of second compartments, wherein the solid supports and the first oligonucleotides are associated with co-localized solid supports; pooling the first solid supports of the precursor type associated with the first oligonucleotides; pooling the second solid supports of the precursor type associated with the first oligonucleotides; allocating the first solid supports of the precursor type and the first population of the second oligonucleotides associated with the first oligonucleotide to a second plurality of first compartments, wherein the solid supports and the second oligonucleotides are associated with co-localized solid supports; and allocating the second solid supports of the precursor type associated with the first oligonucleotides to a second plurality of first compartments, wherein the solid supports and the second oligonucleotides are associated with co-localized solid supports. The method includes the steps of allocating the second solid supports of the precursor type and the second population of the second oligonucleotides into a second plurality of second compartments, wherein the co-localized solid supports and the second oligonucleotides are associated; pooling the plurality of first solid supports of the precursor type associated with the first and second oligonucleotides; pooling the plurality of second solid supports of the precursor type associated with the first and second oligonucleotides; allocating the plurality of first solid supports of the precursor type associated with the first and second oligonucleotides and the first population of the third oligonucleotides into a third plurality of first compartments, wherein the co-localized solid supports and the third oligonucleotides are associated; and allocating the plurality of second solid supports of the precursor type associated with the first and second oligonucleotides and the second population of the third oligonucleotides into a third plurality of second compartments, wherein the co-localized solid supports and the third oligonucleotides are associated.In some embodiments, the method includes pooling a plurality of precursor first solid supports associated with the first, second, and third oligonucleotides to generate a plurality of first solid supports; and pooling a plurality of precursor second solid supports associated with the first, second, and third oligonucleotides to generate a plurality of second solid supports.

[0008] In some embodiments, (i) the first and second population of first oligonucleotides are the same, the first and second population of second oligonucleotides are the same, and the first and second population of third oligonucleotides are different; (ii) the first and second population of first oligonucleotides are the same, the first and second population of second oligonucleotides are different, and the first and second population of third oligonucleotides are the same; (iii) the first and second population of first oligonucleotides are the same, the first and second population of second oligonucleotides are different, and the first and second population of third oligonucleotides are different; or (iv) the first and second population of first oligonucleotides are different, and the second oligonucleotides are different. (v) the first and second populations of the first oligonucleotides are different, the first and second populations of the second oligonucleotides are different, and the first and second populations of the third oligonucleotides are the same; (vi) the first and second populations of the first oligonucleotides are different, the first and second populations of the second oligonucleotides are the same, and the first and second populations of the third oligonucleotides are different; or (vii) the first and second populations of the first oligonucleotides are different, the first and second populations of the second oligonucleotides are the same, and the first and second populations of the third oligonucleotides are the same.

[0009] The first and second populations of first oligonucleotides may each comprise about 192 first cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of second oligonucleotides each comprise about 192 second cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of third oligonucleotides each comprise about 192 third cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of first oligonucleotides are the same, the first and second populations of second oligonucleotides are the same, and the first and second populations of third oligonucleotides are different. In some embodiments, the first and second populations of third oligonucleotides comprise non-overlapping subsets of cell-labeling subsequences of the first and second populations of first oligonucleotides and / or the first and second populations of second oligonucleotides.

[0010] The first and second populations of first oligonucleotides may each comprise about 384 first cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of second oligonucleotides each comprise about 384 second cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of third oligonucleotides each comprise about 48 third cell-labeling moieties having distinct sequences. In some embodiments, the method includes the steps of: allocating a plurality of precursor-type first solid supports associated with the first, second, and third oligonucleotides and the fourth oligonucleotide into a fourth plurality of first compartments, wherein the solid supports and the fourth oligonucleotide are associated, and wherein the co-localized solid supports and the fourth oligonucleotide are associated; allocating a plurality of precursor-type second solid supports associated with the first, second, and third oligonucleotides and the fifth oligonucleotide into a fourth plurality of second compartments, wherein the co-localized solid supports and the fifth oligonucleotide are associated; pooling the plurality of precursor-type first solid supports associated with the first, second, third, and fourth oligonucleotides to generate a plurality of first solid supports; and pooling the plurality of precursor-type second solid supports associated with the first, second, third, and fifth oligonucleotides to generate a plurality of second solid supports.

[0011] In some embodiments, the first and second populations of first oligonucleotides are the same; the first and second populations of second oligonucleotides are the same; the first and second populations of third oligonucleotides are the same; and the fifth and sixth oligonucleotides are different. In some embodiments, the first oligonucleotide comprises a first cell-labeling moiety and a first linker or its complement. In some embodiments, the second oligonucleotide comprises a first linker, a second cell-labeling moiety, and a second linker or its complement. In some embodiments, the third oligonucleotide comprises a second linker and a third cell-labeling moiety or its complement, and the second oligonucleotide may further comprise a third linker or its complement. In some embodiments, the fourth oligonucleotide comprises a third linker and a fourth cell-labeling moiety or its complement.

[0012] In some embodiments, the first oligonucleotide and the second oligonucleotide are configured to be connected via a first linker. In some embodiments, the second oligonucleotide and the third oligonucleotide are configured to be connected via a second linker. In some embodiments, the third oligonucleotide and the fourth oligonucleotide are configured to be connected via a third linker. In some embodiments, the plurality of first solid supports and / or the plurality of second solid supports comprise at least about 1,000, about 10,000, about 100,000, about 1,000,000, about 7,000,000, about 10,000,000, or about 56,000,000 unique cell-labeling sequences. In some embodiments, the sequences of the first second cell-labeling moiety, the second cell-labeling moiety, and / or the third cell-labeling moiety are the same. In some embodiments, the sequences of the first second cell-labeling moiety, the second cell-labeling moiety, and / or the third cell-labeling moiety 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 a plurality of oligonucleotide barcodes, each comprising a cell-labeling sequence, and each cell-labeling sequence comprises a predetermined chamber-indexing subsequence.

[0013] In some embodiments, the oligonucleotide barcodes associated with a plurality of first solid supports have a first predetermined chamber-indexing subsequence and the oligonucleotide barcodes associated with a plurality of second solid supports have a second predetermined chamber-indexing subsequence, and the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different.

[0014] The first predetermined chamber-indexing subsequences of the plurality of first solid supports can be selected from a first set of chamber-indexing subsequences, and the second predetermined chamber-indexing subsequences of the plurality of second solid supports can be selected from a second set of chamber-indexing subsequences, and each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from the chamber-indexing subsequences of the second set of chamber-indexing subsequences. The methods provided herein can produce the compositions provided herein.

[0015] In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments; and distributing a plurality of first solid supports and a plurality of second solid supports into specific first chambers and specific second chambers of the plurality of chambers, respectively. In some embodiments, distributing the oligonucleotides into the plurality of compartments includes providing a plurality of compartments containing the oligonucleotides. In some embodiments, the plurality of compartments comprises a 384-well plate, a 288-well plate, a 192-well plate, a 96-well plate, or a 48-well plate.

[0016] In some embodiments, the solid support comprises a synthetic particle. In some embodiments, at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, partially encapsulated within a synthetic particle, or a combination thereof. In some embodiments, the synthetic particle is disintegratable. In some embodiments, the synthetic particle comprises a bead. In some embodiments, the bead comprises 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 material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof, hi some embodiments, the synthetic particles comprise breakable hydrogel particles.

[0017] The present disclosure includes a method for assigning sequencing data to chambers. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments; distributing each of two or more types of a plurality of solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support includes a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and assigning each of two or more populations of single cells to a specific chamber among the plurality of chambers. The method includes the steps of: distributing the single cells into chamber compartments, each of two or more populations of single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying a chamber indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber indexing subsequence of each cell labeling sequence in the sequencing data.

[0018] The present disclosure includes a method for assigning sequencing data to chambers. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, each solid support including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and assigning each of two or more populations of single cells to a specific one of the plurality of chambers. The method includes: distributing the single cells into compartments of predetermined chambers, each of two or more populations of single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data.

[0019] The present disclosure includes a method for assigning sequencing data to populations of single cells. In some embodiments, the method includes the steps of: preparing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; and distributing each of two or more populations of single cells to a compartment of a specific chamber among the plurality of chambers, wherein each of the two or more populations of single cells is assigned to a compartment of a specific chamber among the plurality of chambers. the plurality of single cells comprises a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a population among two or more populations of single cells based on the chamber assigned to the sequencing read.

[0020] The present disclosure includes a method for assigning sequencing data to populations of single cells. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, the solid supports each including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and distributing each of two or more populations of single cells into a specific chamber compartment of the plurality of chambers, the single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a population among two or more populations of single cells based on the chamber assigned to the sequencing read.

[0021] The present disclosure includes a method for associating sequencing data and phenotypic data of populations of single cells. In some embodiments, the method includes the steps of: obtaining phenotypic data for each of two or more populations of single cells; preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments; distributing each of two or more solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support includes a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support include the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports include different cell labeling sequences, wherein the oligonucleotide barcodes located in the same chamber include the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers include different chamber-indexing subsequences; and obtaining phenotypic data for each of the two or more populations of single cells. the method includes the steps of: distributing each of the single cells to a specific chamber compartment among a plurality of chambers, wherein each of two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying a chamber-indexed subsequence of each cell label sequence in the sequencing data; and associating the sequencing data of the at least one population of single cells with phenotypic data based on the chamber-indexed subsequence of each cell label sequence in the sequencing data.

[0022] The present disclosure includes a method for associating sequencing data and phenotypic data of populations of single cells. In some embodiments, the method includes the steps of: obtaining phenotypic data for each of two or more populations of single cells; preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, each solid support including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber indexing subsequences; obtaining phenotypic data for each of two or more populations of single cells; The method includes the steps of: distributing each of the single cells to a specific chamber compartment among a plurality of chambers, wherein each of two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying chamber index subsequences of each cell label sequence in the sequencing data; and associating the sequencing data of the at least one population of single cells with phenotypic data based on the chamber index subsequences of each cell label sequence in the sequencing data.

[0023] The method may include assigning each of a plurality of sequencing reads to a population of two or more populations of single cells based on a chamber assigned to the sequencing read. The method may include obtaining phenotypic data for each of the two or more populations of single cells; and associating the sequencing data and the phenotypic data for at least one population of single cells based on a chamber-indexed subsequence of each cell label sequence in the sequencing data.

[0024] In some embodiments, the two or more plurality of solid supports are two or more plurality of solid supports of the compositions provided herein and / or produced by the methods provided herein. In some embodiments, the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports. In some embodiments, the oligonucleotide barcodes associated with the plurality of first solid supports have a first predetermined chamber-indexing subsequence. In some embodiments, the oligonucleotide barcodes associated with the plurality of second solid supports have a second predetermined chamber-indexing subsequence. In some embodiments, the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different.

[0025] In some embodiments, the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports. In some embodiments, the first predetermined chamber-indexing subsequence of the plurality of first solid supports is selected from a first set of chamber-indexing subsequences. In some embodiments, the second predetermined chamber-indexing subsequence of the plurality of second solid supports is selected from a second set of chamber-indexing subsequences. In some embodiments, each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from each chamber-indexing subsequence of the second set of chamber-indexing subsequences. In some embodiments, a user can determine whether an oligonucleotide barcode is associated with a plurality of first solid supports or a plurality of second solid supports based on the predetermined chamber-indexing subsequence of a sequencing read derived from the oligonucleotide barcode or its product. The step of distributing each of the two or more types of a plurality of solid supports into a compartment of a particular chamber among the plurality of chambers can include distributing a predetermined plurality of solid supports selected from the two or more types of a plurality of solid supports into a particular chamber among the plurality of chambers. In some embodiments, the step of barcoding the copies of the nucleic acid target includes contacting a plurality of oligonucleotide barcodes with the copies of the nucleic acid target for hybridization, and extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid target to generate a plurality of barcoded nucleic acid targets.

[0026] The section is approximately 1,000 μm 3 ~Approx. 786,000μm 3In some embodiments, the oligonucleotide barcodes each comprise a molecular label sequence. In some embodiments, the oligonucleotide barcodes of the same plurality of solid supports comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes of different plurality of solid supports comprise different chamber-indexing subsequences. In some embodiments, each cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides. In some embodiments, the cell label comprises a plurality of moieties and one or more linkers. In some embodiments, the cell label comprises a first cell-labeling moiety, a first linker, and a second cell-labeling moiety; the cell label may comprise a second linker and a third cell-labeling moiety; and the cell label may further comprise a third linker and a fourth cell-labeling moiety. In some embodiments, the first cell-labeling moiety comprises a chamber-indexing subsequence. The chamber-indexing subsequence may be 2 to 15 nucleotides in length.

[0027] In some embodiments, the populations of single cells are separate samples. In some embodiments, each population of two or more populations of single cells is a biological replicate sample, a technical replicate sample, a control sample, an experimental sample, or a combination thereof. In some embodiments, the two or more populations of single cells are derived from one or more samples separated based on phenotypic data, and the plurality of single cells may comprise T cells, B cells, tumor cells, bone marrow cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof. In some embodiments, the plurality of chambers 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, the plurality of compartments is at least about 100 compartments, about 500 compartments, about 1000 compartments, about 5000 compartments, about 10000 compartments, about 25000 compartments, about 50000 compartments, about 75000 compartments, or about 100000 compartments. In some embodiments, sequencing of the cell labels identifies the chamber of origin of each sequenced barcoded nucleic acid target or its product within the microwell array.

[0028] In some embodiments, a compartment of the plurality of compartments comprises a single cell of the plurality of single cells and a single solid support of the plurality of solid supports. In some embodiments, the plurality of barcoded nucleic acid targets each comprise sequences complementary to at least a portion of the nucleic acid target and the molecular label. In some embodiments, each of the plurality of sequencing reads comprises (1) a cell label sequence and (2) a molecular label sequence. In some embodiments, each oligonucleotide barcode comprises a first universal sequence. In some embodiments, the oligonucleotide barcode comprises a target binding region comprising a capture sequence. In some embodiments, the target binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. In some embodiments, distributing the plurality of solid supports into respective compartments of the plurality of chambers comprises distributing each of the plurality of solid supports into a specific chamber of a microwell array by flow cytometry. In some embodiments, distributing each of two or more populations of single cells into compartments of specific chambers of the plurality of chambers comprises distributing each population of single cells into a specific chamber of the microwell array by flow cytometry. The method may include aligning a sorting component of a flow cytometer with the microwell array.

[0029] The phenotypic data may include event data. The event data may include quantitative biological event data derived from the sorting device. In some embodiments, the event data includes side scatter signals, forward scatter signals, one or more fluorescent signals, or any combination thereof. The method may include correlation analysis of the phenotypic data and 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 the therapeutic agents, and / or cellular targets of the candidate therapeutic agents. The method may include lysing one or more single cells. Viability of the single cells may be maintained for a period of time after distribution and before lysis. This period may be at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 100 minutes, 250 minutes, 500 minutes, 750 minutes, 1000 minutes, 2500 minutes, 5000 minutes, 7500 minutes, or 10000 minutes.

[0030] The method may include determining the copy number of a nucleic acid target in one or more of a plurality of single cells of at least one population of single cells. In some embodiments, determining the copy number of the nucleic acid target in one or more of the plurality of single cells includes determining the copy number of the nucleic acid target in the plurality of single cells based on the number of molecular labels having distinct sequences, their complements, or combinations thereof associated with the plurality of barcoded nucleic acid targets or products thereof.

[0031] The method may include contacting random primers with a plurality of barcoded nucleic acid targets, each of the random primers comprising a second universal sequence or its complement; and extending the random primers hybridized to the plurality of barcoded nucleic acid targets to generate a plurality of extension products. The method may include amplifying the plurality of extension products 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 generating a first plurality of barcoded amplicons. In some embodiments, amplifying the plurality of extension products includes adding sequences of sequencing primers and / or sequencing adapter binding sites, their complementary sequences, and / or portions thereof to the plurality of extension products. The method may include determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the first plurality of barcoded amplicons or products thereof. In some embodiments, determining the copy number of the nucleic acid target in one or more of the plurality of single cells comprises determining the number of each of the plurality of nucleic acid targets in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with barcoded amplicons in a first plurality of barcoded amplicons comprising the sequences of each of the plurality of nucleic acid targets. In some embodiments, the sequences of each of the plurality of nucleic acid targets comprise subsequences of each of the plurality of nucleic acid targets. In some embodiments, the sequences of the nucleic acid targets in the first plurality of barcoded amplicons comprise subsequences of the nucleic acid targets. The method may include amplifying the first plurality of barcoded amplicons 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 generating a second plurality of barcoded amplicons.In some embodiments, amplifying the first plurality of barcoded amplicons comprises adding sequences of sequencing primer and / or sequencing adapter binding sites, their complementary sequences, and / or portions thereof to the first plurality of barcoded amplicons. The method may include determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the second plurality of barcoded amplicons or products thereof. In some embodiments, the first plurality of barcoded amplicons and / or the second plurality of barcoded amplicons comprise whole transcriptome amplification (WTA) products.

[0032] The method may include synthesizing a third plurality of barcoded amplicons using the plurality of barcoded nucleic acid targets as templates to generate a third plurality of barcoded amplicons. In some embodiments, synthesizing the third plurality of barcoded amplicons includes performing polymerase chain reaction (PCR) amplification of the plurality of barcoded nucleic acid targets. In some embodiments, synthesizing the third plurality of barcoded amplicons includes PCR amplification using a primer capable of hybridizing to the first universal sequence or its complement and a target-specific primer. The method may include obtaining sequence data of the third plurality of barcoded amplicons or their products, and obtaining the sequence information may include attaching sequencing adapters to the third plurality of barcoded amplicons or their products. The method may include determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the third plurality of barcoded amplicons or their products.

[0033] 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 indexing oligonucleotide, and the sample indexing oligonucleotide may comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. In some embodiments, the nucleic acid target comprises a cellular component binding reagent-specific oligonucleotide. In some embodiments, the cellular component binding reagent-specific oligonucleotide comprises a unique identifier sequence for the cellular component binding reagent. In some embodiments, extending the plurality of oligonucleotide barcodes comprises extending the plurality of oligonucleotide barcodes 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. In some embodiments, the DNA polymerase comprises a Klenow fragment. In some embodiments, the reverse transcriptase comprises a viral reverse transcriptase, and the viral reverse transcriptase may be a murine leukemia virus (MLV) reverse transcriptase or a 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 comprise a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof. In some embodiments, the sequencing primer comprises a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof.In some embodiments, at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences. In some embodiments, each cell label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides.

[0034] In some embodiments, the solid support comprises a synthetic particle. In some embodiments, the solid support comprises a planar surface. In some embodiments, at least one of the plurality of oligonucleotide barcodes is immobilized on, partially immobilized on, encapsulated within, or partially encapsulated within the synthetic particle. In some embodiments, the synthetic particle is disintegrable. In some embodiments, the synthetic particle comprises a bead. In some embodiments, the bead comprises 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 material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The synthetic particles can be disintegrable hydrogel particles.

[0035] The disclosure herein includes compositions. In some embodiments, the compositions include a microwell array, the microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments, each compartment having a size of about 1,000 μm 3 ~Approx. 786,000μm 3The composition can include a cartridge, the cartridge including at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

[0036] The present disclosure includes compositions. In some embodiments, the compositions include a cartridge, the cartridge including at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, the cartridge including a microwell array, the microwell array including a plurality of chambers, each chamber including a plurality of compartments, each compartment having a size of about 1,000 μm 3 ~Approx. 786,000μm 3 The microwells have volumes in the range of The present disclosure includes compositions. In some embodiments, the compositions include two or more solid supports, each of which includes a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences, wherein the oligonucleotide barcodes of the same plurality of solid supports comprise the same chamber-indexing subsequence, and wherein the oligonucleotide barcodes of the different plurality of solid supports comprise different chamber-indexing subsequences. In some embodiments, the two or more solid supports are the two or more solid supports of the compositions disclosed herein and / or are produced by the methods disclosed herein.

[0037] In some embodiments, a compartment of the plurality of compartments comprises a single solid support of the plurality of solid supports, each solid support comprising a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support comprising the same cell labeling sequence and the oligonucleotide barcodes associated with different solid supports comprising different cell labeling sequences, the oligonucleotide barcodes located within the same chamber comprising the same chamber-indexing subsequence and the oligonucleotide barcodes located in different chambers comprising different chamber-indexing subsequences. The composition can include a chamber-indexing subsequence lookup table, which can, for example, identify a chamber-indexing subsequence associated with each solid support distributed in each microwell of the array. In some embodiments, the cartridge is configured to maintain viability of single cells distributed within the microwells, which may be for a period of at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 100 minutes, 250 minutes, 500 minutes, 750 minutes, 1000 minutes, 2500 minutes, 5000 minutes, 7500 minutes, or 10000 minutes. In some embodiments, the cartridge includes a transparent window for optical imaging of the microwells. The composition may include an imaging system configured to capture and process images of all or a portion of the microwells, the imaging system further including an illumination subsystem, an imaging subsystem, and a processor. In some embodiments, the imaging system is configured to perform bright-field, dark-field, fluorescence, or quantitative phase imaging. The composition may include a buffer. The composition may include one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or both.

[0038] In some embodiments, each oligonucleotide barcode comprises a molecular label sequence. In some embodiments, each cell label of the plurality of oligonucleotide barcodes comprises at least 6 nucleotides. In some embodiments, the cell label comprises a plurality of moieties and one or more linkers. In some embodiments, the cell label comprises a first cell-labeling moiety, a first linker, and a second cell-labeling moiety; the cell label may comprise a second linker and a third cell-labeling moiety; and the cell label may further comprise a third linker and a fourth cell-labeling moiety. In some embodiments, the first cell-labeling moiety comprises a chamber-indexing moiety sequence. In some embodiments, the chamber-indexing moiety sequence is 2-15 nucleotides in length. In some embodiments, the plurality of chambers 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, the plurality of compartments is at least about 100 compartments, about 500 compartments, about 1000 compartments, about 5000 compartments, about 10,000 compartments, about 25,000 compartments, about 50,000 compartments, about 75,000 compartments, or about 100,000 compartments.

[0039] The composition may include a plurality of solid supports, each comprising a plurality of oligonucleotide barcodes. In some embodiments, the oligonucleotide barcodes each comprise a molecular label and a cellular label. In some embodiments, oligonucleotide barcodes associated with the same solid support comprise the same cellular label sequence, and oligonucleotide barcodes associated with different solid supports comprise different cellular label sequences. In some embodiments, each oligonucleotide barcode comprises a first universal sequence. In some embodiments, the oligonucleotide barcode comprises a target binding region comprising a capture sequence. In some embodiments, the target binding region comprises a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. In some embodiments, each molecular label of the plurality of oligonucleotide barcodes comprises at least six nucleotides.

[0040] In some embodiments, the solid support comprises a planar surface. In some embodiments, the solid support comprises a synthetic particle. In some embodiments, at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, partially encapsulated within a synthetic particle, or a combination thereof. In some embodiments, the synthetic particle is disintegratable. In some embodiments, the synthetic particle comprises a bead. In some embodiments, the bead comprises 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, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and combinations thereof. The synthetic particles can be disintegrable hydrogel particles. In some embodiments, the composition further comprises instructions for use. [Brief explanation of the drawings]

[0041] [Figure 1] 1 shows a non-limiting exemplary barcode. [Figure 2] 1 illustrates a non-limiting exemplary workflow for barcoding and electronic counting. [Figure 3] 1 is a schematic diagram showing a non-limiting exemplary process for generating an indexed library of 3′-barcoded targets from multiple targets. [Figure 4A]4A and 4B show non-limiting, exemplary schematic diagrams of the microwell arrays (FIG. 4A) and chambers (FIG. 4B) provided herein. [Figure 4B] 4A and 4B show non-limiting, exemplary schematic diagrams of the microwell arrays (FIG. 4A) and chambers (FIG. 4B) provided herein. [Figure 5] 1 shows a non-limiting exemplary method for producing oligonucleotide barcodes containing chamber-indexing subsequences provided herein. [Figure 6] 1 shows a non-limiting exemplary method for producing oligonucleotide barcodes containing chamber-indexing subsequences provided herein. [Figure 7] 1 shows a non-limiting exemplary method for producing oligonucleotide barcodes containing chamber-indexing subsequences provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0042] In the following detailed description, reference is made to the accompanying drawings, which form a part of this specification. In the drawings, like symbols typically identify like components unless context dictates otherwise. The exemplary 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 drawings, may be arranged, substituted, combined, separated, and designed in a wide variety of different configurations, all of which are expressly contemplated herein and make part of the disclosure herein. All patents, published patent applications, other publications, and sequences from GenBank and other databases referenced herein are incorporated by reference in their entirety for relevant art.

[0043] Quantifying a small number of nucleic acids, such as messenger ribonucleotide acid (mRNA) molecules, is clinically important, for example, to determine the genes expressed in cells at different developmental stages or under different environmental conditions. However, determining the absolute number of nucleic acid molecules (e.g., mRNA molecules) can be very difficult, 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 identical molecular copies in each cycle. However, PCR can have drawbacks because each molecule replicates with a stochastic probability, which varies depending on the PCR cycle and gene sequence, resulting in amplification bias and inaccurate gene expression measurements. Stochastic barcodes with unique molecular labels (also called molecular indexes (MIs)) can be used to count the number of molecules and correct for amplification bias. Stochastic barcoding, such as the Precise™ assay (Cellular Research, Inc., Palo Alto, CA) and the Rhapsody™ assay (Becton, Dickinson and Company, Franklin Lakes, NJ), can correct for biases induced by the library preparation step by using molecular beacons (MLs) to label mRNA during PCR and reverse transcription (RT).

[0044] The Precise™ assay utilizes a non-depleting pool of stochastic barcodes with a large number of unique molecular tag sequences, e.g., 6561-65536, on poly(T) oligonucleotides to hybridize to all poly(A)-mRNAs in a sample during the RT step. The stochastic barcodes may contain universal PCR priming sites. During RT, target gene molecules react randomly with the stochastic barcodes. Each target molecule hybridizes to a stochastic barcode, resulting in the generation of a stochastically barcoded complementary ribonucleotide acid (cDNA) molecule. After labeling, the stochastically barcoded cDNA molecules from the microwells of a microwell plate may be pooled into a single tube for PCR amplification and sequencing. Raw sequencing data can be analyzed to obtain the number of reads, the number of stochastic barcodes with unique molecular tag sequences, and the number of mRNA molecules.

[0045] The present disclosure includes compositions. In some embodiments, the compositions include two or more solid supports. In some embodiments, each solid support includes a plurality of oligonucleotide barcodes, each including a cell labeling sequence, and each cell labeling sequence includes a predetermined chamber-indexing subsequence. In some embodiments, the oligonucleotide barcodes associated with the same solid support include the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports include different cell labeling sequences.

[0046] The disclosure herein includes a method. In some embodiments, the method includes the steps of: allocating a first solid support of a plurality of precursor types and a first population of first oligonucleotides to a first plurality of first compartments, wherein the solid supports and the first oligonucleotides are associated with co-localized solid supports; allocating a second solid support of a plurality of precursor types and a second population of first oligonucleotides to a first plurality of second compartments, wherein the solid supports and the first oligonucleotides are associated with co-localized solid supports; pooling the first solid supports of the precursor types associated with the first oligonucleotides; pooling the second solid supports of the precursor types associated with the first oligonucleotides; allocating the first solid supports of the plurality of precursor types and the first population of second oligonucleotides associated with the first oligonucleotides to a second plurality of first compartments, wherein the solid supports and the second oligonucleotides are associated with co-localized solid supports; and allocating the second solid supports of the plurality of precursor types associated with the first oligonucleotides to a second plurality of first compartments, wherein the solid supports and the second oligonucleotides are associated with co-localized solid supports. The method includes the steps of allocating the second solid supports of the precursor type and the second population of the second oligonucleotides into a second plurality of second compartments, wherein the co-localized solid supports and the second oligonucleotides are associated; pooling the plurality of first solid supports of the precursor type associated with the first and second oligonucleotides; pooling the plurality of second solid supports of the precursor type associated with the first and second oligonucleotides; allocating the plurality of first solid supports of the precursor type associated with the first and second oligonucleotides and the first population of the third oligonucleotides into a third plurality of first compartments, wherein the co-localized solid supports and the third oligonucleotides are associated; and allocating the plurality of second solid supports of the precursor type associated with the first and second oligonucleotides and the second population of the third oligonucleotides into a third plurality of second compartments, wherein the co-localized solid supports and the third oligonucleotides are associated.

[0047] The present disclosure includes a method for assigning sequencing data to chambers. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments; distributing each of two or more types of a plurality of solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support includes a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and assigning each of two or more populations of single cells to a specific chamber among the plurality of chambers. The method includes the steps of: distributing the single cells into chamber compartments, each of two or more populations of single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying a chamber indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber indexing subsequence of each cell labeling sequence in the sequencing data.

[0048] The present disclosure includes a method for assigning sequencing data to chambers. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, each solid support including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and assigning each of two or more populations of single cells to a specific one of the plurality of chambers. The method includes: distributing the single cells into compartments of predetermined chambers, each of two or more populations of single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data.

[0049] The present disclosure includes a method for assigning sequencing data to populations of single cells. In some embodiments, the method includes the steps of: preparing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; and distributing each of two or more populations of single cells to a compartment of a specific chamber among the plurality of chambers, wherein each of the two or more populations of single cells is assigned to a compartment of a specific chamber among the plurality of chambers. the plurality of single cells comprises a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a population among two or more populations of single cells based on the chamber assigned to the sequencing read.

[0050] The present disclosure includes a method for assigning sequencing data to populations of single cells. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, the solid supports each including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and distributing each of two or more populations of single cells into a specific chamber compartment of the plurality of chambers, the single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a population among two or more populations of single cells based on the chamber assigned to the sequencing read.

[0051] The present disclosure includes a method for associating sequencing data and phenotypic data of populations of single cells. In some embodiments, the method includes the steps of: obtaining phenotypic data for each of two or more populations of single cells; preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments; distributing each of two or more solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support includes a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support include the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports include different cell labeling sequences, wherein the oligonucleotide barcodes located in the same chamber include the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers include different chamber-indexing subsequences; and obtaining phenotypic data for each of the two or more populations of single cells. the method includes the steps of: distributing each of the single cells to a specific chamber compartment among a plurality of chambers, wherein each of two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying a chamber-indexed subsequence of each cell label sequence in the sequencing data; and associating the sequencing data of the at least one population of single cells with phenotypic data based on the chamber-indexed subsequence of each cell label sequence in the sequencing data.

[0052] The present disclosure includes a method for associating sequencing data and phenotypic data of populations of single cells. In some embodiments, the method includes the steps of: obtaining phenotypic data for each of two or more populations of single cells; preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, each solid support including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber indexing subsequences; obtaining phenotypic data for each of two or more populations of single cells; The method includes the steps of: distributing each of the single cells to a specific chamber compartment among a plurality of chambers, wherein each of two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying chamber index subsequences of each cell label sequence in the sequencing data; and associating the sequencing data of the at least one population of single cells with phenotypic data based on the chamber index subsequences of each cell label sequence in the sequencing data.

[0053] The method may include assigning each of a plurality of sequencing reads to a population of two or more populations of single cells based on a chamber assigned to the sequencing read. The method may include obtaining phenotypic data for each of the two or more populations of single cells; and associating the sequencing data and the phenotypic data for at least one population of single cells based on a chamber-indexed subsequence of each cell label sequence in the sequencing data.

[0054] The disclosure herein includes compositions. In some embodiments, the compositions include a microwell array, the microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments, each compartment having a size of about 1,000 μm 3 ~Approx. 786,000μm 3 The composition can include a cartridge, the cartridge including at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

[0055] The present disclosure includes compositions. In some embodiments, the compositions include a cartridge, the cartridge including at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, the cartridge including a microwell array, the microwell array including a plurality of chambers, each chamber including a plurality of compartments, each compartment having a size of about 1,000 μm 3 ~Approx. 786,000μm 3 The microwells have volumes in the range of

[0056] The disclosure herein includes compositions. In some embodiments, the compositions include two or more solid supports, each of which includes a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes of the same plurality of solid supports including the same chamber-indexing subsequence, and the oligonucleotide barcodes of the different plurality of solid supports including different chamber-indexing subsequences.

[0057] definition Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. 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 purposes of this disclosure, the following terms are defined below.

[0058] As used herein, the term "adapter" may refer to a sequence for facilitating amplification or sequencing of an associated nucleic acid. The associated nucleic acid may include a target nucleic acid. The associated nucleic acid may include one or more of a spatial label, a target label, a sample label, an indexing label, or a barcode sequence (e.g., a molecular label). The adapter may be linear. The adapter may be a pre-adenylated adapter. The adapter may be double-stranded or single-stranded. One or more adapters may be positioned at the 5' or 3' end of a nucleic acid. When an adapter includes known sequences at the 5' and 3' ends, the known sequences may be the same or different sequences. The adapters positioned at the 5' and / or 3' ends of a polynucleotide may be capable of hybridizing to one or more oligonucleotides immobilized on a surface. In some embodiments, the adapter may include a universal sequence. A universal sequence may be a region of nucleotide sequence that is common to two or more nucleic acid molecules. The two or more nucleic acid molecules may also have regions of different sequences. Thus, for example, the 5' adapters can contain identical and / or universal nucleic acid sequences, and the 3' adapters can contain identical and / or universal sequences. The presence of a universal sequence in different members of a plurality of nucleic acid molecules can allow for the replication or amplification of multiple different sequences 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 can be present in different members of a collection of nucleic acid molecules, allowing for the replication or amplification of multiple different sequences using at least one, two (e.g., a pair), or more single universal primers that are complementary to the universal sequence. Thus, a universal primer includes a sequence that can hybridize to such a universal sequence. A molecule having a target nucleic acid sequence can be modified to add universal adapters (e.g., non-target nucleic acid sequences) to one or both ends of the different target nucleic acid sequences.The one or more universal primers bound to the target nucleic acid may provide a site for hybridization of the universal primer. The one or more universal primers bound to the target nucleic acid may be the same or different from each other.

[0059] As used herein, the terms "associated" or "associated with" can mean that two or more species are identifiable as being located together at a time. Association can mean that two or more species are or were located in similar containers. Association can also be an informational association. For example, digital information about two or more species can be stored and used to determine that one or more of the species were located together at a time. Association can also be a physical association. In some embodiments, two or more associated species are "tethered," "bound," or "immobilized" to each other or to a common solid or semi-solid surface. Association can refer to covalent or non-covalent means for attaching a label to a solid or semi-solid support, such as a bead. Association can also be a covalent bond between a target and a label. Association can include hybridization between two molecules (e.g., a target molecule and a label).

[0060] As used herein, the term "complementary" may refer to the ability for precise pairing between two nucleotides. For example, if a nucleotide at a given position in a nucleic acid can hydrogen bond with a nucleotide in 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 may be "partial," in which only some of the nucleotides bind, or may be complete, in which total complementarity exists between the single-stranded molecules. A first nucleotide sequence may be referred to as the "complement" of a second sequence if the first nucleotide sequence is complementary to the second nucleotide sequence. A first nucleotide sequence may be referred to as the "reverse complement" of a second sequence if the first nucleotide sequence is complementary to a sequence that is the reverse of the second sequence (i.e., the order of the nucleotides is reversed). As used herein, a "complementary" sequence may refer to the "complement" or "reverse complement" of a sequence. It is understood from this disclosure that when a molecule is capable of hybridizing to another molecule, it may be complementary or partially complementary to the hybridizing molecule.

[0061] 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 determining the number of unique labels associated with targets in a sample. This methodology, which can be probabilistic in nature, transforms the molecular counting problem into one of locating and identifying identical molecules into a series of yes / no digital questions regarding the detection of a predefined set of labels. As used herein, the term "label" or "labels" 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 wholly or partially amplifiable label. The label can be a wholly or partially sequenceable label. The label can be a portion 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 a nucleic acid sequence, e.g., a junction of a naturally occurring and non-naturally occurring sequence. As used herein, the term "label" can be used interchangeably with the terms "index," "tag," or "label tag." The label can carry information. For example, in various embodiments, the label can be used to determine the identity of the sample, the source of the sample, the identity of the cell, and / or the target.

[0062] As used herein, the term "non-depletion reservoir" can refer to a pool of barcodes (e.g., stochastic barcodes) composed of many different labels. A non-depletion reservoir can contain many different barcodes so that when the non-depletion reservoir is associated with a pool of targets, each target is likely to associate with a unique barcode. The uniqueness of each labeled target molecule can be determined by random selection statistics and depends on the copy number of identical target molecules in the population compared to the diversity of the labels. The size of the resulting labeled target molecule can be determined by the stochastic nature of the barcoding process, and analysis of the number of detected barcodes then allows for calculation of the number of target molecules present in the original population or sample. If the ratio of the number of target molecules present to the number of unique barcodes is low, the labeled target molecule is highly unique (i.e., the probability that more than one target molecule will be labeled with a given label is very low).

[0063] As used herein, the term "nucleic acid" refers to a polynucleotide sequence or a fragment thereof. A nucleic acid may comprise nucleotides. A nucleic acid may be exogenous or endogenous to a cell. A nucleic acid may exist in a cell-free environment. A nucleic acid may be a gene or a fragment thereof. A nucleic acid may be DNA. A nucleic acid may be RNA. A nucleic acid may comprise one or more analogs (e.g., modified backbones, sugars, or nucleobases). Some non-limiting examples of analogs include 5-bromouracil, peptide nucleic acid, xenonucleic acid, morpholino, locked nucleic acid, glycol nucleic acid, threose nucleic acid, dideoxynucleotide, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol-containing nucleotides, biotin-linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queusine, and wyosine. "Nucleic acid," "polynucleotide," "target polynucleotide," and "target nucleic acid" can be used interchangeably.

[0064] Nucleic acids may contain one or more modifications (e.g., base modifications, backbone modifications) to result in nucleic acids with new or enhanced properties (e.g., improved stability). Nucleic acids may also contain nucleic acid affinity tags. A nucleoside may be a base-sugar combination. The base portion of a nucleoside may be a heterocyclic base. The two most common classes of such heterocyclic bases are purines and pyrimidines. A nucleotide may be a nucleoside further comprising a phosphate group covalently linked to the sugar portion of the nucleoside. For nucleosides containing a pentofuranosyl sugar, the phosphate group may be linked to the 2', 3', or 5' hydroxyl moiety of the sugar. In forming nucleic acids, the phosphate group may covalently link adjacent nucleosides to one another to form a linear polymeric compound. The respective ends of this linear polymeric compound may then be further joined to form a circular compound, although linear compounds are generally preferred. In addition, linear compounds may have internal nucleotide base complementarity and therefore may fold in such a manner as to yield fully or partially double-stranded compounds. Within nucleic acids, the phosphate groups may commonly be referred to as forming the internucleoside backbone of the nucleic acid. The linkage or backbone may be a 3' and 5' phosphodiester linkage.

[0065] The nucleic acids can contain 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 containing a phosphorus atom therein include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates, such as 3'-alkylene phosphonates, 5'-alkylene phosphonates, chiral phosphonates, phosphinates, phosphoramidates, such as 3'-amino phosphoramidate and aminoalkyl phosphoramidates, phosphorodiamidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphates, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs, and those with reverse polarity, wherein one or more internucleotide linkages are 3'-3', 5'-5', or 2'-2' linkages.

[0066] Nucleic acids can contain polynucleotide backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more heteroatom or heterocyclic internucleoside linkages, including those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, riboacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S, and CH moieties.

[0067] Nucleic acids may include nucleic acid mimetics. The term "mimetics" is intended to include polynucleotides in which only the furanose ring, or both the furanose ring and the internucleotide linkage, are replaced with non-furanose groups; replacement of only the furanose ring may also be referred to as a sugar surrogate. The heterocyclic base moiety or modified heterocyclic base moiety may be maintained for hybridization with an appropriate target nucleic acid. One such nucleic acid may be a peptide nucleic acid (PNA). In a PNA, the sugar backbone of a polynucleotide may be replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleotides may be retained and are directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The backbone in a PNA compound may contain two or more linked aminoethylglycine units, thereby providing the PNA with an amide-containing backbone. The heterocyclic base moiety may be directly or indirectly bound to the aza nitrogen atoms of the amide portion of the backbone. The nucleic acid may include a morpholino backbone structure. For example, the nucleic acid may include a six-membered morpholino ring instead of a ribose ring. In some of these embodiments, phosphorodiamidates or other non-phosphodiester internucleoside linkages may replace the phosphodiester linkages.

[0068] Nucleic acids can contain linked morpholino units (e.g., morpholino nucleic acids) with heterocyclic bases attached to the morpholino ring. Linking groups can link the morpholino monomer units in morpholino nucleic acids. Nonionic morpholino-based oligomeric compounds may have fewer undesirable interactions with intracellular proteins. Morpholino-based polynucleotides can be nonionic mimics of nucleic acids. Various compounds within the morpholino class can be attached using different linking groups. A further class of polynucleotide mimics can be called cyclohexenyl nucleic acids (CeNA). The furanose ring normally present in nucleic acid molecules can be replaced with a cyclohexenyl ring. CeNA DMT-protected phosphoramidite monomers can be prepared and used to synthesize oligomeric compounds using phosphoramidite chemistry. Incorporation of CeNA monomers into nucleic acid chains 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. Further modifications include locked nucleic acids (LNAs), in which a 2'-hydroxyl 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 a methylene (-CH2) 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 with complementary nucleic acids (Tm = +3 to +10°C), stability against 3'-exonuclease degradation, and good solubility characteristics.

[0069] Nucleic acids can also include nucleobase (often simply referred to as "base") modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases can include purine bases (e.g., adenine (A) and guanine (G)) and pyrimidine bases (e.g., thymine (T), cytosine (C), and uracil (U)). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl 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 alkyl derivatives of the pyrimidine base, 6-azouracil, cytosine ... Mention may be made of tosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and 3-deazaadenine.Modified nucleobases 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 cytidines (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 cytidines (e.g., 9-(2-aminoethoxy)-H-pyrimido(5,4-(b)(1,4)benzoxazin-2(3H)-one), carbazole cytidines (2H-pyrimido(4,5-b)indol-2-one), and pyridoindole cytidines (H-pyrido(3',2':4,5)pyrrolo[2,3-d]pyrimidin-2-one).

[0070] As used herein, the term "sample" can refer to a composition that contains a target. Samples suitable for analysis by the disclosed methods, devices, and systems include cells, tissues, organs, or organisms. As used herein, the terms "sample collection device" or "device" may refer to a device capable of collecting a section of a sample and / or placing the section on a substrate. A sample device may refer to, for example, a fluorescence activated cell sorter (FACS) machine, a cell sorter, a biopsy needle, a biopsy device, a tissue sectioning device, a microfluidic device, a blade grid, and / or a microtome.

[0071] As used herein, the term "solid support" may refer to a discrete solid or semi-solid surface to which multiple barcodes (e.g., stochastic barcodes) can be attached. A solid support may encompass any type of solid, porous, or hollow sphere, ball, bearing, cylinder, or other similar structure composed 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 may comprise discrete particles that may be spherical (e.g., microspheres) or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. A bead may be non-spherical in shape. A plurality of solid supports spaced apart in an array may not include a substrate. A solid support may be used interchangeably with the term "bead."

[0072] As used herein, the term "stochastic barcode" may refer to a polynucleotide sequence comprising a label of the present disclosure. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target. As used herein, the term "gene-specific stochastic barcode" may refer to a polynucleotide sequence comprising a label and a gene-specific target-binding region. A stochastic barcode may be a polynucleotide sequence that can be used for stochastic barcoding. A stochastic barcode may be used to quantify a target within a sample. A stochastic barcode may be used to control errors that may occur after associating a label with a target. For example, a stochastic barcode may be used to evaluate amplification or sequencing errors. A stochastic barcode associated with a target may be referred to as a stochastic barcode-target or a stochastic barcode-tag-target.

[0073] As used herein, the term "stochastic barcoding" can refer to random labeling (e.g., barcoding) of nucleic acids. Stochastic barcoding can utilize a Poisson recursion strategy to associate labels and quantify the labels associated with targets. As used herein, the term "stochastic barcoding" can be used interchangeably with "stochastic labeling." As used herein, the term "target" may refer to a composition that can be associated with a barcode (e.g., a stochastic barcode). Exemplary targets suitable for analysis by the methods, devices, and systems of the present disclosure include oligonucleotides, DNA, RNA, mRNA, microRNA, tRNA, and the like. Targets may be single-stranded or double-stranded. In some embodiments, targets may be proteins, peptides, or polypeptides. In some embodiments, targets are lipids. As used herein, "target" may be used interchangeably with "species."

[0074] As used herein, the term "reverse transcriptase" can refer to a group of enzymes that have reverse transcriptase activity (i.e., that catalyze 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, retron 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, retron reverse transcriptases, and group II intron reverse transcriptases. Examples of group II intron reverse transcriptases include the Lactococcus lactis LI.LtrB intron reverse transcriptase, the Thermosynechococcus elongatus TeI4c intron reverse transcriptase, or the Geobacillus stearothermophilus GsI-IIC intron reverse transcriptase. Other classes of reverse transcriptases include the numerous classes of non-retroviral reverse transcriptases (i.e., retrons, group II introns, and diversity-generating retroelements, among others).

[0075] The terms "universal adapter primer," "universal primer adapter," or "universal adapter sequence" are used interchangeably to refer to a nucleotide sequence that can hybridize to a barcode (e.g., a stochastic barcode) and be used to generate a gene-specific barcode. A universal adapter sequence can be, for example, a known sequence that is universal across all barcodes used in the methods of the present disclosure. For example, when multiple targets are labeled using the methods disclosed herein, each of the target-specific sequences can be ligated to the same universal adapter sequence. In some embodiments, two or more universal adapter sequences can be used in the methods disclosed herein. For example, when multiple targets are labeled using the methods disclosed herein, at least two of the 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, a universal adapter sequence can be part of an oligonucleotide that contains a target-specific sequence to generate a nucleotide sequence that is complementary to a target nucleic acid. A second oligonucleotide comprising the complement of the barcode and universal adapter sequence can hybridize to the nucleotide sequence to generate a target-specific barcode (target-specific stochastic barcode). In some embodiments, the universal adapter primer has a different sequence than the universal PCR primer used in the disclosed methods.

[0076] Barcode Barcoding, e.g., probabilistic barcoding, is described, for example, in Fu et al., Proc Natl Acad Sci USA, 2011 May 31, 108(22):9026-31; US2011 / 0160078; Fan et al., Science, 2015 February 6, 347(6222):1258367; US2015 / 0299784; and WO2015 / 031691, the contents of each of which, including any supplemental or additional information, are incorporated herein by reference in their entirety. In some embodiments, the barcodes disclosed herein may be probabilistic barcodes, which may be polynucleotide sequences that can be used to stochastically label (e.g., barcode, tag) targets. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences in the stochastic barcode to the number of occurrences of any of the targets to be labeled can be 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, or can be approximately these values ​​or such numbers or ranges. The targets may be mRNA species that include mRNA molecules with identical or nearly identical sequences. A barcode may be referred to as a stochastic barcode if the ratio of the number of distinct barcode sequences of the stochastic barcode to the number of occurrences of any of the targets to be labeled is at least or at most 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. The barcode sequences of a stochastic barcode may be referred to as molecular labels.

[0077] A barcode, e.g., a stochastic barcode, can include one or more labels. Exemplary labels can include a universal label, a cell label, a barcode sequence (e.g., a molecular label), a sample label, a plate label, a spatial label, and / or a pre-spatial label. FIG. 1 shows an exemplary barcode 104 having a spatial label. The barcode 104 can include a 5' amine that can link the barcode to a solid support 105. The barcode can include a universal label, a dimensional label, a spatial label, a cell label, and / or a molecular label. The order of different labels (including, but not limited to, the universal label, the dimensional label, the spatial label, the cell label, and the molecular label) within the barcode can vary. For example, as shown in FIG. 1, the universal label can be the 5'-most label and the molecular label can be the 3'-most label. The spatial label, the dimensional label, and the cell label can be in any order. In some embodiments, the universal label, the spatial label, the dimensional label, the cell label, and the molecular label are in any order. The barcode can include a target binding region. The target binding region can interact with a target (e.g., a target nucleic acid, RNA, mRNA, DNA) in a sample. For example, the target binding region can include an oligo(dT) sequence that can interact with the poly(A) tail of an mRNA. In some cases, the labels of the barcode (e.g., the universal label, dimensional label, spatial label, cellular label, and barcode sequence) can be spaced 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleotides apart.

[0078] 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-correcting codes), that can be designed to provide error-correcting capabilities. An error-correcting subsequence set including seven-nucleotide sequences can be designed so that any pairwise combination of sequences in the set exhibits a defined "genetic distance" (or number of mismatched bases); for example, an error-correcting subsequence set can be designed to exhibit a genetic distance of three nucleotides. In this case, consideration of the error-correcting sequences in a sequence dataset of a labeled target nucleic acid molecule (described in more detail below) can enable detection or correction of amplification or sequencing errors. In some embodiments, the length of the nucleic acid subsequences used to create the error-correcting code can vary, e.g., be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 31, 40, 50 nucleotides, or a number or range between or about any two of these values. In some embodiments, nucleic acid subsequences of other lengths may be used to create error-correcting codes.

[0079] The barcode may include a target binding region. The target binding region may interact with a target in the sample. The target may be or include ribonucleic acid (RNA), messenger RNA (mRNA), microRNA, small interfering RNA (siRNA), RNA degradation products, RNAs each including a poly(A) tail, or any combination thereof. In some embodiments, the multiple targets may include deoxyribonucleic acid (DNA).

[0080] In some embodiments, the target binding region may include an oligo(dT) sequence that can interact with the poly(A) tail of mRNA. One or more of the labels of the barcode (e.g., universal label, dimensional label, spatial label, cellular label, and barcode sequence (e.g., molecular label)) may be separated from another one or two of the remaining labels of the barcode by a spacer. The spacer may 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.

[0081] Universal Signage A barcode may include one or more universal labels. In some embodiments, the one or more universal labels may be the same for all barcodes in a set of barcodes bound to a given solid support. In some embodiments, the one or more universal labels may be the same for all barcodes bound to a plurality of beads. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer. The sequencing primer can be used to sequence barcodes that include a universal label. The sequencing primer (e.g., a universal sequencing primer) may include a sequencing primer associated with a high-throughput sequencing platform. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a PCR primer. In some embodiments, the universal label may include a nucleic acid sequence that can hybridize to a sequencing primer and a PCR primer. The nucleic acid sequence of a universal label that can hybridize to a sequencing primer or a PCR primer may be referred to as a primer binding site. The universal label may include a sequence that can be used to initiate transcription of the barcode. A universal label can include a sequence that can be used to extend a barcode or a region within the barcode. A universal label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between or about any two of these values. For example, a universal label can include at least about 10 nucleotides. A universal label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. In some embodiments, a cleavable linker or modified nucleotides can be part of the universal label sequence to allow the barcode to be cleaved from the support.

[0082] dimensional indicator A barcode may include one or more dimensional labels. In some embodiments, a dimensional label may include a nucleic acid sequence that provides information about the dimension in which labeling (e.g., stochastic labeling) occurred. For example, a dimensional label can provide information about the time a target was barcoded. A dimensional label may be associated with the time of barcoding (e.g., stochastic barcoding) in a sample. A dimensional label may be activated at the time of labeling. Different dimensional labels may be activated at different time points. A dimensional label provides information about the order in which a target, a group of targets, and / or a sample was barcoded. For example, a cell population may be barcoded in the G0 phase of the cell cycle. Cells may be pulsed again with a barcode (e.g., a stochastic barcode) in the G1 phase of the cell cycle. Cells may be pulsed again with a barcode in the S phase of the cell cycle, and so on. The barcode in each pulse (e.g., each stage of the cell cycle) may include a different dimensional label. In this way, the dimensional label provides information about which targets were labeled at which stage of the cell cycle. Dimensional labeling can examine many different biological time periods. Exemplary biological time periods can include, but are not limited to, cell cycle, transcription (e.g., transcription initiation), and transcript degradation. In another example, a sample (e.g., a cell, a cell population) can be stochastically labeled before and / or after treatment with a drug and / or therapy. Changes in copy number of distinct targets can indicate the sample's response to the drug and / or therapy.

[0083] Dimensional labels may be activatable. Activatable dimensional labels can be activated at a specific time. Activatable labels can, for example, be continuously activated (e.g., not turned off). Activatable dimensional labels can, for example, be reversibly activatable (e.g., they can be turned on and turned off). Dimensional labels can be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. Dimensional labels can be reversibly activatable, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, dimensional labels can be activated by fluorescence, light, chemical events (e.g., cleavage, ligation of another molecule, addition of a modification (e.g., pegylation, sumoylation, acetylation, deacetylation, demethylation), photochemical events (e.g., photocaging), and introduction of unnatural nucleotides.

[0084] In some embodiments, the dimension labels may be the same for all barcodes (e.g., stochastic barcodes) attached to a given solid support (e.g., a bead), but may be 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 may comprise the same dimension label. In some embodiments, at least 60% of the barcodes on the same solid support may comprise the same dimension label. In some embodiments, at least 95% of the barcodes on the same solid support may comprise the same dimension label. On multiple solid supports (e.g., beads), 6Many unique dimension label sequences, even more than 10, may be presented. Dimension labels can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Dimension labels can be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Dimension labels can comprise from about 5 to about 200 nucleotides. Dimension labels can comprise from about 10 to about 150 nucleotides. Dimension labels can comprise from about 20 to about 125 nucleotides in length.

[0085] spatial sign A barcode may include one or more spatial labels. In some embodiments, a spatial label may include a nucleic acid sequence that provides information about the spatial orientation of a target molecule associated with the barcode. A spatial label may be associated with a coordinate in a sample. The coordinate may be a fixed coordinate. For example, the coordinate may be fixed relative to a substrate. A spatial label may reference a two-dimensional or three-dimensional grid. The coordinate may be fixed relative to a landmark. The landmark may be identifiable in space. The landmark may be a structure that can be imaged. The landmark may be a biological structure, e.g., an anatomical landmark. The landmark may be a cellular landmark, e.g., an organelle. The landmark may be a non-natural landmark, e.g., an identifiable identifier, e.g., a color code, a barcode, a magnetic property, fluorescence, radioactivity, or a structure with a unique size or shape. A spatial label may be associated with a physical compartment (e.g., a well, a container, or a droplet). In some embodiments, multiple spatial labels are used together to encode one or more locations in space.

[0086] Spatial labels may be the same for all barcodes attached to a given solid support (e.g., beads), but may be different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values ​​or such numbers or ranges. In some embodiments, the percentage of barcodes containing the same spatial label on the same solid support may 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 may contain the same spatial label. In some embodiments, at least 95% of the barcodes on the same solid support may contain the same spatial label.

[0087] On multiple solid supports (e.g., beads), 6 Many unique spatial marker sequences, even more than 10, may be presented. Spatial markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Spatial markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. Spatial markers can comprise between about 5 and about 200 nucleotides. Spatial markers can comprise between about 10 and about 150 nucleotides. Spatial markers can comprise between about 20 and about 125 nucleotides in length.

[0088] cell labeling A barcode (e.g., a stochastic barcode) may include one or more cell labels. In some embodiments, the cell label may include a nucleic acid sequence that provides information for determining which target nucleic acid originated from which cell. In some embodiments, the cell label is the same for all barcodes attached to a given solid support (e.g., a bead) but different for different solid supports (e.g., beads). In some embodiments, the percentage of barcodes containing the same cell label on the same solid support may be, or approximately, 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 label on the same solid support may be, or approximately, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. For example, at least 60% of the barcodes on the same solid support may contain the same cell label. As another example, at least 95% of the barcodes on the same solid support may contain the same cell label. On multiple solid supports (e.g., beads), 6 Many unique cell marker sequences, even more than 10, may be presented. Cell markers can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. Cell markers can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length. For example, a cell marker can comprise between about 5 and about 200 nucleotides. As another example, a cell marker can comprise between about 10 and about 150 nucleotides. As yet another example, a cell marker can comprise between about 20 and about 125 nucleotides in length.

[0089] Barcode sequence The barcode may include one or more barcode sequences. In some embodiments, the barcode sequence may include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. The barcode sequence includes a nucleic acid sequence that provides a counter (e.g., provides a rough approximation) for the specific occurrence of the target nucleic acid species hybridized to the barcode (e.g., target binding region).

[0090] In some embodiments, a diverse set of barcode sequences is attached to a given solid support (e.g., a bead). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 There may be at least 10, or a number or range between, or about, any two of these values, or such number or range of unique molecular label sequences. For example, the plurality of barcodes may include about 6561 barcode sequences having distinct sequences. As another example, the plurality of barcodes may include about 65536 barcode sequences having distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There may be multiple unique barcode sequences. The unique molecular beacon sequences may be attached to a given solid support (e.g., a bead). In some embodiments, the unique molecular beacon sequences are partially or entirely encompassed by a particle (e.g., a hydrogel bead).

[0091] The length of the barcode may vary in different implementations. For example, the barcode may be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, or may be about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or a number or range between any two of these values, in length. As another example, the barcode may be at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0092] molecular label A barcode (e.g., a stochastic barcode) can include one or more molecular labels. A molecular label can include a barcode sequence. In some embodiments, a molecular label can include a nucleic acid sequence that provides information about the specific type of target nucleic acid species hybridized to the barcode. A molecular label includes a nucleic acid sequence that provides a counter for the specific occurrence of a target nucleic acid species hybridized to the barcode (e.g., a target binding region). In some embodiments, a diverse set of molecular labels is attached to a given solid support (e.g., beads). 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 or a number or range between any two of these values, or about 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9There may be at least 10 unique molecular label sequences, or a number or range between any two of these values. For example, the plurality of barcodes may include about 6561 molecular labels with distinct sequences. As another example, the plurality of barcodes may include about 65536 molecular labels with distinct sequences. In some embodiments, there may be at least or at most 10 2 pieces, 10 3 pieces, 10 4 pieces, 10 5 pieces, 10 6 pieces, 10 7 pieces, 10 8 Pieces or 10 9 There can be a number of unique molecular beacon sequences. A barcode with a unique molecular beacon sequence can be attached to a given solid support (e.g., a bead).

[0093] For barcoding using multiple stochastic barcodes (e.g., stochastic barcoding), the ratio of the number of distinct molecular label sequences to the number of occurrences of any of the targets can be 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, or may be about 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 may be an mRNA species that includes mRNA molecules with identical or nearly identical sequences. In some embodiments, the ratio of the number of different molecular beacon sequences to the number of occurrences of any of the targets is at least or at most 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.

[0094] A molecular label can be 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or a number or range of nucleotides approximately equal to or equal to these values ​​or such number or range. A molecular label can be, for example, at least or at most 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, or 300 nucleotides in length.

[0095] Target binding region The barcode may comprise one or more target binding regions, e.g., capture probes. In some embodiments, the target binding region may hybridize with a target of interest. In some embodiments, the target binding region may comprise a nucleic acid sequence that specifically hybridizes to a target (e.g., a target nucleic acid, target molecule, e.g., a cellular nucleic acid to be analyzed), e.g., a specific gene sequence. In some embodiments, the target binding region may comprise a nucleic acid sequence that can bind (e.g., hybridize) to a specific position of a specific target nucleic acid. In some embodiments, the target binding region may comprise a nucleic acid sequence that is capable of specific hybridization to a restriction enzyme site overhang (e.g., an EcoRI sticky end overhang). The barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang.

[0096] In some embodiments, the target binding region may comprise a non-specific target nucleic acid sequence. A non-specific target nucleic acid sequence may refer to a sequence that can bind to multiple target nucleic acids independently of the specific sequence of the target nucleic acid. For example, the target binding region may comprise a random multimer 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 may be an oligo(dT) sequence that hybridizes to a poly(A) tail on an mRNA molecule. The random multimer sequence may be, for example, a random dimer, trimer, tetramer, pentamer, hexamer, heptamer, octamer, nonamer, decamer, or any longer multimer sequence of any length. In some embodiments, the target binding region is the same for all barcodes bound to a given bead. In some embodiments, the target binding regions of multiple barcodes bound to a given bead may comprise two or more different target binding sequences. The target binding region can be 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or a number or range between any two of these values, or approximately these values ​​or such number or range of nucleotides. The target binding region can be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 nucleotides in length, or more. For example, an mRNA molecule can be reverse transcribed using a reverse transcriptase, such as Moloney murine leukemia virus (MMLV) reverse transcriptase, to generate a cDNA molecule with a poly(dC) tail. The barcode can include a target binding region with a poly(dG) tail. Upon 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 replication to the 5' end of the barcode. In doing so, the resulting cDNA molecules contain a barcode sequence (eg, a molecular tag) on ​​the 3' end of the cDNA molecule.

[0097] In some embodiments, the target binding region may comprise an oligo(dT) capable of hybridizing to an mRNA containing a polyadenylated end. The target binding region may be gene-specific. For example, the target binding region may be configured to hybridize to a specific region of the target. The target binding region may be 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 between any two of these values, or approximately these values ​​or such number or range, in length. The target binding region can be at least or at most 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 in length. The target binding region can be about 5-30 nucleotides in length. When a barcode includes a gene-specific target binding region, the barcode may be referred to herein as a gene-specific barcode.

[0098] Orientation Characteristics A stochastic barcode (e.g., a stochastic barcode) may include one or more orientation properties that can be used to orient (e.g., align) the barcode. The barcode may include a moiety for isoelectric focusing. Different barcodes may include different isoelectric focusing points. When these barcodes are introduced into a sample, the sample may undergo isoelectric focusing to orient the barcodes in a known manner. In this manner, the orientation properties can be used to develop a known map of the barcodes in the sample. Exemplary orientation properties can include electrophoretic mobility (e.g., based on the size of the barcode), isoelectric point, spin, conductivity, and / or self-assembly. For example, a barcode with an orientation property for self-assembly may self-assemble into a specific orientation (e.g., a nucleic acid nanostructure) when activated.

[0099] affinity properties A barcode (e.g., a stochastic barcode) can include one or more affinity features. For example, a spatial label can include an affinity feature. Affinity features can include chemical and / or biological moieties that can facilitate binding of the barcode to another entity (e.g., a cellular receptor). For example, an affinity feature can include an antibody, e.g., an antibody specific to a particular moiety (e.g., a receptor) on a sample. In some embodiments, the antibody can direct the barcode to a particular cell type or molecule. Targets on and / or near a particular cell type or molecule can be labeled (e.g., stochastically labeled). In some embodiments, the affinity feature can provide spatial information in addition to the nucleotide sequence of the spatial label, 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.

[0100] An antibody can be a full-length (i.e., naturally occurring or formed by conventional immunoglobulin gene fragment recombination processes) immunoglobulin molecule (e.g., an IgG antibody), or an immunologically active (i.e., specific binding) portion of an immunoglobulin molecule, such as an antibody fragment. An antibody fragment can be, for example, a portion of an antibody, such as F(ab')2, Fab', Fab, Fv, sFv, etc. In some embodiments, an 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 an antibody, such as an "Fv" fragment consisting of the variable regions of the heavy and light chains, and recombinant single-chain polypeptide molecules in which the variable regions of the light and heavy chains are connected by a peptide linker ("scFv protein"). Exemplary antibodies can include, but are not limited to, antibodies against cancer cells, antibodies against viruses, antibodies that bind to cell surface receptors (CD8, CD34, CD45), and therapeutic antibodies.

[0101] Universal Adapter Primer A barcode may include one or more universal adapter primers. For example, a gene-specific barcode, such as a gene-specific stochastic barcode, may include a universal adapter primer. A universal adapter primer may refer to a nucleotide sequence that is universal across all barcodes. A universal adapter primer can be used to construct a gene-specific barcode. A universal adapter primer may be 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 in length, or a number or range between any two of these, or approximately these values ​​or such number or range of nucleotides. The universal adapter primer can be at least or at most 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 in length. The universal adapter primer can be 5 to 30 nucleotides in length.

[0102] Linker When a barcode includes more than one type of label (e.g., more than one cellular label or more than one barcode sequence, e.g., one molecular label), the labels may be interspersed with linker label sequences. The linker label sequence may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. The linker label sequence may be up to about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more nucleotides in length. In some cases, the linker label sequence is 12 nucleotides in length. The linker label sequence may be used to facilitate synthesis of the barcode. The linker label may include an error-correcting (e.g., Hamming) code.

[0103] solid support In some embodiments, the barcodes disclosed herein, e.g., stochastic barcodes, may be associated with a solid support. The solid support may be, for example, a synthetic particle. In some embodiments, some or all of the barcode sequences, e.g., molecular labels of stochastic barcodes (e.g., first barcode sequences) of a plurality of barcodes (e.g., a first plurality of barcodes) on a solid support, differ by at least one nucleotide. Cell labels of barcodes on the same solid support may be the same. Cell labels of barcodes on different solid supports may differ by at least one nucleotide. For example, a first cell label of a first plurality of barcodes on a first solid support may have the same sequence, and a second cell label of a second plurality of barcodes on a second solid support may have the same sequence. A first cell label of a first plurality of barcodes on a first solid support and a second cell label of a second plurality of barcodes on a second solid support may differ by at least one nucleotide. Cell labels may be, for example, about 5 to 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.

[0104] The beads can be, for example, silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The beads can include materials such as polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic materials, ceramic, plastic, glass, methylstyrene, acrylic polymers, titanium, latex, Sepharose, cellulose, nylon, silicone, or any combination thereof. In some embodiments, the beads can be polymer beads, such as deformable beads or gel beads (e.g., gel beads from 10X Genomics (San Francisco, CA)) functionalized with barcodes or stochastic barcodes. In some implementations, the gel beads can comprise a polymer-based gel. Gel beads can be generated, for example, by encapsulating one or more polymer precursors into droplets. Gel beads can be generated when the polymer precursors are exposed to an accelerator (e.g., tetramethylethylenediamine (TEMED)).

[0105] The particles can be disintegrable (e.g., dissolvable, degradable). For example, polymer beads can dissolve, melt, or decompose under desired conditions. The desired conditions can include environmental conditions. The desired conditions can result in the dissolution, melt, or decomposition of the polymer beads in a controlled manner. Gel beads can dissolve, melt, or decompose due to a chemical stimulus, a physical stimulus, a biological stimulus, a thermal stimulus, a magnetic stimulus, an electrical stimulus, a light stimulus, or any combination thereof.

[0106] Analytes and / or reagents, e.g., oligonucleotide barcodes, may be linked / immobilized, for example, to the interior surface of a gel bead (e.g., the interior accessible through diffusion of the oligonucleotide barcodes and / or the material used to generate the oligonucleotide barcodes) and / or to the exterior surface of a gel bead or any other microcapsule described herein. Linkage / immobilization may be via any form of chemical bond (e.g., covalent bond, ionic bond) or physical phenomenon (e.g., van der Waals forces, dipole-dipole interactions, etc.). In some embodiments, the linkage / immobilization of reagents to a gel bead or any other microcapsule described herein may be reversible, such as, for example, via a labile moiety (e.g., via a chemical crosslinker, including those described herein). Upon application of a stimulus, the labile moiety can be cleaved, releasing the immobilized reagent. In some embodiments, the labile moiety is a disulfide bond. For example, in cases where an oligonucleotide barcode is immobilized 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 may be included as part of the gel bead or microcapsule, as part of a chemical linker connecting 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 the plurality of barcodes may be immobilized to the particle, partially immobilized to the particle, encapsulated in the particle, partially encapsulated in the particle, or any combination thereof.

[0107] In some embodiments, the gel beads may comprise a wide variety of different polymers, including, but not limited to, polymers, thermosensitive polymers, light-sensitive polymers, magnetic polymers, pH-sensitive polymers, salt-sensitive polymers, chemically sensitive polymers, polyelectrolytes, polysaccharides, peptides, proteins, and / or plastics. Polymers can include, but are not limited to, materials such as poly(N-isopropylacrylamide) (PNIPAAm), poly(styrenesulfonate) (PSS), poly(allylamine) (PAAm), poly(acrylic acid) (PAA), poly(ethyleneimine) (PEI), poly(diallyldimethylammonium 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) (PTHF), poly(hexylviologen) (PHV), poly(L-lysine) (PLL), poly(L-arginine) (PARG), and poly(lactic-co-glycolic acid) (PLGA).

[0108] A number of chemical stimuli can be used to trigger bead collapse, dissolution, or degradation. Examples of these chemical changes include, but are not limited to, pH-mediated changes to the bead wall, bead wall collapse via chemical cleavage of cross-links, triggering bead wall depolymerization, and bead wall switching reactions. Bulk changes can also be used to trigger bead collapse. Bulk or physical changes to microcapsules through various stimuli also offer many advantages in designing capsules for releasing reagents. Bulk or physical changes occur on a macroscopic scale, with bead rupture being the result of mechanical-physical forces induced by the stimulus. These processes can include, but are not limited to, pressure-induced rupture, bead wall melting, or changes in bead wall porosity.

[0109] Biological stimuli can also be used to trigger bead disintegration, dissolution, or degradation. Generally, biological triggers resemble chemical triggers, but many examples use biomolecules, or molecules commonly found in biological systems, such as enzymes, peptides, sugars, fatty acids, nucleic acids, and the like. For example, beads can contain polymers with peptide crosslinks that are susceptible to cleavage by specific proteases. More specifically, one example can include microcapsules containing GFLGK peptide crosslinks. Addition of a biological trigger, such as the protease cathepsin B, cleaves the peptide crosslinks in the shell wall, releasing the contents of the bead. In other cases, the protease can be heat-activated. In another example, beads contain a shell wall containing cellulose. Addition of the hydrolytic enzyme chitosan serves as a biological trigger for cleavage of the cellulose bonds, depolymerization of the shell wall, and release of its contents.

[0110] Beads can also be induced to release their contents upon application of a thermal stimulus. A change in temperature can cause various changes in the beads. A change in heat can cause the beads to melt, causing the bead walls to collapse. In other cases, heat can increase the internal pressure of the beads' internal components, causing the beads to collapse or explode. In still other cases, heat can transform the beads into a compressed, dehydrated state. Heat can also act on heat-sensitive polymers within the bead walls, causing the beads to collapse. The inclusion of magnetic nanoparticles in the bead walls of microcapsules can trigger the collapse of the beads as well as guide the beads in an array. The devices of the present disclosure can include magnetic beads for either purpose. In one example, the incorporation of Fe3O4 nanoparticles into polyelectrolyte-containing beads triggers collapse in the presence of an oscillating magnetic field stimulus.

[0111] Beads can also be disintegrated, dissolved, or decomposed as a result of electrical stimulation. Similar to the magnetic particles described in the previous section, electrically sensitive beads can trigger both bead disintegration and other functions, such as alignment in an electric field, electrical conduction, or redox reactions. In one example, beads containing electrically sensitive materials are aligned in an electric field to control the release of internal reagents. In another example, an electric field can induce redox reactions within the bead wall itself, which can increase porosity. Light stimulation can also be used to disrupt the beads. Numerous optical triggers are possible, including systems using various molecules such as nanoparticles and chromophores that can absorb photons of specific wavelengths. For example, metal oxide coatings can be used as capsule triggers. UV irradiation of SiO2-coated polyelectrolyte capsules can result in the collapse of the bead wall. In yet another example, photoswitchable materials, such as azobenzene groups, can be incorporated into the bead wall. Upon application of UV or visible light, chemicals such as these absorb photons and undergo reversible cis-to-trans isomerization. In this embodiment, the incorporation of a photoswitch results in a bead wall that can collapse or become more porous upon application of a light trigger. For example, in a non-limiting example of barcoding (e.g., stochastic barcoding) shown in FIG. 2, after cells, e.g., single cells, are introduced into multiple microwells of a microwell array in block 208, beads can be introduced into multiple microwells of the microwell array in block 212. Each microwell can contain one bead. The beads can contain multiple barcodes. The barcodes can include 5' amine regions attached to the beads. The barcodes can include a universal label, a barcode sequence (e.g., a molecular label), a target binding region, or any combination thereof.

[0112] The barcodes disclosed herein may be associated with (e.g., attached to) a solid support (e.g., a bead). The barcodes associated with the solid support may comprise a barcode sequence selected from a group comprising at least 100 or 1000 barcode sequences, each having a unique sequence. In some embodiments, different barcodes associated with a solid support may comprise barcodes with different sequences. In some embodiments, a percentage of the barcodes associated with a solid support comprise the same cell label. For example, the percentage may be 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, 100%, or a number or range between any two of these values, or may be approximately these values ​​or such a number or range. As another example, the percentage may be at least or at most 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99%, or 100%. In some embodiments, barcodes associated with a solid support may have the same cell label. Barcodes associated with different solid supports may have different cell labels selected from a group comprising at least 100 or 1000 cell labels having unique sequences.

[0113] The barcodes disclosed herein may be associated with (e.g., bound to) a solid support (e.g., a bead). In some embodiments, barcoding a plurality of labels in a sample can be performed using a solid support comprising a plurality of synthetic particles associated with a plurality of barcodes. In some embodiments, the solid support may comprise a plurality of synthetic particles associated with a plurality of barcodes. The spatial labeling of the plurality of barcodes on different solid supports may differ by at least one nucleotide. The solid support may comprise a plurality of barcodes, for example, in two or three dimensions. The synthetic particles may be beads. The beads may be silica gel beads, controlled pore glass beads, magnetic beads, Dynabeads, Sephadex / Sepharose beads, cellulose beads, polystyrene beads, or any combination thereof. The solid support may include a polymer, a matrix, a hydrogel, a needle array device, an antibody, or any combination thereof. In some embodiments, the solid support may be free-floating. In some embodiments, the solid support may be embedded in a semi-solid or solid array. The barcodes may not be associated with a solid support. The barcodes may be individual nucleotides. The barcode may be associated with the substrate.

[0114] As used herein, the terms "tethered," "attached," and "immobilized" are 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 to attach pre-synthesized barcodes or as a solid support for in situ solid phase synthesis of barcodes. In some embodiments, the solid support is a bead. Beads may include one or more types of solid, porous, or hollow spheres, balls, bearings, cylinders, or other similar structures to which nucleic acids can be immobilized (e.g., covalently or non-covalently). Beads may be composed of, for example, plastic, ceramic, metal, polymeric material, or any combination thereof. Beads may be or include discrete particles that are spherical (e.g., microspheres), or may have a non-spherical or irregular shape, such as a cube, cube-like, pyramidal, cylindrical, conical, rectangular, or discoid. In some embodiments, beads may be non-spherical in shape.

[0115] The beads may 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 alloys thereof, and some rare earth metal compounds), ceramic, plastic, glass, polystyrene, silica, methylstyrene, acrylic polymers, titanium, latex, sepharose, agarose, hydrogels, polymers, cellulose, nylon, or any combination thereof. In some embodiments, the beads (e.g., the beads to which the label is attached) are hydrogel beads. In some embodiments, the beads comprise a hydrogel.

[0116] Some embodiments disclosed herein include one or more particles (e.g., beads). Each of the particles may include a plurality of oligonucleotides (e.g., barcodes). Each of the plurality of oligonucleotides may include a barcode sequence (e.g., a molecular label sequence), a cell label, and a target binding region (e.g., an oligo(dT) sequence, a gene-specific sequence, a random multimer, or a combination thereof). The cell label sequence of each of the plurality of oligonucleotides may be the same. The cell label sequences of oligonucleotides on different particles may be different so that the oligonucleotides on different particles can be identified. The number of different cell label sequences may vary in different implementations. In some embodiments, the number of cell labeling sequences is 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 pieces, 10 7 pieces, 10 8 pieces, 10 9 In some embodiments, the number of cell labeling sequences may be at least or up to 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 pieces, 10 7 pieces, 10 8 Pieces 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 of the plurality of particles comprise oligonucleotides having the same cellular sequence. In some embodiments, up to 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 of the plurality of particles comprise oligonucleotides with the same cellular sequence, hi some embodiments, none of the plurality of particles have the same cellular targeting sequence.

[0117] The multiple oligonucleotides on each particle can include different barcode sequences (e.g., molecular labels). In some embodiments, the number of barcode sequences is 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 pieces, 10 7 pieces, 10 8 pieces, 10 9In some embodiments, the number of barcode sequences may be at least or at most 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 pieces, 10 7 pieces, 10 8 Pieces or 10 9 For example, at least 100 of the plurality of oligonucleotides comprise 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 plurality of oligonucleotides comprise different barcode sequences. Some embodiments provide a plurality of particles comprising barcodes. In some embodiments, the ratio of occurrences (or copies or numbers) of targets to be labeled to distinct 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 more. In some embodiments, each of the plurality of oligonucleotides further comprises a sample label, a universal label, or both. The particles can be, for example, nanoparticles or microparticles.

[0118] The size of the beads can vary. 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 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 micrometers, or a number or range between any two of these values, or can be approximately these values ​​or such numbers or ranges. The diameter of the beads may be related to the diameter of the wells of the substrate. In some embodiments, the diameter of the beads may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a number or range between any two of these values, longer or shorter than the diameter of the wells, or may be approximately these values ​​or any such number or range, longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer or shorter than the diameter of the wells. The diameter of the beads may be related to the diameter of a cell (e.g., a single cell surrounded by a well of the substrate). In some embodiments, the diameter of the beads may be 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, longer or shorter than the diameter of the cells, or may be approximately these values ​​or any such number or range, longer or shorter than the diameter of the cells. In some embodiments, the diameter of the beads may be at least or up to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, or 300% longer or shorter than the diameter of the cells.

[0119] The beads may be bound and / or embedded in a substrate. The beads may be bound and / or embedded in a gel, hydrogel, polymer, and / or matrix. The spatial location of the beads within the substrate (e.g., gel, matrix, scaffold, or polymer) can be identified using spatial labels present in barcodes on the beads, which can serve as locational addresses. Examples of beads include, but are not limited to, streptavidin beads, agarose beads, magnetic beads, Dynabeads®, MACS® 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, anti-biotin microbeads, anti-fluorescent dye microbeads, and BcMag™ carboxyl-terminated magnetic beads.

[0120] The beads can be associated with (e.g., impregnated with) quantum dots or fluorescent dyes to make them fluorescent in one fluorescent optical channel or multiple optical channels. The beads can be associated with iron oxide or chromium oxide to make them paramagnetic or ferromagnetic. The beads can be identifiable. For example, the beads can be imaged using a camera. The beads can have a detectable code associated with them. For example, the beads can include a barcode. The beads can change size, for example, due to swelling in an organic or inorganic solution. The beads can be hydrophobic. The beads can be hydrophilic. The beads can be biocompatible. The solid support (e.g., beads) can be visualized. The solid support can include a visualization tag (e.g., a fluorescent dye). The solid support (e.g., beads) can be etched with an identifier (e.g., a number). The identifier can be visualized through imaging of the beads.

[0121] A solid support can comprise an insoluble, semi-soluble, or insoluble material. A solid support can be referred to as "functionalized" if it includes a linker, scaffold, building block, or other reactive moiety attached thereto, while a solid support can be "non-functionalized" if it lacks such reactive moieties attached thereto. A solid support can be freely used in solution, e.g., in a microtiter well format, in a flow-through format, e.g., in a column, or in a dipstick.

[0122] The solid support may comprise a membrane, paper, plastic, coated surface, flat surface, glass, slide, chip, or any combination thereof. The solid support may take the form of a resin, gel, microsphere, or other geometric configuration. The solid support may comprise a silica chip, microparticle, nanoparticle, plate, array, caliper, flat support, such as a glass fiber filter, glass surface, metal surface, metal surface (steel, gold silver, aluminum, silicone, and copper), glass support, plastic support, silicone support, chip, filter, membrane, microwell plate, slide, multiwell plate, or plastic material (e.g., formed from polyethylene, polypropylene, polyamide, polyvinylidene difluoride), including membrane, and / or wafer, comb, pin, or needle (e.g., an array of pins suitable for combinatorial synthesis or analysis), or an array of holes or nanoliter wells on a flat surface, such as a wafer (e.g., a silicone wafer), a wafer with holes or without a filter bottom, or beads. The solid support can include a polymer matrix (e.g., a gel, a hydrogel). The polymer matrix can be capable of penetrating intracellular spaces (e.g., around organelles). The polymer matrix can be capable of being pumped through the circulatory system.

[0123] Substrates and Microwell Arrays As used herein, a substrate may refer to a type of solid support. A substrate may refer to a solid support that may include a barcode or stochastic barcode of the present disclosure. A substrate may include, for example, a plurality of microwells. For example, a substrate may be a well array including two or more microwells. In some embodiments, a microwell may include a small reaction chamber having a defined volume. In some embodiments, a microwell may incorporate one or more cells. In some embodiments, a microwell may incorporate only one cell. In some embodiments, a microwell may incorporate one or more solid supports. In some embodiments, a microwell may incorporate only one solid support. In some embodiments, a microwell incorporates a single cell and a single solid support (e.g., a bead). A microwell may include a barcode reagent of the present disclosure.

[0124] Barcoding methods The present disclosure provides methods for estimating the number of distinct targets in distinct locations of a body sample (e.g., tissue, organ, tumor, cell). The method may include placing a barcode (e.g., a stochastic barcode) in proximity to the sample, lysing the sample, associating distinct targets with the barcode, amplifying the targets, and / or digitally counting the targets. The method may further include analyzing and / or visualizing information obtained from the spatial labeling of the barcode. In some embodiments, the method includes visualizing a plurality of targets in the sample. Mapping the plurality of targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps may be created before or after barcoding (e.g., stochastically barcoding) the plurality of targets in the sample. Visualizing a plurality of targets in the sample may include mapping the plurality of targets to a map of the sample. Mapping the plurality of targets to a map of the sample may include creating a two-dimensional or three-dimensional map of the sample. The two-dimensional and three-dimensional maps can be generated before or after barcoding multiple targets in a sample. In some embodiments, the two-dimensional and three-dimensional maps can be generated before or after lysing the sample. Lysing the sample before or after generating the two-dimensional or three-dimensional map can include heating the sample, contacting the sample with a detergent, changing the pH of the sample, or any combination thereof.

[0125] In some embodiments, barcoding the plurality of targets comprises hybridizing a plurality of barcodes to the plurality of targets to create barcoded targets (e.g., stochastically barcoded targets). Barcoding the plurality of targets may comprise generating an indexed library of barcoded targets. Generating an indexed library of barcoded targets may be performed using a solid support comprising a plurality of barcodes (e.g., stochastic barcodes).

[0126] Contacting the sample with the barcode The present disclosure provides methods for contacting a sample (e.g., cells) with a substrate of the present disclosure. For example, a sample including cells, an organ, or a thin section of tissue can be contacted with a barcode (e.g., a stochastic barcode). For example, the cells can be contacted by gravity flow, where they can settle and form a monolayer. The sample can be a tissue slice. The slice can be disposed on a substrate. The sample can be one-dimensional (e.g., forming a planar surface). For example, the sample (e.g., cells) can be spread across the substrate by growing / culturing the cells on the substrate. When the barcode is in close proximity to the target, the target can hybridize to the barcode. The barcodes can be contacted in a non-depleting ratio so that each distinct target can associate with a distinct barcode of the present disclosure. To ensure efficient association between the target and the barcode, the target can be cross-linked to the barcode.

[0127] cell lysis After partitioning the cells and barcodes, the cells can be lysed to liberate the target molecules. Cell lysis can be achieved by any of a variety of means, such as chemical or biochemical means, osmotic shock, or thermal, mechanical, or optical lysis. Cells can also be lysed by adding a cell lysis buffer containing a detergent (e.g., SDS, Li-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 of the target with the barcode, the diffusion rate of the target molecule can be altered, for example, by lowering the temperature and / or increasing the viscosity of the lysate.

[0128] In some embodiments, the sample may be lysed using filter paper, which can be soaked with a lysis buffer over the filter paper, and pressure can be applied to the sample, which can promote lysis of the sample and hybridization of the sample's targets to the substrate. In some embodiments, lysis can be performed by mechanical lysis, thermal lysis, optical lysis, and / or chemical lysis. Chemical lysis can include the use of 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 include Tris-HCl. The lysis buffer can include at least about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include up to about 0.01, 0.05, 0.1, 0.5, or 1 M or more Tris-HCl. The lysis buffer can include 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 more. The pH of the lysis buffer can be up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, the pH of the lysis buffer is about 7.5. The lysis buffer may include a salt (e.g., LiCl). The salt concentration in the lysis buffer may be at least about 0.1, 0.5, or 1 M or higher. The salt concentration in the lysis buffer may be up to about 0.1, 0.5, or 1 M or higher. In some embodiments, the salt concentration in the lysis buffer is about 0.5 M. The lysis buffer may include a detergent (e.g., SDS, Li-dodecyl sulfate, triton X, tween, NP-40). The concentration of the detergent in the lysis buffer may 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 concentration of detergent in the lysis buffer may be up to 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 concentration of detergent in the lysis buffer is about 1% Li-dodecyl sulfate. The time used in the lysis method may depend on the amount of detergent used. In some embodiments, the more detergent used, the less time is required for lysis.The lysis buffer may include a chelating agent (e.g., EDTA, EGTA). The concentration of the chelating agent in the lysis buffer may be at least about 1, 5, 10, 15, 20, 25, or 30 mM or more. The concentration of the chelating agent in the lysis buffer may be up to about 1, 5, 10, 15, 20, 25, or 30 mM or more. In some embodiments, the concentration of the chelating agent in the lysis buffer is about 10 mM. The lysis buffer may include a reducing reagent (e.g., beta-mercaptoethanol, DTT). The concentration of the reducing reagent in the lysis buffer may be at least about 1, 5, 10, 15, or 20 mM or more. The concentration of the reducing reagent in the lysis buffer may be up to about 1, 5, 10, 15, or 20 mM or more. In some embodiments, the concentration of the reducing reagent in the lysis buffer is about 5 mM. In some embodiments, the lysis buffer may 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.

[0129] Lysing can be performed at a temperature of about 4, 10, 15, 20, 25, or 30° C. Lysing can be performed for about 1, 5, 10, 15, 20 minutes, or longer. Lysed cells may contain at least about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules. Lysed cells may contain up to about 100,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, or more target nucleic acid molecules.

[0130] Binding of barcodes to target nucleic acid molecules After cell lysis and release of nucleic acid molecules therefrom, the nucleic acid molecules may randomly associate with the barcodes on the co-localized solid support. Association may involve hybridization of the target recognition region of the barcode to a complementary portion of the target nucleic acid molecule (e.g., the oligo(dT) of the barcode may interact with the poly(A) tail of the target). Assay conditions (e.g., buffer pH, ionic strength, temperature, etc.) used for hybridization may be selected to promote the formation of specific, stable hybrids. In some embodiments, nucleic acid molecules released from lysed cells may associate with multiple probes on a substrate (e.g., hybridize to the probes on a substrate). If the probes contain oligo(dT), mRNA molecules may hybridize to the probes and be reverse transcribed. The oligo(dT) portion of the oligonucleotide may act as a primer for first-strand synthesis of cDNA molecules. For example, in the non-limiting example of barcoding shown in block 216 of FIG. 2, mRNA molecules may hybridize to barcodes on beads. For example, a single-stranded nucleotide fragment may hybridize to the target binding region of the barcode.

[0131] The binding may further include ligating the target recognition region of the barcode with a portion of the target nucleic acid molecule. For example, the target binding region may include a nucleic acid sequence capable of specific hybridization to a restriction site overhang (e.g., an EcoRI sticky end overhang). The assay procedure may further include treating the target nucleic acid with a restriction enzyme (e.g., EcoRI) to generate a restriction site overhang. The barcode can then be ligated to any nucleic acid molecule that contains a sequence complementary to the restriction site overhang. A ligase (e.g., T4 DNA ligase) can be used to connect the two fragments.

[0132] For example, in a non-limiting example of barcoding shown in block 220 of Figure 2, labeled targets (e.g., target-barcode molecules) from multiple cells (or multiple samples) can then be pooled, e.g., in a tube. For example, the labeled targets can be pooled by collecting beads to which barcodes and / or target-barcode molecules are bound. Solid support-based collection recovery of bound target-barcode molecules can be achieved through the use of magnetic beads and an externally applied magnetic field. Once the target-barcode molecules are pooled, all further processing can proceed within a single reaction vessel. Further processing can include, for example, reverse transcription, amplification, cleavage, dissociation, and / or nucleic acid extension reactions. Further processing reactions can be performed within microwells, i.e., without first pooling labeled target nucleic acid molecules from multiple cells.

[0133] Reverse transcription or nucleic acid extension The present disclosure provides methods for generating target-barcode conjugates using reverse transcription (e.g., block 224 of Figure 2) or nucleic acid extension. The target-barcode conjugates can include a barcode and a complementary sequence of all or a portion of a target nucleic acid (i.e., a barcoded cDNA molecule, e.g., a stochastically 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 oligo(dT) primer can be 12-18 nucleotides in length, or about 12-18 nucleotides in length, 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. In some embodiments, reverse transcription of mRNA molecules into labeled RNA molecules can occur by the addition of a reverse transcription primer. In some embodiments, the reverse transcription primer is an oligo(dT) primer, a random hexanucleotide primer, or a target-specific oligonucleotide primer. Typically, oligo(dT) primers are 12-18 nucleotides in length and bind to the endogenous poly(A) tail at the 3' end of mammalian mRNAs. Random hexanucleotide primers can bind to mRNAs at various complementary sites. Target-specific oligonucleotide primers typically selectively prime the mRNA of interest.

[0134] 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 generate a cDNA molecule with a poly(dC) tail. The barcode can include a target binding region with a poly(dG) tail. When base pairing occurs 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 replicating to the 5' end of the barcode. In this way, the resulting cDNA molecule contains the barcode sequence (e.g., a molecular tag) on ​​the 3' end of the cDNA molecule. Reverse transcription can occur repeatedly to generate multiple labeled cDNA molecules. 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 methods 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.

[0135] amplification One or more nucleic acid amplification reactions (e.g., block 228 of FIG. 2 ) can be performed to generate multiple copies of the labeled target nucleic acid molecule. Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are 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 may 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 the plurality of nucleic acids. The method may further include performing one or more cDNA synthesis reactions to generate one or more cDNA copies of the target-barcode molecule comprising the sample label, cell label, spatial label, and / or barcode sequence (e.g., molecular label).

[0136] In some embodiments, amplification can be performed using polymerase chain reaction (PCR). As used herein, PCR can refer to a reaction for amplifying specific DNA sequences in vitro by simultaneous primer extension of 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, multiplex PCR, digital PCR, and assembly PCR.

[0137] Amplification of labeled nucleic acids may include non-PCR-based methods. Examples of non-PCR-based methods include, but are not limited to, multiplex 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-circle amplification. Other non-PCR-based amplification methods include 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), and Qβ replicase (Qβ) methods, the use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved before extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and branched extension amplification (RAM). In some embodiments, amplification does not produce circularized transcripts.

[0138] In some embodiments, the methods disclosed herein further include performing a polymerase chain reaction on the labeled nucleic acid (e.g., labeled RNA, labeled DNA, labeled cDNA) to generate labeled amplicons (e.g., stochastically labeled amplicons). The labeled amplicons may be double-stranded molecules. The double-stranded molecules may comprise double-stranded RNA molecules, double-stranded DNA molecules, or RNA molecules hybridized to DNA molecules. One or both strands of the double-stranded molecules may comprise a sample label, a spatial label, a cell label, and / or a barcode sequence (e.g., a molecular label). The labeled amplicons may be single-stranded molecules. The single-stranded molecules may comprise DNA, RNA, or a combination thereof. The nucleic acids of the present disclosure may include synthetic or modified nucleic acids.

[0139] Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0140] Performing one or more amplification reactions may include the use of one or more primers. The one or more primers may contain, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 or more nucleotides. The one or more primers may contain fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the multiple labeled targets (e.g., stochastically labeled targets). The one or more primers may anneal to the 3' or 5' ends of the multiple labeled targets. The one or more primers may anneal to an internal region of the multiple labeled targets. The internal region may 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 from the 3' end of the multiple labeled targets. The one or more primers may comprise a constant panel of primers. The one or more primers may include at least one or more custom primers, the one or more primers may include at least one or more control primers, or the one or more primers may include at least one or more gene-specific primers.

[0141] The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may 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. The one or more primers may include a universal primer and a custom primer. The custom primers may be designed to amplify one or more targets. The targets may comprise a subset of all nucleic acids in one or more samples. The targets may comprise a subset of all labeled targets in one or more samples. The one or more primers may include at least 96 or more custom primers. The one or more primers may include at least 960 or more custom primers. The one or more primers may include at least 9600 or more custom primers. The one or more custom primers may anneal to two or more different labeled nucleic acids. The two or more different labeled nucleic acids may correspond to one or more genes.

[0142] Any amplification scheme can be used in the disclosed methods. For example, in one scheme, a first PCR can amplify the molecules bound to the beads using a gene-specific primer and a primer for the sequence of universal Illumina sequencing primer 1. A second PCR can amplify the first PCR product using a nested gene-specific primer adjacent to the sequence of Illumina sequencing primer 2 and a primer for the sequence of universal Illumina sequencing primer 1. A third PCR adds P5 and P7 and a sample index to place the PCR product into an Illumina sequencing library. Sequencing using 150 bp x 2 sequencing can reveal cell label and barcode sequences (e.g., molecular labels) on read 1, genes on read 2, and a sample index on index 1 read.

[0143] In some embodiments, chemical cleavage can be used to remove nucleic acids from a substrate. For example, chemical groups or modified bases present in the nucleic acid can be used to facilitate its removal from a 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, nucleic acids containing dUTP or ddUTP can be removed from a substrate using uracil-d-glycosylase (UDG) treatment. For example, enzymes that perform nucleotide excision, such as base excision repair enzymes, for example, 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, a cleavable linker can be used to remove nucleic acids from a substrate. For example, the cleavable linker can comprise at least one of biotin / avidin, biotin / streptavidin, biotin / neutravidin, Ig-Protein A, a photolabile linker, an acid or base labile linker group, or an aptamer.

[0144] If the probe is gene-specific, the molecule can be hybridized to the probe and reverse transcribed and / or amplified. In some embodiments, the nucleic acid can be amplified after it is synthesized (e.g., reverse transcribed). Amplification can be performed in a multiplexed manner, where multiple target nucleic acid sequences are amplified simultaneously. Amplification can add sequencing adapters to the nucleic acid.

[0145] In some embodiments, amplification can be performed on the substrate using, for example, bridge amplification. Homopolymer tails can be added to cDNA to generate ends compatible with bridge amplification using oligo(dT) probes on the substrate. In bridge amplification, a primer complementary to the 3' end of the template nucleic acid can be the first primer of each pair covalently attached to solid particles. When a sample containing the template nucleic acid is contacted with the particles and a single thermal cycle is performed, the template molecule anneals to the first primer, and the first primer can be extended in the forward direction by adding nucleotides to form a double-stranded molecule consisting of the template molecule and a newly formed DNA strand complementary to the template. In the heating step of the next cycle, the double-stranded molecule can be denatured, releasing the template molecule from the particle and leaving the complementary DNA strand attached to the particle through the first primer. In the annealing stage of the subsequent annealing and extension step, the complementary strand can hybridize to a second primer complementary to the segment of the complementary strand at the position removed from the first primer. Through this hybridization, the complementary strand can form a bridge between the first and second primers, immobilized by covalent bonding to the first primer and by hybridization to the second primer. In the extension step, the second primer can be extended in the opposite direction by adding nucleotides to the same reaction mixture, thereby converting the bridge into a double-stranded bridge. The next cycle then begins, and the double-stranded bridge is denatured to yield two single-stranded nucleic acid molecules, each with one end bound to the particle surface via the first and second primers, and the other end unbound. In the annealing and extension step of this second cycle, each strand can hybridize to a previously unused additional complementary primer on the same particle to form a new single-stranded bridge. The two previously unused hybridized primers then extend, converting the two new bridges into double-stranded bridges.

[0146] 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 the plurality of nucleic acids. Amplification of the labeled nucleic acid may include PCR-based or non-PCR-based methods. Amplification of the labeled nucleic acid may include exponential amplification of the labeled nucleic acid. Amplification of the labeled nucleic acid may include linear amplification of the labeled nucleic acid. Amplification may be performed by polymerase chain reaction (PCR). PCR may refer to a reaction for in vitro amplification of specific DNA sequences by simultaneous primer extension of complementary strands of DNA. PCR may encompass 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-suppressive PCR, and assembly PCR.

[0147] Amplification of labeled nucleic acids can include non-PCR-based methods, including, but not limited to, MDA, TMA, NASBA, SDA, real-time SDA, rolling circle amplification, circle-circle amplification, multiple cycles of DNA-dependent RNA polymerase-driven RNA transcription amplification or RNA-directed DNA synthesis and transcription to amplify DNA or RNA targets, ligase chain reaction (LCR), Qβ replicase (Qβ), use of palindromic probes, strand displacement amplification, oligonucleotide-driven amplification using restriction endonucleases, amplification methods in which a primer is hybridized to a nucleic acid sequence and the resulting duplex is cleaved prior to extension and amplification, strand displacement amplification using a nucleic acid polymerase lacking 5' exonuclease activity, rolling circle amplification, and / or RAM.

[0148] In some embodiments, the methods disclosed herein further comprise performing a nested polymerase chain reaction on the amplified amplicon (e.g., target). The amplicon may be a double-stranded molecule. The double-stranded molecule may comprise 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 may comprise a sample tag or molecular identifier label. Alternatively, the amplicon may be a single-stranded molecule. The single-stranded molecule may comprise DNA, RNA, or a combination thereof. The nucleic acids of the invention may comprise synthetic or modified nucleic acids. In some embodiments, the methods include repeatedly amplifying a labeled nucleic acid to generate multiple amplicons. The methods disclosed herein may 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 methods include performing at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amplification reactions.

[0149] The amplification may further include adding one or more control nucleic acids to one or more samples containing the plurality of nucleic acids. The amplification may further include adding one or more control nucleic acids to the plurality of nucleic acids. The control nucleic acids may include a control label. Amplification may include the use of one or more non-natural nucleotides. Non-natural nucleotides may include photolabile and / or trigger nucleotides. Examples of non-natural nucleotides include, but are not limited to, peptide nucleic acids (PNAs), morpholino nucleic acids, locked nucleic acids (LNAs), glycol nucleic acids (GNAs), and threose nucleic acids (TNAs). Non-natural nucleotides may be added to one or more cycles of the amplification reaction. The addition of non-natural nucleotides may be used to identify products at specific cycles or time points of the amplification reaction.

[0150] Performing one or more amplification reactions may involve the use of one or more primers. The one or more primers may comprise one or more oligonucleotides. The one or more oligonucleotides may comprise at least about 7 to 9 nucleotides. The one or more oligonucleotides may comprise fewer than 12 to 15 nucleotides. The one or more primers may anneal to at least a portion of the plurality of labeled nucleic acids. The one or more primers may anneal to the 3' and / or 5' ends of the plurality of labeled nucleic acids. The one or more primers may anneal to an internal region of the plurality of labeled nucleic acids. 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 from the 3' end of the plurality of labeled nucleic acids. The one or more primers can comprise a fixed panel of primers. The one or more primers may include at least one or more custom primers. The one or more primers may include at least one or more control primers. The one or more primers may include at least one or more housekeeping gene primers. The one or more primers may include a universal primer. The universal primer may anneal to a universal primer binding site. The one or more custom primers may anneal to a first sample tag, a second sample tag, a molecular identifier label, a nucleic acid, or a product thereof. The one or more primers may include a universal primer and a custom primer. The custom primer may be designed to amplify one or more target nucleic acids. The target nucleic acids may comprise a subset of the total nucleic acids in one or more samples. In some embodiments, the primers are probes bound to an array of the present disclosure.

[0151] In some embodiments, barcoding (e.g., stochastically barcoding) a plurality of targets in a sample further includes generating an indexed library of barcoded targets (e.g., stochastically barcoded targets) or barcoded fragments of those targets. The barcode sequences of different barcodes (e.g., molecular labels of different stochastic barcodes) may differ from each other. Generating an indexed library of barcoded targets includes generating a plurality of indexed polynucleotides from the plurality of targets in the sample. For example, for an indexed library of barcoded targets including a first indexed target and a second indexed target, the labeled region of the first indexed polynucleotide may differ from the labeled region of the second indexed polynucleotide by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nucleotides, or a number or range between, or about, or at least, or at most, any two of these values. In some embodiments, generating an indexed library of barcoded targets includes contacting a plurality of targets, e.g., mRNA molecules, with a plurality of oligonucleotides comprising a poly(T) region and a label region, and performing first-strand synthesis using a reverse transcriptase to generate single-stranded, labeled cDNA molecules, each comprising a cDNA region and a label region, wherein the plurality of targets comprises at least two mRNA molecules of different sequences, and the plurality of oligonucleotides comprises at least two oligonucleotides of different sequences. Generating an indexed library of barcoded targets may further include amplifying the single-stranded, labeled cDNA molecules to generate double-stranded, labeled cDNA molecules, and performing nested PCR on the double-stranded, labeled cDNA molecules to generate labeled amplicons. In some embodiments, the method may include generating adapter-labeled amplicons.

[0152] Barcoding (e.g., stochastic barcoding) can include labeling individual nucleic acid (e.g., DNA or RNA) molecules with nucleic acid barcodes or tags. In some embodiments, this includes adding DNA barcodes or tags to cDNA molecules as they are generated from mRNA. Nested PCR can minimize PCR amplification bias. Adapters can be added for sequencing, e.g., using next-generation sequencing (NGS). For example, sequencing results can be used to determine the sequence of cellular labels, molecular labels, and nucleotide fragments of one or more copies of the target in block 232 of FIG. 2.

[0153] 3 is a schematic diagram illustrating a non-limiting, exemplary process for generating an indexed library of barcoded targets (e.g., stochastically barcoded targets), e.g., barcoded mRNAs or fragments thereof. As shown in step 1, a reverse transcription process can encode each mRNA molecule containing a unique molecular label sequence, a cellular label sequence, and a universal PCR site. In particular, the RNA molecule 302 can be reverse transcribed to generate labeled cDNA molecules 304 containing cDNA regions 306 by hybridization (e.g., stochastic hybridization) of a set of barcodes (e.g., stochastic barcodes) 310 to a poly(A) tail region 308 of the RNA molecule 302. Each of the barcodes 310 can include a target binding region, e.g., a poly(dT) region 312, a label region 314 (e.g., a barcode sequence or molecule), and a universal PCR region 316.

[0154] In some embodiments, the cell label sequence may comprise 3 to 20 nucleotides. In some embodiments, the molecular label sequence may comprise 3 to 20 nucleotides. In some embodiments, each of the plurality of stochastic barcodes further comprises one or more of a universal label and a cell label, wherein the universal label is the same for the plurality of stochastic barcodes on the solid support and the cell label is the same for the plurality of stochastic barcodes on the solid support. In some embodiments, the universal label may comprise 3 to 20 nucleotides. In some embodiments, the cell label comprises 3 to 20 nucleotides.

[0155] In some embodiments, label region 314 may include a barcode sequence or molecular label 318 and a cell label 320. In some embodiments, label region 314 may include one or more of a universal label, a dimensional label, and a cell label. Barcode sequence or molecular label 318 may be 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 in between any of these values, in length, or may be approximately, at least, or at most 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 in length between any of these values. A cell label 320 may be 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 in between, or may be approximately, or may be at least, or may be up to, these values ​​or such number or range of nucleotides in length. A universal label may be 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 in between, or may be approximately, or may be at least, or may be up to, these values ​​or such number or range of nucleotides in length. The universal label may be the same for multiple stochastic barcodes on a solid support, and the cell label may be the same for multiple stochastic barcodes on a solid support.A dimension marker may be 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 in between any of these values, in length, or may be approximately, at least, or at most, these values ​​or numbers or ranges of nucleotides in length.

[0156] In some embodiments, label region 314 may include 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 of these values, of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include approximately these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include at least these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320, or may include up to these values ​​or such number or range of different labels, e.g., barcode sequence or molecular label 318 and cell label 320. Each label may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or a number or range between any of these values, in length, or may be approximately, at least, or at most these values ​​or number or range of nucleotides in length. A set of barcodes or stochastic barcodes 310 may be 10, 20, 40, 50, 70, 80, 90, 100, or a number or range between any of these values. 2 , 10 3 , 10 4 , 10 5 , 10 6 , 107 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 20 , or a number or range of barcodes or stochastic barcodes 310 between any of these values, or about these values ​​or such number or range of barcodes or stochastic barcodes 310, or at least these values ​​or such number or range of barcodes or stochastic barcodes 310, or at most these values ​​or such number or range of barcodes or stochastic barcodes 310. The set of barcodes or stochastic barcodes 310 may also each contain, for example, a unique labeled region 314. The labeled cDNA molecules 304 may be purified to remove excess barcodes or stochastic barcodes 310. Purification may include Ampure bead purification.

[0157] As shown in step 2, the products from the reverse transcription process in step 1 can be pooled in one tube and PCR amplified using a first pool of PCR primers and a first universal PCR primer. Pooling is possible due to the unique label region 314. In particular, the labeled cDNA molecules 304 can be amplified to generate nested PCR-labeled amplicons 322. The amplification can include multiplex PCR amplification. The amplification can include multiplex PCR amplification using 96 multiplex primers in a single reaction volume. In some embodiments, the multiplex PCR amplification can be performed using 10, 20, 40, 50, 70, 80, 90, 10, 25, 30, 45, 50, 60, 75, 80, 90, 100, 150, 250, 300, 450, 500, 600, 750, 800, 900, 1500, 1500, 2500, 3000, 4500, 5000, 6000, 15000, 25000, 30000, 45000, 50000, 50000, 60000, 75000, 8000, 9000, 15000, 15000, 15000, 25000, 30000, 45000, 50000, 15000, 25000, 30000, 45000, 50000, 60000, 150000, 250000, 300000, 450000, 50000, 60000, 150000, 25000 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 1013 , 10 14 , 10 15 , 10 20 , or a number or range of multiplex primers between any of these values, or approximately, or at least, or up to these values. The amplification may include a first PCR primer pool 324 including custom primers 326A-C targeting specific genes and a universal primer 328. The custom primer 326 may hybridize to a region within the cDNA portion 306' of the labeled cDNA molecule 304. The universal primer 328 may hybridize to the universal PCR region 316 of the labeled cDNA molecule 304.

[0158] As shown in step 3 of Figure 3, the product from the PCR amplification in step 2 can be amplified using a nested PCR primer pool and a second universal PCR primer. Nested PCR can minimize PCR amplification bias. In particular, nested PCR-labeled amplicons 322 can be further amplified by nested PCR. Nested PCR can include multiplex PCR including a nested PCR primer pool 330 of nested PCR primers 332a-c and a second universal PCR primer 328' in a single reaction volume. The nested PCR primer pool 328 may contain 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 of these values, of different nested PCR primers 330, or may contain about, or at least, or at most, of different nested PCR primers 330. The nested PCR primers 332 may contain an adaptor 334 and hybridize to a region within the cDNA portion 306" of the labeled amplicon 322. Universal primer 328' contains adapter 336 and can hybridize to universal PCR region 316 of labeled amplicon 322. Thus, step 3 generates adapter-labeled amplicon 338. In some embodiments, nested PCR primer 332 and second universal PCR primer 328' may not contain adapters 334 and 336. Instead, adapters 334 and 336 can ligate to the product of the nested PCR to generate adapter-labeled amplicon 338.

[0159] As shown in step 4, the PCR products from step 3 can be PCR amplified for sequencing using library amplification primers. In particular, adapters 334 and 336 can be used to perform one or more additional assays on adapter-labeled amplicons 338. Adapters 334 and 336 can be hybridized with primers 340 and 342. One or more of primers 340 and 342 can be PCR amplification primers. One or more of primers 340 and 342 can be sequencing primers. One or more of adapters 334 and 336 can be used for further amplification of adapter-labeled amplicons 338. One or more of adapters 334 and 336 can be used for sequencing of adapter-labeled amplicons 338. Primer 342 can contain a plate index 344, which allows amplicons generated using the same set of barcodes or stochastic barcodes 310 to be sequenced in a single sequencing reaction using next-generation sequencing (NGS).

[0160] Chambered single cell barcoding High-throughput screening can be performed by using a sample multiplexing kit, where antibodies containing sample barcoded oligos can be used to stain various cell populations. However, in some embodiments, such additional staining steps are not preferred due to the potential for altering biological responses. Provided herein are compositions, systems, and methods for chambered Rhapsody with unique barcodes (e.g., chamber-indexing subsequences). Compositions and methods are provided for sorting into barcoded chambers on a Rhapsody cartridge for single-cell workflows. To correlate image- or fluorescence-based sorting with single-cell data, it can be important to have a means of sorting cells into chambers such that information about which populations of cells (based on gating) are sorted into which chambers is preserved. Provided herein are compositions and methods including a chambered Rhapsody cartridge, where each chamber can be loaded with beads containing a unique set of cell labels.

[0161] In some embodiments, schemes for manufacturing solid supports (e.g., beads) are provided. In some embodiments, x unique sets of cell labels are provided, with each set containing y unique cell labels. For example, a 384-well chamber cartridge may contain 384 unique sets of cell labels. There may be thousands of microwells within each of these chambers. A cell sorter can sort various cell populations directly into these chambers (e.g., to be sorted into a 384-well plate). In some embodiments, workflows for maintaining cell viability within the cartridge and the remaining single-cell workflows provided herein (e.g., Rhapsody) are provided. Some embodiments of the compositions and methods provided herein include automated liquid handling. Figures 4A-4B show non-limiting, exemplary schematic diagrams of microwell arrays (Figure 4A) and chambers (Figure 4B) provided herein. Figure 4A shows a non-limiting, exemplary schematic diagram of a microwell array 400 including multiple chambers 402. Each of the chambers may include multiple compartments 404 (e.g., microwells).

[0162] In some embodiments, methods and compositions are provided that enable differentiation of library samples after sequencing by cellular barcodes, thereby simplifying user workflow. The disclosure herein includes methods and compositions that use cellular barcodes for sample and lane identification. In some embodiments, the methods include assigning a specific cellular barcode (e.g., including a chamber-specific chamber-indexing subsequence) to each chamber of a multi-chamber cartridge (e.g., an HT Xpress System 8-lane cartridge) while maintaining a high degree of cellular barcode diversity, for example, by manufacturing eight different bead lots. By assigning specific cellular labels to each lane (e.g., chamber), users can identify library samples when performing multi-lane experiments without having to assign specific forward and reverse primers to each lane and library type. This allows users to achieve an efficient workflow for preparing libraries for sequencing.

[0163] Disclosed herein are multiple solid supports (e.g., eight different barcoded beads per HT lane) containing chamber-specific chamber-indexing subsequences, allowing users to distinguish between chambers (e.g., lanes) without the need to use indexed library primers (e.g., eight different reverse primers). Current methods require users to distinguish between library samples using different forward and reverse primers. The methods and compositions provided herein can eliminate the need to use multiple different forward and reverse primers by allowing users to distinguish based on cell barcodes.

[0164] In some embodiments, the cell label diversity is 384*384*384=56,623,104 different cell barcode combinations when there are 384 cell label 1 (first cell labeling moiety; CL1), 384 cell label 2 (second cell labeling moiety; CL2), and 384 cell label 3 (third cell labeling moiety; CL3). It has been simulated that a maximum of 192*192*192=7,077,888 different cell barcode combinations are required to maintain less than 1% cell barcode overlap. In some embodiments, the methods and compositions provided herein use the same 384 cell labeling moieties across three different oligos, but distributed so that each bead lot has 7,077,888 different cell barcodes. The methods provided herein can generate eight different bead lots with specific cell barcodes. A user can assign a specific bead lot to each lane in advance. For example, for eight lanes, a user can use eight different bead lots. Currently, users assign different reverse primers to distinguish between lanes. For example, if a user were to perform eight lanes of Whole Transcriptome Analysis (WTA) using eight different samples, instead of tracking eight different reverse primers, they would ultimately only need to use one reverse primer to bioinformatically separate the samples, as long as they input beads into each lane using the provided compositions and methods. For example, Table 1 shows a current method for distinguishing between lanes of origin using library indexes on primers (for targeted library amplification or WTA library amplification).

[0165] [Table 1]

[0166] Furthermore, as the complexity of library combinations increases, the number of forward and reverse indexes to track using current methods also increases. Thus, for example, with only eight lanes of WTA, eight library indexes are used, while with eight lanes of WTA+AbSeq, the library index increases to 16, and with eight lanes of WTA+AbSeq+ST (e.g., sample tagging), the library index increases to 24. In contrast, Table 2 shows an exemplary setup using the compositions and methods provided herein, where each lane is assigned a different cell barcode, and users only need one forward / reverse primer to track each library type.

[0167] [Table 2]

[0168] Provided herein are methods and compositions for fabricating and using oligonucleotide barcodes (e.g., solid supports comprising chamber-specific chamber-indexing subsequences) comprising the chamber-indexing subsequences provided herein. Figure 5 shows a non-limiting, exemplary method for producing oligonucleotide barcodes comprising the chamber-indexing subsequences provided herein. Each block represents two 96-well plates (192 CLs total). The fabrication of eight solid supports (which users can use to distinguish eight lanes / chambers) is shown. In this method, (192 CL1s) * (192 CL2s) * (192 CL3s) can be used to generate 7,077,888 different combinations. The letters A, B, C, and D represent different populations of oligonucleotides with different cell-labeling subsequences. Figure 6 shows another non-limiting, exemplary method for producing oligonucleotide barcodes comprising the chamber-indexing subsequences provided herein. For example, a user can combine 384 CL1s, 384 CL2s, and 48 different CL3s for each lane. This method can simplify bead production and maintain a cell barcode diversity of 7,077,888 (384*384*48). This production method can be similar to the current process, but only half of the plate is pooled in the Oligo 3 step. Figure 7 shows another non-limiting exemplary method for producing oligonucleotide barcodes containing chamber-indexing subsequences provided herein. In this production method, bulk ligation is added at the end of the current process to add a differentiating cell label in Oligo 4. The cell barcode diversity can be 384*384*384*1 = approximately 56 million. In some embodiments, Oligo 4 is configured to mitigate increased sequencing read length, which in some embodiments may reduce library diversity in R1. The sequence of the cell labeling moiety can be the same among the first, second, and third oligonucleotides.For example, in some embodiments, the first oligonucleotide, the second oligonucleotide, and / or the third oligonucleotide may each have a first cell-labeling moiety, a second cell-labeling moiety, or a third cell-labeling moiety that includes the sequence AAAGG.

[0169] Provided herein are two or more types of multiple solid supports (e.g., a first solid support, a second solid support, a third solid support, etc.), each of which has a unique chamber-specific chamber-indexing subsequence. Each of the multiple solid supports can be introduced into a separate 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 chamber-specific chamber-indexing subsequence unique to each of the multiple solid supports, it can be determined from a sequencing read whether it is, for example, a product of an oligonucleotide barcode associated with the first solid support or the second solid support (thereby determining whether it originates from lane 1 or lane 2). For example, a first solid support of the multiple first solid supports can have a first chamber-indexing subsequence that allows a user to uniquely identify a sequencing read originating from the first solid support. In some embodiments, the first chamber-indexing subsequence is a single subsequence of a cell label. For example, referring to FIG. 7, in some embodiments, the first chamber-indexing subsequence is a single cell-labeling moiety (such as the fourth cell-labeling moiety shown in FIG. 7), and the unique subsequence is the same across all first solid supports (e.g., not selected from the set of chamber-indexing subsequences). In this case, only the fourth cell-labeling moiety of the cell label is used to distinguish between solid supports (and thereby chambers). In some embodiments, as shown in FIG. 6, a single subsequence of the cell label is used to distinguish between solid supports (and thereby chambers), but the chamber-indexing subsequence is selected from the set of chamber-indexing subsequences. In the case of oligonucleotide barcodes derived from the method of FIG. 6, the chamber-indexing subsequence can be the third cell-labeling moiety. For example, if 48 different oligos (e.g., the first half of population A of third oligonucleotides, including CL3) are used to generate the first solid supports, the first chamber-indexing subsequence will have one of the sequences of the set of 48 chamber-indexing subsequences.The second solid support may have CL3 derived from the latter half of population A of the third oligonucleotides. Thus, the second solid support may have a second chamber-indexing subsequence having one of the sequences of another non-overlapping set of 48 chamber-indexing subsequences (the latter half of population A of the third oligonucleotides). Because the third cell-labeling moieties of the first solid support and the second solid support are derived from non-overlapping subsets of the third cell-labeling subsequences, the user can determine whether the sequencing read is derived from the first solid support or the second solid support based on the sequence of the third labeling moiety. Thus, the chamber-indexing subsequence is predetermined because the user knows the set of possible sequences of chamber-indexing subsequences for both the first solid support and the second solid support.

[0170] In some embodiments, a given chamber-indexing subsequence can include multiple (or all) components of a cell label. For example, using the process shown in Figure 5, eight multiple solid supports can be generated (to distinguish different chambers / lanes). Each multiple solid support can be derived from a different combination of a first population of oligonucleotides, a second population of oligonucleotides, and a third population of oligonucleotides. For example, the first solid support (for use in chamber / lane 1) can be derived from a first population of first oligonucleotides, a first population of second oligonucleotides, and a first population of third oligonucleotides, while the second solid support (for use in chamber / lane 2) can be derived from a second population of first oligonucleotides, a second population of second oligonucleotides, and a second population of third oligonucleotides. In this case, the first and second populations of first oligonucleotides are the same (A / B), the first and second populations of second oligonucleotides are the same (A / B), and the first and second populations of third oligonucleotides are different (A / B vs. C / D). Because the CL3 sequences of the first and second populations of third oligonucleotides differ from each other, the first and second solid supports can be distinguished from each other based on the third cell-labeling moiety (chamber-indexing subsequence). All eight lanes differ from each other with respect to at least one of Oligo 1, Oligo 2, or Oligo 3, and thus can be distinguished based on one, two, or three chamber-indexing subsequences.

[0171] The composition disclosed herein can comprise two or more types of solid supports.Each solid support can comprise a plurality of oligonucleotide barcodes, each comprising a cell labeling sequence, and each cell labeling sequence comprises a predetermined chamber indexing subsequence.In some embodiments, the oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences.

[0172] In some embodiments, the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports, wherein the oligonucleotide barcodes associated with the plurality of first solid supports have a first predetermined chamber-indexing subsequence and the oligonucleotide barcodes associated with the plurality of second solid supports have a second predetermined chamber-indexing subsequence, and wherein the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different.

[0173] In some embodiments, the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports, wherein the first predetermined chamber-indexing subsequences of the plurality of first solid supports are selected from a first set of chamber-indexing subsequences, and the second predetermined chamber-indexing subsequences of the plurality of second solid supports are selected from a second set of chamber-indexing subsequences, and each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from the chamber-indexing subsequences of the second set of chamber-indexing subsequences. In some embodiments, a user can determine whether an oligonucleotide barcode is associated with a plurality of first solid supports or a plurality of second solid supports based on the predetermined chamber-indexing subsequences of sequencing reads derived from the oligonucleotide barcode or its product.

[0174] The two or more plurality of solid supports may include a plurality of third solid supports, where (a) the oligonucleotide barcodes associated with the plurality of third solid supports may have a third chamber-indexing subsequence, and / or (b) the third chamber-indexing subsequence of the plurality of third solid supports may be selected from a third set of chamber-indexing subsequences; a plurality of fourth solid supports, where (a) the oligonucleotide barcodes associated with the plurality of fourth solid supports may have a fourth chamber-indexing subsequence, and / or (b) the fourth chamber-indexing subsequence of the plurality of fourth solid supports may be selected from a fourth set of chamber-indexing subsequences; and a plurality of fifth solid supports, where (a) the oligonucleotide barcodes associated with the plurality of fifth solid supports may have a fifth chamber-indexing subsequence. a plurality of fifth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of sixth solid supports may have a sixth chamber-indexing subsequence, and / or (b) the sixth chamber-indexing subsequence of the plurality of sixth solid supports may be selected from the sixth set of chamber-indexing subsequences; a plurality of sixth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of seventh solid supports may have a seventh chamber-indexing subsequence, and / or (b) the seventh chamber-indexing subsequence of the plurality of seventh solid supports may be selected from the seventh set of chamber-indexing subsequences;and / or a plurality of eighth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of eighth solid supports may have an eighth chamber-indexing subsequence, and / or (b) the eighth chamber-indexing subsequence of the plurality of eighth solid supports may be selected from an eighth set of chamber-indexing subsequences. In some embodiments, the first chamber-indexing subsequence, the second chamber-indexing subsequence, the third chamber-indexing subsequence, the fourth chamber-indexing subsequence, the fifth chamber-indexing subsequence, the sixth chamber-indexing subsequence, the seventh chamber-indexing subsequence, and / or the eighth chamber-indexing subsequence do not share sequence with each other. In some embodiments, a user can determine whether an 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 based on a predetermined chamber-indexed subsequence of a sequencing read derived from the oligonucleotide barcode or its product;

[0175] In some embodiments, the cell label comprises multiple cell-labeling moieties and one or more linkers. In some embodiments, the cell label comprises a first cell-labeling moiety, a first linker, and a second cell-labeling moiety; the cell label may comprise a second linker and a third cell-labeling moiety; and the cell label may further comprise a third linker and a fourth cell-labeling moiety. In some embodiments, the predetermined chamber-indexing moiety sequence comprises a first cell-labeling moiety, a second cell-labeling moiety, a third cell-labeling moiety, a fourth cell-labeling moiety, or any combination thereof. In some embodiments, the first, second, third, fourth, fifth, sixth, seventh, and / or eighth sets of chamber-indexing subsequences comprise a set of about 960, about 864, about 768, about 672, about 576, about 480, about 384, about 288, about 192, about 96, or less than about 48 unique sequences that are distinct from the chamber-indexing subsequences of the other sets of chamber-indexing subsequences. In some embodiments, the first cell-labeling moiety, the second cell-labeling moiety, the third cell-labeling moiety, the fourth cell-labeling moiety, or any combination thereof, is selected from a set of about 480, about 384, about 288, about 192, about 96, or less than about 48 unique sequences. In some embodiments, a user can determine whether an 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 based on the sequence of the first cell labeling portion, the second cell labeling portion, the third cell labeling portion, the fourth cell labeling portion, or any combination thereof, of a sequencing read derived from the oligonucleotide barcode or a product thereof.

[0176] The disclosure herein includes a method. In some embodiments, the method includes the steps of: allocating a plurality of first solid supports of a precursor type and a first population of first oligonucleotides to a first plurality of first compartments, wherein the solid supports and the first oligonucleotides are associated with co-localized solid supports; allocating a plurality of second solid supports of a precursor type and a second population of first oligonucleotides to a first plurality of second compartments, wherein the solid supports and the first oligonucleotides are associated with co-localized solid supports; pooling the plurality of first solid supports of a precursor type associated with the first oligonucleotides; pooling the plurality of second solid supports of a precursor type associated with the first oligonucleotides; allocating the plurality of first solid supports of a precursor type associated with the first oligonucleotides and the first population of second oligonucleotides to a second plurality of first compartments, wherein the solid supports and the second oligonucleotides are associated with co-localized solid supports; and allocating the plurality of first solid supports of a precursor type associated with the first oligonucleotides to a second plurality of first compartments, wherein the solid supports and the second oligonucleotides are associated with co-localized solid supports. The method includes the steps of: allocating a second solid support of the precursor type and a second population of the second oligonucleotides into a second plurality of second compartments, wherein the co-localized solid supports and second oligonucleotides are associated; pooling the first solid supports of the precursor type associated with the first and second oligonucleotides; pooling the second solid supports of the precursor type associated with the first and second oligonucleotides; allocating the first solid supports of the precursor type associated with the first and second oligonucleotides and the first population of the third oligonucleotides into a third plurality of first compartments, wherein the co-localized solid supports and third oligonucleotides are associated; and allocating the second solid supports of the precursor type associated with the first and second oligonucleotides and the second population of the third oligonucleotides into a third plurality of second compartments, wherein the co-localized solid supports and third oligonucleotides are associated.In some embodiments, the method includes pooling a plurality of precursor first solid supports associated with the first, second, and third oligonucleotides to generate a plurality of first solid supports; and pooling a plurality of precursor second solid supports associated with the first, second, and third oligonucleotides to generate a plurality of second solid supports.

[0177] In some embodiments, (i) the first and second population of first oligonucleotides are the same, the first and second population of second oligonucleotides are the same, and the first and second population of third oligonucleotides are different; (ii) the first and second population of first oligonucleotides are the same, the first and second population of second oligonucleotides are different, and the first and second population of third oligonucleotides are the same; (iii) the first and second population of first oligonucleotides are the same, the first and second population of second oligonucleotides are different, and the first and second population of third oligonucleotides are different; or (iv) the first and second population of first oligonucleotides are different, and the second oligonucleotides are different. (v) the first and second populations of the first oligonucleotides are different, the first and second populations of the second oligonucleotides are different, and the first and second populations of the third oligonucleotides are the same; (vi) the first and second populations of the first oligonucleotides are different, the first and second populations of the second oligonucleotides are the same, and the first and second populations of the third oligonucleotides are different; or (vii) the first and second populations of the first oligonucleotides are different, the first and second populations of the second oligonucleotides are the same, and the first and second populations of the third oligonucleotides are the same.

[0178] In some embodiments, the first and second populations of first oligonucleotides each comprise about 192 first cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of second oligonucleotides each comprise about 192 second cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of third oligonucleotides each comprise about 192 third cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of first oligonucleotides are the same; the first and second populations of second oligonucleotides are the same; and the first and second populations of third oligonucleotides are different. In some embodiments, the first and second populations of third oligonucleotides comprise non-overlapping subsets of cell-labeling subsequences of the first and second populations of first oligonucleotides and / or the first and second populations of second oligonucleotides.

[0179] In some embodiments, the first and second populations of first oligonucleotides each comprise about 384 first cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of second oligonucleotides each comprise about 384 second cell-labeling moieties having distinct sequences. In some embodiments, the first and second populations of third oligonucleotides each comprise about 48 third cell-labeling moieties having distinct sequences.

[0180] In some embodiments, the method includes the steps of: allocating a plurality of precursor first solid supports associated with the first, second, and third oligonucleotides and the fourth oligonucleotide into a fourth plurality of first compartments, wherein the solid supports and the fourth oligonucleotide are associated co-localized; allocating a plurality of precursor second solid supports associated with the first, second, and third oligonucleotides and the fifth oligonucleotide into a fourth plurality of second compartments, wherein the solid supports and the fifth oligonucleotide are associated co-localized; pooling the plurality of precursor first solid supports associated with the first, second, third, and fourth oligonucleotides to generate a plurality of first solid supports; and pooling the plurality of precursor second solid supports associated with the first, second, third, and fifth oligonucleotides to generate a plurality of second solid supports.

[0181] In some embodiments, the first and second populations of first oligonucleotides are the same; the first and second populations of second oligonucleotides are the same; the first and second populations of third oligonucleotides are the same; and the fifth and sixth oligonucleotides are different.

[0182] In some embodiments, the first oligonucleotide comprises a first cell-labeling moiety and a first linker or its complement. In some embodiments, the second oligonucleotide comprises a first linker, a second cell-labeling moiety, and a second linker or its complement. In some embodiments, the third oligonucleotide comprises a second linker and a third cell-labeling moiety or its complement, and the second oligonucleotide may further comprise a third linker or its complement. In some embodiments, the fourth oligonucleotide comprises a third linker and a fourth cell-labeling moiety or its complement.

[0183] In some embodiments, the first oligonucleotide and the second oligonucleotide are configured to be connected via a first linker. In some embodiments, the second oligonucleotide and the third oligonucleotide are configured to be connected via a second linker. In some embodiments, the third oligonucleotide and the fourth oligonucleotide are configured to be connected via a third linker. In some embodiments, the plurality of first solid supports and / or the plurality of second solid supports comprise at least about 1,000, about 10,000, about 100,000, about 1,000,000, about 7,000,000, about 10,000,000, or about 56,000,000 unique cell-labeling sequences. In some embodiments, the sequences of the first second cell-labeling moiety, the second cell-labeling moiety, and / or the third cell-labeling moiety are the same. In some embodiments, the sequences of the first second cell-labeling moiety, the second cell-labeling moiety, and / or the third cell-labeling moiety 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 a plurality of oligonucleotide barcodes, each comprising a cell-labeling sequence, and each cell-labeling sequence comprises a predetermined chamber-indexing subsequence.

[0184] In some embodiments, the oligonucleotide barcodes associated with a plurality of first solid supports have a first predetermined chamber-indexing subsequence and the oligonucleotide barcodes associated with a plurality of second solid supports have a second predetermined chamber-indexing subsequence, and the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different. In some embodiments, the first predetermined chamber-indexing subsequences of the plurality of first solid supports are selected from a first set of chamber-indexing subsequences, and the second predetermined chamber-indexing subsequences of the plurality of second solid supports are selected from a second set of chamber-indexing subsequences, and each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from the chamber-indexing subsequences of the second set of chamber-indexing subsequences. The methods provided herein can produce the compositions provided herein.

[0185] In some embodiments, the method includes providing a microwell array including a plurality of chambers, each chamber including a plurality of compartments; and distributing a plurality of first solid supports and a plurality of second solid supports into compartments of specific first chambers and specific second chambers of the plurality of chambers, respectively. In some embodiments, distributing the oligonucleotides into the plurality of compartments includes providing a plurality of compartments containing the oligonucleotides. In some embodiments, the plurality of compartments comprises a 384-well plate, a 288-well plate, a 192-well plate, a 96-well plate, or a 48-well plate.

[0186] In some embodiments, the solid support comprises a synthetic particle. In some embodiments, at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes is immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, partially encapsulated within a synthetic particle, or a combination thereof. In some embodiments, the synthetic particle is disintegrable. In some embodiments, the synthetic particle comprises a bead. In some embodiments, the bead comprises 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 material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof, hi some embodiments, the synthetic particles comprise disintegrable hydrogel particles.

[0187] In some embodiments, a method for assigning sequencing data to chambers is provided. In some embodiments, the method includes the steps of: preparing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more types of a plurality of solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support comprising the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports comprising different cell labeling sequences, the oligonucleotide barcodes located in the same chamber comprising the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers comprising different chamber-indexing subsequences; and assigning each of two or more populations of single cells to a specific chamber among the plurality of chambers. The method includes the steps of: distributing the single cells into chamber compartments, each of two or more populations of single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying a chamber indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber indexing subsequence of each cell labeling sequence in the sequencing data.

[0188] In some embodiments, a method for assigning sequencing data to chambers is provided. In some embodiments, the method includes the steps of: preparing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; each chamber comprising a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, the solid supports each comprising a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support comprising the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports comprising different cell labeling sequences, the oligonucleotide barcodes located in the same chamber comprising the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers comprising different chamber-indexing subsequences; and assigning each of two or more populations of single cells to the plurality of chambers. the single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to barcode copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells to a specific chamber compartment among the plurality of single cells, wherein each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of a different population being located in a different chamber; the single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; the single cells of at least one population of single cells comprising a plurality ...

[0189] The present disclosure includes a method for assigning sequencing data to populations of single cells. In some embodiments, the method includes the steps of: preparing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more solid supports to a compartment of a specific chamber among the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell-labeling sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell-labeling sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; and distributing each of two or more populations of single cells to a compartment of a specific chamber among the plurality of chambers, wherein each of the two or more populations of single cells is assigned to a compartment of a specific chamber among the plurality of chambers. the plurality of single cells comprises a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a population among two or more populations of single cells based on the chamber assigned to the sequencing read.

[0190] In some embodiments, a method for assigning sequencing data to populations of single cells is provided. In some embodiments, the method includes the steps of: preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, each solid support including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber-indexing subsequences; and distributing each of two or more populations of single cells to a specific chamber compartment of the plurality of chambers, wherein each of the two or more populations of single cells is the single cells comprising a plurality of single cells, the single cells comprising copies of a nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target from at least one of the plurality of single cells of at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexing subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on the chamber-indexing subsequence of each cell labeling sequence in the sequencing data; and assigning each of the plurality of sequencing reads to a population among two or more populations of single cells based on the chamber assigned to the sequencing read.

[0191] Some embodiments provide a method for associating sequencing data and phenotypic data of populations of single cells. In some embodiments, the method includes the steps of: obtaining phenotypic data for each of two or more populations of single cells; preparing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more solid supports to a compartment of a particular chamber among the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber-indexing subsequence, wherein the oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and the oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences, wherein the oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; and obtaining phenotypic data for each of the two or more populations of single cells. the method includes the steps of: distributing each of the single cells to a specific chamber compartment among a plurality of chambers, wherein each of two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying a chamber-indexed subsequence of each cell label sequence in the sequencing data; and associating the sequencing data of the at least one population of single cells with phenotypic data based on the chamber-indexed subsequence of each cell label sequence in the sequencing data.

[0192] In some embodiments, a method for associating sequencing data and phenotypic data of populations of single cells is provided. In some embodiments, the method includes the steps of: obtaining phenotypic data for each of two or more populations of single cells; preparing a microwell array including a plurality of chambers, each chamber including a plurality of compartments, each chamber including a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chambers, each solid support including a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes located in the same chamber including the same chamber indexing subsequence, and the oligonucleotide barcodes located in different chambers including different chamber indexing subsequences; and obtaining phenotypic data for each of the two or more populations of single cells. The method includes the steps of: distributing each of the single cells to a specific chamber compartment among a plurality of chambers, wherein each of two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the nucleic acid target, the single cells of the same population being located in the same chamber and the single cells of different populations being located in different chambers; barcoding copies of the nucleic acid target derived from at least one of the plurality of single cells of the at least one population of single cells using a plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of the plurality of barcoded nucleic acid targets or their products; identifying chamber index subsequences of each cell label sequence in the sequencing data; and associating the sequencing data of the at least one population of single cells with phenotypic data based on the chamber index subsequences of each cell label sequence in the sequencing data.

[0193] The method may include assigning each of a plurality of sequencing reads to a population of two or more populations of single cells based on a chamber assigned to the sequencing read. The method may include obtaining phenotypic data for each of the two or more populations of single cells; and associating the sequencing data and the phenotypic data for at least one population of single cells based on a chamber-indexed subsequence of each cell label sequence in the sequencing data.

[0194] In some embodiments, distributing each of the two or more types of solid supports into compartments of specific chambers among the plurality of chambers can include distributing a predetermined plurality of solid supports selected from the two or more types of solid supports into specific chambers among the plurality of chambers. In some embodiments, barcoding copies of the nucleic acid targets includes contacting a plurality of oligonucleotide barcodes with copies of the nucleic acid targets for hybridization; and extending the plurality of oligonucleotide barcodes hybridized to the copies of the nucleic acid targets to generate a plurality of barcoded nucleic acid targets.

[0195] The section is approximately 1,000 μm 3 ~Approx. 786,000μm 3The microwells may have volumes ranging from 1 to 100 nucleotides. Each of the oligonucleotide barcodes may comprise a molecular label sequence. In some embodiments, oligonucleotide barcodes on the same plurality of solid supports may comprise the same chamber-indexing subsequence, and oligonucleotide barcodes on different plurality of solid supports may comprise different chamber-indexing subsequences. Each cell label of the plurality of oligonucleotide barcodes may comprise at least six nucleotides. The cell label may comprise multiple moieties and one or more linkers. The cell label may comprise a first cell-labeling moiety, a first linker, and a second cell-labeling moiety; the cell label may comprise a second linker and a third cell-labeling moiety; and the cell label may further comprise a third linker and a fourth cell-labeling moiety. The first cell-labeling moiety may comprise a chamber-indexing subsequence. In some embodiments, the chamber-indexing subsequence may be 2 to 15 nucleotides in length. The two or more plurality of solid supports may comprise from about 2 to about 100 different solid supports. Oligonucleotide barcodes of the same plurality of solid supports may contain the same chamber-indexing subsequence, and oligonucleotide barcodes of different plurality of solid supports may contain different chamber-indexing subsequences.

[0196] The chamber-indexing subsequences can be selected from a diverse set of chamber-indexing subsequences. The diverse set of chamber-indexing 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-indexing subsequences. In some embodiments, the set of chamber-indexing subsequences is designed to have minimal sequence homology to the DNA or RNA sequences of the sample being analyzed. In some embodiments, the sequences of a set of chamber-indexing subsequences differ from each other or 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 nucleotides. In some embodiments, the sequences of a set of chamber-indexing subsequences differ from each other or their complements by at least 3%, at least 5%, at least 8%, at least 10%, at least 15%, at least 20%, or more.

[0197] The populations of single cells can be separate samples. Each population of the two or more populations of single cells can be a biological replicate sample, a technical replicate sample, a control sample, an experimental sample, or a combination thereof. The two or more populations of single cells can be derived from one or more samples separated based on phenotypic data, and the plurality of single cells can include T cells, B cells, tumor cells, bone marrow cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof. The plurality of chambers 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 1000 chambers. The plurality of compartments can be at least about 100 compartments, about 500 compartments, about 1000 compartments, about 5000 compartments, about 10,000 compartments, about 25,000 compartments, about 50,000 compartments, about 75,000 compartments, or about 100,000 compartments.

[0198] In some embodiments, sequencing of the cell label identifies the chamber of origin of each sequenced barcoded nucleic acid target or its product within the microwell array. A compartment of the plurality of compartments may include a single cell of the plurality of single cells and a single solid support of the plurality of solid supports. The plurality of barcoded nucleic acid targets may each include a sequence complementary to at least a portion of the nucleic acid target and the molecular label. Each of the plurality of sequencing reads may include (1) a cell label sequence and (2) a molecular label sequence. Each oligonucleotide barcode may comprise a first universal sequence. The oligonucleotide barcode may comprise a target binding region comprising a capture sequence. The target binding region may comprise a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. In some embodiments, distributing a plurality of solid supports into respective compartments of a plurality of chambers may comprise distributing each of the plurality of solid supports into a specific chamber of a microwell array by flow cytometry. In some embodiments, distributing each of two or more populations of single cells into compartments of specific chambers of the plurality of chambers may comprise distributing each population of single cells into a specific chamber of the microwell array by flow cytometry. The method may include aligning a sorting component of a flow cytometer with the microwell array.

[0199] The phenotypic data may include event data. The event data may include quantitative biological event data derived from the sorting device. The event data may include side scatter signals, forward scatter signals, one or more fluorescent signals, or any combination thereof. The method may include a correlation analysis of the phenotypic data and sequencing data of the single cells. In some embodiments, the correlation analysis identifies one or more of the following: a candidate biomarker, a candidate therapeutic agent, a candidate dose of the therapeutic agent, and / or a cellular target of the candidate therapeutic agent. The method may include lysing one or more single cells. The viability of the single cells may be maintained for a period of time after partitioning and before lysis, which may be at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 100 minutes, 250 minutes, 500 minutes, 750 minutes, 1000 minutes, 2500 minutes, 5000 minutes, 7500 minutes, or 10000 minutes.

[0200] The solid support may include synthetic particles. The solid support may include a planar surface. At least one of the plurality of oligonucleotide barcodes may be immobilized on the synthetic particles, partially immobilized on the synthetic particles, encapsulated within the synthetic particles, or partially encapsulated within the synthetic particles. The synthetic particles may be disintegrable. 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 material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof. The synthetic particles may comprise disintegrable hydrogel particles.

[0201] Methods for determining the copy number of a nucleic acid target The methods provided herein may include determining the copy number of a nucleic acid target in one or more of a plurality of single cells of at least one population of single cells. Determining the copy number of a nucleic acid target in one or more of the plurality of single cells may include determining the copy number of the nucleic acid target in the plurality of single cells based on the number of molecular labels having distinct sequences, their complements, or combinations thereof associated with the plurality of barcoded nucleic acid targets or products thereof. The method may include contacting random primers with the plurality of barcoded nucleic acid targets, each of the random primers comprising a second universal sequence or its complement, and extending the random primers hybridized to the plurality of barcoded nucleic acid targets to generate a plurality of extension products. The method may include amplifying the plurality of extension products 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 generating a first plurality of barcoded amplicons. Amplifying the plurality of extension products may include adding sequences of sequencing primer and / or sequencing adapter binding sites, their complementary sequences, and / or portions thereof to the plurality of extension products. The method may include determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the first plurality of barcoded amplicons or products thereof. In some embodiments, determining the copy number of the nucleic acid target in one or more of the plurality of single cells may include determining the number of each of the plurality of nucleic acid targets in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with barcoded amplicons of the first plurality of barcoded amplicons that include the respective sequences of the plurality of nucleic acid targets. The respective sequences of the plurality of nucleic acid targets may include respective subsequences of the plurality of nucleic acid targets.The sequence of the nucleic acid target within the first plurality of barcoded amplicons may comprise a subsequence of the nucleic acid target. The method may include amplifying the first plurality of barcoded amplicons 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 generating a second plurality of barcoded amplicons. Amplifying the first plurality of barcoded amplicons may include adding sequences of sequencing primers and / or sequencing adapter binding sites, their complementary sequences, and / or portions thereof to the first plurality of barcoded amplicons. The method may include determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the second plurality of barcoded amplicons or products thereof. The first plurality of barcoded amplicons and / or the second plurality of barcoded amplicons may comprise whole transcriptome amplification (WTA) products.

[0202] The method may include synthesizing a third plurality of barcoded amplicons using the plurality of barcoded nucleic acid targets as templates to generate a third plurality of barcoded amplicons. Synthesizing the third plurality of barcoded amplicons may include performing polymerase chain reaction (PCR) amplification of the plurality of barcoded nucleic acid targets. Synthesizing the third plurality of barcoded amplicons may include PCR amplification using a primer capable of hybridizing to a first universal sequence or its complement and a target-specific primer. The method may include obtaining sequence data of the third plurality of barcoded amplicons or their products, and obtaining sequence information may include attaching sequencing adapters to the third plurality of barcoded amplicons or their products. The method may include determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the third plurality of barcoded amplicons or their products.

[0203] The nucleic acid target may comprise a nucleic acid molecule. The nucleic acid molecule may comprise 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 may comprise a sample indexing oligonucleotide, which may comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences. The nucleic acid target may comprise a cellular component binding reagent-specific oligonucleotide. The cellular component binding reagent-specific oligonucleotide may comprise a unique identifier sequence for the cellular 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 cellular component binding reagent-specific oligonucleotide, a sample indexing oligonucleotide). Some embodiments disclosed herein provide a plurality of compositions, each comprising a cellular component-binding reagent (e.g., a protein-binding reagent) conjugated to an oligonucleotide, the plurality of compositions comprising a unique identifier for the cellular component-binding reagent to which the oligonucleotide is conjugated. Cellular component-binding reagents (e.g., barcoded antibodies) and their uses (e.g., cellular sample indexing) are described in U.S. Patent Application Publication Nos. 2018 / 0088112 and 2018 / 0346970, the contents of each of which are incorporated herein by reference in their entireties.

[0204] Extending the plurality of oligonucleotide barcodes may include extending the plurality of oligonucleotide barcodes 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 may include Klenow fragment. The reverse transcriptase may include a viral reverse transcriptase, which may be murine leukemia virus (MLV) reverse transcriptase or Moloney murine leukemia virus (MMLV) reverse transcriptase. The first universal sequence and the second universal sequence may be the same. The first universal sequence and the second universal sequence may be different. The first universal sequence and / or the second universal sequence may include a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof. The sequencing adapter may include a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof. The sequencing primer may include a lead 1 sequencing primer, a lead 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof. At least 10 of the plurality of oligonucleotide barcodes may include different molecular label sequences. Each molecular label of the plurality of oligonucleotide barcodes may include at least 6 nucleotides.

[0205] particle analysis equipment Phenotypic data may include data derived from a sorting device (e.g., a flow cytometer). Phenotypic data may include event data. Event data may include quantitative biological event data derived from a sorting device. Event data may include side scatter signals, forward scatter signals, one or more fluorescent signals, or any combination thereof. As used herein, the terms "event" and "event data" shall be given their ordinary meanings and shall be used interchangeably, and shall also refer to data measured from a single particle, such as a cell or a synthetic particle. Typically, data measured from a single particle includes numerous parameters, including one or more light scatter parameters and at least one fluorescence intensity parameter. Thus, each event is represented as a vector of parameter measurements, with each measured parameter representing one dimension of the data space. In some biological applications, event data may represent quantitative biological data indicative of the expression of a particular protein or gene. The method may include correlation analysis of single-cell phenotypic data and sequencing data. The correlation analysis may identify one or more of the following: candidate biomarkers, candidate therapeutic agents, candidate doses of the therapeutic agents, and / or cellular targets of the candidate therapeutic agents.

[0206] Particle analysis instruments, such as flow cytometers and scanning cytometers, are analytical tools that enable the characterization of particles (e.g., single cells) based on optical parameters such as light scattering and fluorescence. For example, in a flow cytometer, particles such as molecules in fluid suspension, analyte-bound beads, or individual cells pass through a detection region where they are exposed to excitation light, typically from one or more lasers, and their light scattering and fluorescence properties are measured. Particles or their components are typically labeled with fluorescent dyes for ease of detection. By labeling different particles or components with spectrally distinct fluorescent dyes, multiple different particles or components can be detected simultaneously. In some implementations, the analytical instrument includes multiple photodetectors, one for each scattering parameter being measured and one for each distinct dye being detected. The resulting data includes measured signals for each of the light scattering parameters and fluorescence emission.

[0207] Parameters measured using a flow cytometer typically include excitation light scattered primarily along the forward direction by particles, called forward scatter (FSC), excitation light scattered primarily sideways by particles, called side scatter (SSC), and light emitted by fluorescent molecules in one or more channels (ranges of frequencies) of the spectrum, called FL1, FL2, etc., or light emitted by fluorescent dyes that is primarily detected in that channel. Different cell types can be identified by scatter parameters and the fluorescence emission resulting from labeling various cellular proteins with dye-conjugated antibodies.

[0208] Both flow cytometers and scanning cytometers are commercially available, for example, from BD Biosciences (San Jose, Calif.). 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 University Press (1994); Jaroszeski et al. (eds.), Flow Cytometry Protocols, Methods in Molecular Biology No. 91, Humana Press (1997); and Practical Shapiro, Flow Cytometry, 4th ed., 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.

[0209] Data obtained from analyzing cells (or other particles) by multicolor flow cytometry are multidimensional, with each cell corresponding to a point in a multidimensional space defined by the measured parameters. Populations of cells or particles are identified as clusters of points in the data space. Identification of clusters, and therefore populations, can be performed manually by drawing gates around the populations displayed in one or more two-dimensional plots called "scatter plots" or "dot plots" of the data. Alternatively, clusters can be identified and gates defining the boundaries of the populations can be determined automatically. Examples of automated gating methods are described, for example, in U.S. Pat. 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 Application Publication No. 2012 / 0245889, each of which is incorporated herein by reference.

[0210] Flow cytometry is a useful method for analyzing and isolating biological particles, such as cells and constituent molecules. As such, flow cytometry has a wide range of diagnostic and therapeutic applications. This method uses fluid flow to linearly separate particles, allowing them to pass single-file through a detection device. Individual cells can be distinguished depending on their position in the fluid flow and the presence of detectable markers. Thus, a flow cytometer can be used to generate diagnostic profiles of populations of biological particles.

[0211] Isolation of biological particles has been achieved by adding sorting or collection capabilities to flow cytometers. Particles in the segregated flow that are detected as having one or more desired characteristics are individually isolated from the sample flow by mechanical or electrical removal. This flow sorting method has been used to sort different cell types, separate sperm carrying X and Y chromosomes for animal breeding, sort chromosomes for genetic analysis, and isolate specific organisms from complex biological populations. In flow cytometry sorting, the use of index sorting implies that additional information is available that relates individual cell events to their destination location within the plate or slide holder. This information can be used after data acquisition to perform additional analysis of where cells are physically located on the plate device. This information also allows the user to visualize where these cells are located on a bivariate plot.

[0212] Index sorting allows the sorting device to record the sorting decision for each event (typically a cell or other particle suspended in a flow stream), and the data is available for post-sort analysis. Typically, index sorting is performed by detecting a particle's characteristic (such as color) and directing the particle to a collection plate. The plate may include multiple plate destinations (e.g., well locations). Sorting may involve directing particles to a specific plate location (e.g., a well) within the plate. The sorting device can record the destination plate and / or well location in association with the event identifier. Thus, each sorting event includes all measurements from the detector (PMT, photodiode) along with the well location and sorting destination. Users can examine the sorted cell data and correlate it with subsequent operations on the plate (e.g., gene expression derived from sequencing the sorted cells).

[0213] A sorting mode may be specified for a given sample. The sorting mode includes parameters for controlling the sorting events. For example, when a sorting device receives a sample, the sorting device can receive a sorting mode to control which characteristics to use for sorting and where the detected values ​​of those characteristics should be sorted. A sorting mode can include a purity mode, in which the cell sorter can be configured to ensure that the desired cell type and only the desired cell type are present within a gate. A sorting mode can include a single cell mode, in which the cell sorter can be configured to ensure that only a single cell is present within a droplet. Because there may be uncertainty around the droplet boundary of a droplet in which a cell may reside, subsequent droplets are often not sorted even if they are sortable. In the case of single-cell sorting, this may be desirable for certain experiments, such as genomic-based assays, where a user may want to correlate gene expression with measurements derived from flow cytometry. In such cases, the presence of multiple cells in a well can obscure which cell a gene sequence originates from. Sorting modes can configure the cell sorter by setting masks that examine where events fall within a droplet and in surrounding droplets. Some single cell sorting mode configurations may involve examining a state machine or series of events that fall within a droplet.

[0214] In some implementations, a target gate can be used to identify events of interest. The target gate can be provided by selecting a region of a two-dimensional plot. Events detected to have a characteristic value within the selected two-dimensional 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 under certain sorting modes (e.g., purity mode or single cell mode), the event may not be properly sorted, such as if another event is present in the same droplet in the fluid flow as the event. In some implementations, this may be referred to as entrainment or cohesion. 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 may include detected characteristics of the event (e.g., reflected light values, fluorescence information, light scatter information, time of the event, sequence number of the event, sorting device operating characteristics at the time the event was analyzed (e.g., temperature, flow rate, sorting mode, etc.)). In the case of index sorting, the coordinates of the location where the cells of a particular event were located, such as a current tray, plate, microscope slide, or other physical medium with spatially separated pools into which cell-containing droplets can be placed, can also be transmitted.

[0215] In some embodiments, the methods disclosed herein may include enriching a sample containing a plurality of cells for a cell of interest to generate an enriched cell sample containing a plurality of single cells for analysis as provided herein. Enriching the sample may include focusing the cells of interest in the sample; isolating one or more cells of interest in the enriched cell sample using a flow cytometer; and obtaining sequence information of one or more polynucleotides from each of the one or more isolated cells as described herein. Various focusing methods and techniques may be used, such as hydrodynamic focusing, magnetic focusing, electric field focusing, gravitational field focusing, optical field focusing, and any combination thereof. In some embodiments, enriching the sample includes depleting non-target cells in the sample. In some embodiments, enriching the sample includes both acoustic focusing and depleting non-target cells in the sample. In some embodiments, one or more of non-target cells, interfering cells, and debris in the sample may be depleted, for example, using magnetic depletion.

[0216] Compositions and Kits The disclosure herein includes compositions. In some embodiments, the compositions include a microwell array, the microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments, each compartment having a size of about 1,000 μm 3~Approx. 786,000μm 3 The composition can include a cartridge, the cartridge including at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof.

[0217] The present disclosure includes compositions. In some embodiments, the compositions include a cartridge, the cartridge including at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof, the cartridge including a microwell array, the microwell array including a plurality of chambers, each chamber including a plurality of compartments, each compartment having a size of about 1,000 μm 3 ~Approx. 786,000μm 3 The microwells have volumes in the range of The disclosure herein includes compositions. In some embodiments, the compositions include two or more solid supports, each of which includes a plurality of oligonucleotide barcodes each including a cell labeling sequence, each cell labeling sequence including a predetermined chamber-indexing subsequence, the oligonucleotide barcodes associated with the same solid support including the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports including different cell labeling sequences, the oligonucleotide barcodes of the same plurality of solid supports including the same chamber-indexing subsequence, and the oligonucleotide barcodes of the different plurality of solid supports including different chamber-indexing subsequences.

[0218] A compartment of the plurality of compartments comprises a single solid support of the plurality of solid supports, each solid support comprising a plurality of oligonucleotide barcodes each comprising a cell labeling sequence, each cell labeling sequence comprising a predetermined chamber indexing subsequence, the oligonucleotide barcodes associated with the same solid support comprising the same cell labeling sequence, the oligonucleotide barcodes associated with different solid supports comprising different cell labeling sequences, the oligonucleotide barcodes located within the same chamber comprising the same chamber indexing subsequence, and the oligonucleotide barcodes located within different chambers comprising different chamber indexing subsequences.

[0219] The composition may comprise a chamber-indexing subsequence lookup table, in some embodiments, the chamber-indexing subsequence lookup table identifies a chamber-indexing subsequence associated with each solid support distributed in each microwell of the array.

[0220] The cartridge may be configured to maintain viability of single cells distributed within the microwells for a period of at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 100 minutes, 250 minutes, 500 minutes, 750 minutes, 1000 minutes, 2500 minutes, 5000 minutes, 7500 minutes, or 10000 minutes. The cartridge may include a transparent window for optical imaging of the microwells. The composition may include an imaging system configured to capture and process images of all or a portion of the microwells, the imaging system further including an illumination subsystem, an imaging subsystem, and a processor. The imaging system may be configured to perform brightfield, darkfield, fluorescence, or quantitative phase imaging.

[0221] The composition may include a buffer. The composition may include one or more reagents for a reverse transcription reaction, one or more reagents for an amplification reaction, or both. The oligonucleotide barcodes may each include a molecular label sequence. Each cell label of the plurality of oligonucleotide barcodes may include at least six nucleotides. The cell label may include multiple moieties and one or more linkers. The cell label may include a first cell labeling moiety, a first linker, and a second cell labeling moiety; the cell label may include a second linker and a third cell labeling moiety; and the cell label may further include a third linker and a fourth cell labeling moiety. The first cell labeling moiety may include a chamber indexing moiety sequence. The chamber indexing moiety sequence may be 2 to 15 nucleotides in length. The plurality of chambers 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 1000 chambers. The plurality of compartments can be at least about 100 compartments, about 500 compartments, about 1000 compartments, about 5000 compartments, about 10,000 compartments, about 25,000 compartments, about 50,000 compartments, about 75,000 compartments, or about 100,000 compartments.

[0222] The composition may include a plurality of solid supports, each including a plurality of oligonucleotide barcodes. Each of the oligonucleotide barcodes may include a molecular label and a cellular label. In some embodiments, oligonucleotide barcodes associated with the same solid support may include the same cellular label sequence, and oligonucleotide barcodes associated with different solid supports may include different cellular label sequences. Each oligonucleotide barcode may include a first universal sequence. The oligonucleotide barcode may include a target binding region including a capture sequence. The target binding region may include a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof. Each molecular label of the plurality of oligonucleotide barcodes may include at least six nucleotides. The solid support may include a planar surface. The solid support may include synthetic particles. At least one of the plurality of oligonucleotide barcodes may be immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, partially encapsulated within a synthetic particle, or a combination thereof. The synthetic particle may be disintegratable. The synthetic particle may include a bead. 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 include 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 any combination thereof. The synthetic particles may include disintegrable hydrogel particles. In some embodiments, the composition further includes instructions for use.

[0223] term In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment, except where such substitution is technically infeasible. Those skilled in the art will appreciate that various other omissions, additions, and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and variations are intended to fall within the scope of the subject matter as defined by the appended claims. Those skilled in the art will recognize that, for this and other processes and methods disclosed herein, the functions performed in the processes and methods may be implemented in differing order. Furthermore, the outlined steps and operations are provided only as examples, and some of the steps and operations may be arbitrarily combined into fewer steps and operations or expanded into additional steps and operations without diminishing the essential elements of the embodiments of the present disclosure.

[0224] In connection with the use of virtually any plural and / or singular term herein, those of skill in the art can convert from plural to singular and / or from singular to plural where appropriate in context and / or application. Various singular / plural permutations may be expressly stated herein for clarity. 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 herein to "or" is intended to encompass "and / or" unless specified otherwise.

[0225] In general, it will be understood by those skilled in the art that the terms used herein, particularly in the appended claims (e.g., the body 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.). Furthermore, where a specific number of introduced claim recitations is intended, such intention will be explicitly set forth in the claim; it will be understood by those skilled in the art that, in the absence of such a recitation, no such intention exists. For example, as an aid to understanding, the following appended claims may include the use of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed as meaning that introducing a claim recitation with the indefinite article "a" or "an" means that any particular claim containing such introduced claim recitation is limited to embodiments containing only one such recitation, even if the same claim also includes the introductory phrase "one or more" or "at least one" and an indefinite article such as "a" or "an" (e.g., "a" and / or "an" should be construed to mean "at least one" or "one or more"), nor should the use of definite articles used to introduce claim recitations.Furthermore, even if a particular number of introduced claim recitations is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., an unqualified recitation such as "two recitations" without other modifiers means at least two recitations, or more than two recitations). Furthermore, when a convention similar to "such as at least one of A, B, and C" is used, it is generally intended that such a configuration be understood by one of skill in the art (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). When a convention similar to "such as at least one of A, B, or C" is used, it is generally intended that such construction have the meaning that one of ordinary skill in the art would understand that convention (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, a system having A alone, B alone, C alone, both A and B, both A and C, both B and C, and / or all of A, B, and C, etc.). Furthermore, it will be understood by those skilled in the art that virtually any disjunctive word and / or phrase expressing two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both of the terms. For example, the phrase "A or B" is understood to include the possibilities of "A" or "B" or "A and B."

[0226] Furthermore, when features or aspects of the present disclosure are described in terms of a Markush group, one of skill in the art will recognize that the present disclosure is also described in terms of any individual member or subgroup of members of the Markush group.

[0227] As will be understood by those skilled in the art, for all purposes, e.g., with respect to the provision of a specification, all ranges disclosed herein encompass all possible subranges and combinations of such subranges. Any recited range is readily recognizable as being fully descriptive and that the range can be divided into at least 2, 3, 4, 5, 10, etc. equal parts. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third, upper third, etc. Similarly, as will be understood by those skilled in the art, terms such as "up to," "at least," "greater than," "less than," and the like all refer to ranges that are inclusive of the recited numbers and that can be subsequently broken down into subranges as discussed above. Finally, as will be understood by those skilled in the art, ranges include each individual member. Thus, for example, a group having 1 to 3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1 to 5 items refers to groups having 1, 2, 3, 4, or 5 items, and so forth.

[0228] From the foregoing, it will be appreciated that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications may 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, with the true scope and spirit being indicated by the following claims.

Claims

1. 1. A method for assigning sequencing data to chambers, comprising: providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more types of a plurality of solid supports into a specific chamber compartment of the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence and oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; Distributing each of the two or more populations of single cells into a compartment of a particular chamber of the plurality of chambers, each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the 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 a nucleic acid target from at least one of the plurality of single cells of the at least one population of single cells using the plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of a plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexed subsequence of each cell-labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on a chamber-indexed subsequence of each cell labeling sequence in the sequencing data; A method comprising:

2. 1. A method for assigning sequencing data to chambers, comprising: providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments, each chamber comprising a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chamber, each solid support comprising a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence and oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; Distributing each of the two or more populations of single cells into a compartment of a particular chamber of the plurality of chambers, each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the 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 a nucleic acid target from at least one of the plurality of single cells of the at least one population of single cells using the plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of a plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexed subsequence of each cell-labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber among the plurality of chambers based on a chamber-indexed subsequence of each cell labeling sequence in the sequencing data; A method comprising:

3. 1. A method for assigning sequencing data to a population of single cells, comprising: providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more types of a plurality of solid supports into a specific chamber compartment of the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence and oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; Distributing each of the two or more populations of single cells into a compartment of a particular chamber of the plurality of chambers, each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the 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 a nucleic acid target from at least one of the plurality of single cells of the at least one population of single cells using the plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of a plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexed subsequence of each cell-labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber of the plurality of chambers based on a chamber-indexed subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a population of two or more populations of single cells based on the chamber assigned to the sequencing read; A method comprising:

4. 1. A method for assigning sequencing data to a population of single cells, comprising: providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments, each chamber comprising a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chamber, each solid support comprising a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence and oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; Distributing each of the two or more populations of single cells into a compartment of a particular chamber of the plurality of chambers, each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the 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 a nucleic acid target from at least one of the plurality of single cells of the at least one population of single cells using the plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of a plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexed subsequence of each cell-labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a chamber of the plurality of chambers based on a chamber-indexed subsequence of each cell labeling sequence in the sequencing data; assigning each of the plurality of sequencing reads to a population of two or more populations of single cells based on the chamber assigned to the sequencing read; A method comprising:

5. 1. A method for correlating sequencing data and phenotypic data of a population of single cells, comprising: obtaining phenotypic data for each of the two or more populations of single cells; providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; distributing each of two or more types of a plurality of solid supports into a specific chamber compartment of the plurality of chambers, wherein each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence and oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; Distributing each of the two or more populations of single cells into a compartment of a particular chamber of the plurality of chambers, each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the 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 a nucleic acid target from at least one of the plurality of single cells of the at least one population of single cells using the plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of a plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexed subsequence of each cell-labeling sequence in the sequencing data; correlating the sequencing data and phenotypic data of at least one population of single cells based on chamber-indexed subsequences of each cell labeling sequence in the sequencing data; A method comprising:

6. 1. A method for correlating sequencing data and phenotypic data of a population of single cells, comprising: obtaining phenotypic data for each of the two or more populations of single cells; providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments, each chamber comprising a predetermined plurality of solid supports selected from two or more types of a plurality of solid supports, the solid supports being located within the compartments of the chamber, each solid support comprising a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber comprise the same chamber-indexing subsequence and oligonucleotide barcodes located in different chambers comprise different chamber-indexing subsequences; Distributing each of the two or more populations of single cells into a compartment of a particular chamber of the plurality of chambers, each of the two or more populations of single cells comprises a plurality of single cells, the single cells comprising copies of the 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 a nucleic acid target from at least one of the plurality of single cells of the at least one population of single cells using the plurality of oligonucleotide barcodes to generate a plurality of barcoded nucleic acid targets; obtaining sequencing data comprising a plurality of sequencing reads of a plurality of barcoded nucleic acid targets or products thereof; identifying a chamber-indexed subsequence of each cell-labeling sequence in the sequencing data; correlating the sequencing data and phenotypic data of at least one population of single cells based on chamber-indexed subsequences of each cell labeling sequence in the sequencing data; A method comprising:

7. the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports; an oligonucleotide barcode associated with the plurality of first solid supports having a first predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the plurality of second solid supports have a second predetermined chamber-indexing subsequence; the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different; The method according to any one of claims 1 to 6.

8. the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports; a first predetermined chamber-indexing subsequence of a plurality of first solid supports is selected from a first set of chamber-indexing subsequences; second predetermined chamber-indexing subsequences of the plurality of second solid supports are selected from a second set of chamber-indexing subsequences; each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from each chamber-indexing subsequence of the second set of chamber-indexing subsequences; The composition according to any one of claims 1 to 7.

9. 9. The composition of any one of claims 1 to 8, wherein a user can determine whether an oligonucleotide barcode is associated with a plurality of first solid supports or a plurality of second solid supports based on a predetermined chamber-indexed subsequence of a sequencing read derived from the oligonucleotide barcode or a product thereof.

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

11. obtaining phenotypic data for each of two or more populations of single cells; correlating the sequencing data and phenotypic data of at least one population of single cells based on chamber-indexed subsequences of each cell labeling sequence in the sequencing data; The method of any one of claims 1 to 4 or 10, comprising:

12. The method according to any one of claims 1 to 11, wherein the step of distributing each of two or more types of a plurality of solid supports into a compartment of a specific chamber among the plurality of chambers comprises the step of distributing a predetermined plurality of solid supports selected from the two or more types of a plurality of solid supports into a specific chamber among the plurality of chambers.

13. barcoding copies of the nucleic acid targets, contacting a plurality of oligonucleotide barcodes with copies of a nucleic acid target for hybridization; extending the plurality of oligonucleotide barcodes hybridized to copies of the nucleic acid target to generate a plurality of barcoded nucleic acid targets; The method according to any one of claims 1 to 12, comprising:

14. The partition is about 1,000 μm 3 ~Approx. 786,000μm 3 The method according to any one of claims 1 to 13, wherein the microwell has a volume in the range of

15. The method of any one of claims 1 to 14, wherein the oligonucleotide barcodes each comprise a molecular beacon sequence.

16. 16. The method of any one of claims 1 to 15, wherein the oligonucleotide barcodes of the same plurality of solid supports comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes of different plurality of solid supports comprise different chamber-indexing subsequences.

17. each cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; the cell label comprises a plurality of moieties and one or more linkers; the cell label comprises a first cell labeling moiety, a first linker, and a second cell labeling moiety, the cell label may comprise a second linker and a third cell labeling moiety, and further the cell label may comprise a third linker and a fourth cell labeling moiety; and / or the first cell-labeling moiety, the second cell-labeling moiety, the third cell-labeling moiety, and / or the fourth cell-labeling moiety comprise a chamber-indexing moiety; and / or the chamber-indexing subsequence is 2-80 nucleotides in length; The method according to any one of claims 1 to 16.

18. The method of any one of claims 1 to 17, wherein the populations of single cells are separate samples.

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

20. 20. The method of any one of claims 1 to 19, wherein the two or more populations of single cells are derived from one or more samples separated based on phenotypic data, and the plurality of single cells may comprise T cells, B cells, tumor cells, bone marrow cells, blood cells, normal cells, fetal cells, maternal cells, or a mixture thereof.

21. 21. The method of any one of claims 1 to 20, wherein the plurality of chambers 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, and the plurality of chambers optionally comprises 8 lanes.

22. 22. The method of any one of claims 1-21, wherein the plurality of compartments is at least about 100 compartments, about 500 compartments, about 1000 compartments, about 5000 compartments, about 10,000 compartments, about 25,000 compartments, about 50,000 compartments, about 75,000 compartments, or about 100,000 compartments.

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

24. 24. The method of any one of claims 1 to 23, wherein a compartment of the plurality of compartments comprises a single cell of the plurality of single cells and a single solid support of the plurality of solid supports.

25. 25. The method of any one of claims 1 to 24, wherein the plurality of barcoded nucleic acid targets each comprise a sequence complementary to at least a portion of the nucleic acid target and the molecular label.

26. The method of any one of claims 1 to 25, wherein each of the plurality of sequencing reads comprises (1) a cellular marker sequence and (2) a molecular marker sequence.

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

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

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

30. 30. The method of any one of claims 1 to 29, wherein distributing a plurality of solid supports into respective compartments of a plurality of chambers comprises positioning each of the plurality of solid supports into a specific chamber of a microwell array by flow cytometry.

31. 31. The method of any one of claims 1 to 30, wherein distributing each of the two or more populations of single cells into a compartment of a specific chamber of the plurality of chambers comprises disposing each population of single cells into a specific chamber of a microwell array by flow cytometry.

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

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

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

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

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

37. 37. The method of claim 36, wherein the correlation analysis identifies one or more of the following: a candidate biomarker, a candidate therapeutic agent, a candidate dose of the therapeutic agent, and / or a cellular target of the candidate therapeutic agent.

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

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

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

41. 41. The method of Claim 40, wherein determining the copy number of the nucleic acid target in one or more of the plurality of single cells comprises determining the copy number of the nucleic acid target in the plurality of single cells based on the number of molecular labels having distinct sequences, their complements, or combinations thereof, associated with the plurality of barcoded nucleic acid targets or products thereof.

42. contacting random primers with a plurality of barcoded nucleic acid targets, each of the random primers comprising a second universal sequence or a complement thereof; extending random primers hybridized to the plurality of barcoded nucleic acid targets to generate a plurality of extension products; The method according to any one of claims 40 to 41, comprising:

43. 43. The method of Claim 42, comprising amplifying the plurality of extension products 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 generating a first plurality of barcoded amplicons.

44. 44. The method of claim 43, wherein amplifying the plurality of extension products comprises adding sequences of binding sites of sequencing primers and / or sequencing adapters, their complementary sequences, and / or portions thereof to the plurality of extension products.

45. 45. The method of any one of claims 43-44, comprising determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the first plurality of barcoded amplicons or products thereof.

46. 46. ​​The method of any one of claims 43-45, wherein determining the copy number of the nucleic acid target in one or more of the plurality of single cells comprises determining the number of each of the plurality of nucleic acid targets in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with barcoded amplicons in a first plurality of barcoded amplicons comprising the sequences of each of the plurality of nucleic acid targets.

47. 47. The method of claim 46, wherein the sequence of each of the plurality of nucleic acid targets comprises a subsequence of each of the plurality of nucleic acid targets.

48. 48. The method of any one of claims 43 to 47, wherein the sequence of the nucleic acid target in the first plurality of barcoded amplicons comprises a subsequence of the nucleic acid target.

49. 49. The method of any one of claims 43 to 48, comprising amplifying the first plurality of barcoded amplicons 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 generating a second plurality of barcoded amplicons.

50. 50. The method of Claim 49, wherein amplifying the first plurality of barcoded amplicons comprises adding sequences of sequencing primer and / or sequencing adapter binding sites, complementary sequences thereof, and / or portions thereof to the first plurality of barcoded amplicons.

51. 51. The method of any one of claims 49-50, comprising determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the second plurality of barcoded amplicons or products thereof.

52. 52. The method of any one of claims 43-51, wherein the first plurality of barcoded amplicons and / or the second plurality of barcoded amplicons comprise whole transcriptome amplification (WT A) products.

53. synthesizing a third plurality of barcoded amplicons using the plurality of barcoded nucleic acid targets as templates to produce a third plurality of barcoded amplicons; synthesizing the third plurality of barcoded amplicons comprises performing polymerase chain reaction (PCR) amplification of the plurality of barcoded nucleic acid targets; synthesizing a third plurality of barcoded amplicons comprises PCR amplification using a primer capable of hybridizing to the first universal sequence or its complement and a target-specific primer; obtaining sequence data for the third plurality of barcoded amplicons or products thereof, wherein obtaining sequence information may include attaching sequencing adaptors to the third plurality of barcoded amplicons or products thereof; and / or determining the copy number of the nucleic acid target in one or more of the plurality of single cells based on the number of molecular labels having distinct sequences associated with the third plurality of barcoded amplicons or products thereof; 53. The method of any one of claims 1 to 52.

54. the nucleic acid target comprises a nucleic acid molecule, which may comprise 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 comprises a sample indexing oligonucleotide, the sample indexing oligonucleotide may comprise a sample indexing sequence, and the sample indexing sequences of at least two sample indexing compositions of the plurality of sample indexing compositions may comprise different sequences; and / or The nucleic acid target may comprise a cellular component binding reagent-specific oligonucleotide, and the cellular component binding reagent-specific oligonucleotide may comprise a unique identifier sequence for the cellular component binding reagent.

54. The method of any one of claims 1 to 53.

55. wherein extending the plurality of oligonucleotide barcodes comprises extending the plurality of oligonucleotide barcodes 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 comprises a Klenow fragment; the reverse transcriptase comprises a viral reverse transcriptase, which may be a murine leukemia virus (MLV) reverse transcriptase or a 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 comprises a binding site of a sequencing primer and / or a sequencing adapter, a complementary sequence thereof, and / or a portion thereof; the sequencing adapter comprises a P5 sequence, a P7 sequence, a complementary sequence thereof, and / or a portion thereof; the sequencing primers include a read 1 sequencing primer, a read 2 sequencing primer, a complementary sequence thereof, and / or a portion thereof; at least 10 of the plurality of oligonucleotide barcodes comprise different molecular label sequences; and / or each molecular label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; 55. The method of any one of claims 1 to 54.

56. the solid support comprises a planar surface or a synthetic particle; at least one of the plurality of oligonucleotide barcodes may be immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, or partially encapsulated within a synthetic particle; The synthetic particles may be disintegrable; The synthetic particles may comprise beads; The beads may 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, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; The synthetic particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; and / or The synthetic particles may include disintegrable hydrogel particles.

56. The method of any one of claims 1 to 55.

57. A composition comprising two or more solid supports, each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; A composition wherein oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences.

58. the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports; an oligonucleotide barcode associated with the plurality of first solid supports having a first predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the plurality of second solid supports have a second predetermined chamber-indexing subsequence; the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different; 58. The composition of claim 57.

59. the two or more plurality of solid supports comprises a plurality of first solid supports and a plurality of second solid supports; a first predetermined chamber-indexing subsequence of a plurality of first solid supports is selected from a first set of chamber-indexing subsequences; second predetermined chamber-indexing subsequences of the plurality of second solid supports are selected from a second set of chamber-indexing subsequences; each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from each chamber-indexing subsequence of the second set of chamber-indexing subsequences; 59. The composition according to any one of claims 57 to 58.

60. 60. The composition of any one of claims 57-59, wherein a user can determine whether an oligonucleotide barcode is associated with a plurality of first solid supports or a plurality of second solid supports based on a predetermined chamber-indexed subsequence of a sequencing read derived from the oligonucleotide barcode or a product thereof.

61. Two or more types of solid supports, a plurality of third solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of third solid supports may have third chamber-indexing subsequences, and / or (b) the third chamber-indexing subsequences of the plurality of third solid supports may be selected from a third set of chamber-indexing subsequences; a plurality of fourth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of fourth solid supports may have fourth chamber-indexing subsequences, and / or (b) the fourth chamber-indexing subsequences of the plurality of fourth solid supports may be selected from a fourth set of chamber-indexing subsequences; a plurality of fifth solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of fifth solid supports may have a fifth chamber-indexing subsequence, and / or (b) the fifth chamber-indexing subsequence of the plurality of fifth solid supports may be selected from a fifth set of chamber-indexing subsequences; a plurality of sixth solid supports, wherein (a) oligonucleotide barcodes associated with the plurality of sixth solid supports may have sixth chamber-indexing subsequences, and / or (b) the sixth chamber-indexing subsequences of the plurality of sixth solid supports may be selected from a sixth set of chamber-indexing subsequences; a plurality of seventh solid supports, wherein (a) the oligonucleotide barcodes associated with the plurality of seventh solid supports may have seventh chamber-indexing subsequences, and / or (b) the seventh chamber-indexing subsequences of the plurality of seventh solid supports may be selected from a seventh set of chamber-indexing subsequences; and / or an eighth plurality of solid supports, wherein (a) an oligonucleotide barcode associated with the eighth plurality of solid supports may have an eighth chamber-indexing subsequence, and / or (b) the eighth chamber-indexing subsequence of the eighth plurality of solid supports may be selected from an eighth set of chamber-indexing subsequences; Including, the first chamber-indexing subsequence, the second chamber-indexing subsequence, the third chamber-indexing subsequence, the fourth chamber-indexing subsequence, the fifth chamber-indexing subsequence, the sixth chamber-indexing subsequence, the seventh chamber-indexing subsequence, and / or the eighth chamber-indexing subsequence do not share sequences with each other; The composition according to any one of claims 57 to 60.

62. 62. The composition of any one of claims 57-61, wherein a user can determine whether an 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 based on a predetermined chamber-indexed subsequence of a sequencing read derived from the oligonucleotide barcode or a product thereof.

63. 63. The composition of any one of claims 57 to 62, wherein the cell label comprises a plurality of cell labeling moieties and one or more linkers.

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

65. 65. The composition of any one of claims 57-64, wherein the predetermined chamber-indexing moiety sequence comprises a first cell-labeling moiety, a second cell-labeling moiety, a third cell-labeling moiety, a fourth cell-labeling moiety, or any combination thereof.

66. 66. The composition of any one of claims 57-65, wherein the first, second, third, fourth, fifth, sixth, seventh, and / or eighth sets of chamber-indexing subsequences comprise a set of less 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-indexing subsequences of other sets of chamber-indexing subsequences.

67. 67. The composition of any one of claims 57-66, wherein the first cell-labeling moiety, the second cell-labeling moiety, the third cell-labeling moiety, the fourth cell-labeling moiety, or any combination thereof, is selected from a set of less than about 480, about 384, about 288, about 192, about 96, or about 48 unique sequences.

68. 68. The composition of any one of claims 57-67, wherein a user can determine whether an 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 based on the sequence of the first cell labeling portion, the second cell labeling portion, the third cell labeling portion, the fourth cell labeling portion, or any combination thereof, of a sequencing read derived from the oligonucleotide barcode or a product thereof.

69. distributing a plurality of precursor-type first solid supports and a first population of first oligonucleotides into a first plurality of first compartments, wherein co-localized solid supports and first oligonucleotides associate; distributing a plurality of precursor-type second solid supports and a second population of first oligonucleotides into a first plurality of second compartments, wherein co-localized solid supports and first oligonucleotides associate; pooling a plurality of precursor-type first solid supports associated with a first oligonucleotide; pooling a plurality of precursor-type second solid supports associated with the first oligonucleotide; distributing a plurality of precursor-type first solid supports associated with first oligonucleotides and a first population of second oligonucleotides into a second plurality of first compartments, wherein co-localized solid supports and second oligonucleotides are associated; distributing a plurality of precursor-type second solid supports associated with the first oligonucleotides and a second population of second oligonucleotides into a second plurality of second compartments, wherein co-localized solid supports and second oligonucleotides are associated; pooling a plurality of precursor-type first solid supports associated with first and second oligonucleotides; pooling a plurality of precursor-type second solid supports associated with the first and second oligonucleotides; distributing a plurality of precursor-type first solid supports associated with the first and second oligonucleotides and a first population of third oligonucleotides into a third plurality of first compartments, wherein co-localized solid supports and third oligonucleotides are associated; distributing the plurality of precursor-type second solid supports associated with the first and second oligonucleotides and the second population of third oligonucleotides into a third plurality of second compartments, wherein the co-localized solid supports and third oligonucleotides are associated; A method comprising:

70. pooling a plurality of precursor first solid supports associated with the first, second, and third oligonucleotides to generate a plurality of first solid supports; pooling a plurality of precursor second solid supports associated with the first, second, and third oligonucleotides to generate a plurality of second solid supports; 70. The method of claim 69, comprising:

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

72. the first and second populations of first oligonucleotides each comprise about 192 first cell-labeling moieties having distinct sequences; the first and second populations of second oligonucleotides each comprise about 192 second cell-labeling moieties having distinct sequences; the first and second populations of third oligonucleotides each comprise about 192 third cell-labeling moieties having distinct sequences; 72. The method according to any one of claims 69 to 71.

73. the first and second populations of first oligonucleotides are the same; the first and second populations of second oligonucleotides are the same; the first and second populations of third oligonucleotides are different; the first and second populations of third oligonucleotides may comprise non-overlapping subsets of cell-labeling subsequences of the first and second populations of first oligonucleotides and / or the first and second populations of second oligonucleotides; 73. The method of any one of claims 69 to 72.

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

75. distributing a plurality of precursor-type first solid supports associated with the first, second, and third oligonucleotides and the fourth oligonucleotide into a fourth plurality of first compartments, wherein co-localized solid supports and fourth oligonucleotides are associated; distributing a plurality of precursor-type second solid supports associated with the first, second, and third oligonucleotides and the fifth oligonucleotide into a fourth plurality of second compartments, wherein co-localized solid supports and fifth oligonucleotides are associated; pooling a plurality of precursor first solid supports associated with the first, second, third, and fourth oligonucleotides to generate a plurality of first solid supports; pooling a plurality of precursor second solid supports associated with the first, second, third, and fifth oligonucleotides to generate a plurality of second solid supports; 75. The method of any one of claims 69 to 74, comprising:

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

77. the first oligonucleotide comprises a first cell-labeling moiety and a first linker or its complement; the second oligonucleotide comprises a first linker, a second cell-labeling moiety, and a second linker, or a complement thereof; the third oligonucleotide comprises a second linker and a third cell-labeling moiety, or a complement thereof, and the second oligonucleotide may further comprise a third linker or a complement thereof; and / or the fourth oligonucleotide comprises a third linker and a fourth cell-labeling moiety, or a complement thereof; 77. The method of any one of claims 69 to 76.

78. the first oligonucleotide and the second oligonucleotide are configured to be connected via a first linker; the second oligonucleotide and the third oligonucleotide are configured to be connected via a second linker; and / or the third oligonucleotide and the fourth oligonucleotide are configured to be connected via a third linker; 78. The method of any one of claims 69 to 77.

79. 79. The method of any one of claims 69-78, wherein the plurality of first solid supports and / or the plurality of second solid supports comprise at least about 1,000, about 10,000, about 100,000, about 1,000,000, about 7,000,000, about 10,000,000, or about 56,000,000 unique cell-marker sequences.

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

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

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

83. 83. The method of any one of claims 69-82, wherein the first solid support and the second solid support each comprise a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, and each cell-labeling sequence comprises a predetermined chamber-indexing subsequence.

84. an oligonucleotide barcode associated with the plurality of first solid supports having a first predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the plurality of second solid supports have a second predetermined chamber-indexing subsequence; the first predetermined chamber-indexing subsequence and the second predetermined chamber-indexing subsequence are different; 84. The method according to any one of claims 69 to 83.

85. a first predetermined chamber-indexing subsequence of a plurality of first solid supports is selected from a first set of chamber-indexing subsequences; second predetermined chamber-indexing subsequences of the plurality of second solid supports are selected from a second set of chamber-indexing subsequences; each chamber-indexing subsequence of the first set of chamber-indexing subsequences is different from each chamber-indexing subsequence of the second set of chamber-indexing subsequences; The method according to any one of claims 69 to 84.

86. A method according to any one of claims 69 to 85, producing a composition according to any one of claims 57 to 68.

87. providing a microwell array comprising a plurality of chambers, each chamber comprising a plurality of compartments; Distributing a plurality of first solid supports and a plurality of second solid supports into compartments of specific first chambers and specific second chambers of the plurality of chambers, respectively; 87. The method of any one of claims 69 to 86, further comprising:

88. 88. The method of any one of claims 69 to 87, wherein the step of distributing oligonucleotides into a plurality of compartments comprises the step of providing a plurality of compartments containing said oligonucleotides.

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

90. the solid support comprises synthetic particles; At least one oligonucleotide barcode of the plurality of oligonucleotide barcodes may be immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, partially encapsulated within a synthetic particle, or a combination thereof; The synthetic particles may be disintegrable; The synthetic particles may comprise beads, which may optionally 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, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; The synthetic particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; and / or The synthetic particles may include disintegrable hydrogel particles.

90. The method or composition of any one of claims 57 to 89.

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

92. 1. A composition comprising a microwell array, The microwell array includes a plurality of chambers, each chamber including a plurality of compartments, each compartment having a size of about 1,000 μm 3 ~Approx. 786,000μm 3 a microwell having a volume in the range of and may further include a cartridge, which may include at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof. composition.

93. 1. A composition comprising a cartridge, the cartridge comprises at least one of an inlet port, an outlet port, a pump, a valve, an aperture, a reservoir, a sample collection chamber, a temperature control device, or any combination thereof; The cartridge includes a microwell array, the microwell array includes a plurality of chambers, each chamber includes a plurality of compartments, and each compartment is about 1,000 μm 3 ~Approx. 786,000μm 3 and wherein the microwell has a volume in the range of composition.

94. A composition comprising two or more solid supports, each of the solid supports comprising a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; the oligonucleotide barcodes of the same plurality of solid supports comprise the same chamber-indexing subsequence, and the oligonucleotide barcodes of the different plurality of solid supports comprise different chamber-indexing subsequences; composition.

95. a compartment of the plurality of compartments comprises a single solid support of the plurality of solid supports; each solid support comprises a plurality of oligonucleotide barcodes each comprising a cell-labeling sequence, each cell-labeling sequence comprising a predetermined chamber-indexing subsequence; oligonucleotide barcodes associated with the same solid support comprise the same cell labeling sequence, and oligonucleotide barcodes associated with different solid supports comprise different cell labeling sequences; oligonucleotide barcodes located in the same chamber contain the same chamber-indexing subsequence, and oligonucleotide barcodes located in different chambers contain different chamber-indexing subsequences; The composition according to any one of claims 92 to 94.

96. wherein the cartridge is configured to maintain viability of single cells distributed within the microwells for a period of time that may be at least about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 100 minutes, 250 minutes, 500 minutes, 750 minutes, 1000 minutes, 2500 minutes, 5000 minutes, 7500 minutes, or 10000 minutes; the cartridge includes a transparent window for optical imaging of the microwells; the oligonucleotide barcodes each comprise a molecular beacon sequence; each cell label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; the cell label comprises a plurality of moieties and one or more linkers; the cell label comprises a first cell labeling moiety, a first linker, and a second cell labeling moiety; the cell label may comprise a second linker and a third cell labeling moiety; and the cell label may comprise a third linker and a fourth cell labeling moiety; the first cell-labeling moiety comprises a chamber-indexing subsequence; the chamber-indexing subsequence is 2-15 nucleotides in length; the plurality of chambers 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 plurality of compartments is at least about 100 compartments, about 500 compartments, about 1000 compartments, about 5000 compartments, about 10,000 compartments, about 25,000 compartments, about 50,000 compartments, about 75,000 compartments, or about 100,000 compartments; each oligonucleotide barcode comprises a first universal sequence; the oligonucleotide barcode comprises a target binding region comprising a capture sequence, and the target binding region may comprise a gene-specific sequence, an oligo(dT) sequence, a random multimer, or any combination thereof; and / or each molecular label of the plurality of oligonucleotide barcodes comprises at least six nucleotides; The composition according to any one of claims 92 to 95.

97. the solid support comprises a planar surface or a synthetic particle; at least one oligonucleotide barcode of the plurality of oligonucleotide barcodes may be immobilized on a synthetic particle, partially immobilized on a synthetic particle, encapsulated within a synthetic particle, partially encapsulated within a synthetic particle, or a combination thereof; The synthetic particles may be disintegrable; The synthetic particles may comprise beads, which may optionally 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, anti-biotin microbeads, anti-fluorescent dye microbeads, or any combination thereof; The synthetic particles may comprise a material selected from the group consisting of polydimethylsiloxane (PDMS), polystyrene, glass, polypropylene, agarose, gelatin, hydrogel, paramagnetic material, ceramic, plastic, glass, methylstyrene, acrylic polymer, titanium, latex, sepharose, cellulose, nylon, silicone, and any combination thereof; and / or The synthetic particles may include disintegrable hydrogel particles. The composition according to any one of claims 92 to 96.

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

99. Instructions for use, a chamber-indexing subsequence lookup table, which may identify a chamber-indexing subsequence associated with each solid support distributed in each microwell of the array; an imaging system configured to capture and process images of all or a portion of a microwell, the imaging system further comprising an illumination subsystem, an imaging subsystem, and a processor, and optionally configured to perform bright field, dark field, fluorescence, or quantitative phase imaging; buffer solutions, and / or One or more reagents for the reverse transcription reaction, one or more reagents for the amplification reaction, or both The composition of any one of claims 92 to 98, further comprising:

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