Sorting method using barcoded chambers for single cell workflow

A method of assigning sequencing data to chambers, comprising:

• providing a microwell array comprising a plurality of chambers, wherein each chamber comprises a plurality of partitions, wherein each chamber comprises a predetermined plurality of solid supports selected from two or more pluralities of solid supports, wherein the solid supports are situated within the partitions of said chambers, wherein the solid supports each comprise a plurality of oligonucleotide barcodes each comprising a cell label sequence, wherein each cell label sequence comprises predetermined chamber indexing subsequence(s),
• wherein oligonucleotide barcodes associated with the same solid support comprise the same cell label sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell label sequences,
• wherein oligonucleotide barcodes situated within the same chamber comprise the same chamber indexing subsequence(s), and wherein oligonucleotide barcodes situated within different chambers comprise different chamber indexing subsequence(s);
• partitioning each of two or more populations of single cells to the partitions of an identified chamber of the plurality of chambers,
• wherein each of the two or more populations of single cells comprises a plurality of single cells, wherein the single cells comprise copies of a nucleic acid target,
• wherein single cells of the same population are situated within the same chamber, and wherein single cells of different populations are situated within different chambers;
• barcoding the copies of a nucleic acid target from at least one of the plurality of single cells of 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 the plurality of barcoded nucleic acid targets, or products thereof;
• identifying the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• and
• assigning each of the plurality of sequencing reads to a chamber of the plurality of chambers based on the chamber indexing subsequence(s) of each cell label sequence in the sequencing data.

A method of assigning sequencing data to a population of single cells, comprising:

• providing a microwell array comprising a plurality of chambers, wherein each chamber comprises a plurality of partitions;
• partitioning each of two or more pluralities of solid supports to the partitions of an identified chamber of the plurality of chambers, wherein the solid supports each comprise a plurality of oligonucleotide barcodes each comprising a cell label sequence, wherein each cell label sequence comprises predetermined chamber indexing subsequence(s),
• wherein oligonucleotide barcodes associated with the same solid support comprise the same cell label sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell label sequences,
• wherein oligonucleotide barcodes situated within the same chamber comprise the same chamber indexing subsequence(s), and wherein oligonucleotide barcodes situated within different chambers comprise different chamber indexing subsequence(s);
• partitioning each of two or more populations of single cells to the partitions of an identified chamber of the plurality of chambers,
• wherein each of the two or more populations of single cells comprises a plurality of single cells, wherein the single cells comprise copies of a nucleic acid target,
• wherein single cells of the same population are situated within the same chamber, and wherein single cells of different populations are situated within different chambers;
• barcoding the copies of a nucleic acid target from at least one of the plurality of single cells of 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 the plurality of barcoded nucleic acid targets, or products thereof;
• identifying the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• assigning each of the plurality of sequencing reads to a chamber of the plurality of chambers based on the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• and
• 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.

A method of assigning sequencing data to a population of single cells, comprising:

• providing a microwell array comprising a plurality of chambers, wherein each chamber comprises a plurality of partitions, wherein each chamber comprises a predetermined plurality of solid supports selected from two or more pluralities of solid supports, wherein the solid supports are situated within the partitions of said chambers, wherein the solid supports each comprise a plurality of oligonucleotide barcodes each comprising a cell label sequence, wherein each cell label sequence comprises predetermined chamber indexing subsequence(s),
• wherein oligonucleotide barcodes associated with the same solid support comprise the same cell label sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell label sequences,
• wherein oligonucleotide barcodes situated within the same chamber comprise the same chamber indexing subsequence(s), and wherein oligonucleotide barcodes situated within different chambers comprise different chamber indexing subsequence(s);
• partitioning each of two or more populations of single cells to the partitions of an identified chamber of the plurality of chambers,
• wherein each of the two or more populations of single cells comprises a plurality of single cells, wherein the single cells comprise copies of a nucleic acid target,
• wherein single cells of the same population are situated within the same chamber, and wherein single cells of different populations are situated within different chambers;
• barcoding the copies of a nucleic acid target from at least one of the plurality of single cells of 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 the plurality of barcoded nucleic acid targets, or products thereof;
• identifying the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• and
• assigning each of the plurality of sequencing reads to a chamber of the plurality of chambers based on the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• and
• 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.

A method of associating sequencing data and phenotypic data of a population of obtaining phenotypic data of each of two or more populations of single cells;

• providing a microwell array comprising a plurality of chambers, wherein each chamber comprises a plurality of partitions;
• partitioning each of two or more pluralities of solid supports to the partitions of an identified chamber of the plurality of chambers, wherein the solid supports each comprise a plurality of oligonucleotide barcodes each comprising a cell label sequence, wherein each cell label sequence comprises predetermined chamber indexing subsequence(s),
• wherein oligonucleotide barcodes associated with the same solid support comprise the same cell label sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell label sequences,
• wherein oligonucleotide barcodes situated within the same chamber comprise the same chamber indexing subsequence(s), and wherein oligonucleotide barcodes situated within different chambers comprise different chamber indexing subsequence(s);
• partitioning each of two or more populations of single cells to the partitions of an identified chamber of the plurality of chambers,
• wherein each of the two or more populations of single cells comprises a plurality of single cells, wherein the single cells comprise copies of a nucleic acid target,
• wherein single cells of the same population are situated within the same chamber, and wherein single cells of different populations are situated within different chambers;
• barcoding the copies of a nucleic acid target from at least one of the plurality of single cells of 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 the plurality of barcoded nucleic acid targets, or products thereof;
• identifying the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• and
• associating the sequencing data and the phenotypic data of at least one population of single cells based on the chamber indexing subsequence(s) of each cell label sequence in the sequencing data.

A method of associating sequencing data and phenotypic data of a population of single cells, comprising:

• obtaining phenotypic data of each of two or more populations of single cells;
• providing a microwell array comprising a plurality of chambers, wherein each chamber comprises a plurality of partitions, wherein each chamber comprises a predetermined plurality of solid supports selected from two or more pluralities of solid supports, wherein the solid supports are situated within the partitions of said chambers, wherein the solid supports each comprise a plurality of oligonucleotide barcodes each comprising a cell label sequence, wherein each cell label sequence comprises predetermined chamber indexing subsequence(s),
• wherein oligonucleotide barcodes associated with the same solid support comprise the same cell label sequence, wherein oligonucleotide barcodes associated with different solid supports comprise different cell label sequences,
• wherein oligonucleotide barcodes situated within the same chamber comprise the same chamber indexing subsequence(s), and wherein oligonucleotide barcodes situated within different chambers comprise different chamber indexing subsequence(s);
• partitioning each of two or more populations of single cells to the partitions of an identified chamber of the plurality of chambers,
• wherein each of the two or more populations of single cells comprises a plurality of single cells, wherein the single cells comprise copies of a nucleic acid target,
• wherein single cells of the same population are situated within the same chamber, and wherein single cells of different populations are situated within different chambers;
• barcoding the copies of a nucleic acid target from at least one of the plurality of single cells of 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 the plurality of barcoded nucleic acid targets, or products thereof;
• identifying the chamber indexing subsequence(s) of each cell label sequence in the sequencing data;
• and
• associating the sequencing data and the phenotypic data of at least one population of single cells based on the chamber indexing subsequence(s) of each cell label sequence in the sequencing data.

A method, comprising:

• distributing a plurality of precursor first solid supports and a first population of first oligonucleotides to a first plurality of first partitions, wherein colocalized solid supports and first oligonucleotides become associated;
• distributing a plurality of precursor second solid supports and a second population of first oligonucleotides to a first plurality of second partitions, wherein colocalized solid supports and first oligonucleotides become associated;
• pooling the plurality of precursor first solid supports associated with first oligonucleotides;
• pooling the plurality of precursor second solid supports associated with first oligonucleotides;
• distributing the plurality of precursor first solid supports associated with first oligonucleotides and a first population of second oligonucleotides to a second plurality of first partitions, wherein colocalized solid supports and second oligonucleotides become associated;
• distributing the plurality of precursor second solid supports associated with first oligonucleotides and a second population of second oligonucleotides to a second plurality of second partitions, wherein colocalized solid supports and second oligonucleotides become associated;
• pooling the plurality of precursor first solid supports associated with first and second oligonucleotides;
• pooling the plurality of precursor second solid supports associated with first and second oligonucleotides;
• distributing the plurality of precursor first solid supports associated with first and second oligonucleotides and a first population of third oligonucleotides to a third plurality of first partitions, wherein colocalized solid supports and third oligonucleotides become associated;
• and
• distributing the plurality of precursor second solid supports associated with first and second oligonucleotides and a second population of third oligonucleotides to a third plurality of second partitions, wherein colocalized solid supports and third oligonucleotides become associated.

A composition comprising:

• a microwell array,
• wherein the microwell array comprises a plurality of chambers, wherein each chamber comprises a plurality of partitions, wherein each partition is a microwell having a volume ranging from about 1,000 μm3 to about 786,000 μm3, optionally:
• further comprising a cartridge, wherein the cartridge comprises at least one of:
• an inlet port, an outlet port, a pump, a valve, a vent, a reservoir, a sample collection chamber, a temperature control apparatus, or any combination thereof.