METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180179591A1
• Filed: 01/19/2018
• Published: 06/28/2018
• Est. Priority Date: 12/22/2016
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Belgrader, Phillip, Bent, Zachary, Mikkelsen, Tarjei Sigurd, Ryvkin, Paul, Terry, Jessica Michele
• Granted Patent: US 10,323,278 B2
• Time in Patent Office: Days
• CPC Class Codes: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/683 involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2563/159 Microreactors, e.g. emulsion PCR or sequencing, droplet PCR, microcapsules, i.e. non-liquid containers with a range of different permeability's for different reaction components
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

The present disclosure provides compositions, methods, systems, and devices for polynucleotide processing. Such polynucleotide processing may be useful for a variety of applications, including polynucleotide sequencing.

Claims

1. A method for analyzing an analyte in a cell, comprising:

(a) providing a plurality of partitions, wherein a given partition of said plurality of partitions comprises a plurality of analytes and a plurality of barcode molecules coupled to a bead, wherein (i) a first barcode molecule of said plurality of barcode molecules comprises a first nucleic acid barcode sequence capable of coupling to a first analyte of said plurality of analytes, wherein said first analyte is a CRISPR ribonucleic acid (crRNA) or a single guide ribonucleic acid (sgRNA), and wherein (ii) a second barcode molecule of said plurality of barcoded molecules comprises a second nucleic acid barcode sequence capable of coupling to a second analyte of said plurality of analytes;

(b) in said given partition, (i) coupling said first barcode molecule to said first analyte or a derivative thereof, and synthesizing a first nucleic acid molecule comprising at least a portion of said first nucleic acid barcode sequence or a complement thereof and a sequence of said first analyte or a complement thereof; and

(ii) coupling said second barcode molecule to said second analyte or a derivative thereof, and synthesizing a second nucleic acid molecule comprising at least a portion of said second nucleic acid barcode sequence or complement thereof and a sequence of said second analyte or complement thereof;

(c) processing (i) said first nucleic acid molecule or a derivative thereof and (ii) said second nucleic acid molecule or a derivative thereof, to identify said at least said portion of said first nucleic acid barcode sequence and said at least said portion of said second nucleic acid barcode sequence; and

(d) using said at least said portion of said first nucleic acid barcode sequence and said at least said portion of said second nucleic acid barcode sequence to identify said first analyte and said second analyte as originating from said cell.

2. The method of claim 1, further comprising removing said first nucleic acid molecule and said second nucleic acid molecule, or a derivative of said first nucleic acid molecule and/or said second nucleic acid molecule, from said given partition.

3. The method of claim 1, wherein, after (a), said first barcode molecule and/or said second barcode molecule is released from said bead.

4. The method of claim 1, further comprising subjecting said first nucleic acid molecule and said second nucleic acid molecule, or a derivative of said first nucleic acid molecule and/or said second nucleic acid molecule, to sequencing to characterize said first analyte or said second analyte.

5. The method of claim 1, wherein said first barcode molecule or said second barcode molecule comprises a unique molecular identification (UMI) sequence.

6. The method of claim 1, wherein (i) said first barcode molecule comprises a first priming sequence capable of hybridizing to said first analyte, or a derivative thereof;

(ii) said second barcode molecule comprises a second priming sequence capable of hybridizing to said second analyte, or a derivative thereof;

or (iii) said first barcode molecule comprises a first priming sequence capable of hybridizing to said first analyte, or a derivative thereof, and said second barcode molecule comprises a second priming sequence capable of hybridizing to said second analyte, or a derivative thereof.

7. The method of claim 6, wherein said second priming sequence comprises a poly-deoxythymine (poly(dT)) sequence.

8. The method of claim 6, wherein said first analyte comprises an adapter sequence complementary to said first priming sequence.

9. The method of claim 6, wherein said first priming sequence is complementary to a sequence in said crRNA or complementary to a sequence in said sgRNA.

10. The method of claim 1, wherein said second analyte is messenger ribonucleic acid (mRNA).

11. The method of claim 1, wherein said second analyte is genomic deoxyribonucleic acid (gDNA).

12. The method of claim 1, wherein said second analyte is a labelling agent capable of coupling to a protein.

13. The method of claim 1, wherein said first nucleic acid barcode sequence and said second nucleic barcode sequence are identical.

14. The method of claim 1, wherein said bead is a gel bead.

15. The method of claim 14, wherein said plurality of barcode molecules is reversibly coupled to said gel bead.

16. The method of claim 15, wherein said plurality of barcode molecules is reversibly coupled to said gel bead through chemical cross-links.

17. The method of claim 15, wherein said plurality of barcode molecules is reversibly coupled to said gel bead through disulfide bonds.

18. The method of claim 15, wherein said given partition further comprises an agent capable of releasing said first barcode molecule or said second barcode from said bead.

19. The method of claim 18, wherein said agent comprises a reducing agent.

20. The method of claim 1, wherein said given partition is a droplet among a plurality of droplets.

21. The method of claim 1, wherein said given partition is a well among a plurality of wells.

22. The method of claim 2, further comprising performing one or more reactions subsequent to removing said first nucleic acid molecule and said second nucleic acid molecule from said given partition.

23. The method of claim 15, wherein, after (a), said first barcode molecule and said second barcode molecule are released from said bead.

24. The method of claim 15, wherein, before or during (b), said first barcode molecule or said second barcode molecule is released from said bead.

25. The method of claim 1, wherein said plurality of partitions further comprise a template switching oligonucleotide (TSO) and wherein, in (b), synthesizing said first nucleic acid molecule or synthesizing said second nucleic acid molecule, or a derivative of said first nucleic acid molecule or said second nucleic acid molecule, includes the use of said template switching oligonucleotide.

26. The method of claim 25, wherein said TSO comprises a primer sequence and wherein said first nucleic acid molecule or said second nucleic acid molecule, or a derivative of said first nucleic acid molecule or said second nucleic acid molecule, comprise said TSO primer sequence.

27. The method of claim 1, wherein said plurality of partitions comprise a plurality of cells, wherein at least a subset of said plurality of cells comprise one or more sgRNAs, and wherein said given partition of said plurality of partitions comprises a cell comprising said one or more sgRNAs.

28. The method of claim 27, wherein said given partition of said plurality of partitions comprises a single cell.