METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180142292A1
• Filed: 01/16/2018
• Published: 05/24/2018
• Est. Priority Date: 06/26/2014
• Status: Active Grant

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10X Genomics Incorporated
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Saxonov, Serge, Hardenbol, Paul, Bharadwaj, Rajiv, Zheng, Grace, Belgrader, Philip
• Granted Patent: US 10,041,116 B2
• Time in Patent Office: Days
• 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 2525/191 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 2537/149 Sequential reactions
• 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 2565/629 being a microfluidic device

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 identifying an analyte from a single cell, comprising:

(a) causing an analyte of said single cell to contact one or more antibodies, thereby providing an antibody from said one or more antibodies coupled to said analyte, wherein said antibody is associated with a first nucleic acid molecule comprising a first barcode sequence;

(b) using said first nucleic acid molecule and a second nucleic acid molecule to generate a third nucleic acid molecule, wherein said second nucleic acid molecule comprises a second barcode sequence, and wherein said third nucleic acid molecule comprises (i) said first barcode sequence or a complement thereof, and (ii) said second barcode sequence or a complement thereof;

(c) processing said third nucleic acid molecule or a derivative thereof to identify said first barcode sequence and said second barcode sequence; and

(d) using said first barcode sequence and said second barcode sequence identified in (c) to identify said analyte of said single cell.

2. The method of claim 1, wherein said analyte is a protein.

3. The method of claim 2, wherein said protein is coupled to a surface of said single cell.

4. The method of claim 1, wherein (b) is performed in a partition among a plurality of partitions.

5. The method of claim 4, further comprising co-partitioning said single cell or said analyte from said single cell with a single bead from a plurality of beads into said partition, wherein said single bead comprises said second nucleic acid molecule comprising said second barcode sequence.

6. The method of claim 5, wherein said second nucleic acid molecule is releasably attached to said single bead.

7. The method of claim 6, further comprising releasing said second nucleic acid molecule from said single bead.

8. The method of claim 5, wherein said plurality of beads is a plurality of gel beads and said single bead is a single gel bead.

9. The method of claim 8, wherein said second nucleic acid molecule is reversibly coupled to said gel bead through a chemical cross-link.

10. The method of claim 8, wherein said second nucleic acid molecule is reversibly coupled to said gel bead through a disulfide bond.

11. The method of claim 10, wherein said partition further comprises an agent capable of releasing said second nucleic acid molecule from said bead gel bead.

12. The method of claim 11, wherein said agent comprises a chemical reducing agent.

13. The method of claim 5, wherein said second barcode sequence is different than other barcode sequences of other nucleic acid molecules comprised in other beads of said plurality of beads.

14. The method of claim 5, wherein said single bead is co-partitioned with said single cell in said partition.

15. The method of claim 14, further comprising lysing said single cell in said partition.

16. The method of claim 4, wherein said partition is a droplet among a plurality of droplets

17. The method of claim 4, wherein said partition is a well among a plurality of wells.

18. The method of claim 4, further comprising, subsequent to (b), releasing said third nucleic acid molecule or derivative thereof from said partition, and performing (c) subsequent to release of said third nucleic acid molecule or derivative thereof from said partition.

19. The method of claim 1, wherein said second nucleic acid molecule comprises a unique molecular identification (UMI) sequence.

20. The method of claim 1, wherein said second nucleic acid molecule comprises a sequence complementary to a sequence in said first nucleic acid molecule.

21. The method of claim 1, wherein said second nucleic acid molecule comprises a first sequencing primer sequence.

22. The method of claim 21, wherein said first nucleic acid molecule comprises a second sequencing primer sequence.

23. The method of claim 1, further comprising subjecting said third nucleic acid molecule to one or more reactions.

24. The method of claim 23, wherein said one or more reactions comprise polymerase chain reaction.

25. The method of claim 23, wherein said one or more reactions comprise addition of one or more functional sequences to said third nucleic acid molecule or a derivative thereof, wherein said one or more functional sequences are configured to permit attachment to a flow cell of a sequencer.

26. The method of claim 1, further comprising sequencing said third nucleic acid molecule or a derivative thereof to identify said first barcode sequence and said second barcode sequence, wherein said first barcode sequence identifies an analyte to which said antibody is specific and said second barcode sequence identifies nucleic acids originating from said single cell.

27. The method of claim 1, wherein (b) comprises performing one or more nucleic acid extension reactions on said first nucleic acid molecule and/or said second nucleic acid molecule to yield said third nucleic acid molecule.

28. The method of claim 1, wherein (b) comprises performing a nucleic acid amplification reaction on said third nucleic acid molecule.

29. The method of claim 1, wherein (b) comprises performing a template switching reaction.

30. The method of claim 1, wherein said third nucleic acid molecule or a derivative thereof comprises a functional sequence that is configured to permit attachment to a flow cell of a sequencer.