METHODS AND SYSTEMS FOR PROCESSING POLYNUCLEOTIDES

• US 20180016634A1
• Filed: 09/27/2017
• Published: 01/18/2018
• 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, Phillip
• Granted Patent: US 9,951,386 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 single cell processing or analysis, comprising:

(a) bringing said single cell from a plurality of cells in contact with one or more antibodies, wherein an antibody from said one or more antibodies comprises a first nucleic acid molecule comprising a first barcode sequence;

(b) co-partitioning said single cell with a single bead from a plurality of beads in a droplet, wherein said single bead comprises a second nucleic acid molecule comprising a second barcode sequence; and

(c) generating from said first nucleic acid molecule and said second nucleic acid molecule, a third nucleic acid molecule comprising (i) said first barcode sequence or a complement thereof, and (ii) said second barcode sequence or a complement thereof.

2. The method of claim 1, wherein said single bead comprises a plurality of nucleic acid molecules comprising said second nucleic acid molecule, wherein each of said plurality of nucleic acid molecules comprises said second barcode sequence.

3. The method of claim 2, wherein said plurality of nucleic acid molecules comprises at least 1,000 nucleic acid molecules each comprising said second barcode sequence.

4. The method of claim 2, wherein said plurality of nucleic acid molecules comprises at least 1,000,000 nucleic acid molecules each comprising said second barcode sequence.

5. The method of claim 1, wherein (c) is performed in said droplet.

6. The method of claim 5, further comprising disrupting said droplet to release said third nucleic acid molecule from said droplet.

7. The method of claim 6, further comprising subjecting said third nucleic acid molecule to one or more reactions subsequent to releasing said third nucleic acid molecule from said droplet.

8. The method of claim 7, wherein said one or more reactions comprise polymerase chain reaction.

9. The method of claim 7, 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.

10. The method of claim 1, wherein said plurality of beads comprises a plurality of nucleic acid molecules having barcodes sequences that are different across said plurality of beads.

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

12. The method of claim 11, further comprising, prior to (c), releasing said second nucleic acid molecule from said single bead.

13. The method of claim 12, wherein said second nucleic acid molecule is released from said single bead upon exposure to a chemical stimulus in said droplet.

14. The method of claim 1, further comprising using a sequencer to sequence 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 a protein to which said antibody is specific and said second barcode sequence identifies said single cell.

15. The method of claim 14, wherein said protein is a cell surface protein.

16. The method of claim 1, wherein said second nucleic acid molecule further comprises a unique molecular sequence.

17. The method of claim 1, wherein said second nucleic acid molecule further comprises a sequence that is complementary to a portion of said first nucleic acid molecule.

18. The method of claim 17, wherein said sequence is a poly-T sequence.

19. The method of claim 18, wherein (c) comprises hybridizing said poly-T sequence to said portion of said first nucleic acid molecule comprising a poly-A sequence.

20. The method of claim 1, wherein (c) comprises performing a nucleic acid amplification reaction to generate said third nucleic acid molecule.

21. The method of claim 1, wherein (c) comprises performing a reverse transcription reaction to generate said third nucleic acid molecule.

22. The method of claim 1, wherein (c) comprises performing a template switching reaction to generate said third nucleic acid molecule.

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

24. The method of claim 1, further comprising, subsequent to (c), adding one or more priming sequences for priming extension of said third nucleic acid molecule or a derivative thereof in a sequencer.

25. The method of claim 1, wherein said antibody is covalently attached to said first nucleic acid molecule.

26. The method of claim 1, wherein said antibody is non-covalently attached to said first nucleic acid molecule.

27. A method for single cell processing or analysis, comprising:

(a) co-partitioning said single cell from a plurality of cells with a single bead from a plurality of beads in a partition, wherein said single cell is coupled to an antibody comprising a first nucleic acid molecule comprising a first barcode sequence, wherein said single bead comprises a second nucleic acid molecule comprising a second barcode sequence; and

(b) in said partition, generating from said first nucleic acid molecule and said second nucleic acid molecule, a third nucleic acid molecule comprising (i) said first barcode sequence or a complement thereof, and (ii) said second barcode sequence or a complement thereof.

28. The method of claim 27, wherein said partition is a droplet or a well.

29. The method of claim 27, wherein said antibody is coupled to a protein of said single cell.

30. The method of claim 27, wherein said partition is among a plurality of partitions.