Methods and systems for processing polynucleotides

Patent metadata

Field	Value
Publication number	US-9951386-B2
Application number	US-201715717871-A
Country	US
Kind code	B2
Filing date	Sep 27, 2017
Priority date	Jun 26, 2014
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

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.

First claim

What is claimed is:

1. A method for single cell processing or analysis, comprising:
(a) causing a single cell from a plurality of cells to contact one or more antibodies such that an antibody from said one or more antibodies couples to said single cell, wherein said antibody is associated with a first nucleic acid molecule comprising a first barcode sequence;
(b) co-partitioning said single cell having said antibody coupled thereto 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 1,000,000 nucleic acid molecules each comprising said second barcode sequence.

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

5. The method of claim 4 , further comprising disrupting said droplet to release said third nucleic acid molecule from said droplet.

6. The method of claim 5 , further comprising subjecting said third nucleic acid molecule to one or more reactions subsequent to releasing said third nucleic acid molecule from said droplet.

7. The method of claim 6 , wherein said one or more reactions comprise polymerase chain reaction.

8. The method of claim 6 , 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.

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

10. The method of claim 9 , further comprising, prior to (c), releasing said second nucleic acid molecule from said single bead.

11. The method of claim 10 , wherein said second nucleic acid molecule is released from said single bead upon exposure to a chemical stimulus in said droplet.

12. 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 or complement thereof and said second barcode sequence or complement thereof, wherein said first barcode sequence or complement thereof identifies a cell surface feature to which said antibody is specific and said second barcode sequence or complement thereof identifies said single cell.

13. The method of claim 12 , wherein said cell surface feature is a cell surface protein.

14. The method of claim 2 , wherein said second nucleic acid molecule further comprises an identifier sequence, wherein said identifier sequence of said second nucleic acid molecule is different from identifier sequences of other nucleic acid molecules of said plurality of nucleic acid molecules.

15. 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.

16. The method of claim 15 , wherein said sequence is a poly-thymine (poly-T) sequence.

17. The method of claim 16 , wherein (c) comprises hybridizing said poly-T sequence to said portion of said first nucleic acid molecule, wherein said portion of said first nucleic acid molecule comprises a poly-adenine (poly-A) sequence.

18. The method of claim 1 , wherein said generating of (c) comprises performing a nucleic acid amplification reaction.

19. The method of claim 1 , wherein said generating of (c) comprises performing a reverse transcription reaction.

20. The method of claim 1 , wherein said generating of (c) comprises performing a template switching reaction.

21. 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.

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

23. The method of claim 1 , wherein, in at least (a), said antibody is covalently attached to said first nucleic acid molecule.

24. The method of claim 1 , wherein, in at least (a), said antibody is non-covalently attached to said first nucleic acid molecule.

25. A method for single cell processing or analysis, comprising:
(a) co-partitioning a 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.

26. The method of claim 23 , wherein, in at least (a), said antibody is coupled to a cell surface feature of said single cell.

27. The method of claim 23 , wherein said partition is among a plurality of partitions.

28. The method of claim 1 , further comprising releasing said third molecule from said droplet and performing one or more reactions on said third molecule subsequent to release from said droplet.

29. The method of claim 2 , 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.

30. The method of claim 25 , wherein said partition is a droplet.

31. The method of claim 25 , wherein said partition is a well.

32. The method of claim 25 , wherein said second nucleic acid molecule is releasably attached to said single bead.

33. The method of claim 32 , wherein said partition comprises an agent that releases said nucleic acid molecule from said single bead.

34. The method of claim 33 , wherein said agent is a reducing agent.

35. The method of claim 32 , further comprising, after (a), releasing said second nucleic acid molecule from said single bead.

36. The method of claim 25 , wherein said single bead is a gel bead.

37. The method of claim 36 , wherein said gel bead comprises a disulfide bond.

38. The method of claim 25 , wherein said second nucleic acid molecule is covalently attached to said single bead.

39. The method of claim 38 , wherein said second nucleic acid molecule is coupled to said single bead via a disulfide bond.

40. The method of claim 25 , wherein said second nucleic acid molecule is not releasably coupled to said sin

Assignees

• 10X Genomics Inc

Inventors

• Hindson Benjamin
• Hindson Christopher
• Schnall-Levin Michael
• Ness Kevin
• Jarosz Mirna
• Saxonov Serge

Classifications

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q1/6874 Primary

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2563/149

  Particles, e.g. beads · CPC title

• C12Q2563/159

  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 title