Methods and systems for processing polynucleotides

• US 10,337,061 B2
• Filed: 09/21/2018
• Issued: 07/02/2019
• 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, Xinying, Belgrader, Phillip
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/138,448
• Publication Number:
  US 20190024166A1
• Time in Patent Office: 284 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 nucleic acid sequencing, comprising:

(a) co-partitioning a plurality of beads and a plurality of primers in a plurality of droplets, wherein a droplet of said plurality of droplets comprises (i) a ribonucleic acid (RNA) molecule comprising a nucleic acid sequence, (ii) a primer from said plurality of primers, and (iii) a bead from said plurality of beads, wherein said bead comprises a nucleic acid barcode molecule coupled thereto, and wherein said nucleic acid barcode molecule comprises a barcode sequence;

(b) hybridizing said primer to a region at a 3′

end of said RNA molecule;

(c) using an enzyme to extend said primer to generate a nucleic acid product comprising a sequence corresponding to said nucleic acid sequence of said RNA molecule, wherein said enzyme incorporates an end sequence at a 3′

end of said nucleic acid product that is complementary to said nucleic acid barcode molecule;

(d) hybridizing said nucleic acid barcode molecule to said nucleic acid product generated in (c) and extending said nucleic acid product using said nucleic acid barcode molecule as a template, to generate a barcoded nucleic acid molecule comprising, from a 5′

end to a 3′

end, (1) said sequence corresponding to said nucleic acid sequence of said RNA molecule and (2) a complement of said barcode sequence; and

(e) sequencing said barcoded nucleic acid molecule or derivative thereof,

wherein, after (a), said nucleic acid barcode molecule is released from said bead.

2. The method of claim 1, wherein said RNA molecule is from a cell.

3. The method of claim 2, wherein said droplet comprises said cell.

4. The method of claim 3, further comprising releasing said RNA molecule from said cell prior to (b).

5. The method of claim 1, wherein said bead further comprises a plurality of nucleic acid molecules coupled thereto.

6. The method of claim 5, wherein each of said plurality of nucleic acid molecules comprises said barcode sequence.

7. The method of claim 6, wherein each of said plurality of nucleic acid molecules comprises an additional barcode sequence that varies across said plurality of nucleic acid molecules.

8. The method of claim 1, further comprising, prior to (e), subjecting said barcoded nucleic acid molecule or derivative thereof to nucleic acid amplification.

9. The method of claim 8, wherein said nucleic acid amplification is performed subsequent to releasing said barcoded nucleic acid molecule or derivative thereof from said droplet.

10. The method of claim 8, wherein said nucleic acid amplification is polymerase chain reaction.

11. The method of claim 1, wherein said RNA molecule is a messenger ribonucleic acid (mRNA) molecule.

12. The method of claim 1, wherein in (a) said droplet comprises (i) a nucleic acid molecule comprising an additional nucleic acid sequence, and (ii) an additional nucleic acid barcode molecule comprising an additional barcode sequence, and wherein said nucleic acid molecule and said additional nucleic acid barcode molecule are used to generate an additional barcoded nucleic acid molecule comprising, from a 5′

end to a 3′

end, (1) said additional barcode sequence and (2) an additional sequence corresponding to said additional nucleic acid sequence.

13. The method of claim 12, wherein said additional nucleic acid barcode molecule is coupled to said bead.

14. The method of claim 1, wherein (b)-(d) are performed in said droplet.

15. The method of claim 1, further comprising releasing said barcoded nucleic acid molecule or derivative thereof from said droplet.

16. The method of claim 1, wherein said barcoded nucleic acid molecule further comprises, towards a 5′

end, a functional sequence for permitting said barcoded nucleic acid molecule or derivative thereof to couple to a flow cell of a sequencer.

17. The method of claim 1, wherein said sequence corresponding to said nucleic acid sequence of said RNA molecule is a reverse complement of said nucleic acid sequence.

18. The method of claim 1, further comprising, prior to (e), using said barcoded nucleic acid molecule or derivative thereof and a pair of primers to generate barcoded nucleic acid molecules having a target nucleic acid sequence.

19. The method of claim 18, wherein said barcoded nucleic acid molecules having said target nucleic acid sequence or derivatives thereof are sequenced in (e).

20. The method of claim 1, wherein said nucleic acid barcode molecule is released from said bead before said barcoded nucleic acid molecule is generated.

21. The method of claim 1, wherein said nucleic acid barcode molecule is released from said bead while said barcoded nucleic acid molecule is generated.

22. The method of claim 1, wherein said nucleic acid barcode molecule is released from said bead after said barcoded nucleic acid molecule is generated.

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

24. The method of claim 1, wherein said barcode sequence is a combinatorial assembly of a plurality of barcode segments.

25. The method of claim 24, wherein said plurality of barcode segments comprises at least three barcode segments.

26. The method of claim 1, wherein said enzyme has terminal transferase activity.

27. The method of claim 1, wherein said bead is degradable upon application of a stimulus.

28. The method of claim 27, wherein said stimulus is a reducing agent.

29. The method of claim 27, wherein said droplet comprises said stimulus.

30. The method of claim 27, wherein releasing said nucleic acid barcode molecule from said bead comprises degrading said bead.

31. The method of claim 1, wherein said bead comprises disulfide bonds.

32. The method of claim 1, wherein said nucleic acid barcode molecule is coupled to said bead via a disulfide bond.

33. The method of claim 1, wherein said enzyme is a reverse transcriptase.

34. The method of claim 5, wherein said plurality of nucleic acid molecules comprises at least 10,000 nucleic acid molecules.

35. The method of claim 5, wherein said plurality of nucleic acid molecules comprises at least 100,000 nucleic acid molecules.

36. The method of claim 5, wherein said plurality of nucleic acid molecules comprises at least 1,000,000 nucleic acid molecules.

37. The method of claim 5, wherein said plurality of nucleic acid molecules comprises at least 10,000,000 nucleic acid molecules.

38. The method of claim 7, wherein said additional barcode sequence is different for each nucleic acid molecule of said plurality of nucleic acid molecules.

39. The method of claim 1, wherein said barcoded nucleic acid molecule further comprises a functional sequence.

40. The method of claim 39, wherein said functional sequence is a sequencing primer binding sequence or a sequencing primer sequence.

41. The method of claim 1, wherein said end sequence is a poly-cytosine (polyC) sequence.

42. The method of claim 41, wherein said nucleic acid barcode molecule comprises a poly-guanine (polyG) sequence configured to hybridize with said polyC sequence.

43. The method of claim 3, wherein said droplet comprises only one cell.