Methods and systems for processing polynucleotides

• US 10,752,949 B2
• Filed: 12/06/2018
• Issued: 08/25/2020
• Est. Priority Date: 08/14/2012
• 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
• Primary Examiner(s): Thomas, David C
• Application Number:
  US16/212,441
• Publication Number:
  US 20190136316A1
• Time in Patent Office: 628 Days
• 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 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/185 incorporating bases where the precise position of the bases in the nucleic acid string is important
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2531/10 the purpose being amplify/increase the copy number of target nucleic acid
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• 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 2565/629 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 processing, comprising:

(a) providing a partition comprising a plurality of sample nucleic acid molecules and a bead, wherein said bead comprises a plurality of barcode oligonucleotide molecules that are releasable from said bead through cleavage of a photo-labile linkage, and wherein a subset of barcode oligonucleotide molecules of said plurality of barcode oligonucleotide molecules comprises a common barcode sequence and a targeted primer sequence;

(b) applying a photo-stimulus to the partition to release said subset of said plurality of barcode oligonucleotide molecules from said bead, wherein said subset of said plurality of barcode oligonucleotide molecules hybridizes to a subset of said plurality of sample nucleic acid molecules via said targeted primer sequence; and

(c) using said subset of said plurality of sample nucleic acid molecules comprising said subset of said plurality of barcode oligonucleotide molecules hybridized thereto to generate a plurality of barcoded nucleic acid molecules, wherein a barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises said common barcode sequence or a complement thereof.

2. The method of claim 1, wherein said partition further comprises nucleic acid extension reagents.

3. The method of claim 1, wherein said partition further comprises a plurality of primers.

4. The method of claim 1, wherein (c) comprises performing a nucleic acid amplification reaction on said subset of said plurality of sample nucleic acid molecules or a derivative thereof.

5. The method of claim 1, wherein said common barcode sequence comprises at least 6 nucleotides.

6. The method of claim 1, wherein said partition comprises a lysate from a cell.

7. The method of claim 6, wherein said cell is a cancer cell.

8. The method of claim 6, wherein said cell is derived from a solid tumor.

9. The method of claim 6, wherein said cell is derived from a hematological malignancy.

10. The method of claim 6, wherein said cell is a circulating tumor cell.

11. The method of claim 1, wherein said partition is a microwell.

12. The method of claim 1, wherein said partition is a droplet.

13. The method of claim 1, wherein said plurality of sample nucleic acid molecules comprises genomic deoxyribonucleic acid (gDNA).

14. The method of claim 1, further comprising removing said barcoded nucleic acid molecule, or a derivative thereof, from said partition.

15. The method of claim 14, wherein said partition is a droplet in an emulsion, and wherein said removing comprises breaking said emulsion.

16. The method of claim 1, wherein (b) occurs prior to (c).

17. The method of claim 1, wherein said partition comprises a nuclease inactivating agent.

18. The method of claim 17, wherein said nuclease inactivating agent is proteinase K.

19. The method of claim 1, wherein said bead further comprises an additional barcode oligonucleotide molecule having an additional targeted primer sequence.

20. The method of claim 1, further comprising amplifying said barcoded nucleic acid molecule or a derivative thereof to provide a plurality of amplified barcoded nucleic acid molecules.

21. The method of claim 1, further comprising attaching a functional sequence to said barcoded nucleic acid molecule or a derivative thereof.

22. The method of claim 21, wherein said functional sequence is a sequencing primer.

23. The method of claim 21, wherein said functional sequence is a sample index sequence.

24. The method of claim 1, wherein said barcoded nucleic acid molecule, or a derivative thereof, is sequenced.

25. The method of claim 14, wherein said barcoded nucleic acid molecule, or a derivative thereof, is sequenced.

26. The method of claim 20, wherein an amplified barcoded nucleic acid molecule of said plurality of amplified barcoded nucleic acid molecules is sequenced.

27. The method of claim 1, wherein said common barcode sequence is a unique identifier for said partition.

28. A method for nucleic acid processing, comprising:

(a) co-partitioning a plurality of beads with nucleic acid contents of individual cells to provide a plurality of partitions, wherein a partition of said plurality of partitions comprises (i) a bead and (ii) nucleic acid contents of an individual cell, wherein said bead comprises a plurality of barcode oligonucleotide molecules that are releasable from said bead through cleavage of a photo-labile linkage, and wherein a subset of barcode oligonucleotide molecules of said plurality of barcode oligonucleotide molecules comprises a common barcode sequence and a targeted primer sequence;

(b) applying a photo-stimulus to said subset of said plurality of barcode oligonucleotide molecules from said bead, wherein said subset of said plurality of barcode oligonucleotide molecules hybridizes to a subset of said nucleic acid contents of said individual cell via said targeted primer sequence; and

(c) using said subset of said nucleic acid contents of said individual cell comprising said subset of said plurality of barcode oligonucleotide molecules hybridized thereto to generate a plurality of barcoded nucleic acid molecules, wherein a barcoded nucleic acid molecule of said plurality of barcoded nucleic acid molecules comprises said common barcode sequence, or a complement thereof.

29. The method of claim 28, further comprising pooling said plurality of barcoded nucleic acid molecules, or derivative thereof, from said partition with other barcoded nucleic acid molecules, or derivatives thereof, from other partitions of said plurality of partitions.

30. The method of claim 28, wherein said partition further comprises nucleic acid extension reagents.

31. The method of claim 28, wherein said partition further comprises a plurality of primers.

32. The method of claim 28, wherein (c) comprises performing a nucleic acid amplification reaction on said subset of said nucleic acid contents of said individual cell or a derivative thereof.

33. The method of claim 28, wherein said common barcode sequence comprises at least ( nucleotides.

34. The method of claim 28, wherein said cell is a cancer cell.

35. The method of claim 28, wherein said cell is derived from a solid tumor.

36. The method of claim 28, wherein said cell is derived from a hematological malignancy.

37. The method of claim 28, wherein said cell is a circulating tumor cell.

38. The method of claim 28, wherein said partition is a microwell.

39. The method of claim 28, wherein said partition is a droplet.

40. The method of claim 28, wherein said nucleic acid contents of said individual cell comprises genomic deoxyribonucleic acid (gDNA) from said individual cell.

41. The method of claim 28, further comprising removing said barcoded nucleic acid molecule, or a derivative thereof, from said partition.

42. The method of claim 28, wherein said partition is a droplet in an emulsion, and wherein said removing comprises breaking said emulsion.

43. The method of claim 28, wherein (b) occurs prior to (c).

44. The method of claim 28, wherein said partition comprises a nuclease inactivating agent.

45. The method of claim 28, wherein said nuclease inactivating agent is proteinase K.

46. The method of claim 45, wherein said nuclease inactivating agent is proteinase K.

47. The method of claim 28, wherein said bead further comprises an additional barcode oligonucleotide molecule having an additional targeted primer sequence.

48. The method of claim 28, further comprising amplifying said barcoded nucleic acid molecule or a derivative thereof to provide a plurality of amplified barcoded nucleic acid molecules.

49. The method of claim 28, further comprising attaching a functional sequence to said barcoded nucleic acid molecule or a derivative thereof.

50. The method of claim 49, wherein said functional sequence is a sequencing primer.

51. The method of claim 49, wherein said functional sequence is a sample index sequence.

52. The method of claim 28, wherein said barcoded nucleic acid molecule, or a derivative thereof, is sequenced.

53. The method of claim 41, wherein said barcoded nucleic acid molecule, or a derivative thereof, is sequenced.

54. The method of claim 48, wherein an amplified barcoded nucleic acid molecule of said plurality of amplified barcoded nucleic acid molecules is sequenced.

55. The method of claim 28, wherein said common barcode sequence is a unique identifier for said partition.