Methods and systems for processing polynucleotides

• US 10,273,541 B2
• Filed: 08/01/2018
• Issued: 04/30/2019
• 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:
  US16/052,431
• Publication Number:
  US 20180346979A1
• Time in Patent Office: 272 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 2521/107 RNA dependent DNA polymerase,(i.e. reverse transcriptase)
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• 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

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 processing messenger ribonucleic acid (mRNA) molecules from a single cell, comprising:

(a) partitioning a plurality of cells and a plurality of beads in a microwell array comprising a plurality of wells, wherein a well of said plurality of wells comprises said single cell from said plurality of cells and a single bead from said plurality of beads, and wherein said single bead comprises nucleic acid barcode molecules each comprising a common barcode sequence;

(b) in said well comprising said single cell and said single bead, releasing messenger ribonucleic acid (mRNA) molecules from said single cell, wherein upon release from said single cell, said released mRNA molecules attach to said nucleic acid barcode molecules;

(c) subjecting said released mRNA molecules attached to said nucleic acid barcode molecules to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules each comprising said common barcode sequence or a complement thereof; and

(d) subjecting said cDNA molecules to one or more reactions to generate a set of nucleic acid molecules for nucleic acid sequencing.

2. The method of claim 1, wherein (c) is performed in said well comprising said single cell and said single bead, and wherein subsequent to (c), said cDNA molecules, or derivatives thereof, are removed from said well.

3. The method of claim 1, wherein, prior to (c), said released mRNA molecules attached to said nucleic acid barcode molecules are removed from said well comprising said single cell and said single bead.

4. The method of claim 1, wherein each of said nucleic acid barcode molecules comprises a universal primer sequence.

5. The method of claim 1, wherein said single bead is a single magnetic bead.

6. The method of claim 5, wherein said nucleic acid barcode molecules are attached to said single magnetic bead.

7. The method of claim 6, wherein prior to (c), said single magnetic bead comprising said released mRNA molecules attached to said nucleic acid barcode molecules are removed from said well comprising said single cell and said single bead.

8. The method of claim 7, wherein said single magnetic bead is removed using a magnetic field.

9. The method of claim 1, wherein each of said nucleic acid barcode molecules comprises a sequence for priming the synthesis of cDNA.

10. The method of claim 1, wherein said plurality of cells comprises at least 100 cells.

11. The method of claim 1, wherein said plurality of cells comprises at least 1,000 cells.

12. The method of claim 1, wherein said plurality of cells comprises at least 10,000 cells.

13. The method of claim 1, wherein said released mRNA molecules attach to said nucleic acid barcode molecules by hybridization.

14. The method of claim 1, wherein said plurality of beads comprises a plurality of nucleic acid barcode molecules comprising barcode sequences that are different across said plurality of beads.

15. The method of claim 1, further comprising, prior to (c), (i) pooling said released mRNA molecules attached to said nucleic acid barcode molecules and (ii) performing said one or more reactions in bulk.

16. The method of claim 1, wherein said one or more reactions comprise nucleic acid amplification that generates amplified products from said plurality of cDNA molecules.

17. The method of claim 16, wherein said nucleic acid amplification adds functional sequences to said amplified products, wherein said functional sequences permit attachment of said amplified products to a flow cell of a sequencer for said nucleic acid sequencing.

18. The method of claim 16, further comprising ligating functional sequences to said amplified products, wherein said functional sequences permit attachment of said amplified products to a flow cell of a sequencer for said nucleic acid sequencing.

19. The method of claim 16, wherein said nucleic acid amplification is polymerase chain reaction.

20. The method of claim 1, wherein said one or more reactions comprise addition of functional sequences to said plurality of cDNA molecules, wherein said functional sequences permit attachment to a flow cell of a sequencer for said nucleic acid sequencing.

21. The method of claim 1, further comprising performing said nucleic acid sequencing on said set of nucleic acid molecules, or derivatives thereof, to generate a plurality of sequences comprising sequences corresponding to said released mRNA molecules and said common barcode sequence.

22. The method of claim 1, wherein said plurality of beads have substantially monodisperse cross-sectional dimensions.

23. The method of claim 1, wherein said nucleic acid barcode molecules further comprise functional sequences that facilitate sequencing of said set of nucleic acid molecules.

24. The method of claim 4, wherein said universal primer sequence is a random N-mer.

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

26. The method of claim 1, wherein said microwell array comprises 100,000 wells.

27. The method of claim 1, wherein said microwell array comprises 200,000 wells.

28. The method of claim 1, wherein said microwell array comprises 1,000,000 wells.

29. The method of claim 1, wherein a subset of said plurality of wells in said microwell array does not include a cell.

30. The method of claim 1, wherein a subset of said plurality of wells in said microwell array does not include a bead.

31. The method of claim 25, wherein said nucleic acid barcode molecules are attached to said single gel bead.

32. The method of claim 25, wherein prior to (c), said single gel bead comprising said released mRNA molecules attached to said nucleic acid barcode molecules is removed from said well.

33. The method of claim 1, wherein releasing said mRNA molecules from said single cell comprises lysing said single cell.

34. The method of claim 33, wherein lysing said single cell comprises treating said single cell with a detergent.

35. The method of claim 33, wherein lysing said single cell comprises heating said single cell.

36. The method of claim 1, wherein said nucleic acid barcode molecules comprise a sequence complementary to said released mRNA molecules.

37. The method of claim 1, wherein said plurality of cells is a plurality of cancer cells.

38. The method of claim 1, wherein said nucleic acid barcode molecules comprise uracil.

39. The method of claim 1, wherein said nucleic acid barcode molecules comprise 1,000 nucleic acid barcode molecules.

40. The method of claim 1, wherein said nucleic acid barcode molecules comprise 100,000 nucleic acid barcode molecules.

41. The method of claim 1, wherein said plurality of beads comprises 1,000 beads.

42. The method of claim 1, wherein said plurality of beads comprises 100,000 beads.

43. The method of claim 1, wherein said plurality of wells comprises 1,000 wells.

44. The method of claim 1, wherein said plurality of wells comprises 100,000 wells.

45. The method of claim 1, wherein said plurality of wells comprises 1,000,000 wells.

46. The method of claim 1, wherein said well has a volume of less than about 5 nanoliters (nL).

47. The method of claim 1, wherein said well has a volume of less than about 500 nanoliters (nL).

48. The method of claim 14, wherein said barcode sequences are capable of distinguishing nucleic acid molecules in different wells of said plurality of wells.

49. A method for processing messenger ribonucleic acid (mRNA) molecules from single cells, comprising:

(a) partitioning a plurality of cells and a plurality of beads in a microwell array comprising a plurality of wells, wherein said plurality of wells comprises 1,000 occupied wells, each occupied well of said 1,000 occupied wells comprising a single cell of said plurality of cells and a single bead of said plurality of beads, and wherein said single bead comprises nucleic acid barcode molecules comprising (i) a common barcode sequence capable of identifying said single cell and (ii) a capture sequence, wherein said common barcode sequence is different for different occupied wells of said 1,000 occupied wells;

(b) releasing messenger ribonucleic acid (mRNA) molecules from single cells of said 1,000 occupied wells, wherein released mRNA molecules in each occupied well of said 1,000 occupied wells are captured by said nucleic acid barcode molecules via said capture sequence subsequent to release;

(c) subjecting said released mRNA molecules in each occupied well of said 1,000 occupied wells to reverse transcription to yield complementary deoxyribonucleic acid (cDNA) molecules comprising said common barcode sequence or complement thereof; and

(d) subjecting cDNA molecules or derivatives thereof generated in each occupied well of said 1,000 occupied wells to one or more nucleic acid amplification reactions to generate a set of barcoded nucleic acid molecules for nucleic acid sequencing.

50. The method of claim 49, wherein (c) is performed in said plurality of wells, and wherein said cDNA molecules or derivatives thereof are removed from said plurality wells and pooled prior to (d).

51. The method of claim 49, wherein (c) is performed after removing said released mRNA molecules captured by said nucleic acid barcode molecules from said plurality of wells.

52. The method of claim 51, further comprising pooling said released mRNA molecules prior to (d).

53. The method of claim 49, wherein said plurality of beads is a plurality of magnetic beads.

54. The method of claim 53, wherein magnetic beads of said plurality of magnetic beads are removed from said plurality of wells using a magnetic field.

55. The method of claim 49, wherein said released mRNA molecules are captured by hybridization of the released mRNA molecule of to the capture sequence.

56. The method of claim 49, wherein said plurality of wells comprises 5,000 occupied wells.

57. The method of claim 49, wherein said plurality of wells comprises 10,000 occupied wells.

58. The method of claim 49, wherein no more than 5% of occupied wells of said 1,000 occupied wells have more than one cell.

59. The method of claim 58, wherein no more than 1% of occupied wells of said 1,000 occupied wells have more than one cell.

60. The method of claim 49, wherein no more than 5% of occupied wells of said 1,000 occupied wells have more than one bead.

61. The method of claim 60, wherein no more than 1% of occupied wells of said 1,000 occupied wells have more than one bead.

62. The method of claim 49, wherein said nucleic acid barcode molecules further comprise (iii) an additional barcode sequence configured to aid in quantitation of mRNA molecules during said nucleic acid sequencing.

63. The method of claim 62, wherein said additional barcode sequence is a random sequence.

64. The method of claim 62, wherein a bead of said plurality of bead comprises a first nucleic acid barcode molecule having a first additional barcode sequence and a second nucleic acid barcode molecule having a second additional barcode sequence different from said first additional barcode sequence.

65. The method of claim 49, wherein said one or more nucleic acid amplification reactions add functional sequences to barcoded nucleic acid molecules of said set of nucleic acid molecules that permit binding of said barcoded nucleic acid molecules to a flow cell of a sequencer.

66. The method of claim 49, further comprising ligating functional sequences to barcoded nucleic acid molecules of said set of barcoded nucleic acid molecules that permit binding of said barcoded nucleic acid molecules to a flow cell of a sequencer.

67. The method of claim 49, wherein said one or more nucleic acid amplification reactions are polymerase chain reactions.

68. The method of claim 49, wherein wells of said plurality of wells have a volume of less than about 5 nanoliters (nL).

69. The method of claim 49, wherein wells of said plurality of wells have a volume of less than about 500 nanoliters (nL).

70. The method of claim 49, wherein said microwell array comprises 100,000 wells.

71. The method of claim 49, wherein said microwell array comprises 200,000 wells.

72. The method of claim 49, wherein said microwell array comprises 1,000,000 wells.

73. The method of claim 49, wherein a subset of said plurality of wells in said microwell array does not include a cell.

74. The method of claim 49, wherein a subset of said plurality of wells in said microwell array does not include a bead.