Compositions and methods for sample processing

• US 10,221,442 B2
• Filed: 06/26/2014
• Issued: 03/05/2019
• 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
• Primary Examiner(s): Priest, Aaron A
• Assistant Examiner(s): Oyeyemi, Olayinka A
• Application Number:
  US14/316,447
• Publication Number:
  US 20140378350A1
• Time in Patent Office: 1,713 Days
• Field of Search: None
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00547 Bar codes
• CPC Class Codes: B01J 2219/00572 Chemical means
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• 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 2535/122 Massive parallel sequencing
• 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

Abstract

This disclosure provides methods and compositions for sample processing, particularly for sequencing applications. Included within this disclosure are bead compositions, such as diverse libraries of beads attached to large numbers of oligonucleotides containing barcodes. Often, the beads provides herein are degradable. For example, they may contain disulfide bonds that are susceptible to reducing agents. The methods provided herein include methods of making libraries of barcoded beads as well as methods of combining the beads with a sample, such as by using a microfluidic device.

Claims

1. A method of processing a template nucleic acid sequence, comprising:

(a) co-partitioning a template nucleic acid sequence and a bead comprising a plurality of oligonucleotides releasably attached thereto into a partition, wherein each oligonucleotide of the plurality of oligonucleotides comprises a sequence comprising a common barcode sequence and a primer sequence complementary to one or more regions of the template nucleic acid sequence;

(b) annealing the primer sequence of an oligonucleotide of the plurality of oligonucleotides to the template nucleic acid sequence; and

(c) extending the primer sequence to produce an extension product complementary to at least a portion of the template nucleic acid sequence, the extension product comprising the primer sequence and the common barcode sequence,

wherein, after (a), the sequence of the oligonucleotide is released from the bead into the partition, and

wherein, after (c), the extension product is in solution within the partition.

2. The method of claim 1, wherein the primer sequence comprises a variable primer sequence.

3. The method of claim 2, wherein the variable primer sequence comprises a random N-mer.

4. The method of claim 1, wherein the primer sequence comprises a targeted primer sequence.

5. The method of claim 1, wherein the partition is a droplet in an emulsion.

6. The method of claim 1, further comprising providing a polymerase enzyme in the partition.

7. The method of claim 6, wherein the polymerase enzyme comprises an exonuclease deficient polymerase enzyme.

8. The method of claim 1, wherein (c) comprises extending the primer sequence using a strand displacing polymerase enzyme.

9. The method of claim 8, wherein the strand displacing polymerase enzyme comprises a thermostable strand displacing polymerase enzyme.

10. The method of claim 9, wherein the strand displacing polymerase enzyme has substantially no exonuclease activity.

11. The method of claim 1, wherein the oligonucleotide is exonuclease resistant.

12. The method of claim 11, wherein the oligonucleotide comprises one or more phosphorothioate linkages.

13. The method of claim 12, wherein the one or more phosphorothioate linkages comprise a phosphorothioate linkage at a terminal internucleotide linkage in the oligonucleotide.

14. The method of claim 1, wherein the bead is porous.

15. The method of claim 1, wherein the bead is non-porous.

16. The method of claim 1, wherein the oligonucleotide comprises a functional sequence that facilitates determining a sequence of the extension product.

17. The method of claim 1, wherein the bead is a gel bead.

18. The method of claim 17, wherein (b) comprises dissolving the gel bead, thereby releasing the sequence of the oligonucleotide from the gel bead into the partition.

19. The method of claim 1, wherein the plurality of oligonucleotides releasably attached to the bead is formed by coupling multiple oligonucleotides to each other.

20. The method of claim 1, wherein the plurality of oligonucleotides is generated on the bead with the aid of a splint sequence that is in part complementary to at least a portion of a first oligonucleotide sequence and in part complementary to at least a portion of a second oligonucleotide sequence, wherein the first oligonucleotide sequence or the second oligonucleotide sequence is immobilized to the bead.

21. The method of claim 1, wherein the plurality of oligonucleotides releasably attached to the bead is formed with the aid of combinatorial ligation.

22. The method of claim 1, wherein the partition is a well.

23. The method of claim 1, wherein the sequence of the oligonucleotide is released from the bead into the partition prior to (b).

24. The method of claim 1, wherein the sequence of the oligonucleotide remains attached to the bead while annealing the primer sequence of the oligonucleotide to the template nucleic acid sequence.

25. The method of claim 1, further comprising determining a sequence of the extension product.

26. The method of claim 1, further comprising, subsequent to (c), recovering contents of the partition.

27. The method of claim 1, wherein:

in (a), a plurality of template nucleic acid sequences are co-partitioned with the bead into the partition, wherein the plurality of template nucleic acid sequences include the template nucleic acid sequence;

in (b), primer sequences of multiple oligonucleotides of the plurality of oligonucleotides are annealed to template nucleic acid sequences of the plurality of template nucleic acid sequences; and

in (c), the primer sequences are extended to produce a plurality of extension products complementary to at least portions of the template nucleic acid sequences of the plurality of template nucleic acid sequences, wherein the plurality of extension products includes the extension product; and

wherein, after (a), the sequences of the multiple oligonucleotides are released from the bead into the partition.

28. The method of claim 27, further comprising determining sequences of the plurality of extension products.

29. The method of claim 1, wherein the plurality of oligonucleotides is releasably attached to the bead by a labile bond or moiety.

30. The method of claim 29, wherein the labile bond or moiety is a chemically labile bond or moiety.

31. The method of claim 30, wherein the chemically labile bond or moiety is a disulfide bond.

32. The method of claim 1, wherein the sequences of the plurality of oligonucleotides are released upon application of a stimulus.

33. The method of claim 32, wherein the stimulus is a chemical stimulus.

34. The method of claim 33, wherein the chemical stimulus is in the partition.

35. The method of claim 33, wherein the chemical stimulus is a reducing agent.

36. The method of claim 35, wherein the reducing agent is dithiothreitol (DTT).

37. The method of claim 1, further comprising:

(d) annealing the primer sequence of a second oligonucleotide of the plurality of oligonucleotides to the template nucleic acid sequence; and

(e) extending the primer sequence of the second oligonucleotide to produce a second extension product complementary to at least a portion of the template nucleic acid sequence, the second extension product comprising the primer sequence of the second oligonucleotide and the common barcode sequence.

38. The method of claim 37, wherein, after (e), the second extension product is in solution within the partition.

39. The method of claim 1, wherein the oligonucleotide comprises a sequence that is not released from the bead into the partition.

40. The method of claim 1, wherein the oligonucleotide is released from the bead into the partition.

41. A method of processing a template nucleic acid sequence, comprising:

(a) co-partitioning a template nucleic acid sequence and a bead comprising a plurality of oligonucleotides releasably attached thereto into a partition, wherein each oligonucleotide of the plurality of oligonucleotides comprises a sequence comprising a common barcode sequence and a primer sequence complementary to one or more regions of the template nucleic acid sequence, wherein the plurality of oligonucleotides comprises (i) a first oligonucleotide comprising a first sequence comprising the common barcode sequence and a first primer sequence and (ii) a second oligonucleotide comprising a second sequence comprising the common barcode sequence and a variable primer sequence;

(b) annealing the first primer sequence of the first oligonucleotide to the template nucleic acid sequence;

(c) extending the first primer sequence to produce a first extension product complementary to at least a portion of the template nucleic acid sequence, the first extension product comprising the first primer sequence and the common barcode sequence;

(d) annealing the variable primer sequence of the second oligonucleotide to the first extension product; and

(e) extending the variable primer sequence to produce a second extension product complementary to at least a portion of the first extension product, the second extension product comprising the variable primer sequence and the common barcode sequence,

wherein, after (a), the first sequence of the first oligonucleotide is released from the bead into the partition, and

wherein, after (c), the first extension product is in solution within the partition.

42. The method of claim 41, wherein the second extension product further comprises a sequence complementary to the first primer sequence of the first oligonucleotide.

43. The method of claim 41, comprising repeating (a)-(e) to produce additional extension products.

44. The method of claim 41, wherein the second extension product comprises a complement of a first sequence of the first oligonucleotide, and does not comprise a complement of a second sequence of the first oligonucleotide.

45. The method of claim 44, wherein the second sequence of the first oligonucleotide comprises one or more uracil containing nucleotides.

46. The method of claim 41, wherein the second extension product forms a hairpin molecule under annealing conditions.

47. The method of claim 41, wherein the plurality of oligonucleotides releasably attached to the bead is formed by coupling multiple oligonucleotides to each other.

48. The method of claim 41, wherein the plurality of oligonucleotides is generated on the bead with the aid of a splint sequence that is in part complementary to at least a portion of a first oligonucleotide sequence and in part complementary to at least a portion of a second oligonucleotide sequence, wherein the first oligonucleotide sequence or the second oligonucleotide sequence is immobilized to the bead.

49. The method of claim 41, wherein the plurality of oligonucleotides releasably attached to the bead is formed with the aid of combinatorial ligation.

50. The method of claim 41, wherein the partition is a well.

51. The method of claim 41, wherein the partition is a droplet in an emulsion.

52. The method of claim 41, wherein the bead is a gel bead.

53. The method of claim 52, wherein (b) comprises dissolving the gel bead, thereby releasing the first sequence of the first oligonucleotide and the second sequence of the second oligonucleotide from the gel bead into the partition.

54. The method of claim 41, further comprising determining a sequence of the first extension product or the second extension product.

55. The method of claim 41, further comprising, subsequent to (e), recovering contents of the partition.

56. The method of claim 41, wherein:

in (a), a plurality of template nucleic acid sequences are co-partitioned with the bead into the partition, wherein the plurality of template nucleic acid sequences include the template nucleic acid sequence;

in (b), primer sequences of a first plurality of oligonucleotides of the plurality of oligonucleotides are annealed to template nucleic acid sequences of the plurality of template nucleic acid sequences;

in (c), the primer sequences of the first plurality of oligonucleotides are extended to produce a plurality of first extension products complementary to at least portions copies of the template nucleic acid sequences of the plurality of template nucleic acid sequences, wherein the plurality of first extension products includes the first extension product;

in (d), variable primer sequences of a second plurality of oligonucleotides are annealed to the plurality of first extension products; and

in (e) the variable primer sequences of the second plurality of oligonucleotides are extended to produce a plurality of second extension products complementary to at least portions of the plurality of first extension products, wherein the plurality of second extension products includes the second extension product.

57. The method of claim 56, further comprising determining sequences of the plurality of second extension products.

58. The method of claim 41, wherein the plurality of oligonucleotides is releasably attached to the bead by a labile bond or moiety.

59. The method of claim 58, wherein the labile bond or moiety is a chemically labile bond or moiety.

60. The method of claim 59, wherein the chemically labile bond or moiety is a disulfide bond.

61. The method of claim 41, wherein the sequences of the plurality of oligonucleotides are released upon application of a stimulus.

62. The method of claim 61, wherein the stimulus is a chemical stimulus.

63. The method of claim 62, wherein the chemical stimulus is in the partition.

64. The method of claim 62, wherein the chemical stimulus is a reducing agent.

65. The method of claim 64, wherein the reducing agent is dithiothreitol (DTT).

66. The method of claim 41, wherein the first primer sequence of the first oligonucleotide is a variable primer sequence.

67. The method of claim 41, wherein the first primer sequence of the first oligonucleotide and the variable primer sequence of the second oligonucleotide are different.

68. The method of claim 41, wherein the variable primer sequence of the second oligonucleotide is a random N-mer.

69. The method of claim 41, wherein the first oligonucleotide comprises a functional sequence that facilitates determining a sequence of the first extension product.

70. The method of claim 41, wherein the second oligonucleotide comprises a functional sequence that facilitates determining a sequence of the second extension product.

71. The method of claim 41, wherein the first sequence of the first oligonucleotide is released from the bead into the partition prior to (b).

72. The method of claim 41, wherein, after (a), the second sequence of the second oligonucleotide is released from the bead into the partition.

73. The method of claim 41, wherein the first sequence of the first oligonucleotide remains attached to the bead while annealing the first primer sequence to the template nucleic acid sequence.

74. The method of claim 41, wherein the first oligonucleotide and/or the second oligonucleotide comprises a sequence that is not released from the bead into the partition.

75. The method of claim 41, wherein the first and second oligonucleotides are released from the bead into the partition.