COMPOSITIONS AND METHODS FOR SAMPLE PROCESSING

• US 20140378345A1
• Filed: 06/26/2014
• Published: 12/25/2014
• Est. Priority Date: 08/14/2012
• Status: Abandoned Application

• Current Assignee: 10X Genomics Incorporated
• Original Assignee: 10x Technology Inc.
• Inventors: Hindson, Benjamin, Hindson, Christopher, Schnall-Levin, Michael, Ness, Kevin, Jarosz, Mirna, Masquelier, Donald, Saxonov, Serge, Merrill, Landon, Price, Andrew, Hardenbol, Paul, Li, Yuan
• Application Number:
  US14/316,383
• Publication Number:
  US 20140378345A1
• Time in Patent Office: Days
• US Class Current: 506/16
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• 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 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• 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
• CPC Class Codes: C40B 50/16 involving encoding steps
• CPC Class Codes: C40B 50/18 using a particular method of attachment to the solid support

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 barcoding sample materials, comprising:

providing a first partition comprising a plurality of nucleic acid barcode molecules associated therewith, the plurality of nucleic acid barcode molecules comprising the same nucleic acid barcode sequence;

co-partitioning the first partition with components of a sample material into a second partition;

releasing the barcode molecules from the first partition into the second partition; and

attaching the barcode molecules to one or more of the components of the sample material or fragments thereof within the second partition.

2. The method of claim 1, wherein the first partition comprises at least 1,000 barcode molecules associated therewith, the at least 1,000 barcode molecules having the same barcode sequence.

3. The method of claim 1, wherein the first partition comprises at least 10,000 barcode molecules associated therewith, the at least 10,000 barcode molecules having the same barcode sequence.

4. The method of claim 1, wherein the first partition comprises at least 100,000 barcode molecules associated therewith, the at least 100,000 barcode molecules having the same barcode sequence.

5. The method of claim 1, wherein the first partition comprises at least 1,000,000 barcode molecules associated therewith, the at least 1,000,000 barcode molecules having the same barcode sequence.

6. The method of claim 1, wherein the first partition is selected from a bead, a microcapsule and a droplet.

7. The method of claim 1, wherein the first partition comprises a bead, and the barcode molecules are releasably coupled to the bead.

8. The method of claim 7, wherein the bead comprises a gel bead.

9. The method of claim 1, wherein the second partition comprises a droplet.

10. The method of claim 7, wherein the co-partitioning step comprises combining a first aqueous fluid comprising beads with a second aqueous fluid comprising the sample components in a droplet within an immiscible fluid.

11. The method of claim 1, wherein the releasing step comprises degrading the first partition.

12. The method of claim 7, wherein the releasing step comprises degrading the bead to release the barcode molecules into the second partition.

13. The method of claim 7, wherein the releasing step comprises cleaving a chemical linkage between the barcode molecules and the bead.

14. The method of claim 7, wherein at least one of crosslinking of the bead and a linkage between the bead and the barcode molecules comprises a disulfide linkage, and the releasing step comprises exposing the first partition to a reducing agent.

15. The method of claim 14, wherein the reducing agent comprises a reducing agent selected from the group consisting of DTT and TCEP.

16. The method of claim 1, wherein the sample materials comprise one or more template nucleic acid molecules, and the attaching step comprises attaching the barcode molecules to one or more fragments of the template nucleic acid molecules.

17. The method of claim 1, wherein the sample materials comprise one or more template nucleic acid molecules, the barcode molecules further comprise a primer sequence complementary to at least a portion of the template nucleic acid molecules, and the attaching step comprises extending the barcode molecules to replicate at least a portion of the template nucleic acid molecules.

18. The method of claim 1, wherein the sample materials comprise contents of a single cell.

19. The method of claim 18, wherein the single cell comprises a cancer cell.

20. The method of claim 18, wherein the single cell comprises a bacterial cell.

21. The method of claim 20, wherein the bacterial cell is isolated from a human microbiome sample.

22. The method of claim 1, wherein the second partition comprises no more than one first partition.

23. The method of claim 1, wherein:

the providing step comprises providing a plurality of first partitions comprising a plurality of different nucleic acid barcode sequences, wherein each of the plurality of first partitions comprises a plurality of at least 1000 nucleic acid barcode molecules having the same nucleic acid barcode sequence associated therewith;

the co-partitioning step comprises co partitioning the plurality of first partitions with the components of the sample material into a plurality of second partitions;

the releasing step comprises releasing the nucleic acid barcode molecules from the plurality of first partitions into the plurality of second partitions; and

the attaching step comprises attaching the nucleic acid barcode molecules to the components of the sample material or fragments thereof within the plurality of second partitions.

24. The method of claim 23, wherein the plurality of different nucleic acid barcode sequences comprises at least about 1000 different barcode sequences.

25. The method of claim 23, wherein the plurality of different nucleic acid barcode sequences comprises at least about 10,000 different barcode sequences

26. The method of claim 23, wherein the plurality of different nucleic acid barcode sequences comprises at least about 100,000 different barcode sequences

27. The method of claim 23, wherein the plurality of different nucleic acid barcode sequences comprises at least about 500,000 different barcode sequences

28. The method of claim 23, wherein a subset of the plurality of second partitions comprise the same nucleic acid barcode sequence.

29. The method of claim 28, wherein at least about 1% the plurality of second partitions comprise the same nucleic acid barcode sequence.

30. The method of claim 28, wherein at least about 2% of the plurality of second partitions comprise the same nucleic acid barcode sequence.

31. The method of claim 28, wherein at least about 5% of the plurality of second partitions comprise the same nucleic acid barcode sequence.

32. The method of claim 28, wherein the fragments of the components of the sample material comprise one or more fragments of one or more template nucleic acid sequences and the method further comprises sequencing the one or more fragments of the one or more template nucleic acid sequences and characterizing the one or more fragments of the template nucleic acid sequences based at least in part upon a nucleic acid barcode sequence attached thereto.

33. The method of claim 32, wherein the characterizing the one or more fragments of the one or more template nucleic acid sequences comprises mapping a fragment of an individual template nucleic acid sequence of the one or more template nucleic acid sequences to an individual template nucleic acid sequence of the one or more template nucleic acid sequences or a genome from which the individual template nucleic acid sequence was derived.

34. The method of claim 32, wherein the characterizing the one or more fragments of the template nucleic acid sequences comprises at least identifying an individual nucleic acid barcode sequence of said plurality of different nucleic acid barcode sequences, and identifying a sequence of an individual fragment of the one or more fragments of the one or more template nucleic acid sequences attached to the individual nucleic acid barcode sequence.

35. The method of claim 23, wherein at least 50% of the plurality of second partitions contain no more than one first partition.

36. The method of claim 23, wherein at least 70% of the plurality of second partitions contain no more than one first partition.

37. The method of claim 23, wherein at least 90% of the plurality of second partitions contain no more than one first partition.

38. The method of claim 23, wherein at least 50% of the plurality of second partitions comprises exactly one first partition.

39. The method of claim 23, wherein at least 70% of the plurality of second partitions comprises exactly one first partition.

40. The method of claim 23, wherein at least 90% of the plurality of second partitions comprises exactly one first partition.

41. A method of barcoding sample materials, comprising:

providing a plurality of first partitions comprising a plurality of different nucleic acid barcode sequences, wherein each of the plurality of first partitions comprises a plurality of nucleic acid barcode molecules having the same nucleic acid barcode sequence associated therewith;

co-partitioning the plurality of first partitions with components of a sample material into a plurality of second partitions;

releasing the barcode molecules from the plurality of first partitions into the plurality of second partitions; and

attaching the barcode molecules to the components of the sample material within the plurality of second partitions.

42. A method of barcoding sample materials, comprising:

providing an activatable nucleic acid barcode sequence;

partitioning the activatable nucleic acid barcode sequence with one or more components of a sample material into a first partition;

activating the activatable nucleic acid barcode sequence to produce an active nucleic acid barcode sequence in the first partition; and

attaching the active nucleic acid barcode sequence to the one or more components of the sample material.

43. The method of claim 42, wherein the step of activating the activatable nucleic acid barcode sequence comprises releasing the activatable nucleic acid barcode sequence from a second partition within the first partition.

44. The method of claim 42, wherein the step of activating the activatable nucleic acid barcode sequence comprises removing a removable protecting group from the activatable nucleic acid barcode sequence.

45. A composition, comprising:

a first partition, comprising one or more sample components; and

a second partition contained within the first partition, the second partition having a plurality of oligonucleotides releasably associated therewith, the plurality of oligonucleotides comprising a common barcode sequence.

46-63. -63. (canceled)

64. A method, comprising:

combining a sample of nucleic acids with a library of barcoded beads to form a mixture;

partitioning the mixture into a plurality of partitions, wherein at least a subset of the partitions comprises at most one barcoded bead; and

within the partitions, releasing barcodes from the barcoded beads.

65. The method of claim 64, wherein the barcoded beads are capable of being dissolved by a reducing agent.

66. The method of claim 65, wherein the barcoded beads comprise disulfide bonds.

67. The method of claim 64, wherein the sample of nucleic acids is genomic DNA.

68. The method of claim 67, wherein the genomic DNA is fragmented prior to being combined with the barcoded beads.

69. The method of claim 64, further comprising using a microfluidic device to combine the sample of nucleic acids with the library of barcoded beads.

70. The method of claim 64, further comprising using a microfluidic device to partition the mixture into the plurality of partitions.

71. The method of claim 64, wherein the partitions are aqueous droplets within a water-in-oil emulsion.

72. The method of claim 71, further comprising using a microfluidic device to partition the mixture into the plurality of partitions.

73. The method of claim 72, wherein the microfluidic device is a droplet generator.

74. The method of claim 71, wherein the microfluidic device comprises a first input channel and a second input channel that meet at a junction that is fluidly connected to an output channel.

75. The method of claim 74, wherein the method further comprises introducing the sample of nucleic acids into the first input channel and introducing the library of barcoded beads into the second input channel, thereby generating the mixture of the sample of nucleic acids and the library of barcoded beads in the output channel.

76. The method of claim 75, wherein the output channel is fluidly connected to a third input channel at a junction.

77. The method of claim 76, further comprising introducing an oil into the third input channel, such that the aqueous droplets within a water-in-oil emulsion are formed, individual droplets comprising, on average, at most ten barcoded beads.

78. The method of claim 77, further comprising introducing oil into a fourth input channel, wherein the fourth input channel intersects the third input channel and the output channel at a junction.

79. The method of claim 74, further comprising introducing a reducing agent into the first or second input channel.

80. The method of claim 74, wherein the microfluidic device further comprises a third input channel that intersects the first input channel, the second input channel, or the junction of the first input channel and the second input channel, and the method further comprises introducing a reducing agent into the third input channel.

81. The method of claim 74, wherein the first input channel and the second input channel form a substantially perpendicular angle between one another.

82. The method of claim 64, wherein the barcodes are released from the barcoded beads by the action of a reducing agent.

83. The method of claim 64, wherein the barcoded beads comprise a matrix that is crosslinked with disulfide bonds and the barcodes are released from the barcoded beads by the action of a reducing agent that dissolves the barcoded beads.

84. The method of claim 64, further comprising releasing the barcodes from the barcoded beads by heating the partitions.

85. The method of claim 64, wherein the barcodes are pre-synthesized with known sequences.

86. The method of claim 64, wherein the barcodes further comprise a plurality of random Nmers.

87. The method of claim 86, further comprising hybridizing the plurality of random Nmers to the sample of nucleic acids.

88. The method of claim 86, further comprising performing an amplification reaction within the partitions.