Methods of Analyzing Nucleic Acids from Individual Cells or Cell Populations

• US 20150376609A1
• Filed: 06/26/2015
• Published: 12/31/2015
• Est. Priority Date: 06/26/2014
• Status: Abandoned Application

• 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, Belgrader, Phillip, Zheng, Grace
• Application Number:
  US14/752,641
• Publication Number:
  US 20150376609A1
• Time in Patent Office: Days
• US Class Current: 1/1
• 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 1/6804 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 2525/161 incorporating target specific and non-target specific sites
• 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 2545/114 involving a quantitation step
• 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
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 50/16 involving encoding steps

Abstract

Methods, compositions and systems for analyzing individual cells or cell populations through the partitioned analysis of contents of individual cells or cell populations. Individual cells or cell populations are co-partitioned with processing reagents for accessing cellular contents, and for uniquely identifying the contents of a given cell or cell population, and subsequently analyzing the cell'"'"'s contents and characterizing it as having derived from an individual cell or cell population, including analysis and characterization of the cell'"'"'s nucleic acids through sequencing.

Claims

1. A method of analyzing nucleic acids from cells, comprising:

(a) providing nucleic acids derived from an individual cell and oligonucleotides having a common barcode sequence into a discrete partition, wherein the oligonucleotides are releasably attached to a bead;

(b) generating one or more first nucleic acid sequences derived from the nucleic acids within the discrete partition, which one or more first nucleic acid sequences have attached thereto oligonucleotides that comprise the common nucleic acid barcode sequence;

(c) generating a characterization of the one or more first nucleic acid sequences or one or more second nucleic acid sequences derived from the one or more first nucleic acid sequences, which one or more second nucleic acid sequences comprise the common barcode sequence; and

(d) identifying the one or more first nucleic acid sequences or one or more second nucleic acid sequences as being derived from the individual cell based, at least in part, upon a presence of the common nucleic acid barcode sequence in the characterization generated in (c).

2. The method of claim 1, wherein the discrete partition is a discrete droplet.

3. The method of claim 1, wherein, in (a), the oligonucleotides are co-partitioned with the nucleic acids derived from the individual cell into the discrete partition.

4. The method of claim 3, wherein, in (a), at least 10,000 of the oligonucleotides are co-partitioned with the nucleic acids derived from the individual cell into the discrete partition.

9. The method of claim 1, wherein the bead comprises a degradable bead.

10. The method of claim 9, further comprising, prior to or during (b), releasing the oligonucleotides from the bead via degradation of the bead.

11. The method of claim 1, further comprising, prior to (c), releasing the one or more first nucleic acid sequences from the discrete partition.

12. The method of claim 1, wherein (c) comprises sequencing the one or more first nucleic acid sequences or the one or more second nucleic acid sequences.

13. The method of claim 12, further comprising assembling a contiguous nucleic acid sequence for at least a portion of a genome of the individual cell from sequences of the one or more first nucleic acid sequences or the one or more second nucleic acid sequences.

15. The method of claim 1, wherein the nucleic acids are released from the individual cell in the discrete partition.

16. The method of claim 1, wherein the nucleic acids comprise ribonucleic acid (RNA).

17. The method of claim 16, wherein the RNA is messenger RNA (mRNA).

18. The method of claim 16, wherein (b) further comprises subjecting the nucleic acids to reverse transcription under conditions that yield the one or more first nucleic acid sequences.

19. The method of claim 18, wherein the reverse transcription occurs in the discrete partition.

20. The method of claim 18, wherein the oligonucleotides comprise a poly-T sequence.

21. The method of claim 20, wherein the reverse transcription comprises hybridizing the poly-T sequence to at least a portion of each of the nucleic acids and extending the poly-T sequence in template directed fashion.

22. The method of claim 21, wherein the oligonucleotides further comprise an anchoring sequence that facilitates hybridization of the poly-T sequence.

23. The method of claim 1, wherein the oligonucleotides further comprise a random priming sequence.

25. The method of claim 23, wherein the reverse transcription comprises hybridizing the random priming sequence to at least a portion of each of the nucleic acids and extending the random priming sequence in template directed fashion.

26. The method of claim 1, wherein a given one of the one or more first nucleic acid sequences has sequence complementarity to at least a portion of a given one of the nucleic acids.

27. The method of claim 1, wherein the discrete partition at most includes the individual cell among a plurality of cells.

28. The method of claim 1, wherein the oligonucleotides further comprise a unique molecular sequence segment.

29. The method of claim 28, further comprising identifying an individual nucleic acid sequence of the one or more first nucleic acid sequences or of the one or more second nucleic acid sequences as derived from a given nucleic acid of the nucleic acids based, at least in part, upon a presence of the unique molecular sequence segment.

30. The method of claim 29, further comprising determining an amount of the given nucleic acid based upon a presence of the unique molecular sequence segment.

31. The method of claim 1, further comprising, prior to (c), adding one or more additional sequences to the one or more first nucleic acid sequences to generate the one or more second nucleic acid sequences.

32. The method of claim 31, further comprising adding a first additional nucleic acid sequence to the one or more first nucleic acid sequences with the aid of a switch oligonucleotide.

33. The method of claim 32, wherein the switch oligonucleotide hybridizes to at least a portion of the one or more first nucleic acid sequences and is extended in a template directed fashion to couple the first additional nucleic acid sequence to the one or more first nucleic acid sequences.

34. The method of claim 33, further comprising amplifying the one of more first nucleic acid sequences coupled to the first additional nucleic acid sequence.

37. The method of claim 34, further comprising, after the amplifying, adding one or more second additional nucleic acid sequences to the one or more first nucleic acid sequences coupled to the first additional sequence to generate the one or more second nucleic acid sequences.

38. The method of claim 37, wherein the adding the one or more second additional sequences comprises removing a portion of each of the one or more first nucleic acid sequences coupled to the first additional nucleic acid sequence and coupling thereto the one or more second additional nucleic acid sequences.

39. The method of claim 38, wherein the removing is completed via shearing of the one or more first nucleic acid sequences coupled to the first additional nucleic acid sequence.

41. The method of claim 18, further comprising, prior to (c), subjecting the one or more first nucleic acid sequences to transcription to generate one or more RNA fragments.

42. The method of claim 41, wherein the transcription occurs after releasing the one or more first nucleic acid sequences from the discrete partition.

43. The method of claim 41, wherein the oligonucleotides further comprise a T7 promoter sequence.

44. The method of claim 43, further comprising, prior to (c), removing a portion of each of the one or more RNA sequences and coupling an additional sequence to the one or more RNA sequences.

45. The method of claim 44, further comprising, prior to (c), subjecting the one or more RNA sequences coupled to the additional sequence to reverse transcription to generate the one or more second nucleic acid sequences.

46. The method of claim 45, further comprising, prior to (c), amplifying the one or more second nucleic acid sequences.

47. The method of claim 41, further comprising, prior to (c), subjecting the one or more RNA sequences to reverse transcription to generate one or more DNA sequences.

48. The method of claim 47, further comprising, prior to (c), removing a portion of each of the one or more DNA sequences and coupling one or more additional sequences to the one or more DNA sequences to generate the one or more second nucleic acid sequences.

49. The method of claim 48, further comprising, prior to (c), amplifying the one or more second nucleic acid sequences.

50. The method of claim 1, wherein the nucleic acids comprise complementary (cDNA) generated from reverse transcription of RNA from the individual cell.

51. The method of claim 1, wherein the oligonucleotides further comprise a priming sequence.

53. The method of claim 51, wherein (b) comprises hybridizing the priming sequence to the nucleic acids and extending the priming sequence in template directed fashion.

54. The method of claim 1, wherein the discrete partition comprises switch oligonucleotides comprising a complement sequence of the oligonucleotides.

55. The method of claim 54, wherein (b) comprises hybridizing the switch oligonucleotides to at least a portion of nucleic acid fragments derived from the nucleic acids and extending the switch oligonucleotides in template directed fashion.

58. The method of claim 1, wherein the (b) comprises coupling the oligonucleotides to the nucleic acids.

60. The method of claim 1, wherein a plurality of partitions comprises the discrete partition.

61. The method of claim 60, wherein, on average, the plurality of partitions comprises less than one cell per partition.

62. The method of claim 60, wherein less than 25% of partitions of the plurality of partitions do not comprise a cell.

64. The method of claim 60, wherein fewer than 25% of partitions of the plurality of partitions comprise more than one cell.

65. The method of claim 60, wherein at least a subset of the plurality of partitions comprises a bead.

68. The method of claim 60, wherein the plurality of partitions comprises at least 1,000 different partitioned nucleic acid barcode sequences.

71. The method of claim 60, wherein the plurality of partitions comprises at least 1,000 partitions.

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74. A method of characterizing cells in a population of a plurality of different cell types, comprising:

(a) providing nucleic acids from individual cells in the population into discrete partitions;

(b) attaching oligonucleotides that comprise a common nucleic acid barcode sequence to one or more fragments of the nucleic acids from the individual cells within the discrete partitions, wherein a plurality of different partitions comprise different common nucleic acid barcode sequences;

(c) characterizing the one or more fragments of the nucleic acids from the plurality of discrete partitions, and attributing the one or more fragments to individual cells based, at least in part, upon the presence of a common barcode sequence; and

(d) characterizing a plurality of individual cells in the population based upon the characterization of the one or more fragments in the plurality of discrete partitions.

75-82. -82. (canceled)

83. A method of characterizing an individual cell or population of cells, comprising:

(a) incubating a cell with a plurality of different cell surface feature binding group types, wherein each different cell surface binding group type is capable of binding to a different cell surface feature, and wherein each different cell surface binding group type comprises a reporter oligonucleotide associated therewith, under conditions that allow binding between one or more cell surface feature binding groups and its respective cell surface feature, if present;

(b) partitioning the cell into a partition that comprises a plurality of oligonucleotides comprising a barcode sequence;

(c) attaching the barcode sequence to oligonucleotide reporter groups present in the partition;

(d) sequencing the oligonucleotide reporter groups and attached barcodes; and

(e) characterizing cell surface features present on the cell based upon reporter oligonucleotides that are sequenced.

84-88. -88. (canceled)