HIGH-THROUGHPUT RNA-SEQ

• US 20160122753A1
• Filed: 06/12/2014
• Published: 05/05/2016
• Est. Priority Date: 06/12/2013
• Status: Abandoned Application

• Current Assignee: President and Fellows of Harvard College (Harvard Corporation), The Broad Institute, Inc.
• Original Assignee: President and Fellows of Harvard College (Harvard Corporation), The Broad Institute, Inc.
• Inventors: Mikkelsen, Tarjei, Soumillon, Magali
• Application Number:
  US14/898,030
• Publication Number:
  US 20160122753A1
• 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 1/6844 Nucleic acid amplification reactions
• 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 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

The present invention relates generally to methods for single-cell nucleic acid profiling, and nucleic acids useful in those methods. For example, it concerns using barcode sequences to track individual nucleic acids at single-cell resolution, utilizing template switching and sequencing reactions to generate the nucleic acid profiles. These methods and compositions are also applicable to other starting materials, such as cell and tissue lysates or extracted/purified RNA.

Claims

1. A nucleic acid comprising a 5′

poly-isonucleotide sequence, an internal adapter sequence, and a 3′

guanosine tract.

7. The nucleic acid of claim 1, wherein the adapter sequence is 12 to 32 nucleotides in length.

8. The nucleic acid of claim 7, wherein the adapter sequence is 22 nucleotides in length.

9. The nucleic acid of claim 8, wherein the internal adapter sequence is 5′

-ACACTCTTTCCCTACACGACGC-3′

.

23. A kit comprising the nucleic acid of claim 7.

30. The kit of claim 23, further comprising a third nucleic acid primer comprising 12 to 32 nucleotides and a 5′

blocking group.

36. The kit of claim 23, further comprising a phosphorothioate bond-containing nucleic acid comprising an X1*X2*X3*X4*X5*3′

sequence, wherein * is a phosphorothioate bond.

39. The kit of claim 36, wherein the sequence of the phosphorothioate bond-containing nucleic acid is 5′

-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T*-3′

.

47. A method for gene profiling, comprising:

a) providing a plurality of single cells;

b) releasing mRNA from each single cell to provide a plurality of individual mRNA samples, wherein each individual mRNA sample is from a single cell;

c) reverse transcribing the individual mRNA samples, performing a template switching reaction to produce cDNA incorporating a barcode sequence, and contacting each individual mRNA sample with a nucleic acid of claim 1 and a nucleic acid of claim 10;

d) pooling and purifying the barcoded cDNA produced from the separate cells;

e) amplifying the barcoded cDNA to generate a cDNA library comprising double-stranded cDNA;

f) purifying the double-stranded cDNA;

g) fragmenting the purified cDNA;

h) purifying the cDNA fragments; and

i) sequencing the cDNA fragments.

48. A method for gene profiling, comprising:

a) providing an isolated population of cells;

b) releasing mRNA from the population of cells to provide one or more mRNA samples;

c) reverse transcribing the one or more mRNA samples, performing a template switching reaction to produce cDNA incorporating a barcode sequence, and contacting each individual mRNA sample with a nucleic acid of claim 1 and a nucleic acid of claim 10;

d) pooling and purifying the barcoded cDNA;

e) amplifying the barcoded cDNA to generate a cDNA library comprising double-stranded cDNA;

f) purifying the double-stranded cDNA;

g) fragmenting the purified cDNA;

h) purifying the cDNA fragments; and

i) sequencing the cDNA fragments.

49. The method of claim 47, further comprising separating a population of cells to provide the plurality of single cells.

54. The method of claim 47, further comprising contacting the cells with proteinase K.

60. The method of claim 47, further comprising treating the barcoded cDNA with an exonuclease.

71. The method of claim 47, wherein the fragmentation of g) utilizes a transposase.

72. The method of claim 71, wherein the fragmentation of g) utilizes a first fragmentation nucleic acid and a second fragmentation nucleic acid, wherein the first fragmentation nucleic acid comprises a barcode sequence.

73. The method of claim 72, wherein the sequence of the first fragmentation nucleic acid is 5′

-CAAGCAGAAGACGGCATACGAGAT[i7]GTCTCGTGGGCTCGG-3′

, wherein [i7] is a nucleic acid sequence.

94. The method of claim 47, further comprising assembling a database of the sequences of the sequenced cDNA fragments of j).

95. The method of claim 94, further comprising identifying the UMI sequences of the sequences of the database.

96. The method of claim 95, further comprising discounting duplicate sequences that share a UMI sequence, thereby assembling a set of sequences in which each sequence is associated with a unique UMI.

2-6. -6. (canceled)

10. A nucleic acid comprising a 5′

blocking group, an internal adapter sequence, a barcode sequence, a unique molecular identifier (UMI) sequence, a complementarity sequence, and a 3′

dinucleotide sequence comprising a first nucleotide and a second nucleotide, wherein the first nucleotide of the dinucleotide sequence is a nucleotide selected from adenine, guanine, and cytosine, and the second nucleotide of the dinucleotide sequence is a nucleotide selected from adenine, guanine, cytosine, and thymine.

12. The nucleic acid of claim 10, wherein the 5′

blocking group is biotin.

15. The nucleic acid sequence of claim 12, wherein the internal adapter sequence is 5′

-ACACTCTTTCCCTACACGACGC-3′

.

24. The kit of claim 23, further comprising the nucleic acid of claim 10.

77. The method of claim 72, wherein the barcode sequence of the first fragmentation nucleic acid is different than the barcode sequence of the nucleic acid of claim 10.

78. The method of claim 77, wherein the barcode sequence of the first fragmentation nucleic acid uniquely identifies a predetermined subset of cells.

79. The method of claim 78, wherein the predetermined subset of cells is a subset of cells contained in individual wells of a single capture plate.

80. The method of claim 79, wherein the barcode sequence that uniquely identifies the predetermined subset of cells uniquely identifies the capture plate.

81. The method of claim 77, wherein the barcode sequence of the nucleic acid of claim 10 uniquely identifies the cell within the predetermined subset of cells, which cell comprised the mRNA from which the barcoded cDNA of c) was produced.

82. The method of claim 81, wherein the barcode sequence that uniquely identifies the cell within the predetermined subset of cells uniquely identifies an individual well in a capture plate.

83. The method of claim 82, wherein the combination of the barcode sequence that uniquely identifies the predetermined subset of cells and the barcode sequence that uniquely identifies the cell within a predetermined subset of cells uniquely identifies the capture plate and the individual well which comprised the cell, which cell comprised the mRNA from which the barcoded cDNA of c) was produced.

89. The method of claim 83, wherein the sequence of the second fragmentation nucleic acid is 5′

-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCG*A*T*C*T*-3′

.

99. The method of claim 72, wherein the barcode sequence of the first fragmentation nucleic acid and the barcode sequence of the nucleic acid of claim 10 are used to correlate the sequencing data with the predetermined subset of cells and the individual cell.

11. (canceled)

13-14. -14. (canceled)

16-22. -22. (canceled)

25-29. -29. (canceled)

31-35. -35. (canceled)

37-38. -38. (canceled)

40-46. -46. (canceled)

50-53. -53. (canceled)

55-59. -59. (canceled)

61-70. -70. (canceled)

74-76. -76. (canceled)

84-88. -88. (canceled)

90-93. -93. (canceled)

97-98. -98. (canceled)