METHODS AND COMPOSITIONS FOR PREPARING NUCLEIC ACID LIBRARIES

• US 20160265027A1
• Filed: 10/16/2014
• Published: 09/15/2016
• Est. Priority Date: 10/17/2013
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Sanches-Kuiper, Raquel Maria, Smith, Vincent Peter, Humphray, Sean
• Granted Patent: US 10,294,511 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• 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 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid

Abstract

Embodiments provided herein relate to methods and compositions for preparing nucleic acid libraries. Some embodiments include preparing libraries from nucleic acids obtained from degraded samples, such as ancient samples and fixed samples.

Claims

1. A method for preparing a nucleic acid library from a low quality nucleic acid sample comprising:

(a) providing a plurality of nucleic acids obtained from the low quality nucleic acid sample;

(b) removing single-stranded nucleic acids from the plurality of nucleic acids; and

(c) repairing the nucleic acids of the plurality of nucleic acids, thereby preparing a nucleic acid library.

2. The method of claim 1, wherein step (b) is performed before step (c).

3. The method of claim 1, wherein step (c) is performed before step (b).

4. The method of claim 1, wherein (b) comprises removing the single-stranded portion of a nucleic acid comprising a single-stranded portion and a double-stranded portion.

5. The method of claim 1, wherein (b) comprises contacting the nucleic acids with a single-strand exonuclease.

6. The method of claim 5, wherein the single-strand exonuclease is a 5′

-3′

exonuclease.

7. The method of claim 5, wherein the single-strand exonuclease is selected from the group consisting of RecJ, Exonuclease I, Exonuclease T, Exonuclease VII, Exonuclease VIII, and Mung bean Exonuclease.

8. The method of claim 7, wherein (c) comprises contacting the nucleic acids with an enzyme selected from the group consisting of Uracil N-Glycosylase (UNG), Uracil DNA Glycosylase (UDG), Endonuclease IV, Endonuclease VIII, formamidopyrimidine-DNA glycosylase (FPG), DNA-(apurinic or apyrimidinic site) lyase, and Pol β

.

9. The method of claim 1, wherein (a) further comprises shearing the plurality of nucleic acids.

10. The method of claim 9, wherein the shearing comprises selectively shearing the plurality of nucleic acids comprising:

(i) obtaining a first fraction comprising longer nucleic acids of the plurality of nucleic acids, and a second fraction comprising shorter nucleic acids of the plurality of nucleic acids,

(ii) shearing the nucleic acids of the first fraction, and

(iii) combining the first fraction and the second fraction, thereby preparing the nucleic acid library.

11. The method of claim 10, wherein (i) comprises contacting the plurality of nucleic acids with a matrix under conditions wherein nucleic acids greater than about 100 by selectively bind to the matrix.

12. The method of claim 10, wherein (i) comprises contacting the plurality of nucleic acids with a matrix under conditions wherein nucleic acids greater than about 550 by selectively bind to the matrix.

13. The method of claim 11, wherein the matrix comprises a solid phase reversible immobilisation (SPRI) bead solution

14. The method of claim 13, wherein the SPRI bead solution is selected from the group consisting of AMPURE XP, SPRISELECT, AXYPREP MAG FRAGMENTSELECT, and MAGJET Magnetic Beads.

15. The method of claim 9, wherein shearing the plurality of nucleic acids comprises a step selected from the group consisting of sonicating, acoustic shearing, nebulizing, needle shearing, and enzymatic fragmenting.

16. The method of claim 10, wherein the second fraction comprises nucleic acids less than about 550 bp.

17. The method of claim 10, wherein the second fraction comprises nucleic acids less than about 100 bp.

18. The method of claim 10, wherein the second fraction essentially consists of nucleic acids less than about 550 bp.

19. The method of claim 10, wherein the second fraction essentially consists of nucleic acids less than about 100 bp.

20. The method of claim 10, further comprising repeating (i) and (ii).

21. The method of claim 1, further comprising ligating adapters to the nucleic acids of the prepared library.

22. The method of claim 1, wherein the plurality of nucleic acids is selected from the group consisting of DNA, genomic DNA, cDNA, RNA, and circulating tumor DNA (ctDNA).

23. The method of claim 1, wherein the low quality nucleic acid sample comprises a fixed sample.

24. The method of claim 23, wherein the fixed sample is fixed with a compound selected from the group consisting of formalin, glutaraldehyde, alcohol, osmic acid, and paraformaldehyde.

25. The method of claim 1, wherein the sample is paraffin-embedded.

26. The method of claim 1, wherein the sample is a formalin fixed paraffin-embedded sample selected from the group consisting of a fine needle aspirate (FNA), a core biopsy, and a needle biopsy.

82. A nucleic acid library prepared by the method of any one of claims 1-81.

83. A method for sequencing a nucleic acid comprising:

obtaining the nucleic acid library of claim 82; and

sequencing at least a portion of the nucleic acid library, thereby sequencing the nucleic acid.

27. A method for preparing a nucleic acid library from a low quality nucleic acid sample comprising single stranded DNA, the method comprising:

(a) providing a plurality of nucleic acids obtained from the low quality nucleic acid sample, the nucleic acids comprising single stranded DNA;

(b) hybridizing random primers to the single stranded DNA and extending the primers to form double stranded DNA; and

(c) treating the double stranded DNA to remove single strand overhangs to form blunt ended DNA, thereby preparing a nucleic acid library.

28. The method of claim 27, wherein the method further comprises a DNA repair step comprising contacting the nucleic acids with an enzyme selected from the group consisting of Uracil N-Glycosylase (UNG), Uracil DNA Glycosylase (UDG), Endonuclease IV, Endonuclease VIII, formamidopyrimidine-DNA glycosylase (FPG), DNA-(apurinic or apyrimidinic site) lyase, and Pol β

.

29. The method of claim 28, wherein the DNA repair step is performed prior to step (b).

30. The method of claim 28, wherein the DNA repair step is performed after step (b).

31. The method of claim 28, wherein the DNA repair step is performed prior to step (c).

32. The method of claim 28, wherein the DNA repair step is performed after step (c).

33. A method for preparing a nucleic acid library from a low quality nucleic acid sample comprising:

(a) providing a plurality of nucleic acids obtained from the low quality nucleic acid sample; and

(b) selectively shearing the plurality of nucleic acids comprising;

(i) obtaining a first fraction comprising longer nucleic acids of the plurality of nucleic acids, and a second fraction comprising shorter nucleic acids of the plurality of nucleic acids,

(ii) shearing the nucleic acids of the first fraction, and

(iii) combining the first fraction and the second fraction, thereby preparing the nucleic acid library.

34. The method of claim 33, wherein (i) comprises contacting the plurality of nucleic acids with a matrix under conditions wherein nucleic acids greater than about 100 by selectively bind to the matrix.

35. The method of claim 33, wherein (i) comprises contacting the plurality of nucleic acids with a matrix under conditions wherein nucleic acids greater than about 550 by selectively bind to the matrix.

36. The method of claim 34, wherein the matrix comprises a solid phase reversible immobilisation (SPRI) bead solution

37. The method of claim 36, wherein the SPRI bead solution is selected from the group consisting of AMPURE XP, SPRISELECT, AXYPREP MAG FRAGMENTSELECT, and MAGJET Magnetic Beads.

38. The method of claim 33, wherein shearing the nucleic acids comprises a step selected from the group consisting of sonicating, acoustic shearing, nebulizing, needle shearing, and enzymatic fragmenting.

39. The method of claim 33, wherein the second fraction comprises nucleic acids less than about 550 bp.

40. The method of claim 33, wherein the second fraction comprises nucleic acids less than about 100 bp.

41. The method of claim 33, wherein the second fraction essentially consists of nucleic acids less than about 550 bp.

42. The method of claim 33, wherein the second fraction essentially consists of nucleic acids less than about 100 bp.

43. The method of claim 33, further comprising repeating (i) and (ii).

44. The method of claim 33, further comprising (c) removing single-stranded nucleic acids from the combined fractions.

45. The method of claim 44, further comprising removing the single-stranded portion of a nucleic acid comprising a single-stranded portion and a double-stranded portion.

46. The method of claim 44, wherein (c) comprises contacting the nucleic acids with a single-strand exonuclease.

47. The method of claim 46, wherein the single-strand exonuclease is a 5′

-3′

exonuclease.

48. The method of claim 46, wherein the single-strand exonuclease is selected from the group consisting of RecJ, Exonuclease I, Exonuclease T, Exonuclease VII, Exonuclease VIII, and Mung bean Exonuclease.

49. The method of claim 33, further comprising (d) repairing the nucleic acids of the combined fractions.

50. The method of claim 49, wherein (d) comprises contacting the nucleic acids with an enzyme selected from the group consisting of Uracil N-Glycosylase (UNG), Uracil DNA Glycosylase (UDG), Endonuclease IV, Endonuclease VIII, formamidopyrimidine-DNA glycosylase (FPG), DNA-(apurinic or apyrimidinic site) lyase, and Pol β

.

51. The method of claim 33, further comprising ligating adapters to the nucleic acids of the prepared library.

52. The method of claim 33, wherein the plurality of nucleic acids is selected from the group consisting of DNA, genomic DNA, cDNA, RNA, and circulating tumor DNA (ctDNA).

53. The method of claim 33, wherein the low quality nucleic acid sample comprises a fixed sample.

54. The method of claim 53, wherein the fixed sample is fixed with a compound selected from the group consisting of formalin, glutaraldehyde, alcohol, osmic acid, and paraformaldehyde.

55. The method of claim 33, wherein the sample is paraffin-embedded.

56. The method of claim 33, wherein the sample is a formalin fixed paraffin-embedded sample selected from the group consisting of a fine needle aspirate (FNA), a core biopsy, and a needle biopsy.

57. The method of claim 33, wherein the low quality nucleic acid sample comprises ancient nucleic acids.

58. A method for preparing a nucleic acid library from a low quality nucleic acid sample comprising:

(a) providing a plurality of nucleic acids obtained from the low quality nucleic acid sample;

(b) selectively shearing the plurality of nucleic acids comprising;

(i) obtaining a first fraction comprising longer nucleic acids of the plurality of nucleic acids, and a second fraction comprising shorter nucleic acids of the plurality of nucleic acids,

(ii) shearing the nucleic acids of the first fraction, and

(iii) combining the first fraction and the second fraction; and

(c) repairing the nucleic acids of the combined fractions, thereby preparing the nucleic acid library.

59. The method of claim 58, wherein (c) comprises contacting the nucleic acids with an enzyme selected from the group consisting of Uracil N-Glycosylase (UNG), Uracil DNA Glycosylase (UDG), Endonuclease IV, Endonuclease VIII, formamidopyrimidine-DNA glycosylase (FPG), DNA-(apurinic or apyrimidinic site) lyase, and Pol β

.

60. The method of claim 58, wherein (i) comprises contacting the plurality of nucleic acids with a matrix under conditions wherein nucleic acids greater than about 100 by selectively bind to the matrix.

61. The method of claim 58, wherein (i) comprises contacting the plurality of nucleic acids with a matrix under conditions wherein nucleic acids greater than about 550 by selectively bind to the matrix.

62. The method of claim 58, wherein the matrix comprises a solid phase reversible immobilisation (SPRI) bead solution

63. The method of claim 62, wherein the SPRI bead solution is selected from the group consisting of AMPURE XP, SPRISELECT, AXYPREP MAG FRAGMENTSELECT, and MAGJET Magnetic Beads.

64. The method of claim 58, wherein shearing the nucleic acids comprises a step selected from the group consisting of sonicating, acoustic shearing, nebulizing, needle shearing, and enzymatic fragmenting.

65. The method of claim 58, wherein the second fraction comprises nucleic acids less than about 550 bp.

66. The method of claim 58, wherein the second fraction comprises nucleic acids less than about 100 bp.

67. The method of claim 58, wherein the second fraction essentially consists of nucleic acids less than about 550 bp.

68. The method of claim 58, wherein the second fraction essentially consists of nucleic acids less than about 100 bp.

69. The method of claim 58, further comprising repeating (i) and (ii).

70. The method of claim 58, further comprising (d) removing single-stranded nucleic acids from the combined fractions.

71. The method of claim 70, further comprising removing the single-stranded portion of a nucleic acid comprising a single-stranded portion and a double-stranded portion.

72. The method of claim 70, wherein (d) comprises contacting the nucleic acids with a single-strand exonuclease.

73. The method of claim 72, wherein the single-strand exonuclease is a 5′

-3′

exonuclease.

74. The method of claim 72, wherein the single-strand exonuclease is selected from the group consisting of RecJ, Exonuclease I, Exonuclease T, Exonuclease VII, Exonuclease VIII, and Mung bean Exonuclease.

75. The method of claim 58, further comprising ligating adapters to the nucleic acids of the prepared library.

76. The method of claim 58, wherein the plurality of nucleic acids is selected from the group consisting of DNA, genomic DNA, cDNA, RNA, and circulating tumor DNA (ctDNA).

77. The method of claim 58, wherein the low quality nucleic acid sample comprises a fixed sample.

78. The method of claim 77, wherein the fixed sample is fixed with a compound selected from the group consisting of formalin, glutaraldehyde, alcohol, osmic acid, and paraformaldehyde.

79. The method of claim 58, wherein the sample is paraffin-embedded.

80. The method of claim 58, wherein the sample is a formalin fixed paraffin-embedded sample selected from the group consisting of a fine needle aspirate (FNA), a core biopsy, and a needle biopsy.

81. The method of claim 58, wherein the low quality nucleic acid sample comprises ancient nucleic acids.