COMPOSITIONS AND METHODS FOR SEQUENCING NUCLEIC ACIDS

• US 20130244886A1
• Filed: 09/28/2011
• Published: 09/19/2013
• Est. Priority Date: 09/29/2010
• Status: Active Grant

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Incorporated
• Inventors: Rigatti, Roberto, Boutell, Jonathan Mark, Betley, Jason Richard, Gormley, Niall Anthony
• Granted Patent: US 8,759,038 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2523/301 Sonication
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2565/537 characterised by the capture oligonucleotide acting as a primer
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Disclosed herein are compositions and methods for sequencing nucleic acids.

Claims

1. A method of generating overlapping sequencing reads, said method comprising:

providing a plurality of polynucleotides;

processing said plurality of polynucleotides so as to produce a set of overlapping nucleotide regions to be sequenced;

providing a substrate having a surface, said surface comprising a site having said set of overlapping nucleotide regions attached thereto; and

sequencing said set of overlapping nucleotide regions, thereby generating a set of overlapping sequencing reads.

2. The method of claim 1 further comprising the step of assembling sequencing reads from said set of overlapping sequencing reads so as to obtain a contiguous nucleotide sequence of a nucleic acid of interest.

3. The method of claim 2, wherein at least one of said polynucleotides comprises said contiguous nucleotide sequence of the nucleic acid of interest.

4. The method of claim 1, wherein said processing step comprises generating an overlapping set of deletion products from said plurality of polynucleotides.

5. The method of claim 4, wherein said plurality of nucleotides are attached to said site before said processing step.

6. The method of claim 4, wherein said plurality of nucleotides are attached to said site after said processing step.

7. The method of claim 4 further comprising amplifying said set of deletion products on the surface to form amplicons corresponding to the set of deletion products.

8. The method of claim 5, wherein said processing step comprises copying said polynucleotides so as to produce a plurality of polynucleotide copies, wherein said polynucleotide copies comprise a modified nucleotide that indicates the beginning of each nucleotide region to be sequenced.

9. A nucleic acid sequencing method comprising:

providing a substrate having a surface, said surface comprising a site having a plurality of polynucleotides attached thereto;

generating deletion products from said plurality of polynucleotides;

amplifying the deletion products on the surface to form amplicons corresponding to the deletion products; and

obtaining nucleotide sequence information from nucleic acids present in said amplicons.

10. The method of claim 9, wherein said deletion products comprise overlapping deletion products.

11. The method of claim 10 further comprising the step of assembling nucleotide sequence information corresponding to said overlapping deletion products, thereby producing a contiguous nucleotide sequence of a nucleic acid of interest.

12. The method of claim 9, wherein providing said substrate comprises hybridizing a plurality of template nucleic acids to a plurality of primers attached to said site.

13. The method of claim 12, wherein each template nucleic acid comprises an adaptor sequence that is complementary to a primer sequence.

14. The method of claim 13, wherein said plurality of primers comprises first primers and second primers, said first primers and said second primers comprising a different nucleotide sequence.

15. The method of claim 12 further comprising extending said primers, thereby producing extended strands.

16. The method of claim 15 further comprising preamplifying said extended strands prior to generating deletion products.

17. The method of claim 12, wherein generating deletion products comprises extending said primers in the presence of one or more terminator nucleotides such that said primers are extended to lengths that are less than the full length of the template nucleic acid.

18. The method of claim 17 further comprising dehybridizing said template nucleic acids, thereby producing single-stranded deletion products.

19. The method of claim 18 further comprising attaching an adaptor to a free end of the single-stranded deletion products.

20. The method of claim 12, wherein generating deletion products comprises extending said primers in the presence of uracil such that said primers are extended to lengths that are less than the full length of the template nucleic acid.

21. The method of claim 9, wherein generating deletion products comprises digesting said plurality of polynucleotides with an exonuclease.

22. The method of claim 9, wherein generating deletion products comprises physically shearing said polynucleotides.

23. The method of claim 9, wherein said polynucleotides comprise double-stranded polynucleotides having a nick and generating deletion products comprises translating said nick less than the full length of the polynucleotides and cleaving at said nick.

24. The method of claim 9, wherein said deletion products comprise an oligonucleotide tag.

25. The method of claim 24, wherein said oligonucleotide tag is complementary to a sequencing primer.

26. The method of claim 9, wherein the substrate comprises a flow chamber.

27. The method of claim 9, wherein the surface is planar.

28. The method of claim 9, wherein the surface is patterned.

29. The method of claim 9, wherein the surface comprises a channel in a flow chamber.

30. The method of claim 9, wherein said site comprises a well.

31. The method of claim 9, wherein said site comprises a bead in a well.

32. A nucleic acid sequencing method comprising:

providing a substrate having a surface, said surface comprising a site having a plurality of primers attached thereto;

contacting said plurality of primers with a plurality of template nucleic acids;

extending the primers in the presence of a first modified nucleotide such that said first modified nucleotide is incorporated in a plurality of extended strands;

synthesizing complements of the extended strands using a polymerase that incorporates a second modified nucleotide complementary to said first modified nucleotide; and

initiating a sequencing read from the point of incorporation of said first modified nucleotide, thereby producing nucleic acid sequence information.

33. The method of claim 32 further comprising initiating a sequencing read from the point of incorporation of said second modified nucleotide.

34. The method of claim 32 further comprising amplifying said extended strand and said complement to form amplicons prior to initiating said sequencing read.

35. The method of claim 32, wherein said sequence information comprises a set of overlapping sequencing reads.

36. The method of claim 35 further comprising the step of assembling overlapping sequencing reads, thereby producing a contiguous nucleotide sequence of a nucleic acid of interest.

37. The method of claim 32, wherein each template nucleic acid comprises an adaptor sequence that is complementary to a primer sequence.

38. The method of claim 32, wherein said plurality of primers comprises first primers and second primers, said first primers and said second primers comprising a different nucleotide sequence.

39. The method of claim 32, wherein said extended strands or said complements comprise an oligonucleotide tag.

40. The method of claim 39, wherein said oligonucleotide tag is complementary to a sequencing primer.

41. The method of claim 32, wherein said first modified nucleotide comprises d-isoGTP.

42. The method of claim 41, wherein said second modified nucleotide comprises d-isoCTP.

43. The method of claim 32, wherein the substrate comprises a flow chamber.

44. The method of claim 32, wherein the surface is planar.

45. The method of claim 32, wherein the surface is patterned.

46. The method of claim 32, wherein the surface comprises a channel in a flow chamber.

47. The method of claim 32, wherein said site comprises a well.

48. The method of claim 32, wherein said site comprises a bead in a well.

49. A nucleic acid sequencing method comprising:

providing a plurality of polynucleotides;

generating deletion products from said plurality of polynucleotides;

providing a substrate having a surface, said surface comprising a site having said deletion products attached thereto;

amplifying the deletion products on the surface to form amplicons corresponding to the deletion products; and

obtaining nucleotide sequence information from nucleic acids present in said amplicons.

50. The method of claim 49, wherein said deletion products comprise overlapping deletion products.

51. The method of claim 50 further comprising the step of assembling nucleotide sequence information corresponding to said overlapping deletion products, thereby producing a contiguous nucleotide sequence of a nucleic acid of interest.

52. The method of claim 49, wherein providing said substrate comprises hybridizing a plurality of deletion products to a plurality of primers attached to said site.

53. The method of claim 52, wherein each deletion product comprises an adaptor sequence that is complementary to a primer sequence.

54. The method of claim 53, wherein said plurality of primers comprises first primers and second primers, said first primers and said second primers comprising a different nucleotide sequence.

55. The method of claim 52 further comprising extending said primers, thereby producing extended strands.

56. The method of claim 53, wherein generating deletion products comprises extending random primers that hybridize to said polynucleotides and initiating extension at random positions such that said random primers are extended to lengths that are less than the full length of the polynucleotides.

57. The method of claim 56, wherein the random primers comprise a random hexamer oligonucleotide.

58. The method of claim 49, wherein generating deletion products comprises inserting transposon ends into said polynucleotides.

59. The method of claim 58, wherein inserting transposon ends into said polynucleotides produces fragments of said polynucleotides disposed between said transposon ends.

60. The method of claim 59, wherein generating deletion products further comprises amplifying the fragments that comprise the ends of the polynucleotides.

61. The method of claim 60, wherein generating deletion products further comprises amplifying the fragments that correspond to internal regions of the polynucleotides.

62. An array comprising:

a substrate having a surface, said surface having a site comprising a plurality of amplicons distributed thereon, wherein each of said plurality of amplicons comprises a set of amplified polynucleotides comprising a deletion product, said deletion product being different for different amplicons; and

a sequencing enzyme associated with each of said amplicons.

63. The array of claim 62, wherein each amplified polynucleotide comprises an oligonucleotide tag.

64. The array of claim 63, wherein said oligonucleotide tag is complementary to a sequencing primer.

65. The array of claim 62, wherein the substrate comprises a flow chamber.

66. The array of claim 62, wherein the surface is planar.

67. The array of claim 62, wherein the surface is patterned.

68. The array of claim 62 , wherein the surface comprises a channel in a flow chamber.

69. The array of claim 62, wherein said site comprises a well.

70. The array of claim 62, wherein said site comprises a bead in a well

71. The array of claim 62, wherein said sequencing enzyme comprises one or more polymerase activities.

72. An array comprising:

a substrate having a surface, said surface having a site comprising a plurality of amplicons distributed thereon, wherein each of said plurality of amplicons comprises a set of amplified polynucleotides, each polynucleotide comprising a modified nucleotide incorporated at the same sequence location, said location being different for different amplicons; and

a sequencing enzyme associated with each of said amplicons.

73. The array of claim 72, wherein each amplified polynucleotide comprises an oligonucleotide tag.

74. The array of claim 73, wherein said oligonucleotide tag is complementary to a sequencing primer.

75. The array of claim 72, wherein the substrate comprises a flow chamber.

76. The array of claim 72, wherein the surface is planar.

77. The array of claim 72, wherein the surface is patterned.

78. The array of claim 72, wherein the surface comprises a channel in a flow chamber.

79. The array of claim 72, wherein said site comprises a well.

80. The array of claim 72, wherein said site comprises a bead in a well.

81. The array of claim 72, wherein said sequencing enzyme comprises one or more polymerase activities.