METHODS AND COMPOSITIONS FOR RAPID NUCLEIC ACID LIBRARY PREPARATION

• US 20170247689A1
• Filed: 09/09/2015
• Published: 08/31/2017
• Est. Priority Date: 09/09/2014
• Status: Active Grant

• Current Assignee: iGenomX International Genomics Corporation (Twist Bioscience Corporation)
• Original Assignee: iGenomX International Genomics Corporation (Twist Bioscience Corporation)
• Inventors: Brown, Keith
• Granted Patent: US 10,450,562 B2
• Time in Patent Office: Days
• 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 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• 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 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 30/20 Sequence assembly

Abstract

Rapid nucleic acid libraries, methods of generation, kits, and compositions relating to library synthesis, including reagents, intermediaries and final products are disclosed herein. The disclosure enables rapid synthesis of libraries that allow independent verification of sequence information and rapid identification of sequence information with template of origin.

Claims

1. A method of generating a population of non-identical, tagged nucleic acid molecule sequences each comprising a subset of sequence from a target nucleic acid sample, the method comprising:

a) obtaining a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length from said target nucleic acid sample;

b) annealing an oligonucleotide comprising a second molecular tag sequence to said first nucleic acid molecule;

c) extending said oligonucleotide to obtain a first double-stranded nucleic acid molecule comprising a first molecular tag sequence, a first target sequence having a first length, and a second molecular tag sequence;

d) obtaining a second double-stranded nucleic acid molecule comprising a third molecular tag sequence, a second target sequence having a second length, and a fourth molecular tag sequence;

e) sequencing the first nucleic acid molecule and the second nucleic acid molecule; and

f) discarding the sequence of said second double-stranded nucleic acid molecule if;

said third molecular tag sequence is identical to said first molecular tag sequence;

said fourth molecular tag sequence is identical to said second molecular tag sequence;

said second target sequence is identical to said first target sequence; and

said second target sequence length is identical to said first target sequence length.

2. The method of claim 1, further comprising:

g) retaining the sequence of said second double-stranded nucleic acid molecule if;

said third molecular tag sequence is different from said first molecular tag sequence;

said fourth molecular tag sequence is different from said second molecular tag sequence;

said second target sequence is different from said first target sequence;

or

said second target sequence length is different from said first target sequence length, thereby generating a population of non-identical, tagged nucleic acid molecules each comprising a subset of sequence from a target nucleic acid sample.

3. The method of claim 1, wherein said first nucleic acid molecule is obtained through contacting a first primer comprising a first random oligonucleotide sequence to a target nucleic acid sample.

4. The method of claim 3, wherein said contacting a first primer comprises annealing said first primer to a nucleic acid of said target nucleic acid sample.

5. The method of claim 1, wherein said first nucleic acid molecule comprises a molecular ligand.

6. The method of claim 5, wherein said molecular ligand comprises biotin.

7. The method of claim 3, wherein said second nucleic acid molecule is generated through contacting a second primer comprising a second random oligonucleotide sequence to said first nucleic acid molecule.

8. The method of claim 3, wherein said first random oligonucleotide sequence of said first nucleic acid molecule consists of a number of nucleic acid bases selected from the list consisting of 6, 7, 8, 9, and 10 nucleotide bases.

9. The method of claim 8, wherein said first nucleic acid molecule comprises an adapter sequence positioned 5′

to said first random oligonucleotide sequence.

10. The method of claim 3, comprising contacting said first nucleic acid and said first primer to a nucleic acid polymerase and a nucleotide triphosphate.

11. The method of claim 10, wherein said nucleotide triphosphate is selected by said nucleic acid polymerase from a pool comprising deoxynucleotide triphosphates and dideoxynucleotide triphosphates.

12. The method of claim 11, wherein said pool comprises dideoxynucleotide triphosphates in an amount ranging from 0.01% to 5%.

13. The method of claim 11, wherein said pool comprises dideoxynucleotide triphosphates in an amount ranging from 0.05% and 1.0%.

14. The method of claim 10, wherein said nucleic acid polymerase has strand displacement activity.

15. The method of claim 11, wherein said pool comprises at least one of said dideoxynucleotide triphosphates bound to a molecular ligand.

16. The method of claim 15, wherein said molecular ligand comprises biotin.

17. The method of any one of claims 1-16, comprising contacting said first double-stranded nucleic acid molecule to a ligand binding agent.

18. The molecule of claim 17, wherein said ligand binding agent is avidin.

19. The molecule of claim 17, wherein said ligand binding agent is streptavidin.

20. The method of claim 11, wherein said pool comprises a population of a natural dNTP pair and a population of a semi-complementary dNTP pair.

21. The method of claim 20, wherein the population of a natural dNTP pair comprises dCTP and dGTP.

22. The method of claim 20, wherein the population of a semi-complementary pair comprises 2,6-diaminopurine and 2-thiothymidine.

23. The method of claim 20, wherein the pool comprises deoxynucleotides dCTP, dGTP, d(2,6-aminopurine)TP and d(2-thiothymidine)TP.

24. The method of any one of claims 20-23, wherein the pool comprises a population of ddNTP.

25. The method of claim 7, wherein each of said first random oligonucleotide sequence and said second random oligonucleotide sequence is selected to reflect the GC content of the target nucleic acid sample.

26. The method of any of claims 1-25, wherein at least one of said nucleic acids is a deoxyribonucleic acid.

27. The method of any of claims 1-25, wherein at least one of said nucleic acids is a ribonucleic acid.

28. The method of claim 22, wherein said target nucleic acid sample is ribonucleic acid.

29. The method of claim 23, wherein said first nucleic acid molecule is a complementary deoxyribonucleic acid molecule generated from said ribonucleic acid.

30. The method of claim 22, wherein said nucleic acid polymerase is an RNA-dependent DNA polymerase.

31. The method of claim 10, wherein said nucleic acid polymerase lacks strand displacement activity.

32. The method of claim 1, wherein said first nucleic acid molecule is a complementary nucleic acid molecule generated through contacting a first primer comprising an oligo(dT) sequence to said target nucleic acid sample.

33. The method of claim 21, wherein said deoxyribonucleic acid is fragmented into fragments greater than 10 kilobases.

34. The method of claim 1, comprising assigning all sequences from a given contig having the same molecular tag to a specific chromosome.

35. The method of any one of claim 1-6 or 8-16, wherein said second nucleic acid molecule is generated through contacting a second primer comprising a locus-specific oligonucleotide sequence and a second molecular tag sequence to said first nucleic acid molecule.

36. The method of claim 1, wherein steps a)-d) are performed in a single tube.

37. The method of any one of claims 1-35, wherein steps a)-d) are performed in a single tube.

38. The method of claim 1, wherein substantially no covalent bonds of said nucleic acid sample are disrupted.

39. The method of any one of claims 1-37, wherein substantially no covalent bonds of said nucleic acid sample are disrupted.

40. A composition comprising:

a first nucleic acid molecule comprising a first molecular tag sequence and a first target sequence having a first length; and

an oligonucleotide comprising a second molecular tag sequence.

41. The composition of claim 40, wherein said first nucleic acid molecule comprises a 3′

deoxynucleotide.

42. The composition of claim 40, wherein said first nucleic acid molecule comprises a 3′

dideoxynucleotide.

43. The composition of claim 40, wherein said first nucleic acid molecule comprises a semi-compatible base pair set.

44. The composition of claim 40, wherein said first nucleic acid molecule comprises 2,6-diaminopurine.

45. The composition of claim 40, wherein said first nucleic acid molecule comprises 2-thiothymidine.

46. The composition of claim 40, wherein said first nucleic acid molecule comprises 2,6-diaminopurine, 2-thiothymidine, cytosine and guanine.

47. The composition of claim 46, wherein said first nucleic acid molecule comprises 2,6-diaminopurine, 2-thiothymidine, cytosine and guanine, and wherein said first nucleic acid molecule does not comprise adenine or thymidine in sequence resulting from primer extension.

48. The composition of claim 40, wherein said first nucleic acid molecule comprises an adapter sequence positioned 5′

to said first molecular tag sequence.

49. The composition of claim 40, wherein said first adapter comprises SEQ ID NO:

1.

50. The composition of claim 49, wherein said molecular ligand comprises biotin.

51. The composition of claim 40, wherein said composition comprises a ligand binding agent.

52. The composition of claim 51, wherein said ligand binding agent comprises avidin.

53. The composition of claim 51, wherein said ligand binding agent comprises streptavidin.

54. The composition of claim 51, wherein said ligand binding agent is bound to a magnetic bead.

55. The composition of any one of claims 40-53, comprising unincorporated nucleotides.

56. The composition of any one of claims 40-53, comprising unincorporated deoxynucleotides.

57. The composition of any one of claims 40-53, comprising unincorporated dideoxynucleotides.

58. The composition of any one of claims 40-53, wherein said a first nucleic acid molecule is hybridized to said oligonucleotide comprising a second molecular tag sequence.

59. The composition of claim 58, wherein said first nucleic acid molecule is completely hybridized to said second molecular tag sequence of said oligonucleotide.

60. The composition of claim 58, wherein said first nucleic acid molecule is incompletely hybridized to said second molecular tag sequence of said oligonucleotide.

61. The composition of any one of claims 40-60, comprising a ligand-ligand binding agent wash buffer.

62. The composition of any one of claims 40-61, comprising a biotin wash buffer.

63. A composition comprising a population of nucleic acid molecules, wherein each molecule of said population independently comprises:

a first strand comprising

a first adapter sequence,

a molecular tag sequence, and

an independent target sequence,

wherein said each independent target sequence comprises a subset of a sample nucleic acid sequence, and wherein at least a first molecule of said population comprises an independent target sequence comprising a first subset of said sample nucleic acid sequence, and wherein at least a second molecule of said population comprises an independent target sequence that comprises a second subset of said sample nucleic acid sequence.

64. The composition of claim 63, wherein said adapter of each first strand of said population is identical.

65. The composition of claim 63, wherein said molecular tag sequence of each molecule of said population comprises at least 6 nucleotide bases.

66. The composition of claim 63, wherein a first member of said population and a second member of said population comprise non-identical molecular tag sequences.

67. The composition of any one of claims 63-66, wherein each first strand comprises a 3′

- deoxynucleotide base at its 3′

end.

68. The composition of any one of claims 63-67, wherein each first strand comprises a molecular ligand at its 5′

end.

69. The composition of any one of claims 63-68, wherein each first strand comprises a molecular ligand attached at a non-terminal position.

70. The composition of any one of claims 63-69, wherein each first strand comprises a molecular ligand at its 3′

end.

71. The composition of any one of claims 63-70, wherein said molecular ligand is biotin.

72. The composition of any one of claims 63-71, wherein each molecule of said population comprises a second strand comprising:

a second adapter sequence, and

a second molecular tag sequence.

73. The composition of claim 72, wherein said second strand of at least one molecule of said population is annealed to a first strand via at least partial base pairing of a second molecular tag sequence of said second strand to the independent target sequence of said first strand.

74. The composition of any one of claims 72-73, wherein said adapter of each second strand of said population is identical.

75. The composition of any one of claims 63-74, wherein at least one molecule of said population is bound to a molecular ligand binder.

76. The composition of claim 75, wherein said molecular ligand binder comprises avidin.

77. The composition of claim 75, wherein said molecular ligand binder comprises streptavidin.

78. The composition of any one of claims 63-77, wherein said composition comprises unincorporated nucleic acid triphosphates.

79. The composition of any one of claims 63-77, wherein said composition comprises molecular ligand binder wash buffer.

80. The composition of any one of claims 63-77, wherein said composition comprises polymerase extension buffer.

81. The composition of any one of claims 63-77, wherein said composition comprises nucleic acid polymerase.

82. The composition of claim 81, wherein said composition comprises nucleic acid polymerase possessing nucleic acid helicase activity.

83. The composition of claim 81, wherein said composition comprises nucleic acid polymerase possessing nucleic acid strand displacement activity.

84. The composition of claim 81, wherein said composition comprises nucleic acid comprising SEQ ID NO:

1 and SEQ ID NO;

2.

85. The composition of claim 63, wherein the composition is contained in a single tube.

86. The composition of any one of claims 63-84, wherein the composition is contained in a single tube.