REDUCED REPRESENTATION BISULFITE SEQUENCING WITH DIVERSITY ADAPTORS
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Abstract
Described herein are methods, compositions and kits for the generation of bisulfite-converted libraries useful for conducting reduced representation bisulfite sequencing (RRBS). The methods described herein can be employed to generate RRBS libraries in a manner that is easier and more cost-efficient than conventional RRBS methods, and can be efficiently sequenced with next generation sequencing (NGS) techniques without the need for genomic, higher diversity sequencing controls such as PhiX spike-ins.
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Citations
143 Claims
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1-44. -44. (canceled)
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45. A method for generating a bisulfite converted library, the method comprising:
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a.) introducing a pool of oligonucleotide sequences to each end of a plurality of polynucleotides from a sample, i. wherein each oligonucleotide sequence in the pool of oligonucleotide sequences comprises a sequence complementary to a sequencing primer and a sequence element adjacent to the sequence complementary to a sequencing primer, ii. wherein the pool of oligonucleotide sequences comprises a first sequence, wherein the first sequence terminates at its 3′
end with the sequence element, wherein following ligation the 3′
end of the sequence element is adjacent to a 5′
end of a polynucleotide from the plurality of polynucleotides,iii. wherein the pool of oligonucleotide sequences comprises a second sequence, wherein the second oligonucleotide sequence terminates at its 3′
end with an additional base relative to the 3′
end of the first sequence, wherein following ligation the 3′
end of the second sequence is adjacent to a 5′
end of a polynucleotide from the plurality of polynucleotides;iv. wherein the pool of oligonucleotide sequences comprises a third sequence, wherein the third sequence terminates at its 3′
end with two additional bases relative to the 3′
end of the first sequence, wherein following ligation the 3′
end of the third sequence is adjacent to a 5′
end of a polynucleotide from the plurality of polynucleotides;v. wherein the pool of oligonucleotide sequences comprises a fourth sequence, wherein the fourth sequence terminates at its 3′
end with three additional bases relative to the 3′
end of the first sequence, wherein following ligation the 3′
end of the fourth sequence is adjacent to a 5′
end of a polynucleotide from the plurality of polynucleotides;vi. wherein the introducing generates a plurality of oligonucleotide sequence-polynucleotide complexes; and b.) treating the plurality of oligonucleotide sequence-polynucleotide complexes with bisulfite, thereby generating a bisulfite converted polynucleotide library. - View Dependent Claims (48, 49, 51, 52, 53, 54, 55, 56, 62, 65, 66, 67, 69, 77, 78, 81, 82)
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46-47. -47. (canceled)
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50. (canceled)
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57-61. -61. (canceled)
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63-64. -64. (canceled)
- 68. (canceled)
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70-74. -74. (canceled)
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76. (canceled)
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79-80. -80. (canceled)
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83-102. -102. (canceled)
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103. A method for analyzing sequence reads, the method comprising:
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a. generating a plurality of sequence reads, wherein the generating comprises conducting sequencing by synthesis on a plurality of clusters, wherein each cluster of the plurality of clusters comprises copies of one oligonucleotide sequence-polynucleotide from a plurality of oligonucleotide sequence-polynucleotides, wherein the sequencing by synthesis comprises hybridizing a sequencing primer to sequence complementary to the sequencing primer present in each cluster of the plurality of clusters, wherein the plurality of clusters are produced from solid-phase nucleic acid amplification of the plurality of oligonucleotide sequence-polynucleotides, wherein each of the plurality of oligonucleotide sequence-polynucleotides comprises a single nucleotide base extender sequence, a dinucleotide base extender sequence, a trinucleotide base extender sequence or no nucleotide base extender sequence between the sequence complementary to the sequencing primer of an oligonucleotide sequence and an end of a polynucleotide; and b. analyzing each of the plurality of sequence reads, wherein the analyzing comprises trimming or removing the extender sequence in silico from each of the plurality of sequence reads.
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104-142. -142. (canceled)
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143. A method for increasing diversity of sequences at ends of a plurality of polynucleotide fragments in a library, the method comprising:
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a. fragmenting polynucleotides to generate polynucleotide fragments; b. ligating a pool of adaptor sequences to 3′
ends of the plurality of polynucleotide fragments, wherein each adaptor sequence in the pool of adaptor sequences comprises a sequence complementary to a sequencing primer, wherein a 3′
end of the sequencing primer anneals at least four bases from the 5′
end of the adaptor sequence, and wherein each of four different types of nucleotide bases are present in each of the at least three terminal nucleotides at the 5′
ends of the adaptors, thereby increase diversity of sequences at ends of the plurality of polynucleotide fragments.
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Specification