Reduced representation bisulfite sequencing with diversity adaptors
First Claim
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1. A method for generating a bisulfite converted library, the method comprising:
- a) ligating a first oligonucleotide sequence from a pool of oligonucleotide sequences to a 5′
end of a first polynucleotide from a plurality of polynucleotides from a sample to generate a first oligonucleotide-polynucleotide complex,wherein following the ligating of a), a 3′
end of the first oligonucleotide sequence is immediately adjacent to a 5′
end of the first polynucleotide from the plurality of polynucleotides;
b) ligating a second oligonucleotide sequence from the pool of oligonucleotide sequences to a 5′
end of a second polynucleotide from the plurality of polynucleotides to generate a second oligonucleotide-polynucleotide complex, wherein the second oligonucleotide sequence terminates at its 3′
end with one random base, wherein following the ligating of b), the one random base at the 3′
end of the second oligonucleotide sequence is immediately adjacent to the 5′
end of the second polynucleotide from the plurality of polynucleotides;
c) ligating a third oligonucleotide sequence from the pool of oligonucleotide sequences to a 5′
end of a third polynucleotide from the plurality of polynucleotides to generate a third oligonucleotide-polynucleotide complex, wherein the third oligonucleotide sequence terminates at its 3′
end with two random bases, wherein following the ligating of c), a 3′
most base of the two random bases at the 3′
end of the third oligonucleotide sequence is immediately adjacent to the 5′
end of the third polynucleotide from the plurality of polynucleotides;
d) ligating a fourth oligonucleotide sequence from the pool of oligonucleotide sequences to a 5′
end of a fourth polynucleotide from the plurality of polynucleotides to generate a fourth oligonucleotide-polynucleotide complex, wherein the fourth oligonucleotide sequence terminates at its 3′
end with three random bases, wherein following the ligating of d), a 3′
most base of the three random bases at the 3′
end of the fourth oligonucleotide sequence is immediately adjacent to a 5′
end of the fourth polynucleotide from the plurality of polynucleotides;
e) treating the first, second, third, and fourth oligonucleotide-polynucleotide complexes with bisulfite, thereby generating a bisulfite converted polynucleotide library.
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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
21 Claims
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1. A method for generating a bisulfite converted library, the method comprising:
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a) ligating a first oligonucleotide sequence from a pool of oligonucleotide sequences to a 5′
end of a first polynucleotide from a plurality of polynucleotides from a sample to generate a first oligonucleotide-polynucleotide complex,wherein following the ligating of a), a 3′
end of the first oligonucleotide sequence is immediately adjacent to a 5′
end of the first polynucleotide from the plurality of polynucleotides;b) ligating a second oligonucleotide sequence from the pool of oligonucleotide sequences to a 5′
end of a second polynucleotide from the plurality of polynucleotides to generate a second oligonucleotide-polynucleotide complex, wherein the second oligonucleotide sequence terminates at its 3′
end with one random base, wherein following the ligating of b), the one random base at the 3′
end of the second oligonucleotide sequence is immediately adjacent to the 5′
end of the second polynucleotide from the plurality of polynucleotides;c) ligating a third oligonucleotide sequence from the pool of oligonucleotide sequences to a 5′
end of a third polynucleotide from the plurality of polynucleotides to generate a third oligonucleotide-polynucleotide complex, wherein the third oligonucleotide sequence terminates at its 3′
end with two random bases, wherein following the ligating of c), a 3′
most base of the two random bases at the 3′
end of the third oligonucleotide sequence is immediately adjacent to the 5′
end of the third polynucleotide from the plurality of polynucleotides;d) ligating a fourth oligonucleotide sequence from the pool of oligonucleotide sequences to a 5′
end of a fourth polynucleotide from the plurality of polynucleotides to generate a fourth oligonucleotide-polynucleotide complex, wherein the fourth oligonucleotide sequence terminates at its 3′
end with three random bases, wherein following the ligating of d), a 3′
most base of the three random bases at the 3′
end of the fourth oligonucleotide sequence is immediately adjacent to a 5′
end of the fourth polynucleotide from the plurality of polynucleotides;e) treating the first, second, third, and fourth oligonucleotide-polynucleotide complexes with bisulfite, thereby generating a bisulfite converted polynucleotide library. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21)
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Specification