Reduced representation bisulfite sequencing with diversity adaptors

• US 9,745,614 B2
• Filed: 02/27/2015
• Issued: 08/29/2017
• Est. Priority Date: 02/28/2014
• Status: Active Grant

• Current Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Original Assignee: NuGEN Technologies, Inc. (Tecan Group AG)
• Inventors: Schroeder, Benjamin G.
• Primary Examiner(s): Bhat, Narayan
• Application Number:
  US14/634,326
• Publication Number:
  US 20150284769A1
• Time in Patent Office: 914 Days
• Field of Search: 435 61, 435 611, 435 912, 435 9152, 536 231, 536 242, 422430, 506 16, 506 26
• 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 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing

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.

Claims

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.

2. The method of claim 1, further comprising fragmenting the plurality of polynucleotides prior to steps a), b), c), d), and e).

3. The method of claim 2, wherein the fragmenting comprises contacting the plurality of polynucleotides with an enzyme.

4. The method of claim 3, wherein the enzyme is a methylation insensitive restriction enzyme.

5. The method of claim 4, wherein the methylation insensitive restriction enzyme is MspI.

6. The method of claim 5, wherein the one random base of the second oligonucleotide sequence comprises an adenine, a thymine, or a guanine base.

7. The method of claim 5, wherein each of the two random bases of the third oligonucleotide sequence comprises an adenine, a thymine, or a guanine base.

8. The method of claim 5, wherein a 5′

most base of the three random bases of the fourth oligonucleotide sequence comprises an adenine or a guanine base, and each of the two other bases of the three random bases of the fourth oligonucleotide sequence comprises an adenine, a thymine, or a guanine base.

9. The method of claim 1, wherein each oligonucleotide sequence in the pool of oligonucleotide sequences is within a strand of a duplexed adaptor.

10. The method of claim 9, wherein the plurality of polynucleotides are double-stranded nucleic acids, wherein each 5′

end of the double-stranded nucleic acids comprises a ligation end and each 3′

end of the double-stranded nucleic acids comprises a non-ligation end.

11. The method of claim 9, wherein the duplexed adaptor comprises a ligation strand and a non-ligation strand, wherein the ligation strand is capable of ligating to a ligation end of the plurality of polynucleotides, and wherein a nick is formed between the non-ligation strand and a non-ligation end of the plurality of polynucleotides.

12. The method of claim 11, further comprising an extending step prior to step e), wherein the non-ligation end of the plurality of polynucleotides is extended using a polymerase in the presence of a mixture of dNTPs using the ligation strand of the duplexed adaptor as template.

13. The method of claim 12, wherein the ligation strand of the duplexed adaptor comprises one or more cytosine bases sensitive to bisulfite treatment and the mixture of dNTPs comprises dATP, dTTP, dGTP, and a dCTP analog resistant to bisulfite treatment.

14. The method of claim 13, wherein the dCTP analog resistant to bisulfite treatment comprises 5-methylcytosine, 5-hydroxymethylcytosine, or 5-propynylcytosine.

15. The method of claim 1, further comprising amplifying the first, second, third, and fourth oligonucleotide-polynucleotide complexes to generate amplified oligonucleotide-polynucleotide complexes.

16. The method of claim 15, wherein the amplifying comprises solid-phase nucleic acid amplification to generate a plurality of clusters, wherein different clusters of the plurality of clusters comprise a plurality of copies of the first, second, third, and fourth oligonucleotide-polynucleotide complexes.

17. The method of claim 16, further comprising sequencing each of the plurality of clusters, wherein the sequencing comprises hybridizing a sequencing primer to a sequence complementary to the sequencing primer in the amplified oligonucleotide-polynucleotide complexes, and conducting sequencing by synthesis.

18. The method of claim 17, wherein the sequencing comprises imaging.

19. The method of claim 1, wherein the first oligonucleotide sequence further comprises a sequence element, wherein the sequence element comprises a barcode, a universal sequence, a linker sequence, or a random sequence.

20. The method of claim 1, wherein the second oligonucleotide sequence further comprises sequence complementary to a sequencing primer.

21. The method of claim 20, wherein the sequence complementary to a sequencing primer is immediately adjacent to the one random base at the 3′

end of the second oligonucleotide sequence.