Preparation of templates for methylation analysis

• US 9,868,982 B2
• Filed: 02/07/2008
• Issued: 01/16/2018
• Est. Priority Date: 02/07/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated), Massachusetts Institute of Technology
• Inventors: Gormley, Niall, Gnirke, Andreas, Jaffe, David, Nusbaum, Harris
• Primary Examiner(s): SALMON, KATHERINE D
• Application Number:
  US12/069,174
• Publication Number:
  US 20090148842A1
• Time in Patent Office: 3,631 Days
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides

Abstract

The invention relates to a method of preparing and using a library of template polynucleotides suitable for use as templates in solid-phase nucleic acid amplification and sequencing reactions to determine the methylation status of the cytosine bases in the library. In particular, the invention relates to a method of preparing and analyzing a library of template polynucleotides suitable for methylation analysis.

Claims

1. A method of genome-wide analysis of the methylation status of cytosine bases in a whole genome sample, comprising:

a. providing a whole genome sample and fragmenting the whole genome sample to produce fragmented double stranded nucleic acid target fragments, wherein the fragments are obtained from said whole genome, and wherein the fragments span across said whole genome;

b. ligating forked universal adaptors to the fragmented double stranded nucleic acid target fragments to produce adaptor-ligated double stranded nucleic acid target fragments comprising identical nucleic acid bases at each termini, wherein after said ligation step, said adaptor-ligated double stranded nucleic acid target fragments comprise a region of double stranded nucleic acids and at least one region of single stranded nucleic acids and wherein all cytosine bases in said forked universal adaptors are methylated, and wherein said forked universal adaptors are phosphorylated at the 5′

end and said at least one region of single stranded nucleic acids is capable of hybridizing to SEQ ID NO;

6;

c. treating the adaptor-ligated double stranded nucleic acid target fragments with bisulfite to convert non-methylated cytosine bases to uracil thereby producing treated adaptor-ligated double stranded nucleic acid target fragments;

d. optionally performing an amplification step to produce amplicons of the treated adapter-ligated double stranded nucleic acid target fragments;

e. attaching the treated adaptor-ligated double stranded nucleic acid fragments or the amplicons thereof to a solid support;

f. sequencing the treated adaptor-ligated double stranded nucleic acid target fragments or the amplicons thereof to generate sequences; and

g. analyzing the sequences of step to determine which cytosine bases were converted to uracil bases, thereby determining the methylation status of the whole genome sample.

2. The method according to claim 1, wherein the method comprises the step of performing an amplification step to produce amplicons of the treated adapter-ligated double stranded nucleic acid target fragments.

3. The method according to claim 2, wherein said amplification uses two or more amplification primers, at least one of said amplification primers comprising a region that extends beyond the 5′

end of the at least one region of single stranded nucleic acids of the forked universal adaptor sequences.

4. The method according to claim 2, wherein said amplification uses two or more amplification primers, at least one of said amplification primers comprising a region that hybridises to the at least one region of single stranded nucleic acids of the forked universal adaptor.

5. The method according to claim 4, wherein said amplification uses two or more amplification primers, at least one of said amplification primers comprising a region that hybridises to, and extends beyond the 5′

end of the at least one region of single stranded nucleic acids of the forked universal adaptor.

6. The method according to claim 2, wherein the amplification is a polymerase chain reaction and amplification products of the polymerase chain reaction are collected to provide a 5′

and 3′

modified library of template polynucleotide molecules comprising known sequences at their 5′ and

3′

ends.

7. The method according to claim 6, wherein the polymerase chain reaction is carried out using first oligonucleotide primers capable of annealing to adaptor sequences of each of the adaptor-ligated double stranded nucleic acid target fragments and second oligonucleotide primers capable of annealing to a region of extended strands produced by extension of the first oligonucleotide primers.

8. The method according to claim 7, wherein the first and second oligonucleotide primers have different nucleotide sequences.

9. The method according to claim 8, wherein the first and second oligonucleotide primers are capable of annealing to one of the strands in the region of the double stranded nucleic acids in the adaptor sequences of the adaptor-ligated double stranded nucleic acid target fragments.

10. The method according to claim 1, wherein the forked universal adaptors are formed by annealing partially complementary first methylated and second methylated polynucleotide strands, wherein a sequence of 5 or more consecutive nucleotides at 3′

end of the first strand is complementary to a sequence of 5 or more consecutive nucleotides at the 5′

end of the second strand, wherein a duplex region of 5 or more consecutive base pairs is formed by annealing the first and second strands and wherein a sequence of at least 10 consecutive nucleotides at the 5′

end of the first strand and a sequence of at least 10 consecutive nucleotides at the 3′

end of the second strand are not complementary such that a mismatched single stranded region of at least 10 consecutive nucleotides on each strand remains single stranded when the duplex region is annealed.

11. The method according to claim 10, wherein the duplex region formed when the two strands are annealed is 5 to 20 consecutive base pairs in length.

12. The method according to claim 10, wherein the mismatched single stranded region comprises 10 to 50 consecutive unpaired nucleotides on each strand.

13. The method according to claim 1 wherein the fragments are generated by random shearing.

14. The method according to claim 13 wherein the random shearing is hydroshearing or nebulisation.

15. The method according to claim 1 wherein the fragments are generated using an enzymatic treatment.

16. The method according to claim 15 wherein the enzymatic treatment is a restriction endonuclease that is selective for CG dinucleotides.

17. The method according to claim 16 wherein the enzyme is MspI.

18. The method according to claim 1, wherein the analysis is performed by comparing the sequence of the treated sample against a known reference sequence.

19. The method according to claim 1, wherein the analysis is performed by comparing the sequence of the treated sample against the sequence of an untreated sample.

20. The method according to claim 1, wherein the treated adaptor-ligated double stranded nucleic acid target fragments, or the amplicons thereof are further amplified on the solid support after step e and prior to step f.

21. The method according to claim 1, wherein the solid support is a planar array.

22. The method according to claim 21, wherein the planar array is a clustered array of amplified single target molecules.

23. The method according to claim 22, wherein the clustered array is formed by solid-phase nucleic acid amplification using immobilised amplification primers.

24. The method according to claim 23, wherein the clustered array is formed by isothermal solid-phase nucleic acid amplification using immobilised amplification primers.

25. The method according to claim 19, wherein the treated and untreated portions are combined and immobilised on the same support.

26. The method according to claim 1, wherein said sequencing involves cycles of addition of nucleotides.

27. The method according to claim 26, wherein said nucleotides are labelled.

28. The method according to claim 27, wherein nucleotide labels are fluorophores.

29. The method of claim 1, wherein said forked universal adaptors further comprise SEQ ID NO:

2.

30. The method of claim 7, wherein said first oligonucleotide primers and said second oligonucleotide primers comprise one or more of SEQ ID NO:

3 and SEQ ID NO;

4.

31. The method of claim 24, wherein said immobilised amplification primers comprise one or more of SEQ ID NO:

5 and SEQ ID NO;

6.