Preparation of templates for methylation analysis

• US 20090148842A1
• Filed: 02/07/2008
• Published: 06/11/2009
• Est. Priority Date: 02/07/2007
• Status: Active Grant

• Current Assignee: Massachusetts Institute of Technology
• Original Assignee: Massachusetts Institute of Technology
• Inventors: Gnirke, Andreas, Nusbaum, Harris, Jaffe, David, Gormley, Niall
• Granted Patent: US 9,868,982 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 analysing a library of template polynucleotides suitable for methylation analysis.

Claims

1. A method of analysing methylation status of cytosine bases in a nucleic acid, comprising:

a. providing a sample of fragmented double stranded nucleic acid target fragments derived from said nucleic acid;

b. ligating 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 cytosine bases in said universal adaptors are methylated and said universal adaptors comprise a region of double stranded nucleic acids and at least one region of single stranded nucleic acids;

c. treating the adaptor-ligated double stranded nucleic acid target fragments with a reagent that converts the non-methylated cytosine bases to uracil to produce a treated sample of adaptor-ligated double stranded nucleic acid target fragments;

d. sequencing the treated adaptor-ligated double stranded nucleic acid target fragments;

e. analysing the sequences of the treated sample to determine which cytosine bases were converted to uracil bases, thereby determining the methylation status of the nucleic acid.

2. The method according to claim 1, wherein said reagent comprises bisulfite.

3. The method according to claim 1, wherein the method comprises an additional step of amplification in solution prior to the sequencing step.

4. The method according to claim 3, 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 universal adaptor sequences.

5. The method according to claim 3, 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 universal adaptor.

6. The method according to claim 5, 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 universal adaptor.

7. The method according to claim 3, 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.

8. The method according to claim 7, 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.

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

10. The method according to claim 9, 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.

11. The method according to claim 1, wherein the universal adaptors are forked adaptors 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.

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

13. The method according to claim 11, wherein the mismatched single stranded region comprises 10 to 50 consecutive unpaired nucleotides on each strand.

14. The method according to claim 1, wherein the nucleic acid is genomic DNA.

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

16. The method according to claim 15 wherein the random shearing is hydroshearing or nebulisation.

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

18. The method according to claim 17 wherein the enzymatic treatment is a restriction endonuclease that is selective for CG dinucleotides.

19. The method according to claim 18 wherein the enzyme is MspI.

20. The method according to claim 1, wherein the sequencing is performed on a solid support.

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

22. 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.

23. The method according to claim 1, wherein the adaptor-ligated double stranded nucleic acid target fragments, or amplicons thereof are immobilised on a solid support and further amplified prior to sequencing.

24. The method according to claim 23, wherein the solid support is a planar array.

25. The method according to claim 24, wherein the planar array is a clustered array of amplified single target molecules.

26. The method according to claim 25, wherein the clustered array is formed by solid-phase nucleic acid amplification using immobilised amplification primers.

27. The method according to claim 26, wherein the clustered array is formed by isothermal solid-phase nucleic acid amplification using immobilised amplification primers.

28. The method according to claim 22, wherein the treated and untreated portions are combined and immobilised on the same support.

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

30. The method according to claim 29, wherein said nucleotides are labelled.

31. The method according to claim 30, wherein nucleotide labels are fluorophores.

32. A kit for preparing a 5′

and 3′

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

3′

ends, the kit comprising methylated adaptor polynucleotides as defined in claim 1 and oligonucleotide primers capable of annealing to the methylated adaptor polynucleotides.

33. The kit according to claim 32, wherein the oligonucleotide primers are capable of annealing to the at least one region of single stranded nucleic acids of the universal adaptor polynucleotides.

34. The kit according to claim 33, wherein the methylated adaptor polynucleotides are forked adaptors 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 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.