High-throughput sequencing method for methylated DNA and use thereof

• US 9,518,295 B2
• Filed: 08/11/2010
• Issued: 12/13/2016
• Est. Priority Date: 08/11/2010
• Status: Active Grant

• Current Assignee: BGI Shenzhen, Institute of Microelectronics Chinese Academy of Sciences
• Original Assignee: BGI Shenzhen, Institute of Microelectronics Chinese Academy of Sciences
• Inventors: Wang, Yan, Ye, Mingzhi, Han, Xu, Zhang, Xiuqing, Sun, Zhongsheng
• Primary Examiner(s): Bertagna, Angela M
• Application Number:
  US13/814,925
• Publication Number:
  US 20130244885A1
• Time in Patent Office: 2,316 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• 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 1/6869 Methods for sequencing
• 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 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor

Abstract

The present invention provides a high-throughput sequencing method for methylated DNA, and use thereof. Particularly, the present invention provides a high-throughput sequencing method for methylated DNA, which combines methylated DNA immunoprecipitation, removal of repetitive sequences, and bisulfite treatment. The site of sequencing library will be decreased, and the cost will be reduced by using the method disclosed in the present invention.

Claims

1. A method for constructing a methylated DNA library, consisting of:

A) fragmenting a DNA sample;

B) ligating auxiliary adapters to the ends of the fragmented DNA obtained in A);

C) enriching methylated DNA fragments;

D) removing fragments comprising moderately and highly repetitive sequences from the DNA fragments obtained in C);

E) converting unmethylated cytosines in the product obtained in D) into uracils by bisulfite treatment;

F) amplifying the DNA obtained in E) with primers; and

G) removing the auxiliary adapters by digesting the product obtained in step F) with a restriction enzyme,

wherein for step B), the ends of the auxiliary adapters that link to the fragmented DNA are joining ends, the other ends are non-joining ends, and the auxiliary adapters are selected from at least one of the following a-d;

a) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is an overhang structure and a joining end that is a blunt end;

b) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is a forked structure and a joining end that is a blunt end;

c) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is an overhang structure and a joining end that is a sticky end;

or

d) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is a forked structure and a joining end that is a sticky end;

and wherein (i) the primers in step F) are complementary to the sequence of the auxiliary adapters after conversion in step E) and have a restriction enzyme recognition site near the 5′

end, (ii) the cutting sites for said restriction enzyme are located within five base pairs downstream of the linking site of the auxiliary adapters to the fragmented DNA, and (iii) step G) is carried out using a single restriction enzyme with a recognition site that corresponds to the restriction enzyme recognition site contained in the primers.

3. The method of claim 1 or 2, wherein step A) comprises:

a) fragmenting genomic DNA into DNA fragments;

b) end repairing the DNA fragments obtained in step a) to obtain DNA fragments with blunt ends; and

c) adding an “

A”

base to the 3′

-end of the DNA fragments obtained in step b).

4. The method of claim 1, wherein step G) further comprises:

end repairing the DNA without auxiliary adapters and ligating sequencing adapters to the DNA after end repairing.

5. The method according to claim 1 or 2, wherein step C) is performed by the MeDIP technique or the MBD technique.

6. The method according to claim 1 or 2, wherein step D) is performed by hybridization based on Cot-1 DNA binding.

7. The method according to claim 1 or 2, wherein the auxiliary adapters are each labeled at the 5′

-end with a biotin.

2. A method for sequencing methylated DNA, consisting of:

A) fragmenting a DNA sample;

B) ligating auxiliary adapters to the ends of the fragmented DNA obtained in A);

C) enriching methylated DNA fragments;

D) removing moderately and highly repetitive sequences from the product obtained in C);

E) converting unmethylated cytosines in the product obtained in D) into uracils by bisulfite treatment;

F) amplifying the DNA obtained in E) with primers;

G) removing the auxiliary adapters by digesting the product obtained in step F) with a restriction enzyme; and

H) sequencing the DNA obtained in G);

optionally, comprising aligning the sequencing results of H) with the sequence of sample DNA or a reference sequence to identify the numbers and positions of methylated cytosine bases;

wherein in step B), the ends of the auxiliary adapters that link to the fragmented DNA are joining ends, the other ends are non-joining ends, and the auxiliary adapters are selected from at least one of the following a-d;

a) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is an overhang structure and a joining end that is a blunt end;

b) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is a forked end and a joining end that is a blunt end;

c) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is an overhang structure and a joining end that is a sticky end;

or

d) an adapter that is free of restriction enzyme cutting sites and has a non-joining end that is a forked structure and a joining end that is a sticky end;

and wherein (i) the primers in step F) are complementary to the sequence of the auxiliary adapters after conversion in step E) and have a restriction enzyme recognition site near the 5′

end, (ii) the cutting sites for said restriction enzyme are located within five base pairs downstream of the linking site of the auxiliary adapters to the fragmented DNA, and (iii) step G) is carried out using a single restriction enzyme with a recognition site that corresponds to the restriction enzyme recognition site contained in the primers.