HIGH-THROUGHPUT SEQUENCING METHOD FOR METHYLATED DNA AND USE THEREOF

• US 20130244885A1
• Filed: 08/11/2010
• Published: 09/19/2013
• Est. Priority Date: 08/11/2010
• Status: Active Grant

• Current Assignee: BGI Shenzhen, Institute of Microelectronics Chinese Academy of Sciences
• Original Assignee: Han Xu
• Inventors: Wang, Yan, Ye, Mingzhi, Han, Xu, Zhang, Xiuqing, Sun, Zhongsheng
• Granted Patent: US 9,518,295 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• 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

1-9. -9. (canceled)

10. A method for constructing a methylated DNA library, comprises the following steps:

A) fragmenting a DNA sample;

B) ligating cleavable 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 single-stranded DNA fragments obtained in C);

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

F) amplifying the single-stranded DNA obtained in E) with primers to obtain double-stranded DNA;

G) removing the cleavable auxiliary adapters.

12. The method of claim 10 or 11, wherein the step A) comprises:

a) fragmenting genomic DNA into DNA fragments;

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

c) adding an “

A”

base to the 3′

-end of the DNA fragments obtained in b).

13. The method of claim 10, wherein step G) further comprises:

end repairing of the DNA without auxiliary adapters, ligating sequencing adaptors to the DNA after end repairing to obtain a methylated DNA library.

14. The method of claim 10 or claim 11, wherein step E) is bisulfite treatment.

15. The method according to claim 10 or 11, wherein the step G) is performed by designing a restriction enzyme cutting site in the auxiliary adapter and/or primer.

16. The method according to claim 15, wherein in step B), the end of auxiliary adapter that links to the fragmented DNA is a joining end, the other end is a non-joining end, and the auxiliary adapter is selected from at least one of the following a-h:

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

b). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being an overhang structure and a joining end being a blunt end;

c). an adapter that is free of restriction enzyme cutting site and has a non-joining end being a forked structure and a joining end being a blunt end;

d). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being a forked structure and a joining end being a blunt end;

e). an adapter that is free of restriction enzyme cutting site and has a non-joining end being an overhang structure and a joining end being a sticky end;

f). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being an overhang structure and a joining end being a sticky end;

g). an adapter that is free of restriction enzyme cutting site and has a non joining end being a forked structure and a joining end being a sticky end;

h). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being a forked structure and a joining end being a sticky end;

according to the auxiliary adapter in the step B), the primer in the step F) and the digestion enzyme in the step G) are designed as follows;

(I) when an auxiliary adapter is a) and/or c), the primer is designed to be complementary to the sequence of auxiliary adapter after conversion in E) and to have recognition sites of restriction enzyme on/near its 5′

-end, wherein the restriction enzyme recognition site are located on the primer, and the restriction enzyme cutting site are assured to locate within 5 bp of the upstream and downstream of the linking site of the auxiliary adapter to the DNA to be detected;

when step G) is carried out, single enzymatic digestion is performed using an enzyme corresponds to the designed restriction enzyme cutting site;

(II) when an auxiliary adapter is b) and d), a restriction enzyme cutting site is designed to locate within 5 bp of upstream of the linking site of the auxiliary adapter to the DNA to be detected;

the restriction enzyme cutting site is required to have the following three characteristics;

(1) the restriction enzyme cutting site comprises at least one methylated cytosine;

(2) the restriction enzyme cutting site does not comprise unmethylated cytosines; and

(3) the restriction enzyme cutting site does not comprise CpG dinucleotide site;

the primer is designed to be complementary to the sequence of auxiliary adapter after conversion in E);

when the step G) is carried out, a single enzymatic digestion is performed using the enzyme corresponds to the designed methylated restriction enzyme cutting site;

optionally, another restriction enzyme cutting site is designed on the 5′

-end of the primer, and the two restriction enzyme cutting sites are overlapped by adjusting the length of the auxiliary adapter;

when the step G) is carried out, a double enzymatic digestion is performed using enzymes correspond to the designed restriction enzyme cutting sites.

(III) when an auxiliary adapter is e) and g), the design principle of the auxiliary adapter and primer is the same as that in the solution wherein an auxiliary adapter is a) and c), and the difference between them only resides in linkage manner to target DNA fragment, that is, the linkage manner for a) and c) is “

T-A”

linkage, while for e) and g) is sticky end linkage;

(IV) when an auxiliary adapter is f) and h), the design principle of the auxiliary adapter and primer is the same as that in the solution wherein an auxiliary adapter is b) and d), and the difference between themonly residesin linkage manner to target DNA fragment, that is, the linkage manner for b) and d) is “

T-A”

linkage, while for f) and h) is sticky end linkage;

(V) the design of auxiliary adapter is the same as that of b) and d) adapters, a restriction enzyme cutting site is further designed on the position within 20-30 bp of upstream close to the linkage site of DNA to be detected to the auxiliary adapter, and the restriction enzyme cutting site has the following four characteristics;

(1) the restriction enzyme cutting site comprises at least one methylated cytosine;

(2) the restriction enzyme cutting site does not comprise unmethylated cytosines;

(3) the restriction enzyme cutting site does not comprises CpG dinucleotide site; and

(4) the restriction enzyme cutting site is designed on the position within 5 bp of upstream or downstream close to the linking site of DNA to be detected to the auxiliary adapter;

the primer is designed as a sequence complementary to the sequence of auxiliary adapter after E) conversion;

when the step G) is carried out, a single enzyme digestion is performed by using an enzyme corresponds to the designed methylated restriction enzyme cutting site;

(VI) a combination of (V) and b), d), f) and h), wherein the two restriction enzyme cutting sites are overlapped by adjusting the length of the auxiliary adapter, and the auxiliary adapter is removed by means of double enzyme digestion;

the primer is designed into a sequence complementary to the sequence of auxiliary adapter after E) conversion, when the step G) is carried out, a double enzyme digestion is performed by using the enzymes correspond to the designed restriction enzyme cutting sites.

17. The method according to claim 10 or 11, wherein the enriching of methylated DNA in step C) is performed by MeDIP or MBD technique;

18. The method according to claim 10 or 11, wherein step D) is performed by hybridization based on C0T1 DNA binding.

19. The method according to claim 16, wherein the restriction enzyme cutting site methylation in step F) refers to that the cytosine in the restriction enzyme cutting site designed in the auxiliary adapter is methylated.

20. The method according to claim 10 or 11, wherein the auxiliary adapter is designed into a structure for preventing self-connection and having a biotin labeled at the 5′

-end.

21. A methylated DNA library constructed by the method according to claim 10.

11. A sequencing method of methylated DNA, comprises the following steps:

A) fragmenting a DNA sample;

B) ligating cleavable 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;

F) amplifying the single-stranded DNA obtained in E) with primers to obtain double-stranded DNA;

G) removing the cleavable auxiliary adapters;

H) sequencing the double-stranded DNA obtained in G);

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

27. An automatic device for carrying out the method according to claim 11, comprises:

A) an element for fragmenting a DNA sample;

B) a linkage element for ligating the auxiliary adapters to the two ends of the double-stranded DNA obtained in A);

C) an element for enriching the methylated DNA of the product from B);

D) an removal element for removing moderately and highly repetitive sequences from the product in C);

E) an element for converting unmethylated cytosines in the product obtained in D) into uracils;

F) an element for amplification of single stranded-DNA obtained in E) via a primer to obtain a double-stranded DNA;

G) an element for removing the cleavable auxiliary adapter;

H) a sequencing element for sequencing double stranded DNA in G).

28. The device according to claim 27, wherein A) further comprises an element for end repairing;

G) further comprises an element for end repairing of the double stranded without auxiliary adapter, an element for ligating sequencing adaptor to the ends of repaired DNA.

22. A kit comprising at least a reagent for DNA end repair, an auxiliary adapter reagent, a reagent for enriching methylated DNA by MeDIP or MBD technique, a reagent for converting methylated cytosines into uracils, a reagent for amplification, and a reagent for cleaving auxiliary adapter;

wherein the auxiliary adapter comprises partially or fully complementary forward/reverse oligonucleotides and can form a double-stranded auxiliary adapter after annealing;

said auxiliary adapter is designed to be a double-stranded DNA sequence capable of linking to the end of the double-stranded DNA, wherein the end of auxiliary adapter that links to the repaired double-stranded DNA is a joining end, the other end is a non-joining end;

said reagent for amplification includes a primer;

the design of the sequence of auxiliary adapter along with the design of the sequence of primer is such that the auxiliary adapter is disgestable.

23. The kit according to claim 22, wherein

the end of auxiliary adapter that links to the double-stranded DNA is a joining end, the other end is a non-joining end;

the digestion reagent is a restriction endonuclease;

said auxiliary adapter is selected from at least one of the following a-h adapters;

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

b). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being an overhang structure and a joining end being a blunt end;

c). an adapter that is free of restriction enzyme cutting site and has a non-joining end being a forked structure and a joining end being a blunt end;

d). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being a forked structure and a joining end being a blunt end;

e). an adapter that is free of restriction enzyme cutting site and has a non-joining end being an overhang structure and a joining end being a sticky end;

f). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being an overhang structure and a joining end being a sticky end;

g). an adapter that is free of restriction enzyme cutting site and has a non joining end being a forked structure and a joining end being a sticky end;

h). an adapter that comprises restriction enzyme cutting sites and has a non-joining end being a forked structure and a joining end being a sticky end;

wherein the amplification reagent is selected from one of (I)-(VI);

(I) when an auxiliary adapter is a) and c), the primer in the PCT amplification reagent is designed to be complementary to the sequence of auxiliary adapter after bisulfite treatment and to have recognition sites of restriction enzyme additionally attached to the 5′

-end, wherein the restriction enzyme cutting site are located on the primer, and the restriction enzyme cutting site are assured to locate within 5 bp of the upstream and downstream of the linking site of the auxiliary adapter to the DNA to be detected;

(II) when an auxiliary adapter is b) and d), a restriction enzyme cutting site is designed to locate within 5 bp of upstream of the linking site of the auxiliary adapter to the DNA to be detected;

the restriction enzyme cutting site is required to have the following three characteristics;

(1) the restriction enzyme cutting site comprises at least one methylated cytosine;

(2) the restriction enzyme cutting site does not comprise unmethylated cytosines; and

(3) the restriction enzyme cutting site does not comprise CpG dinucleotide site;

the primer is designed to be complementary to the sequence of auxiliary adapter after bisulfite treatment;

(III) when an auxiliary adapter is e) and g), the design principle of the auxiliary adapter and primer is the same as that in the solution wherein an auxiliary adapter is a) and c), and the difference between them only resides in linkage manner to target DNA fragment, that is, the linkage manner for a) and c) is “

T-A”

linkage, while for e) and g) is sticky end linkage;

(IV) when an auxiliary adapter is f) and h), the design principle of the auxiliary adapter and primer is the same as that in the solution wherein an auxiliary adapter is b) and d), and the difference between them only resides in linkage manner to target DNA fragment, that is, the linkage manner for b) and d) is “

T-A”

linkage, while for f) and h) is sticky end linkage;

(V) the design of auxiliary adapter is the same as that of b) and d) adapters, a restriction enzyme cutting site is further designed on the position within 20-30 bp of upstream close to the linkage site of DNA to be detected to the auxiliary adapter, and the restriction enzyme cutting site has the following four characteristics;

(1) the restriction enzyme cutting site comprises at least one methylated cytosine;

(2) the restriction enzyme cutting site does not comprise unmethylated cytosines;

(3) the restriction enzyme cutting site does not comprises CpG dinucleotide site; and

(4) the restriction enzyme cutting site is designed on the position within 5 bp of upstream or downstream close to the linking site of DNA to be detected to the auxiliary adapter;

the primer is designed as a sequence complementary to the sequence of auxiliary adapter after bisulfite treatment;

(VI) a combination of (V) and b), d), f) and h), wherein the two restriction enzyme cutting sites are overlapped by adjusting the length of the auxiliary adapter, and the auxiliary adapter is removed by means of double enzyme digestion;

the primer is designed into a sequence complementary to the sequence of auxiliary adapter after bisulfite treatment.

24. The kit according claim 22, wherein the reagent for enriching methylated DNA comprises 5′

-methylcytosine antibody or Methyl-CpG Binding Domain Protein (MBD).

25. The kit according to claim 22, wherein the reagent for converting methylated cytosines into uracils is bisulfite.

26. Use of the kit according to claim 22 for the construction of a methyalted DNA library or for sequencing methylated DNA.