Targeted sequencing technique for whole genome DNA methylation

• US 10,011,867 B2
• Filed: 11/13/2014
• Issued: 07/03/2018
• Est. Priority Date: 11/15/2013
• Status: Active Grant

• Current Assignee: Fudan University
• Original Assignee: Fudan University
• Inventors: Yu, Wenqiang, Li, Yan, Wu, Feizhen
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US14/902,509
• Publication Number:
  US 20160376644A1
• Time in Patent Office: 1,328 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/331 Methylation site specific nuclease
• CPC Class Codes: C12Q 2523/125 Bisulfite(s)
• CPC Class Codes: C12Q 2531/113 PCR

Abstract

This invention is directed to a guide positioning sequencing technology of whole-genome DNA methylation. The invention provides a new detection method of nucleic acid methylation. In particular, a concept of “positioning” in the detection of nucleic acid methylation is provided. Specifically, a portion of a sequence is used for genome wide positioning and the other portion of the sequence is used for methylation detection in sequencing, thereby solving/defeating previously existing challenges in methylation detection and bioinformatics analysis of a genome.

Claims

1. A method for detection of nucleic acid methylation status, wherein said nucleic acid is double-stranded, said method comprises:

(1) treating the nucleic acid double strands with a polymerase with 3′

→

5′

cleavage function or a 3′

→

5′

exonuclease, so that there is a deletion of 80 to 200 bases at the 3′

end of both strands;

(2) adding dNTP, wherein cytosine (C) is methylated cytosine (5mC), so that the deletion at the 3′

end of both strands is end-filled and cytosine thereof is methylated cytosine;

(3) treating the double strands in step (2), so that un-methylated cytosine is converted into uracil (U) while the methylated cytosine remains unchanged;

(4) PCR amplifying and sequencing the nucleic acid to determine the methylation status of the nucleic acid, wherein the un-methylated cytosine has been converted into thymine in a portion of a sequence for determining a methylation site;

the other portion of the sequence is the same as the original nucleic acid sequence because the cytosine has been methylated.

2. The method according to claim 1, wherein in step (3), treating the double strands in step (2) comprises treatment with bisulfate, bisulfite, hydrosulfite or bihydrosulfite.

3. The method according to claim 1, wherein the nucleic acid is a nucleic acid longer than 2 kb or comprises a whole-genome, and prior to step (1), the method further comprises:

breaking the nucleic acid sequence to generate double-stranded fragments of 200 to 1000 bp.

4. The method according to claim 1, wherein the method is used for detection in a gene site of interest, wherein step (4) comprises:

designing PCR amplification primers for amplifying the gene site of interest, in which one primer is located in a sequence position where cytosine is methylated;

the other primer is located in a sequence position where un-methylated cytosine have been converted into thymine, performing PCR amplification, obtaining the sequence of the gene site of interest and performing methylation analysis.

5. The method according to claim 1, wherein in step (2), after end-filling, the method further comprises:

adding a sequence adapter at both ends of the double strands in step (2) for high-throughput sequencing.

6. The method according to claim 5, wherein the adapter is linked as follows:

adding a protruding A at the 3′

end of the double strands, and linking the double strands to a sequencing adapter with a protruding T at the 5′

end.

7. The method according to claim 6, wherein the sequencing adapter is a methyl adapter, and wherein the sequencing of step (4) comprises high-throughput sequencing methods.

8. The method according to claim 1, wherein the methylation of cytosine comprises one or more selected from the group consisting of:

CpG methylation, CHG methylation and CHH methylation.

9. The method according to claim 1, wherein the polymerase with 3′

→

5′

cleavage function comprises one or more enzymes selected from the group consisting of;

T4 DNA polymerase, T7 DNA polymerase, and Klenow enzyme; and

wherein the 3′

→

5′

exonuclease comprises exonuclease III.

10. The method according to claim 9, wherein when the 3′

→

5′

exonuclease is used in step (1), the polymerase is also added in step (2).

11. The method according to claim 10, wherein the polymerase comprises one or more enzymes selected from the group consisting of:

Taq enzyme, Pfu, and reverse transcriptase.

12. The method according to claim 1, wherein in step (4), the same sequence as the original nucleic sequence is one read of paired-end sequencing, or a portion of a read of single-end sequencing.

13. The method according to claim 12, wherein the paired-end sequencing is performed, wherein one end of a nucleic acid sequence in which the un-methylated cytosine has been converted into thymine is used for determining the methylation sites and the other end of the sequence, which retains the original nucleic acid sequence because of the methylated cytosine is used for sequence positioning.

14. The method according to claim 1, wherein in step (4), the sequencing method of a nucleic acid is paired-end sequencing, wherein one end is used for primary positioning and the other end is used for searching, the method comprises:

(a) localizing one end of a sequence to the genome, allowing the positioning of each sequence in several positions within the genome;

(b) matching the other end sequence to the sequence that has been positioned in genome;

(c) searching the position of the other end nearby according to the primary positioning in genome;

(d) selecting the best alignment position when several alignment positions are available;

(e) removing an amplified redundant sequence produced by PCR;

(f) analyzing methylation level of the genome and calculating the percentage of methylation.