CN113584600A

CN113584600A - Whole genome methylation single-stranded DNA library building method

Info

Publication number: CN113584600A
Application number: CN202110917984.XA
Authority: CN
Inventors: 曹振; 秦雪梅; 曹欣茹; 任静; 宋东亮
Original assignee: Yisheng Biotechnology Shanghai Co ltd
Current assignee: Yisheng Biotechnology Shanghai Co ltd
Priority date: 2021-08-11
Filing date: 2021-08-11
Publication date: 2021-11-02

Abstract

The invention discloses a whole genome methylation single-stranded DNA library building method, which comprises the following steps: treating genome DNA of a library to be built by bisulfite to obtain single-stranded DNA; adding a primer 1, wherein a random primer of the primer 1 is complementary to the single-stranded DNA, and synthesizing a first strand under the action of DNA polymerase 1; after the product is purified, firstly melting at high temperature, and then adding 2-4 ATP at the 3' end of a newly synthesized first chain to form a tail of ribonucleotide; under the catalysis of RNA ligase, connecting a primer 2 behind the tail of the ribonucleotide of the first strand; adding a primer 3 which is complementary to the primer 2, and synthesizing a second strand under the action of DNA polymerase 2 to obtain double-stranded DNA; and (4) amplifying the library. The single-stranded DNA library building method can effectively utilize the DNA sample treated by the bisulfite to smoothly amplify the treated single-stranded DNA, the conversion rate of the library is higher than that of the traditional method, and the required initial DNA dosage can be as low as 1 ng.

Description

Whole genome methylation single-stranded DNA library building method

Technical Field

The invention relates to a method for establishing a database of full-genome methylated single-stranded DNA, belonging to the technical field of biology.

Background

DNA methylation (DNA methylation) is a form of chemical modification of DNA that can alter genetic behavior without altering the DNA sequence.

DNA methylation refers to the covalent bonding of the cytosine 5' carbon atom of genomic CpG dinucleotides to a methyl group under the action of DNA methyltransferase (DNMT). CpG dinucleotide sequences occur in much lower proportions in the genome than other dinucleotide sequences in the genome. However, there are some regions in the genome of about 0.5-4 kb in length, and these regions have a high CpG density, which are called CpG islands. The CpG island is carried in the promoter region adjacent to 56% of the genes. CpG islands are usually unmethylated in normal cells. However, in many types of cancer, certain genes in cancer cells are methylated and thus are not normally expressed. Therefore, researchers have begun to investigate the use of gene methylation to predict cancer.

Currently, libraries based on second generation high throughput sequencing are used for the research work of methylated DNA, and the common methods are BS-seq (bisulfite conversion sequencing) and RRBS (enzyme digestion-bisulfite conversion sequencing), and the working principle is that samples are fragmented and then treated with bisulfite, wherein bisulfite can convert unmethylated cytosine residues in DNA into uracil residues, and the uracil residues can be replaced by thymine residues in subsequent amplification. While 5-methylcytosine (5-mC) and 5 hydroxymethylcytosine (5-hmC) remain as cytosine residues. The methylation state of the DNA can be evaluated by second-generation sequencing with single base resolution. The conventional DNA library construction scheme is based on double-stranded DNA (dsDNA) samples, and can not directly perform library construction on single-stranded DNA (ssDNA), most of the existing methylation library construction is performed with bisulfite treatment after adaptor connection is completed, and Bisulfite (BS) treatment can cause DNA molecules to break and form single strands, so that the treated library is seriously damaged, the number of amplified templates is only about 10%, the final library conversion rate is poor, and the complexity of sequencing samples is low. If the conversion rate of the library is to be improved, the amplification cycle number needs to be increased to compensate, so that the repeated data of sequencing is increased, and the application cost is increased due to phase change. In the prior art, in order to avoid that cytosine in the connected joint is also converted into uracil by bisulfite, a special methylation modified joint needs to be connected, wherein cytosine in a common joint is replaced by 5-methylcytosine, and the methylation modified joint has higher price and improves the application cost.

Disclosure of Invention

The invention aims to provide a method for constructing a whole genome methylated single-stranded DNA library, which can effectively utilize the single-stranded DNA generated after bisulfite treatment to carry out joint connection, and the library yield is far higher than that of the conventional whole genome methylated library construction scheme.

The technical scheme adopted by the invention is as follows:

a whole genome methylated single-stranded DNA library building method comprises the following steps:

(1) treating genome DNA of a library to be built by bisulfite to obtain single-stranded DNA;

(2) adding a primer 1, wherein a random primer of the primer 1 is complementary to the single-stranded DNA, and synthesizing a first strand under the action of DNA polymerase 1;

(3) after purifying the product obtained in the step (2), firstly melting at high temperature, and then adding 2-4 ATP at the 3' end of a newly synthesized first chain under the action of TdT enzyme and ATP to form a ribonucleotide tail;

(4) under the catalysis of RNA ligase, connecting a primer 2 behind the tail of the ribonucleotide of the first strand;

(5) adding a primer 3 which is complementary to the primer 2, and synthesizing a second strand under the action of DNA polymerase 2 to obtain double-stranded DNA;

(6) performing PCR library amplification by using the synthesized double-stranded DNA as a template;

wherein the sequence of the primer 1 is as follows: 5 '-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNN-3';

the sequence of primer 2 is: 5 '- [ phosphate ] AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT [ phosphate ] -3';

the sequence of primer 3 is: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' are provided.

Preferably, the DNA polymerase 1 is Klenow 3 '-5' exo-, Taq DNA polymerase, phi 29 DNA polymerase, Bst DNA polymerase large fragment or Bsu DNA polymerase.

Preferably, the DNA polymerase 1 is Klenow 3 '-5' exo-.

Preferably, the RNA ligase is TS2126 RNA ligase, circligase II, T4 RNA ligase 1, 5' AppDNA/RNA thermostable ligase.

Preferably, the RNA ligase is TS2126 RNA ligase.

Preferably, the DNA polymerase 2 is Taq DNA polymerase, or KAPA HiFi Uracil + high fidelity DNA polymerase.

Preferably, the DNA polymerase 2 is KAPA HiFi Uracil + high fidelity DNA polymerase.

Preferably, in step (6), the library amplification is performed by using a primer 4 and a primer 5, wherein the sequence of the primer 4 is: 5 '-CAAGCAGAAGACGGCATACGAGAT [ Barcode Sequence ] GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3'; the sequence of primer 5 is: 5'-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' are provided.

The invention has the beneficial effects that:

the single-stranded DNA library building method can effectively utilize the DNA sample treated by the bisulfite to smoothly amplify the treated single-stranded DNA, the library conversion rate is higher than that of the traditional method, the required initial DNA dosage can be as low as 1ng, and the traditional scheme is at least more than 100 ng. In addition, the single-stranded DNA library can adopt a common joint instead of an expensive methylation modified joint, and meanwhile, the data repetition rate of high-throughput sequencing is low, and the cost of the whole library construction sequencing is lower than that of the conventional scheme.

Detailed Description

Further features and advantages of the present invention will be understood from the following detailed description. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

Preparation work in advance: the primer sequences in table 1 were synthesized.

Table 1: primer sequences

Name (R)	Sequence 5 '-3'
		Primer 1	ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNN
Primer 2	[phosphate]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT[phosphate]
		Primer 3	ACACTCTTTCCCTACACGACGCTCTTCCGATCT
Primer 4	CAAGCAGAAGACGGCATACGAGAT[Barcode Sequence]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC
		Primer 5	AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT

Example 1:

the whole genome methylated single-stranded DNA library building method comprises the following steps:

(1) human gDNA was extracted from 5 ml of whole blood using a blood genome extraction kit from Tiangen. Treating gDNA to be treated with EZ DNA Methylation-Glod Kit sulfite reagent of ZYMO company to obtain single-stranded DNA;

(2) adding a primer 1, wherein a random primer of the primer 1 is complementary to the single-stranded DNA, and synthesizing a first strand under the action of DNA polymerase 1, wherein the reaction system is as follows:

components	Volume of
		10×NEB Buffer 2	5 ul
10 mM dNTP	1.25 ul
		Primer 1 (100 mu M)	4 ul
Bisulfite treatment of gDNA	50 ng
		H2O	To 40 ul

The reaction procedure is as follows:

step (ii) of	Time
		94 ℃ (unzipping)	5 min
4 ℃	5 min
		4 ℃	Addition of klenow (3 '-5' exo)^-）
4 ℃	3 min
		37 ℃ (extension)	30 min
70 deg.C (deactivation)	10 min
		4 ℃	--

The reaction product was purified 1.0 × AMPure XP beads using 12 μ l ddH₂Eluting with oxygen;

(4) under the catalysis of RNA ligase, connecting a primer 2 after the tail of the ribonucleotide of the first strand, and carrying out the step (3) and the step (4) in the same reaction system as follows:

components	Volume of
		Purifying the product in the last step	10 µl
2.5 XRNA ligase reaction buffer	10 µl
		10 mM ATP	1 µl
Primer 2	0.3 µl
		H2O	To 23 μl

The reaction procedure was as follows:

step (ii) of	Time
		94 ℃ (unzipping)	3 min
4 ℃	5 min
		4 ℃	Mu.l TS2126 RNA ligase and 1. mu.l TDT DNA polymerase were added
37 ℃	30 min
		65 ℃	30 min
95 ℃	5 min
		4 ℃	--

The reaction product was purified on 1.0 × AMPure XP magnetic beads using 14 μ l ddH₂Eluting with oxygen;

(5) adding a primer 3 which is complementary to the primer 2, and synthesizing a second strand under the action of DNA polymerase 2 to obtain double-stranded DNA, wherein the reaction system is as follows:

components	Volume of
		The above-mentioned ligation product	12.5 µl
10× PCR buffer	5 µl
		10 mM dNTPs	1 µl
Primer 3	1.5 µl
		KAPA HiFi Uracil + high fidelity DNA polymerase	1 µl
H2O	To 50 µl

The reaction procedure was as follows:

components	Volume of
		94 ℃	3 min
45 ℃	5 min
		72 ℃	30 min
4 ℃	--

The reaction product was purified 1.0 × AMPure XP beads using 22. mu.l ddH₂Eluting with oxygen;

(6) and (3) performing PCR library amplification by using the synthesized double-stranded DNA as a template, wherein the reaction system for library amplification is as follows:

components	Volume of
		Purifying the product in the last step	20 µl
2× KAPA HiFi Readymix	25 µl
		Primer 4	2.5 µl
Primer 5	2.5 µl

The reaction product was purified on 0.8 × AMPure XP magnetic beads using 22. mu.l ddH₂And (4) eluting with O. The library was quantified using a Qubit 4.0, and the library size was checked with an Agilent 2100 nucleic acid analyzer, followed by high throughput sequencing.

The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.

Claims

1. a whole genome methylation single-stranded DNA library building method, is characterized in that its step comprises:

(1) Bisulfite treatment of the genomic DNA to be established to obtain single-stranded DNA;

(2) Add primer 1, the random primer of primer 1 is complementary to single-stranded DNA, and the first strand is synthesized under the action of DNA polymerase 1;

(3) Step (2) After the product is purified, it is first melted at high temperature, and then under the action of TdT enzyme and ATP, 2 to 4 ATPs are added to the 3' end of the newly synthesized first strand to form a ribonucleotide. Tail;

(4) Under the catalysis of RNA ligase, primer 2 is connected to the tail of the first-strand ribonucleotide;

(5) Add primer 3 complementary to primer 2, and synthesize the second strand under the action of DNA polymerase 2 to obtain double-stranded DNA;

(6) PCR library amplification is carried out using the above-mentioned synthetic double-stranded DNA as a template;

The sequence of primer 1 is: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNN-3';

The sequence of primer 2 is: 5'-[phosphate]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT[phosphate]-3';

The sequence of primer 3 is: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'.

2. whole genome methylation single-stranded DNA library building method according to claim 1, is characterized in that: described DNA polymerase 1 is Klenow 3'-5' ^exo- , Taq DNA polymerase, phi 29 DNA polymerase enzyme, Bst DNA polymerase large fragment or Bsu DNA polymerase.

3. The method for building a whole genome methylated single-stranded DNA library according to claim 2, wherein the DNA polymerase 1 is Klenow 3'-5' ^exo- .

4. whole genome methylation single-stranded DNA library building method according to claim 1, is characterized in that: described RNA ligase is TS2126 RNA ligase, circligase II, T4 RNA ligase 1, 5 ' AppDNA/RNA Thermostable ligase.

5 . The method for building a whole genome methylated single-stranded DNA library according to claim 4 , wherein the RNA ligase is TS2126 RNA ligase. 6 .

6 . The method for building a whole genome methylated single-stranded DNA library according to claim 1 , wherein the DNA polymerase 2 is Taq DNA polymerase or KAPA HiFi Uracil+ high-fidelity DNA polymerase. 7 .

7 . The method for building a whole-genome methylated single-stranded DNA library according to claim 6 , wherein the DNA polymerase 2 is KAPA HiFi Uracil+ high-fidelity DNA polymerase. 8 .

8. The method for building a whole genome methylated single-stranded DNA library according to claim 1, characterized in that: in step (6), primer 4 and primer 5 are used for library amplification, and the sequence of primer 4 is: 5'- CAAGCAGAAGACGGCATACGAGAT[BarcodeSequence]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATC-3'; the sequence of primer 5 is: 5'-AATGATACGGCGACCACCGAGATCTACACACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'.