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Single-chain library building method of whole-genome methylated library and obtained whole-genome methylated library

A genome-wide, methylation technology, applied in the field of genome methylation research, can solve the problems of waste, too much sequencing data, low efficiency, etc., and achieve the effect of reducing the cost of library construction and improving the coverage.

Pending Publication Date: 2020-04-28
MGI TECH CO LTD
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Problems solved by technology

This library construction method uses single-stranded DNA plus double-stranded adapters for the connection of the 3' end and the 5' end in sequence, and the efficiency is relatively low.
In this method, both the original strand and the newly synthesized complementary strand processed by bisulfite conversion will be sequenced, which means that the same original DNA template is sequenced twice. Double the waste of data

Method used

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  • Single-chain library building method of whole-genome methylated library and obtained whole-genome methylated library
  • Single-chain library building method of whole-genome methylated library and obtained whole-genome methylated library
  • Single-chain library building method of whole-genome methylated library and obtained whole-genome methylated library

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Experimental program
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Embodiment

[0054] The sample in this embodiment is unfragmented genomic DNA.

[0055] (1) Bisulfite treatment of DNA:

[0056] Prepare the reaction mixture shown in Table 1 below:

[0057] Table 1

[0058]

[0059]

[0060] The reaction conditions are shown in Table 2 below:

[0061] Table 2

[0062] Bisulfite Treatment Conditions time 98℃ 10min 64℃ 2.5h 4℃ ∞

[0063] After the reaction, purify with a purification column and dissolve back in 10 μL.

[0064] (2) 5' end dephosphorylation treatment:

[0065] Prepare the reaction mixture shown in Table 3 below:

[0066] table 3

[0067]

[0068] The reaction conditions are shown in Table 4 below:

[0069] Table 4

[0070]

[0071]

[0072] After the reaction, place on ice for 3-5 minutes.

[0073] (3) Two-strand synthesis reaction:

[0074] Prepare the reaction mixture shown in Table 5 below:

[0075] table 5

[0076] Reagent volume 10X NEB2 buffer 3μL 2.5mM dN...

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Abstract

The invention relates to a whole genome methylation library single-chain library building method and the obtained whole genome methylation library. The method comprises the following steps: (a) carrying out bisulfite treatment on broken or unbroken genome DNA to convert a non-methylated C base into a U base, and forming a random AP site on the DNA; (b) carrying out 5' end dephosphorylation treatment on the reaction product in the previous step by adopting dephosphorylase to remove the phosphate group at the 5' end; (c) amplifying the denatured single strand of the reaction product in the previous step under the action of DNA polymerase and random primers to synthesize a second strand; and (d) removing the AP site by using endonuclease with an AP site removal effect, wherein the step (d) iscarried out at any stage after the step (a). The method has the advantages of low initial quantity, high library quality, high data utilization rate and high data reliability.

Description

technical field [0001] The invention relates to the technical field of genome methylation research, in particular to a single-strand library construction method for a genome-wide methylation library and the obtained genome-wide methylation library. Background technique [0002] There are many types of epigenetic modifications, mainly including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and N6-methyladenine (6mA) modifications. Genome-wide methylation modification affects genomic imprinting, gene expression, RNA alternative splicing, nucleosome positioning and dynamic combination of transcription factors at different levels of precision, and thus is an important regulatory factor for biological traits. Whole genome methylation sequencing (WGBS) is a basic and important research method in epigenetics research. At the genome-wide level, single-base resolution detection of methylation sites can not only detect changes in methylation levels in common regions such as ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C40B40/08
CPCC40B50/06C40B40/08
Inventor 李艳耿春雨杨心石蒋慧
Owner MGI TECH CO LTD
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