Novel low-initial-quantity DNA methylation library building method

A methylation and sequencing library technology, applied in the field of low-initial DNA methylation library construction, can solve the problems of poor uniformity, low GC coverage, large damage, etc., to improve the unbalanced distribution of GC bases, operation Simple steps and less damage

Pending Publication Date: 2021-07-09
BGI GENOMICS CO LTD
View PDF7 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Regardless of the input level of library construction, bisulfite treatment is required, and bisulfite treatment can damage DNA, resulting in fragmentation, loss and bias
Although it is possible to build a library from a small amount to the level of the initial amount of a single cell, it is damaged, the GC coverage is low, and the uniformity is poor.
[0004] For the methylation library construction based on the enzymatic method, the existing technology can only realize the conventional initial amount of library construction, and the initial amount of DNA needs to reach more than 10ng

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Novel low-initial-quantity DNA methylation library building method
  • Novel low-initial-quantity DNA methylation library building method
  • Novel low-initial-quantity DNA methylation library building method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Embodiment 1, establishment of method

[0056] 1. Oxidative deamination treatment

[0057] Genomic DNA was taken for oxidative deamination treatment.

[0058] Oxidative deamination method: use EM-seq Conversion Module Kit and operate according to the kit instructions. EM-seq Conversion Module Kit, full name Enzymatic Methyl-seqConversion Module, NEB Corporation, Cat. No. #E7125. Internal control DNA is included in the EM-seq Conversion Module Kit. EM-seq Conversion Module Kit uses TET2 (dioxygenase) for oxidation treatment and APOBEC (cytosine deaminase) for deamination treatment to deaminate unmethylated cytosine C to uracil U.

[0059] 2. Whole Genome Amplification

[0060] The product of step 1 was taken for whole genome amplification.

[0061] Whole-genome amplification method: use REPLI-g Single Cell Kit and perform multiple displacement amplification reactions according to the kit instructions. REPLI-g Single Cell Kit: QIAGEN, Cat. No. 150343; website link...

Embodiment 2

[0072] Embodiment 2, the application of method

[0073] Take Hela cells and extract genomic DNA.

[0074] Take 100pg of genomic DNA and operate according to Example 1.

[0075] Take 10 pg of genomic DNA, and operate according to Example 1.

[0076] The electropherogram of the reaction product of the multiple displacement amplification reaction that completed step 2 is shown in Figure 5 .

[0077] The sequencing results are shown in Table 1.

[0078] Agilent 2100 was used to detect the size of library fragments, the results are shown in Figure 6 (100pg genomic DNA) and Figure 7 (10 pg genomic DNA).

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a novel low-initial-quantity DNA methylation library building method. The invention provides a method for preparing a methylation sequencing library, which sequentially comprises the following steps of: (1) taking genome DNA, and carrying out oxidative deamination treatment; (2) carrying out whole genome amplification; and (3) constructing a sequencing library. In the step (1), the oxidative deamination treatment is carried out by adopting an enzyme chemical method, the oxidation treatment is carried out by adopting dioxygenase (TET2), and the deamination treatment is carried out by adopting cytosine deaminase (APOBEC). The purpose of whole genome amplification is to convert DNA at an ng level into DNA at a mu g level. The method provided by the invention has the following beneficial effects: genome DNA methylation treatment with extremely low initial quantity can be realized, and the initial quantity can be as low as a single cell level; the operation steps are simple; the damage to DNA is minimum; and the phenomena of unbalanced GC base distribution, low coverage, high duplication rate and the like of the existing trace methylation library establishment are improved.

Description

technical field [0001] The invention relates to a novel low-initial DNA methylation library construction method. Background technique [0002] DNA methylation of the fifth carbon of cytosine is a stable epigenetic modification that occurs in many species ranging from bacteria to higher eukaryotes. This modification is what we often call DNA methylation modification, which plays a role in transcriptional regulation during embryonic development, such as genomic imprinting, transposon silencing, and also plays an important role in stem cell differentiation and X chromosome inactivation role. Moreover, changes in DNA methylation levels were also found in tumor research to be associated with tumor progression. Therefore, the detection of DNA methylation provides an important means for the study of the physiological functions of organisms. [0003] Methylation library sequencing based on the principle of bisulfite has become the gold standard for DNA methylation analysis. Afte...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C12Q1/6886C40B50/06
CPCC12Q1/6806C40B50/06C12Q1/6869C12Q1/6886C12Q2600/154
Inventor 唐冲王娟朱欠华杨林峰张弛高强
Owner BGI GENOMICS CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap