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Method for preparing novel genome simplified methylation sequencing library

A sequencing library and genome technology, applied in the field of genome simplified methylation sequencing library preparation, can solve the problems of difficult preparation of detection chips, lack of large-scale application, and few methylation data detection methods

Inactive Publication Date: 2016-01-20
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chip method combined with restriction enzymes has high sensitivity, while the immunoprecipitation method has high specificity, but currently only a few research groups in the world are conducting research on DNA methylation chips. The high-throughput of DNA methylation provides a technical platform, but the research on the detection chip is still late, and the preparation of the detection chip is difficult, and the preparation of the chip still needs the support of a large amount of experimental data
[0007] At present, there are not many effective detection methods for methylation data at the genome level, and WGBS and RRBS methods have not been applied on a large scale

Method used

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  • Method for preparing novel genome simplified methylation sequencing library
  • Method for preparing novel genome simplified methylation sequencing library
  • Method for preparing novel genome simplified methylation sequencing library

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 MSAP-seq library preparation of photothermosensitive male sterile line rice Wuxiang S (WXS) and conventional rice 9311

[0046] Material background: Wuxiang S (WXS) is a light-temperature-sensitive male sterile line of rice. It is sterile under high temperature in long days and fertile in low temperature in short days. Fertility is regulated by light and temperature, which belongs to the category of epigenetics. Epigenetics is a branch of genetics that studies the heritable changes in gene expression without changing the nucleotide sequence of the gene. 9311 is an indica rice conventional rice, which is fertile under long-day high temperature and short-day low temperature, and 9311 is a positive control.

[0047] Sample processing and collection:

[0048] Table 2. Sample Handling

[0049]

[0050] 9311 two groups of materials are negative controls, which are used as background impurities; WXS two groups of materials are positive controls. The three tissu...

Embodiment 2

[0072] Example 2 MSAP-seq library preparation of camphor tree young leaves and mature leaves

[0073] Sample collection: Three healthy camphor trees were selected next to the School of Life Sciences of Wuhan University, three fresh leaves and three mature leaves were taken respectively, and MSAP-seq was prepared to study the methylation status changes of the same tissue at different developmental stages library.

[0074] Table 4. Barcode (Barcode) and indicator label information table in the MSAP-seq library preparation of new leaves and mature leaves of camphor tree

[0075]

[0076]

[0077] Note: Due to the small number of samples, the samples can be mixed into one pool and share the same indicator PCR primer to save the cost of library construction and sequencing.

[0078] The specific MSAP-seq library preparation process is as follows:

[0079] (1) Extraction of DNA samples. Genomic DNA was extracted from fresh and mature leaves of camphor tree with 2×CTAB extrac...

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Abstract

The invention discloses a method for preparing a novel genome simplified methylation sequencing library. The method comprises the following steps: respectively performing enzyme digestion on genome DNA by using two dual enzyme digestion combinations of EcoRI / MspI and EcoRI / HapII, and purifying fragments after enzyme digestion; designing a specific connection primer for connecting the purified DNA fragments; dividing the connected products into two groups, and mixing into two tanks; purifying mixing tank samples and recycling fragments of 300-400bp through agarose gel electrophoresis; and performing PCR enrichment and agarose gel electrophoresis recycling on the recycled fragments, thereby obtaining the sequencing library. The method has the advantages of high cover rate, low redundancy, high sensitivity, large-scale sample, high cost performance ratio, high result reliability, good experiment repeatability, and the like, and is generally applicable to various genome methylation difference studies in ordinary molecular biological laboratories.

Description

technical field [0001] The invention relates to the technical field of molecular biology and epigenetics genome DNA methylation profile changes, in particular to a novel method for preparing a genome simplified methylation sequencing library. Background technique [0002] Epigenetic mechanisms, including DNA methylation, are important determinants of the control of gene expression in higher plants and animals. In eukaryotes, genomic DNA methylation is a very important epigenetic mark, which can affect the structure of chromatin and gene expression. As early as 1975, Holliday and Riggs et al. had discovered that the methylation of cytosine at the CpG site on vertebrate DNA can be used as a genetic marker, and this methylation can be inherited through the division of somatic cells. Methylation at carbon 5 of cytosine residues is the most widely studied epigenetic modification in plants and mammals. In addition to CG methylation, there are CHG and CHH methylation (H=A, C or T...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12N15/10
Inventor 丁毅张洪源
Owner WUHAN UNIV
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