Library establishing method for unicellular RRBS sequencing and application thereof

Abstract

The invention relates to the technical field of molecular biologics and specifically discloses a library establishing method for unicellular RRBS sequencing and an application thereof. The library establishing method for unicellular RRBS sequencing, disclosed by the invention, comprises the following steps: (1) performing digestion, terminal filling, A and joint adding and bisulfate conversion on genomic DNA of a sample in turn; (2) performing linear amplification on the genomic DNA converted in the step (1); (3) performing index amplification on the amplicon subjected to linear amplification in the step (2), wherein the amplicon of the index amplification is used for sequencing the library. The DNA required by the sequencing according to the method is low in initial mass; methylation sequencing can be performed on the genome of a single cell; the method contains an effective library segment control step; the segment of the end product is controlled according to the linear amplification step; complex operation is avoided while the coverage rate is increased.

Description

technical field [0001] The present invention relates to the technical field of molecular biology, in particular to a method for establishing a library suitable for representative bisulfite sequencing (Reduced Representation Bisulfite Sequencing, RRBS) sequencing of single cells and its application. Background technique [0002] DNA methylation refers to the covalent modification of the fifth carbon atom of cytosine (C) on DNA into 5' methylcytosine (5mC). DNA methylation is one of the earliest discovered epigenetic modification pathways, which can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression. It has a variety of important biological functions, including X chromosome inactivation, transposon silencing, gene imprinting, etc. The vast majority of DNA methylation modifications occur at CpG sites. In mammals, CpG exists in two forms: one is dispersed in the DNA sequence; the o...

AI Technical Summary

Problems solved by technology

[0008] The existing restriction endonuclease-bisulfite sequencing method requires complex experimental procedures: MspI digestion, electrophoresis, and purification steps to enrich the promoter and CpG island regions, followed by end repair, A addition, adapter ligation, Bisulfite conversion, purification, PCR amplification, PAGE gel fragment selection and other steps to construct a sequencing library will take about 5-6 days

Moreover, after adding adapters and performing bisulfite treatment, the drug will randomly interrupt the DNA, which will cause a great loss of sample information, especially when the input sample volume is extremely low at the single-cell level, with only a very small copy number

At the same time, the use of PAGE gel to recover the library of the required size is cumbersome, and it is easy to cause cross-contamination between samples. The impact of recovery efficiency will also greatly lose useful information.

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