Construction method of high-throughput simplified methylation sequencing library without reference genome

Abstract

The invention provides a method for constructing a high-throughput simplified methylation sequencing library without a reference genome. The sample genome is digested and A is added to obtain a DNA fragment with phosphorylation modification at the 5' end and A at the 3' cohesive end. It was connected with methylated adapters containing different barcode sequences; the ligation products were divided into two groups, one group was amplified by PCR, and the other group was amplified after bisulfite conversion, and two groups containing the same barcode, only in Amplified product sequences with different cytosine sites without methylation; two sets of PCR products are mixed separately, the same specific length fragment sequence is selected for low-cycle PCR, and the amplified products are separated and purified separately to construct high-throughput simplified methylation sequencing library. This method has high throughput, low cost, consistent sequence, and does not require a reference genome. It has broad application prospects in the field of DNA methylation epigenetics research in a large number of species without reference genomes or with incomplete assembly of reference genomes.

Description

technical field [0001] The invention relates to the technical field of bioinformatics, in particular to a method for constructing a simplified methylation sequencing library of species without a reference genome. Background technique [0002] DNA methylation is an important epigenetic modification, which plays an important role in cell development and differentiation, regulation of gene expression, X chromosome inactivation, gene silencing, disease occurrence, etc. It is one of the new research hotspots . The DNA methylation detection technology developed based on next-generation sequencing technology can provide a large amount of high-throughput, genome-wide DNA methylation data, which has greatly promoted the development of epigenetics research. Bisulfite conversion combined with next-generation sequencing technology is currently the most accurate DNA methylation detection method, which can detect the methylation status at the single base level, and is known as the "gold ...

AI Technical Summary

Problems solved by technology

Since bisulfite-converted sequences must be compared with a reference genome to determine the sites of methylated cytosines, these two techniques are currently only available in humans, mice, rats, etc. with high-quality For species with reference genome sequences, there are also problems such as high cost of BS-seq library construction and low efficiency of restriction endonuclease MspI required for RRBS

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