Whole-genome methylation non-bisulfite sequencing library, construction and applications thereof

Abstract

The invention relates to the technical field of bioinformatics, particularly to a whole-genome methylation non-bisulfite sequencing library, a construction and applications thereof, wherein the sequencing library comprises a TET enzyme reaction solution, the TET enzyme reaction solution comprises the following independently packaged components: a TET enzyme oxidation buffer solution, and the TET enzyme oxidation buffer solution comprises, by micromole, (20-167)*10<3> parts of HEPES or Tris-Cl, (100-333)*10<3> parts of NaCl, 3.3*10<3> parts of alpha-KG or 2-oxoglutarate, 6.67*10<3> parts of ascorbic acid and 4*10<3> parts of adenosine triphosphate. According to the invention, by combining the kit, Fe(NH4)2(SO4)2 and TET enzyme, 5mc can be oxidized into 5cac, the 5cac is reduced into dihydrouracil under the action of a reducing agent, and T is identified through PCR sequencing, so that the the DNA methylation C-to-T conversion under the non-bisulfite condition is achieved, the defects ofbase imbalance and low sequencing data use rate of the existing methylation sequencing library constructed based on the bisulfite conversion are solved; and the formula further has effects of simplecomponents, extremely low TET enzyme use amount and significant cost reducing.

Description

technical field [0001] The invention relates to the technical field of bioinformatics, in particular to a genome-wide methylated non-bisulfite sequencing library and its construction. Background technique [0002] DNA methylation is a major form of epigenetic modification of genomic DNA. In recent years, a large number of studies have shown that DNA methylation modification plays a vital role in maintaining normal cell function, transmitting genomic imprinting, embryonic development and human tumorigenesis. [0003] A gold standard for methylation sequencing is whole genome bisulfite sequencing (WGBS), referred to as BS-seq. The detection principle is to use bisulfite treatment to deaminate the unmethylated cytosine (C) in the genome into uracil (U). In the subsequent PCR amplification process, the U base is used The tolerant polymerase recognizes the U base as thymine (T), realizes the conversion of C->T, and separates it from the original C base with methylation modif...

AI Technical Summary

Problems solved by technology

The single-strand ligation strategy can realize the methylation detection of low-input substances, but the established library still has the defects of AT ratio >80% and base imbalance, and it connects Illumina's P5 and P7 truncated sequences in the On a single strand, it is difficult to achieve sequencing compatibility with other platforms

[0006] It can be seen from the above that the AT ratio of the library constructed based on the bisulfite conversion method is >80%, and the base is unbalanced, and the detection on the machine needs to be mixed with other whole-genome libraries for sequencing, which is inconvenient to operate. The data utilization rate is low, and valid data only accounts for 20-30% of the sequencing volume

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