Single-cell methylation sequencing technology and application thereof

Abstract

The invention discloses a single-cell methylation sequencing technology and an application thereof. The technology has the following advantages: (1) the technology has the characteristics of simplicity in operation, short manual operating time, and no pollution; (2) a pre-amplification primer is designed for the characteristics of a bisulfate treated genome sequence, the 3' end of the pre-amplification primer is an H base, and the H base does not complement or bind to C, so binding of the pre-amplification primer to the bisulfite converted sequence is not affected, and the probability of binding of a random primer and a linker sequence is reduced; and (3) the technology has a high Mapping rate, and effectively improves the use rate of sequencing data, and the use rate is 20-30% higher thanthe use rate (average 25%) of existing scRRBS and the use rate (average 10-30%) of scBS. The technology is of great significance for the study of single-cell DNA methylation.

Description

technical field [0001] The invention relates to a single-cell methylation sequencing technology and its application. Background technique [0002] Recent studies have shown that DNA methylation plays a key regulatory role in many biological processes, such as the regulation of gene expression, the activity of transposons, the inactivation of X chromosome, and the maintenance of genomic imprinting. Genomic DNA methylation is tissue-specific and is closely related to development and aging. Abnormal DNA methylation usually occurs in the genome of many diseases and even cancer cells. At present, the study of genome-wide methylation at the single-cell level has become a research hotspot, which is of great significance for the discovery of epigenetic information in highly heterogeneous cells. Large-scale single-cell epigenetic analysis can be used in embryonic development, Complicated diseases such as the regulation mechanism of cancer occurrence, cell heterogeneity, early detect...

AI Technical Summary

Problems solved by technology

However, this method is extremely complicated, and the main steps include cell lysis, BS transformation, pre-amplification with biotin-labeled pre-amplification primers, magnetic bead capture and recovery of the pre-amplified first strand, ExoI primer digestion, and Oligo2 primer pre-amplification And subsequent PCR amplification and indexing

This process is very time-consuming and complicated. During each cycle of the amplification process, Klenow enzyme needs to be newly added. The steps are complicated, time-consuming and labor-intensive.

Responding to multiple opening operations, it is easy to cause pollution, bring great difficulties to subsequent analysis, and cause data waste. The mapping rate of sequencing data is low. The mapping rate reported in the literature is only about 10-30%, resulting in a large amount of data. waste

Images