Double-stranded nucleic acid cyclization method, methylation sequencing library construction method and kit

Abstract

The invention discloses a nucleic acid double-chain cyclization method, a methylation sequencing library construction method and a kit. The nucleic acid double-strand cyclization method comprises the following steps: providing a forward primer and a reverse primer, wherein each of the forward primer and the reverse primer comprises a U basic group, the nucleotide number difference of the upstream sequences of the 5'ends of the U basic groups of the forward primer and the reverse primer is N, and the upstream sequences of the 5 'ends of the U basic groups of the forward primer and the reverse primer are at least partially and reversely complementary; amplifying the nucleic acid fragment by using the primer pair to obtain an amplification product; a USER enzyme is used for cutting off U basic groups in the amplification product, then a mononucleotide gap is formed, chain dissociation occurs to the residual nucleotide sequence from the gap to the 5'end due to the unstable structure, and then partially complementary cohesive terminal structures are formed at the two ends of the amplification product; the sticky tail end structures at the two ends are complementarily connected to form a double-chain structure, one chain of the double-chain structure is closed, and the other chain of the double-chain structure has N deleted basic groups. The method is used for constructing the WGBS library, and the phenomenon that the methylation rate of the WGBS library is high due to single-chain cyclization deviation can be reduced.

Description

technical field [0001] The invention relates to the technical field of sequencing, in particular to a double-stranded nucleic acid circularization method, a methylation sequencing library construction method and a kit. Background technique [0002] As an open platform, BGISEQ-500 and MGI2000, the sequencing platform of BGI, provide a one-stop sequencing operation process with a clear and clear sequencing process. It not only provides optional library preparation system and sample loading system, but also supports a variety of supporting library preparation The solution, using optimized combined probe-anchored aggregation technology (cPAS) and improved DNA nanoball (DNB) core sequencing technology, is one of the industry-leading high-throughput sequencing platforms. [0003] When constructing the sequencing library corresponding to this type of platform, the PCR library needs to be denatured into a single strand, and the single strand DNA is self-circularized with the help of...

AI Technical Summary

Problems solved by technology

The efficiency of the circularization process is low, not all single-stranded DNA can self-link into a circle, and the shape of single-stranded DNA is more likely to form a secondary structure, resulting in the bias of the final circularization product, especially for the methyl group Whole genome bisulfite sequencing (WGBS), a library rich in AT bases, has a more prominent bias, and ultimately shows a high methylation rate

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