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.