Rapid full-length amplicon library construction method, primers and sequencing method applicable to PacBio platform

Abstract

The invention relates to a rapid full-length amplicon library construction method and primers applicable to PacBio platform and a full-length amplicon sequencing method based on the library construction method. According to the characteristic that PCR is required for full-length amplicon sequencing, an amplicon primer with a general binding sequence and a dumbbell-shaped primer matched with the general binding sequence are designed in combination with a dumbbell-shaped library joint structure of the PacBio sequencing platform, the rapid full-length amplicon library construction method applicable to the PacBio platform is provided based on the two primers with special structures, through library construction by the method, addition of sequencing joints can be realized by increasing two cycles of annular primer amplification only after conventional amplification, and meanwhile, a PCR product becomes a standard library applicable to the PacBio sequencing platform under the action of damage repair enzyme. The method optimizes an original PacBio standard library construction process into only one-step amplification and repair, the library construction process of a full-length amplicon is greatly simplified, and the sequencing cost is significantly reduced.

Description

technical field [0001] The present invention relates to the technical field of high-throughput sequencing library construction, and more specifically, relates to a rapid full-length amplicon library construction method suitable for the PacBio platform, primers and a full-length amplicon sequencing method based on the library construction method. Background technique [0002] The amplicon is a nucleotide sequence after DNA or RNA amplification, such as an amplified fragment of a certain gene obtained by PCR amplification. More simply, amplicons are the amplification products of artificially amplified DNA fragments or RNA fragments. Amplicon sequencing is a highly targeted approach for the analysis of genetic variants in specific genomic regions. [0003] Amplicon sequencing mainly includes 16S rDNA sequencing, 18S rDNA sequencing, ITS sequencing and target region amplicon sequencing. 16S / 18S rDNA contains variable regions and conserved regions. The 16S rDNA gene length of p...

AI Technical Summary

Problems solved by technology

However, due to the limitation of the length of short-segment sequencing on the Illumina platform, it is difficult to accurately identify species to the taxonomic level below the genus based on the sequencing analysis of some hypervariable regions on this platform.

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