Three-generation library building sequencing method for whole genome full-length amplification based on novel coronavirus

Abstract

The invention discloses a three-generation library building sequencing method for whole genome full-length amplification based on novel coronavirus. The three-generation library building sequencing method comprises the following steps of 1) designing and synthesizing primers according to a novel coronavirus genome consistency sequence; 2) carrying out full-length amplification on the genome of thenovel coronavirus; 3) performing mixing on the whole genome amplicon samples; 4) performing library construction; and 5) performing third-generation sequencing. According to the method, the odd primers and the even primers are combined to respectively amplify, and the odd amplification fragments and the even amplification fragments are mixed so as to obtain the amplicon fragment covering the whole novel coronavirus genome, such that only two amplification reactions are required, and the experiment efficiency is substantially improved. In addition, through the PacBio library building process,sequencing connectors with barcodes are added to the two ends of an amplicon, PacBio sequencing can be carried out, and HiFiReads with the accuracy rate being 99% or above are obtained after splittingand data correction.

Description

technical field [0001] The invention relates to the technical field of gene sequencing, in particular to a three-generation library construction and sequencing method based on the full-length amplification of the whole genome of the new coronavirus. Background technique [0002] The new coronavirus is an enveloped, non-segmented positive-sense single-stranded RNA virus with round or oval particles and a diameter of about 80-120 nm. The virus particle is wrapped by the lipid bilayer provided by the host cell, which contains nucleic acid and nucleocapsid protein. There are three main proteins: envelope protein (E protein), membrane protein (M protein) and spike protein (S protein). . The length of each genome of this virus is about 30,000 nucleotides. The gene sequence shows that SARS-CoV-2 belongs to a virus with a long branch in the evolutionary tree of the Betacoronavirus Lineage β (Sarbecovirus), which is similar to that of the Chinese Coronaviruses found in horseshoe ba...

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Problems solved by technology

[0006] However, at present, the accuracy of PCR amplification technology for the new coronavirus gene sequence needs to be improved, and the long-read advantage of PacBio's third-generation sequencing has not been fully utilized. No research has been done on the basis of PacBio third-generation sequencing to obtain more than 99% of long-read sequences, which can be used for virus mutation detection, assembly, traceability analysis, etc.

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