基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立OA北大核心CSTPCD

African swine fever(ASF)is a highly contagious and deadly disease caused by the African swine fever virus(ASFV),which has dealt a heavy blow to the healthy development of China's pig industry in recent years.The large genome of ASFV makes it difficult to obtain whole-genome sequence about epidemic strains in a timely manner.This study aims to establish a simple and reliable ASFV whole-genome sequencing method using Nanopore third-generation sequencing technology.Thirty-one primer pairs covering the entire ASFV genome were designed and divided into 4 primer pools to amplify the sample.The amplified product was sequenced by Nanopore sequencing technology,and the relevant bioinformatics analysis methods were further optimized,and finally the ASFV whole-genome sequencing method was successfully established.Whole-genome sequence of ASFV with a total length of 189 416 bp was successfully obtained from an environmental swab sample by this method.Validation through first-generation sequencing has shown that the result of this method is 100％consistent with first-generation sequencing results in key genes and certain variant positions,including B646L,EP402R,E183L,MGF_360-12L,MGF_505-3R,and I177L gene.At the whole-genome level,the consistency between the result of this method and next-generation sequencing(NGS)result is 99.94％.In addition,the utilization of Nanopore sequencing technology in this study revealed a 56 bp repeat sequence insertion within the intergenic region flanked by the NP1450L and NP419L genes.This insertion was subsequently confirmed via first-generation sequencing techniques.Intriguingly,NGS methods failed to detect this distinct variant feature.This study successfully established an ASFV whole-genome sequencing method based on Nanopore technology.This method demonstrates excellent simplicity and reliability,providing an essential tool for the current prevention and control and molecular epidemiological research of ASF.