基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立

doi:10.11843/j.issn.0366-6964.2024.05.026

Abstract

Abstract: 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.

Key words: nanopore sequencing, African swine fever virus, bioinformatics analysis

CLC Number:

S852.659.1

ZHOU Yang, WU Weizi, CAO Weisheng, WANG Fuguang, XU Xiuqiong, ZHONG Wenxia, WU Liyang, YE Jian, LU Shousheng. A Whole Genome Sequencing Method for African Swine Fever Virus based on Nanopore Sequencing Technology was Established[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 2080-2089.

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A Whole Genome Sequencing Method for African Swine Fever Virus based on Nanopore Sequencing Technology was Established

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