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

摘要/Abstract

摘要： 非洲猪瘟(ASF)是由非洲猪瘟病毒(ASFV)引起的一种高度传染性和致死性疫病，近年来给我国生猪产业的健康发展造成了沉重打击。ASFV庞大的基因组导致人们难以及时掌握流行毒株的全基因组序列。本研究旨在利用Nanopore三代测序技术建立一种简便可靠的ASFV全基因组测序方法。设计覆盖ASFV全基因组的31对引物并分为4个引物池对样本进行扩增，通过Nanopore测序技术对扩增产物进行测序，进一步优化相关生物信息学分析方法，最终成功建立了ASFV 全基因组测序方法。应用该方法成功从某环境拭子样本中获取一株全长为189 416 bp的ASFV全基因组测序。经一代测序验证表明，在B646L、EP402R、E183L、MGF_360-12L、MGF_505-3R和I177L等关键基因及部分变异位点上，本方法结果与一代测序结果一致性100%；在全基因组水平上，本方法结果与二代测序结果一致性为99.94%。此外，在这项研究中，采用Nanopore测序技术发现了NP1450L基因与NP419L基因间区存在56 bp的重复序列插入(通过一代测序技术进行了验证)，但是二代测序未能发现这一显著的变异特征。本研究成功建立了基于Nanopore技术的ASFV全基因组测序方法，该方法具有良好的简便性和可靠性，为当前ASF的防控和分子流行病学研究提供了一个重要手段。

关键词: Nanopore测序, 非洲猪瘟病毒, 生物信息学分析

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

中图分类号:

S852.659.1

周扬, 吴炜姿, 曹伟胜, 王福广, 许秀琼, 钟文霞, 吴立炀, 叶健, 卢受昇. 基于Nanopore测序技术的非洲猪瘟病毒全基因组测序方法建立[J]. 畜牧兽医学报, 2024, 55(5): 2080-2089.

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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