扩增子与宏基因组策略在猴痘病毒全基因组测序考核中的应用比较  

Comparison of amplicon sequencing and metagenomic sequencing strategies in MPXV whole-genome sequencing testing

作　　者：黄枝妙 翁育伟[1] 陈炜[1] 游丽斌 王金章[1] 俞婷婷 林琦[1] HUANG Zhi-miao;WENG Yu-wei;CHEN Wei;YOU Li-bin;WANG Jin-zhang;YU Ting-ting;LIN Qi(Fujian Provincial Center for Disease Control and Prevention,Fujian Provincial Key Laboratory of Zoonosis Research,Fuzhou 350012,China)

机构地区：[1]福建省疾病预防控制中心,福建省人兽共患病研究重点实验室,福州350012

出　　处：《中国人兽共患病学报》2024年第10期944-949,共6页Chinese Journal of Zoonoses

基　　金：福建省卫健委科技计划项目青年科研课题(No.2022QNA062);福建省卫生健康中青年科研重大项目(No.2021ZQNZD006)。

摘　　要：目的比较扩增子与宏基因组测序在猴痘病毒全基因组测序考核中的应用效果,为猴痘病毒测序溯源与疫情防控提供技术参考。方法扩增子测序:对猴痘DNA先进行全基因组靶向扩增,再对扩增产物进行二代测序;宏基因组测序:对猴痘DNA直接进行二代测序。获得序列后使用CLC、IGV等软件分析2种不同测序方法的有效数据百分比、测序深度及不同测序深度下的病毒全基因组测序覆盖度等质量参数,评估测序质量。使用Nextclade进行病毒分型、序列突变及缺失情况分析,比较2种测序方法所获得序列的一致性及完整性。结果使用猴痘参比毒株MPXV-M5312_HM12_Rivers全基因序列(GenBank登录号:NC_063383.1)进行拼接后,扩增子测序和宏基因测序所获得有效数据百分比分别为99.72%和7.54%,测序深度范围分别为0×~334839×和44×~1000×,测序深度10×以上的位点覆盖度分别为90.3%和100%。通过IGV查看全基因组在不同测序深度下的覆盖情况发现,宏基因测序的全基因组位点测序深度覆盖均匀,而扩增子测序的全基因组位点测序深度覆盖不均、差异明显。病毒分型及序列一致性分析显示,2种方法所获得序列均为猴痘病毒IIb B.1分支,与参比序列相比宏基因测序结果存在73个突变位点,扩增子测序存在68个突变位点,深入分析发现扩增子测序有7个IIb B.1分支的共有突变位点未测到,还存在2个假阳性私有突变位点。结论在猴痘病毒全基因测序中可灵活使用扩增子测序或宏基因组测序方法,其中扩增子测序可以获得更多的有效数据量,而宏基因组测序在序列覆盖的均一性和准确性方面较好,本研究旨在为提高猴痘病毒全基因组测序成功率提供一点启发与技术参考。The implementation of amplicon sequencing and metagenomic sequencing methods in the whole-genome sequencing for MPXV testing was compared,to provide a technical reference for sequencing,tracing,and epidemic prevention and control of MPXV.For amplicon sequencing,targeted amplification of the viral whole genome was performed on MPXV DNA,and was followed by next-generation sequencing of the amplification products.For metagenomic sequencing,next-generation sequencing was performed directly on MPXV DNA.After the sequences were obtained,software such as CLC and IGV were used to analyze the effective data percentage,sequencing depth,and whole-genome sequencing coverage under different sequencing depths for both sequencing methods,to evaluate sequencing quality.Nextclade was used to analyze virus typing,mutations,and deletions.Subsequently,the similarity and completen ess of sequences obtained through both sequencing methods were further compared.On the basis of mapping to the reference sequence of strain MPXV-M5312_HM12_Rivers(GenBank number NC_063383.1),the percentage effective data obtained from amplicon sequencing and metagenomic sequencing was 99.72%and 7.54%,respectively,with a sequencing depth range of 0×to 334839×,and 44×to 1000×.On the basis of a sequencing depth of 10×,the site coverage of the above was 90.3%and 100%,respectively.IGV was used to validate the whole-genome coverage under different sequencing depths.The depth coverage of whole-genome sites for metagenomic sequencing was uniform,whereas that of the whole-genome sites for amplicon sequencing was uneven and significantly differed.Virus typing and sequence similarity analysis indicated that the viral sequences obtained with the two sequencing methods all belonged to the IIb B.1 lineage of MPXV.Comparison with the reference sequence indicated that metagenomic sequencing identified 73 nucleotide mutation sites,whereas amplicon sequencing identified 68 mutation sites.Further analysis demonstrated that seven common mutation sites of IIb B.1 were not

关 键 词：猴痘病毒 全基因组测序 扩增子测序 宏基因测序 

分 类 号：R373[医药卫生—病原生物学]