摘要
目的基于扩增子测序技术结合高通量测序平台和生物信息分析技术,建立猴痘病毒(MpxV)全基因组测序流程和分子溯源方法,为大连市猴痘病例分子流行病学研究提供技术支持。方法以猴痘病例的痘疱液样本总核酸为模板,通过全基因组测序技术在Ion Torrent测序平台构建cDNA文库,完成模板制备和测序,通过Ion GeneStudio S5 Plus内置插件,以NC_063383.1作为内参序列,通过数据质控、碱基识别、序列比对、变异分析和全基因组序列组装,获得一致性序列,即Fasta文件。采用在线分析工具分析MpxV的全基因组特征、基因分型,遗传进化关系。结果2例患者的痘疱液样本MpxV基因核酸检测Ct值分别为28.03,29.60。1号样本获得的一致性序列全长为197084 bp的MpxV全基因组序列,总reads数3470722,平均测序深度3205,平均读长为182 bp,覆盖率为98.8%;2号样本获得的一致性序列全长为197099 bp的MpxV全基因组序列,总reads数1440296,平均测序深度746.7,平均读长为105 bp,覆盖率为97.8%。1号样本存在84个单核苷酸突变和40个氨基酸突变,2号样本存在85个单核苷酸突变和42个氨基酸突变。1号和2号样本均属于MpxVⅡb分支(西非分支)的C.1.1亚分支。hMpxV/China/DLCDC-01与中国广东省提交序列EPI_ISL_18213375、EPI_ISL_19058867、EPI_ISL_18213374、EPI_ISL_19058866、EPI_ISL_19058873,中国云南省提交序列EPI_ISL_18059184的进化关系较近。hMpxV/China/DLCDC-02与hMpxV/China/DLCDC-01及GISAID上传的中国20条有效序列不同源。结论从2例患者痘疱液样本获得MpxV的一致性序列,成功构建了大连市MpxV全基因组测序技术和生物信息分析及分子溯源方法。
Objective To establish a whole-genome sequencing(WGS)workflow and molecular tracing method for monkeypox virus(MpxV)based on amplicon sequencing technology combined with a high-throughput sequencing platform and bioinformatics analysis techniques,providing technical support for molecular epidemiological research of monkeypox cases in Dalian city.Methods By taking total nucleic acid extracted from vesicle fluid samples of monkeypox cases as templates,a cDNA library was constructed on the Ion Torrent sequencing platform via WGS technology.Template preparation and sequencing were completed.Using the built-in plug-ins of Ion GeneStudio S5 Plus and NC063383.1 as the reference sequence,data quality control,base identification,sequence alignment,variant analysis,and whole-genome sequence assembly were performed to obtain consensus sequences(Fasta files).Online analysis tools were used to analyze the whole-genome characteristics,genotyping,and genetic evolutionary relationships of MpxV.Results The cycle threshold(Ct)values of MpxV nucleic acid detection in vesicle fluid samples from the two patients were 28.03 and 29.60,respectively.The consensus sequence obtained from sample 1 was a 197084 bp MpxV wholegenome sequence with a total of 3470722 reads,an average sequencing depth of 3205,an average read length of 182 bp,and approximately 98.8%coverage.The consensus sequence obtained from sample 2 was a 197099 bp MpxV wholegenome sequence with a total of 1440296 reads,an average sequencing depth of 746.7,an average read length of 105 bp,and approximately 97.8%coverage.Sample 1 had 84 single nucleotide mutations and 40 amino acid mutations,while sample 2 had 85 single nucleotide mutations and 42 amino acid mutations.Both samples belonged to the C.1.1 subbranch of the MpxV IIb clade(West African clade).hMpxV/China/DLCDC-01 was closely related to the sequences EPI_ISL18213375,EPI_ISL19058867,EPI_ISL18213374,EPI_ISL19058866,and EPI_ISL19058873 submitted from Guangdong province,China,and EPIISL_18059184 submitted from Yunnan province,China.hMpxV/China/DLCDC-02 was not homologous to hMpxV/China/DLCDC-01 or the 20 valid sequences from China uploaded to GISAID.Conclusions Consensus sequences of MpxV were obtained from vesicle fluid samples of two patients,successfully establishing WGS technology,bioinformatics analysis,and molecular tracing methods for MpxV in Dalian city.
作者
郎兴莹
孙楠
滕雪
刘颖丽
栾明春
LANG Xingying;SUN Nan;TENG Xue;LIU Yingli;LUAN Mingchun(Dalian Center for Disease Control and Prevention,Dalian,116035,China)
出处
《中国公共卫生》
北大核心
2025年第7期792-796,共5页
Chinese Journal of Public Health
基金
大连市科技创新基金(2023JJ13SN040)。
关键词
猴痘病毒(MpxV)
高通量测序
全基因组特征
大连市
monkeypox virus(MPXV)
high-throughput sequencing
whole-genome characteristics
Dalian city
