福建省一起经货轮输入的新冠肺炎病毒全基因组测序分析

Whole genome sequencing and analysis of 2019 novel coronavirus imported from a freighter in Fujian province,China

目的应用高通量测序和生物信息学分析技术,从一起经货轮输入的新冠肺炎(COVID-19)聚集性病例标本中直接测定新型冠状病毒(2019 novel coronavirus,2019-nCoV)全基因组序列,并从全基因组水平分析2019-nCoV的基因组变异特征,追溯病毒的潜在来源。方法采用2019-nCoV全基因组靶向扩增结合Ion S5二代测序的技术,对来源于同一艘货轮的8例COVID-19确诊病例咽拭子标本进行病毒全基因组测序。应用在线分析平台,判断病毒型别,分析病毒突变位点。利用进化分析软件,构建系统发育树,结合病例流行病学资料,推测病毒的来源。结果成功测序获得8条长度为29822~29865 bp的2019-nCoV全基因组序列,平均测序深度为11928×~33588×,基因组覆盖度为99.73%~99.87%;Pangolin分型结果显示8个2019-nCoV基因组均属于VOC/Delta(B.1.617.2)进化分支;全基因组突变分析显示,与武汉参考株(NC_045512.2)相比,8个2019-nCoV基因组序列核苷酸突变的中位数为35个(31个~38个),氨基酸突变的中位数为26个(24个~28个),突变位点分布于8个编码区(ORF1a、ORF1b、S、ORF3a、M、ORF7a、ORF8、N);进一步分析发现8个2019-nCoV基因组中含有23个属于2019-nCoV Delta(B.1.617.2·AY.2)变异株的特征性突变位点;但因8个2019-nCoV基因组间的突变位点并非完全重合,且流调报告显示货轮中途经停多个口岸且有人员更替,故推测该起聚集性COVID-19疫情可能有不同传播来源;进化分析显示,8个2019-nCoV序列共同处于B.1.617.2进化分支的AY.2子分支上,同Pangolin分型及突变分析结果一致。结论本研究从一起经货轮输入的COVID-19聚集性疫情病例标本中测序获得8个Delta变异株全基因组序列,本研究所构建的测序方法和分析结果可在COVID-19防控中为2019-nCoV的变异分析和病例溯源提供参考。

Objective To investigate the whole genome characteristics and mutation of imported 2019-nCoV and trace potential source at the genomics level,the viral genomes from the specimens of a cluster of COVID-19 cases which were imported from a freighter were directly sequenced and determined by high-throughput sequencing technology and bioinformatics analysis.Methods Throat swab specimens from the 8 confirmed cases of COVID-19 from the same freighter were collected to perform the whole genome sequencing for 2019-nCoV using the targeted genome amplification,combined with Ion S5 next-generation sequencing(NGS)technology.Varieties of online virus analysis platforms was used to classify the viruses and analyze mutation sites in the whole genome.Phylogenetic analysis software was used to construct a phylogenetic tree for the viruses,combined with the epidemiological data of the case,to speculate about the source of the viruses.Results Eight complete genome sequences of 2019-nCoV was successfully obtained.The complete genomes of the viruses were 29,822-29,865 bp in length,with the average sequencing depth of 11928×-33588×and the coverage of 99.73%-99.87%;the result of Pangolin classification showed that all the eight 2019-nCoV genomes belong to VOC/Delta(B.1.617.2)lineage.The result of whole genome mutation analysis showed that compared with the Wuhan reference strain,the median number of nucleotide mutations in the eight 2019-nCoV genome sequences was 35(31 to 38),and the median number of amino acid mutations was 26(24-28);the mutation sites were distributed in 8 gene coding regions(ORF1a,ORF1b,S,ORF3a,M,ORF7a,ORF8,N).Further analysis revealed that eight 2019-nCoV genomes contained 23 characteristic mutation sites belonging to the Delta(B.1.617.2·AY.2)variants of 2019-nCoV.Since the mutation sites among the eight 2019-nCoV genomes were not completely overlapped,and the epidemiological survey report showed that the freighter stopped at multiple ports and had personnel alternation,it was speculated that the clustered COVID-19 cases might have different origins.Phylogenetic analysis showed that all the eight 2019-nCoV genomes were on the AY.2 sub-lineage of the B.1.617.2 lineage.This result was consistent with the result of Pangolin classification and mutation analysis.Conclusions In this study,8 whole genome sequences of Delta variants were obtained through NGS technology from the clustered COVID-19 cases which were imported from a freighter.The sequencing method and analysis result in this study could provide reference for the 2019-nCoV mutation analysis and tracing the source of the COVID-19 cases for the prevention and control of the COVID-19 epidemic.