两种高通量测序平台应用于不同SARS-CoV-2变异株的对比研究

Comparative analysis of SARS-CoV-2 variants with two high-throughput sequencing platforms

目的利用Illumina MiSeq和Oxford Nanopore高通量测序平台对河南省新型冠状病毒肺炎(COVID-19)确诊病例的上呼吸道样本进行全基因组测序,为新型冠状病毒(SARS-CoV-2)全基因组监测工作提供参考。方法收集河南省2021年6月至2022年1月共10份COVID-19确诊病例的上呼吸道样本,分别采用二代和三代测序技术进行测序,获得SARS-CoV-2全基因组序列,运用生物信息学软件CLC Genomics Workbench(CLC)进行序列比对分析。结果与武汉参考株(Wuhan-Hu-1)相比,10份样本中3份属于Omicron(BA.1)变异株,核苷酸变异位点55个和61个;1份属于Alpha(B.1.1.7)变异株,核苷酸变异位点41个;6份属于Delta(B.1.617.2)变异株,核苷酸变异位点35个、42个和47个。二代测序识别碱基变异位点的准确率更高,6份样本的二代和三代测序变异位点100%同源,7份样本S基因编码区共享一致数目的变异位点。Ct值<33的样本,Illumina MiSeq平台和Oxford Nanopore平台均能获得较高的基因组覆盖度和测序深度。二代测序和三代测序覆盖度差异有统计学意义(t=-2.037,P<0.05);三代测序不同时间覆盖度差异无统计学意义(F=2.498,P>0.05)。结论两种高通量测序平台均能满足SARS-CoV-2变异株的检测需求,Illumina MiSeq平台对SARS-CoV-2变异位点识别更精准;Oxford Nanopore平台可用于SARS-CoV-2的快速鉴定分型。

Whole-genome sequencing of upper respiratory tract specimens from patients with confirmed COVID-19 in Henan Province was performed to compare the performance of the Illumina and Oxford Nanopore sequencing platforms,thus providing a reference for whole-genome monitoring of the novel coronavirus(SARS-CoV-2).Ten samples from COVID-19 cases in Henan Province from June 2021 to January 2022 were collected and sequenced with Illumina and Nanopore high-throughput sequencing technology to obtain full genome sequences of the novel coronavirus,which were compared with the Wuhan reference sequence(Wuhan-Hu-1).Bioinformatics software(CLC)was used for sequence alignment analysis.Three of the ten samples were Omicron(BA.1)variants with 55,61 nucleotide variation sites.One sample was an Alpha(B.1.1.7)variant with 41 nucleotide variation sites.Six samples were Delta(B.1.617.2)variants with 35,42,47 nucleotide variation sites.The sequence identity of mutation sites in six samples was 100%,and the mutation sites in the S genome segment of seven samples were consistent.For samples with a Ct value<33,both next-generation and third-generation sequencing achieved high genome coverage and sequencing depth.A significant difference in coverage was observed between second-generation sequencing and third-generation sequencing(t=-2.037,P<0.05).However,the coverage at different time points of the third-generation sequencing did not significantlydiffer(F=2.498,P>0.05).The needs for SARS-CoV-2 mutant detection could be met through use of either high-throughput sequencing platform.The identification of mutations in the novel coronavirus through Illumina high-throughput sequencing was more accurate,whereas Nanopore high-throughput sequencing technology could be used for rapid detection and typing of different novel coronaviruses.