不同样本测序前处理方案对冠状病毒基因组高通量测序结果的影响

目的以基因组最大RNA病毒(冠状病毒)为代表,研究不同测序前样本处理模式对高通量测序获得病毒全基因组序列信息质量的影响。方法以细胞培养的人冠状病毒HCoV-OC43样本为代表,分为4种测序前样本处理模式,即:未处理组、核酸提取前DNase和RNase处理组、核酸提取后DNase处理组、核酸提取前DNase和RNase处理且核酸提取后DNase处理组。不同模式处理后的核酸分为两份,一份直接RNA测序(未扩增),另一份经序列非依赖的单引物扩增(SISPA)后DNA测序。结果尽管不同处理方式下获得的病毒基因组覆盖率差别不大,但是样本核酸提取后经DNase处理组直接测序获得了最高的基因覆盖度和测序准确性,而SISPA扩增可有效提高病毒测序读长(reads)比例与基因组各位点的测序深度。结论本研究为优化冠状病毒等RNA病毒全基因组测序策略提供了技术参考。

Effects of sample processing modes on high-throughput sequencing of coronavirus whole genome

Objective To study the effects of different pre-sequencing sample processing modes on the results of whole genome sequencing with high-throughput sequencing(HTS)by taking the largest RNA virus(human coronavirus,HCoV)as the representative.Methods Cell-cultured human coronavirus HCoV-OC43 strains were used as the representative samples and divided into different groups based on pre-sequencing processing modes as follows:untreated group,DNase and RNase treatment before nucleic acid extraction group,DNase treatment after nucleic acid extraction group,and DNase and RNase treatment before nucleic acid extraction and DNase treatment after nucleic acid extraction group.Nucleic acid samples of each group were analyzed by direct RNA sequencing(without amplification)and DNA sequencing after sequence independent single primer amplification(SISPA),respectively.Results No significant difference in viral genome coverage rates was observed between different groups.The highest genome coverage and sequencing accuracy were obtained in DNase treatment after nucleic acid extraction group by direct RNA sequencing,and the ratio of viral reads and the sequencing depth of each locus were effectively improved by SISPA amplification.Conclusions This study provided an optimized technical strategy for whole genome sequencing of RNA viruses such as coronavirus.