利用宏基因组纳米孔测序方法检测模拟临床样本中的基孔肯雅病毒和辛德毕斯病毒

Detection of the Chikungunya Virus and Sindbis Viruses in Simulated Clinical Samples Using Metagenomic Nanopore Sequencing

基于二代测序的宏基因组测序方法(m-NGS)在理论上可以无偏性地检测样品中的所有生物,在感染性疾病的分子诊断领域彰显了突出的作用。但其实验流程复杂,需要操作者具备足够的知识和经验才能获得准确而丰富的诊断信息。因此,需要建立更简单,更快速而且更低成本的宏基因组测序方法,以满足公共卫生实验室的需求。本研究基于MinION建立了宏基因组纳米孔测序(m-NanoS)方法,并成功用于基孔肯雅病毒(CHIKV)和辛德毕斯病毒(SINV)模拟临床样本的检测。将CHIKV和SINV培养物与发热病人咽拭子混合,灭活后提取核酸用于构建m-NanoS文库。MinION共产生了25147条reads,平均质量得分为10.20,平均读长为1334 bp。来自CHIKV和SINV的reads分别有47条和1371条,占总数据量的0.19%和5.45%。CHIKV和SINV的基因组覆盖度分别为99.45%和99.34%,经过拼接获得一致性序列,与参考序列比对的一致性分别为95%和90%。在公共卫生领域,快速地对样本中潜在的全部病原体进行准确鉴定是一个重要又困难的工作,而m-NanoS方法的成功建立无疑为该工作提供了一个有力的诊断工具。

Metagenomic sequencing method based on next generation sequencing(m-NGS) can detect all organisms in samples without bias in theory,and plays a prominent role in the field of molecular diagnosis of infectious diseases. However,its operation process is complex,and it requires the operator to have enough knowledge and experience to obtain accurate and rich diagnostic information. Therefore,it is necessary to establish a simpler,faster and lower-cost metagenomic sequencing method to meet the needs of public health laboratories. We established a metagenomic nanopore sequencing(m-NanoS)method based on MinION and applied it to detection of the Chikungunya virus(CHIKV) and Sindbis virus(SINV) in simulated clinical samples. CHIKV and SINV cultures were mixed with the throat swabs from fever patients. Nucleic acids were extracted after inactivation to construct m-NanoS libraries. MinION produced 25,147 reads with a mean quality score of 10.20 and an average read length of 1,334 bp. There were 47 and 1,371 reads from the CHIKV and SINV respectively,which accounted for 0.19% and 5.45% of the total data volume. The genome coverage of the CHIKV and SINV were 99.45% and 99.34%,respectively. Consensus sequences were obtained,and the identity with reference sequences were 95% and 90%,respectively. With respect to public health,rapid and accurate identification of all potential pathogens in a sample is important,but difficult. Successful establishment of the m-NanoS method provides a powerful diagnostic tool for such work.