高通量测序平台测定混合病毒样品基因组序列(英文)

De novo sequencing of cultured virus pool on NGS platform

目的建立RNA病毒基因组的从头测序方法,验证高通量测序平台同时测定不同RNA病毒基因组序列的能力。方法选取日常监测工作中分离的一株EV71毒株和一株季节性流感病毒,提取病毒RNA,制备测序文库,按标准步骤在GS-Junior完成测序反应,分析数据。结果共得到超过14万序列片段,总计46.9M碱基,平均读长334bp。初步拼接可以得到914个片段(contig),而以相应参考毒株基因组为标准,一次反应得到EV71全长基因组的99.84%及流感病毒的96.7%。结论高通量测序仪能够同时快速、准确地从头测定多株RNA病毒基因组序列,适合应用于多个病原生物学领域。

The advent of next generation sequencing （NGS） technology has shed lights on comprehensive investigation of unusual or emerging pathogens for global microbe-hunters. In this study, protocols for de novo sequencing of RNA viruses were established in our laboratory, the preliminary performance of genomic sequencing of a cultured virus pool was assessed on a GS-Junior sequencer. Viral RNAs of a hand-foot-mouth disease （HFMD） associated EV71 strain and a seasonal H1N1 sub- type influenza virus were extracted, artificially pooled and reversely transcribed with random primers, an NGS library was con- structed and subjected to routine pyrosequencing on the GS Junior platform. It was indicated from raw sequence reads that over 140 K reads generated approximately 46.9 M base-pairs, with an average length of 334 bp and a median read-length of 352 bp. De novo assembly produced 914 contigs, while reference mapping also showed that 99.8% of full-length EV71 genome and 96.7 % of influenza virus genome were achieved in a single run. The observations demonstrated that NGS platforms are capable of rapidly and precisely de novo sequencing of multiple genomes for RNA viruses, suggesting that NGS technologies are applicable to a variety of fields in future pathogen biology.