Full genome direct RNA sequencing of influenza A virus

For the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to the highly conserved terminal ends of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrated successful sequencing of the full influenza virus genome with 100% coverage, 99% consensus identity, and 99.8% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

本研究首次实现了RNA病毒完整基因组的原始形态测序。此前，RNA测序需通过对放射性标记RNA进行化学降解完成，该方法操作复杂且仅能获得短序列读段。而常规RNA测序通常采用间接策略：先将RNA反转录为互补DNA（cDNA, complementary DNA），再通过聚合酶链式反应（PCR）扩增为双链DNA（dsDNA, double-stranded DNA），最终依托各类DNA测序技术完成分析。本研究针对流感病毒基因组高度保守的末端序列设计了专属接头，将负义RNA（(-) sense RNA）锚定至牛津纳米孔（Oxford Nanopore）MinION测序平台的蛋白质纳米孔中。本研究利用该方法结合从感染鸡胚尿囊液中提取的总RNA，成功完成了流感病毒全基因组测序：测序覆盖度达100%，一致性序列同源性为99%，且99.8%的测序读段（reads）比对至流感病毒基因组。依托相同的实验策略，我们可通过重新设计接头，将检测靶点拓展至病毒生命周期必需的病毒信使RNA（mRNA）及正义互补RNA（(+) sense cRNA）。该技术有望实现现有方法难以实际检测的剪接变异体与碱基修饰的识别与定量分析。