Oak Ridge Institute of Science and Education (ORISE), Oak Ridge, Tennessee, USA.
Battelle Memorial Institute, Atlanta, Georgia, USA.
Sci Rep. 2018 Sep 26;8(1):14408. doi: 10.1038/s41598-018-32615-8.
For the first time, a coding complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabeled 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 short highly conserved termini of the influenza A virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method with total RNA extracted from the allantoic fluid of influenza rA/Puerto Rico/8/1934 (H1N1) virus infected chicken eggs (EID 6.8 × 10), we demonstrate successful sequencing of the coding complete influenza A virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza A virus. By utilizing the same methodology one 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, or other pathogens. This approach also has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.
首次以原始形式对 RNA 病毒的完整编码基因组进行了测序。以前，通过放射性标记 RNA 的化学降解对 RNA 进行测序，这是一种困难的方法，只能产生短序列。相反，通常通过将 RNA 复制成 cDNA 来间接对其进行测序，cDNA 通常通过 PCR 扩增成双链 DNA，然后使用多种 DNA 测序方法对其进行分析。我们设计了一个衔接子，用于流感病毒基因组的短高度保守末端，将（-）义 RNA 靶向 Oxford Nanopore MinION 测序平台上的蛋白纳米孔。利用这种方法，对从感染鸡卵的流感 rA/Puerto Rico/8/1934（H1N1）病毒的尿囊液中提取的总 RNA（EID6.8×10）进行测序，我们成功地对编码完整的流感 A 病毒基因组进行了测序，核苷酸覆盖率为 100%，一致性为 99%，99%的读段映射到流感 A 病毒。通过利用相同的方法学，人们可以重新设计适配器，以便将目标扩展到包括病毒 mRNA 和（+）义 cRNA，这对于病毒生命周期是必不可少的，或者其他病原体。这种方法还有可能识别和量化剪接变体和碱基修饰，这是当前方法无法实际测量的。
