PT  - JOURNAL ARTICLE
AU  - Matthew W. Keller
AU  - Benjamin L. Rambo-Martin
AU  - Malania M. Wilson
AU  - Callie A. Ridenour
AU  - Samuel S. Shepard
AU  - Thomas J. Stark
AU  - Elizabeth B. Neuhaus
AU  - Vivien G. Dugan
AU  - David E. Wentworth
AU  - John R. Barnes
TI  - Complete genome direct RNA sequencing of influenza A virus
AID  - 10.1101/300384
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 300384
4099  - http://biorxiv.org/content/early/2018/04/12/300384.short
4100  - http://biorxiv.org/content/early/2018/04/12/300384.full
AB  - 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 short highly conserved termi 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 demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% 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.