Direct RNA Sequencing of the Coding Complete Influenza A Virus Genome

Matthew W Keller,John R Barnes,Vivien G Dugan,Elizabeth B Neuhaus,David E Wentworth,Samuel S Shepard,Malania M Wilson,Thomas J Stark,Callie A Ridenour,Benjamin L Rambo-Martin

doi:10.1038/s41598-018-32615-8

Abstract

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 (EID50 6.8 × 109), 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.

Highlights

For the first time, a coding complete genome of an RNA virus has been sequenced in its original form
In 2009, a method for RNA sequencing was developed on the Helicos Genetic Analysis System where poly(A) mRNA is sequenced by the step-wise synthesis and imaging of nucleotides labeled with an interfering but cleavable fluorescent dye[8]
We describe direct RNA sequencing of five influenza A virus genomes through modification of recently released RNA methods from Oxford Nanopore Technologies[19] (Fig. 1C) by targeting the conserved 3′ end of the influenza A virus genome with an adapter to capture it (Fig. 1D), rather than a primer to amplify it

Summary

Introduction

A coding complete genome of an RNA virus has been sequenced in its original form. 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 (EID50 6.8 × 109), 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. We describe direct RNA sequencing of five influenza A virus genomes through modification of recently released RNA methods from Oxford Nanopore Technologies[19] (Fig. 1C) by targeting the conserved 3′ end of the influenza A virus genome with an adapter to capture it (Fig. 1D), rather than a primer to amplify it.

Methods

Results

Conclusion