Rapid and efficient deep sequencing and bioinformatic speciation of RNA viruses. Viruses

Sequencing of RNA viruses provides crucial insight into viral infection and evolution. However, whole genome sequencing of viruses can be particularly challenging for second-generation platforms due to genome size, structure, and the presence of large amounts of host nucleic acids. Most protocols rely on either gene specific or global RNA amplification to produce sufficient template quantities for ligation-based sequencing library preparation, a process that can potentially introduce errors interpreted as viral quasispecies or major variants. Conversely, total RNAseq, while agnostic to input, requires co-sequencing of host RNA at the cost of depth of coverage over the virus of interest. We have developed an efficient and robust alternative viral RNA sequencing (vRNAseq) protocol that addresses these shortcomings by direct priming of highly conserved regions of RNA viruses for double-stranded cDNA synthesis without genomic amplification, thereby eliminating potential sources of amplification-induced error and obviating the need for host ribosomal RNA depletion. The resulting material is suitable for transposon-mediated library preparation and sequencing on Illumina platforms. We additionally demonstrate the use of the ultrafast read classifier Kraken for accurate viral speciation when using universal priming approaches.