A primer-free whole genomic sequencing approach for single stranded RNA viruses in faecal and blood samples
In the past, very large scale sequencing of RNA viruses, such as Norovirus and Hepatitis C virus using Sanger sequencing technology, has been precluded by their high mutation rate and substantial genetic variation which poses difficulties for primer design. This is a particular problem for many RNA viruses that are difficult or impossible to culture. Here, we describe a simple, novel, high-throughput approach to Norovirus and Hepatitis C virus whole genome sequence determination based on RNA-Seq. We demonstrate the effectiveness of this method by sequencing 3 Norovirus and 2 Hepatitis C samples from faeces and blood, respectively, through a rapid benchtop sequencer. More than 90% of genomes were recovered using both platforms in all but one sample, and no nucleotide differences were found on comparison with Sanger sequencing. To confirm scaleability of our method, an additional 77 Norovirus samples were sequenced on a high throughput sequencer, with similar results. By generating viral genomic sequences that are not biased by primer-specific amplification or enrichment, this method offers the prospect of large-scale, affordable studies of RNA viruses which could be adapted to routine diagnostic laboratory workflows in the near future.