Abstract
BackgroundWith the advent of Next Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Most RNA viruses have relatively small genomes in comparison to other organisms and as such, would appear to be an obvious success story for the use of NGS technologies. However, due to the relatively low abundance of viral RNA in relation to host RNA, RNA viruses have proved relatively difficult to sequence using NGS technologies. Here we detail a simple, robust methodology, without the use of ultra-centrifugation, filtration or viral enrichment protocols, to prepare RNA from diagnostic clinical tissue samples, cell monolayers and tissue culture supernatant, for subsequent sequencing on the Roche 454 platform.ResultsAs representative RNA viruses, full genome sequence was successfully obtained from known lyssaviruses belonging to recognized species and a novel lyssavirus species using these protocols and assembling the reads using de novo algorithms. Furthermore, genome sequences were generated from considerably less than 200 ng RNA, indicating that manufacturers’ minimum template guidance is conservative. In addition to obtaining genome consensus sequence, a high proportion of SNPs (Single Nucleotide Polymorphisms) were identified in the majority of samples analyzed.ConclusionsThe approaches reported clearly facilitate successful full genome lyssavirus sequencing and can be universally applied to discovering and obtaining consensus genome sequences of RNA viruses from a variety of sources.
Highlights
With the advent of Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field
RNA viruses have small, simple genomes, which have a high level of diversity, due to the low-fidelity viral polymerase used for replication
Due to their small genomes, ‘genome-walking’ was used to obtain a reference sequence, from which primer pairs can be designed for down-stream use on similar viruses [1,2,3,4,5,6]
Summary
With the advent of Generation Sequencing (NGS) technologies, the ability to generate large amounts of sequence data has revolutionized the genomics field. Known positive samples have been ‘deep sequenced’ using NGS technologies, for a plethora of uses including determining viral heterogeneity, or the effect of the immune system or pharmaceutical drugs on viruses [15,16,17,18,19] These deep sequencing methodologies employ the use of viral PCR amplicons to obtain a depth of coverage sufficient to detect variants which occur down to 0.1% frequency. Between these two approaches, a methodology to obtain full genome sequences directly from known positive clinical specimens or cultured material, without the use of amplicons, viral enrichment or virus concentration is still lacking. We describe a broadly applicable approach to obtaining full genome sequences from clinical or cultured samples, which we have successfully applied to lyssavirus infected samples
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