Next generation sequencing of viral RNA genomes

Denise A. Marston, Lorraine M. McElhinney*, Richard J. Ellis, Daniel L. Horton, Emma L. Wise, Stacey L. Leech, Dan David, Xavier de Lamballerie, Anthony R. Fooks

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

94 Citations (Scopus)

Abstract

Background: With 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.Results: As 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.Conclusions: The 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.

Original languageEnglish
Article number444
JournalBMC Genomics
Volume14
Issue number1
DOIs
Publication statusPublished - 4 Jul 2013
Externally publishedYes

Bibliographical note

Funding Information:
The authors would like to thank Saira Crawthraw and Colin Black for technical assistance, Don King, Dirk Höper, Thomas Mϋller, Sylvia Grieson, Bhudipa Choudhury, Chad Fuller and Brandon Londt for fruitful discussions. We are grateful to our collaborators in the US, Estonia, Morocco, Tanzania, Iraq and Denmark, for providing the lyssavirus positive samples. This work was funded by the Department for Environment, Food and Rural Affairs (Defra), UK (grant ROAME SE0427) and by the EU FP7–funded Research Infrastructure Grant European Virus Archive (no.19 228292).

Keywords

  • Genome
  • Lyssavirus
  • Next generation sequencing
  • Pyrosequencing
  • RNA
  • Virus

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