Complete viral RNA genome sequencing of ultra-low copy samples by sequence-independent amplification

Malboeuf, Christine M.; Yang, Xiao; Charlebois, Patrick; Qu, James; Berlin, Aaron M.; Casali, Monica; Pesko, Kendra N.; Boutwell, Christian L.; DeVincenzo, John P.; Ebel, Gregory D.; Allen, Todd M.; Zody, Michael C.; Henn, Matthew R.; Levin, Joshua Z.

Sequence-independent methods (21) (22) (23) (24) (25) are attractive for sequencing viral genomes in that they do not rely on prior viral sequence knowledge. This allows sequencing of viral genomes with little or no genomic information and of highly divergent viruses for which robust primers targeting conserved regions are difficult to design. To date these methods have difficulty with sequencing complete genomes from clinical samples due to high levels of host contamination and low viral amounts. The SISPA method described by Djikeng et al. is capable of capturing complete genomes from viral RNA samples, but requires high viral amounts and removal of host RNA contamination with RNases prior to viral RNA extraction (22) . Work by Victoria et al. demonstrated the ability to capture complete genomes from stool samples, but it appears these samples had high viral titers (25) . Recent work by Ninomiya et al. has demonstrated success of capturing hepatitis C virus (HCV) total RNA sequencing from clinical samples (23) , but this method required 200 ng of input RNA which exceeds the amount typically present in most clinical samples. Moore et al. evaluated the sensitivity of detection of infectious agents using RNA sequencing (24) . Their method worked well for detecting low amounts of virus but was limited to input amounts of 30,000 copies of viral RNA per sample.