Matranga, Christian B.Gladden-Young, AdrianneQu, JamesWinnicki, SarahNosamiefan, DoloLevin, Joshua Z.Sabeti, Pardis2016-11-182016Matranga, Christian B., Adrianne Gladden-Young, James Qu, Sarah Winnicki, Dolo Nosamiefan, Joshua Z. Levin, and Pardis C. Sabeti. 2016. “Unbiased Deep Sequencing of RNA Viruses from Clinical Samples.” Journal of Visualized Experiments : JoVE (113): 54117. doi:10.3791/54117. http://dx.doi.org/10.3791/54117.1940-087Xhttp://nrs.harvard.edu/urn-3:HUL.InstRepos:29407858Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA — including poly(rA) carrier and ribosomal RNA — from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based 'tagmentation' step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.en-USMedicineIssue 113RNA virusesEbola virusLassa virusintra-host variantsLassa feverpoly(rA) carrierrRNARNase HRT-PCRJournal Article2016-11-1810.3791/54117