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. 2016 Jul 2;(113):54117.
doi: 10.3791/54117.

Unbiased Deep Sequencing of RNA Viruses From Clinical Samples

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Free PMC article

Unbiased Deep Sequencing of RNA Viruses From Clinical Samples

Christian B Matranga et al. J Vis Exp. .
Free PMC article

Abstract

Here 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.

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