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J Vis Exp DOI:10.3791/54117

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples.

Publication TypeJournal Article
Year of Publication2016
AuthorsMatranga, CB, Gladden-Young, A, Qu, J, Winnicki, S, Nosamiefan, D, Levin, JZ, Sabeti, PC
JournalJ Vis Exp
Date Published2016 Jul 02
KeywordsGenome, Viral, High-Throughput Nucleotide Sequencing, RNA Viruses, RNA, Viral, Sequence Analysis, RNA

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.


Alternate JournalJ Vis Exp
PubMed ID27403729
PubMed Central IDPMC4993327
Grant ListDP2 OD006514 / OD / NIH HHS / United States
HHSN272200900018C / AI / NIAID NIH HHS / United States
HHSN272200900049C / AI / NIAID NIH HHS / United States
U19 AI110818 / AI / NIAID NIH HHS / United States