Unbiased deep sequencing of rna viruses from clinical samples

Matranga, Christian B.; Gladden-Young, Adrianne; Qu, James; Winnicki, Sarah; Nosamiefan, Dolo; Levin, Joshua Z.; Sabeti, Pardis C.

Unbiased deep sequencing of rna viruses from clinical samples

2016

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.

显示更多 [+]