title: Unbiased Deep Sequencing of RNA Viruses from Clinical Samples.
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.
authors.affiliation: Broad Institute of MIT and Harvard; matranga@broadinstitute.org.
authors.forename: Christian B
authors.initials: CB
authors.lastname: Matranga
authors.affiliation: Broad Institute of MIT and Harvard.
authors.forename: Adrianne
authors.initials: A
authors.lastname: Gladden-Young
authors.affiliation: Broad Institute of MIT and Harvard.
authors.forename: James
authors.initials: J
authors.lastname: Qu
authors.affiliation: Broad Institute of MIT and Harvard.
authors.forename: Sarah
authors.initials: S
authors.lastname: Winnicki
authors.affiliation: Broad Institute of MIT and Harvard.
authors.forename: Dolo
authors.initials: D
authors.lastname: Nosamiefan
authors.affiliation: Broad Institute of MIT and Harvard.
authors.forename: Joshua Z
authors.initials: JZ
authors.lastname: Levin
authors.affiliation: Broad Institute of MIT and Harvard; Harvard University.
authors.forename: Pardis C
authors.initials: PC
authors.lastname: Sabeti
mesh_terms: D016679:Genome, Viral; D059014:High-Throughput Nucleotide Sequencing; D012328:RNA Viruses; D012367:RNA, Viral; D017423:Sequence Analysis, RNA
chemical_list: D012367:RNA, Viral