Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

作者：Matranga Christian B^*; Gladden Young Adrianne; Qu James; Winnicki Sarah; Nosamiefan Dolo; Levin Joshua Z; Sabeti Pardis C

来源：Jove-Journal of Visualized Experiments, 2016, (113): e54117.

DOI：10.3791/54117

• Medicine
• Issue 113
• RNA viruses
• Ebola virus
• Lassa virus
• intra-host variants
• Lassa fever
• poly(rA) carrier
• rRNA
• RNase H
• RT-PCR

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.

• 出版日期2016-7
• 单位MIT