Researchers at the Broad Institute have developed a robust RNA sequencing method for generating complete de novo assemblies with intra-host variant calls of Lassa and Ebola virus genomes in clinical and biological samples. This method uses targeted RNase H-based digestion to remove contaminating poly(rA) carrier and ribosomal RNA. This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries. The ressearchers have also developed a hybrid-selection protocol to further enrich the viral content of sequencing libraries. These protocols have enabled rapid deep sequencing of both Lassa and Ebola virus and are broadly applicable to other viral genomics studies.
Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples
Matranga CB, Andersen KG, Winnicki S, Busby M, Gladden AD, Tewhey R, Stremlau M, Berlin A, Gire SK, England E, Moses LM, Mikkelsen TS, Odia I, Ehiane PE, Folarin O, Goba A, Kahn S, Grant DS, Honko A, Hensley L, Happi C, Garry RF, Malboeuf CM, Birren BW, Gnirke A, Levin JZ, Sabeti PC. (2014) Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples. Genome Biol 15(11):519. [abstract]