Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples.
Bottom Line: This depletion step improves both the quality of data and quantity of informative reads in unbiased total RNA sequencing libraries.We 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.
We 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. Our 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. We 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.
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Mentions: We demonstrated the utility of host rRNA depletion on tissue samples collected from LASV-infected rodents and non-human primate disease models. These tissue samples contain higher levels of 18S rRNA than human plasma or serum (on average 5 times more - data not shown). Using the same human rRNA probes, we depleted rRNA and enriched unique LASV reads approximately five-fold in a Mastomys natalensis spleen sample (Figure 3A). Most of the remaining 10% (approximately) rRNA reads aligned to 28S rRNA sequences which are divergent between humans and rodents . Similarly, our protocol reduced the rRNA content in six different tissue samples from cynomolgous macaques to approximately 10% (Figure 3B). Depletion of rRNA led to an increase in LASV content in all macaque samples, reaching the highest levels in adrenal gland and spleen, two tissues known to accumulate LASV during infection .Figure 3