Enhanced methods for unbiased deep sequencing of Lassa and Ebola RNA viruses from clinical and biological samples

Abstract

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.

Metavisitor, a Suite of Galaxy Tools for Simple and Rapid Detection and Discovery of Viruses in Deep Sequence Data

“…Having illustrated that Metavisitor is able to generate robust genome assemblies from known and novel viruses in Drosophila and Anopheles sequencing datasets, we tested whether it can be used to diagnose viruses in RNA sequencing datasets of human patients from three different studies [29–31]. …”

“…recently improved library preparation methods for deep sequencing of Lassa and Ebola viral RNAs in clinical and biological samples [31]. Accordingly, they were able to generate sequence datasets of 150 nt reads providing high coverage of the viral genomes.…”

“…In order to take into account longer reads and higher viral sequencing depths in the available datasets [31], we adapted a Metavisitor workflow for Use Case 3–3 (S17 Fig) as follows: (i) sequencing reads were directly aligned to vir1 sequences using bowtie2, without prior depletion by alignment to the human or rodent hosts; (ii) the Trinity de novo assembler [23] that performs well with longer reads was used instead of Oases (S1 Table); (iii) reconstruction of Lassa and Ebola genomes from the sequences of the blast hits with the nucleotide viral blast database was directly performed with the “blast to scaffold” tool without CAP3 assembly since the Trinity contigs were already covering a significant part of the viral genomes; (iv) the reports generated by our “Parse blast output and compile hits” tool as well as the reconstructed genome generated for each sample were merged in single datasets for easier browsing and subsequent phylogenetic or variant analyses; (v) for adaptability of this workflow to any type of virus, we allowed users to specify two input variables at runtime: the name of the virus to be searched for in the analysis and the identifier of the sequence to be used as guide in genome reconstruction steps.…”

Genomic Analysis of Viral Outbreaks

“…Dramatic improvements in high-throughput (also known as next-generation) sequencing technologies and in virus-specific sequencing (14–17) in the past decade have enabled sequencing of viruses, known and novel, from all kinds of samples. We briefly review current sequencing technologies and discuss methods for sequence processing.…”

“…Hybrid selection has been used to enrich the viral content of sequencing libraries with high host contamination even after RNase H digestion (17), and is an active area of development. Refining this technology will improve viral genomic analysis during outbreaks, when sample quality may be variable.…”