Evaluation of convenient pretreatment protocols for RNA virus metagenomics in serum and tissue samples

Abstract

Viral metagenomic approaches are increasingly being used for viral discovery. Various strategies are applied to enrich viral sequences, but there is often a lack of knowledge about their effective influence on the viral discovery sensitivity. We evaluate some convenient and widely used approaches for RNA virus discovery in clinical samples in order to reveal their sensitivity and potential bias introduced by the enrichment or amplifications steps. An RNA virus was artificially spiked at a fixed titer in serum and lung tissue, respectively, low and high nucleic acid content matrices. For serum, a simple DNase treatment on the RNA extract gave the maximum gain in proportion of viral sequences (83×), and a subsequent ribosomal RNA removal nearly doubled once more the proportion of viral sequences. For lung tissue, a ribosomal RNA depletion step on the RNA extract had the biggest gain in proportion of viral sequences (32×). We show also that direct sequencing of cDNA is recommended above an extra random PCR amplification step, and a that the virion enrichment strategy (filtration and nuclease treatment) has a beneficial effect for sequencing-based virus discovery. Our findings provide sample-dependent guidelines for targeted virus discovery strategies.

Keywords: Clinical samples; Next generation sequencing; Pretreatment; RNA virus discovery; Viral metagenomics; rRNA depletion.