Evaluation of extraction and enrichment methods for recovery of respiratory RNA viruses in a metagenomics approach.

Viral metagenomics is increasingly applied in viral detection and virome characterization. Different extraction and enrichment techniques may be adopted, however, reports on their effective influence on viral recovery is often conflicting. Using a three step enrichment steps, the effect of three extraction kits and the influence of DNase treatment with or without rRNA removal for respiratory RNA virus recovery from nasopharyngeal swab samples was evaluated. The viral cocktail containing six different RNA viruses pooled in equal volume were subjected to the different extraction and enrichment methods, sequenced using the Illumina MiSeq, and analysed using Genome Detective. The PureLink® Viral RNA/DNA Mini Kit (PureLink) was highly efficient with better recovery of all the viral agents in the cocktail. The use of rRNA treatment resulted in increased viral recovery with PureLink and QIAamp® Viral RNA Mini kit, while having comparable recovery rate as DNase only with the QIAamp® MinElute Virus Spin Kit. The observed low reads and genome coverage of some of the viruses could be attributed to their low abundance. Depending on sample matrix, extraction choice and enrichment strategy may influence recovery of respiratory RNA virus in metagenomics studies, therefore individual evaluation and adoption may be necessary for a robust result.