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Any etiological agent containing nucleic acids should be identifiable using random next-generation sequencing (NGS) of human clinical materials. Unlike PCR based and baiting strategies, random NGS metagenomics is able to identify unknown and genetically drifted agents without relying on prior knowledge. This makes it suitable for the identification of RNA-viruses, which naturally drift due to their error-prone RNA-dependent RNA polymerase. However, NGS applied to virome investigation (viral metagenomics) presents biological and technical barriers. Viruses often cannot be cultured or isolated, therefore, low viral load samples are common. Furthermore, during NGS, all nucleic acid molecules compete for limited sequencing capacity (whether viral or non-viral) and the costs of NGS increases proportional with required sequencing depth. Therefore, to efficiently sequence low viral load samples, a protocol has to be developed that enriches viruses/viral nucleic acid. We first focused on single stranded RNA-viruses in serum and faeces. Different stages of the protocol were tested in the process from RNA-virus positive sample to dsDNA input for NGS: centrifugation, filtration, endonuclease treatment, RNA extraction, reverse transcription, second strand synthesis and library preparation. Different combinations of these methods were applied to human faeces and serum and assessed using qPCR. Subsequently, the optimal method was applied to Chikungunya virus positive serum and norovirus positive faeces ranging from Ct eight and eleven up to Ct 35 and 30, respectively. Lastly, these samples were sequenced using an Illumina MiSeq (PE300, 10 reads/sample) and analyzed to determine detection limits. Our method reliably generates full (>95 per cent) viral genomes up to Ct 26 in both serum and faeces, while allowing identification of viral agent up to Ct 30. Viral metagenomics proved its merit by also identifying sapovirus, coxsackievirus, parechovirus, and picobirnavirus in faeces. The coxsackievirus and parechovirus were confirmed using qPCR with a Ct of 28 and 29.18, respectively. The identified sapovirus could not be confirmed using our diagnostic qPCR, although NGS data coverage indicated a high viral load. Further analysis of this sapovirus showed many mutations in the qPCR primer binding site, explaining the negative result in our diagnostic assay. These results emphasize the power and promise of viral metagenomics.

A54 Viral metagenomics: Relative viral enrichment and detection limits in clinical serum and faeces