In-depth comparison of untargeted and targeted sequencing for detecting virus diversity in wastewater.

Sequencing approaches may enable monitoring of a broad range of viruses in wastewater, including potential emerging and non-reportable human viruses. Considering the fact that metagenomic sequencing may be non-specific for low-abundance human viruses, integration of viral amplification and enrichment strategies are proposed to enhance the accurate detection of a broad range of human viruses in municipal wastewater. In this study, we focused on the in-depth comparison analysis of three untargeted amplification methods (Multiple Displace Amplification [MDA], Reverse Transcription - MDA [RT-MDA], and a PCR-based random amplification [PCR-based]) and one targeted method (Twist Comprehensive Viral Research Panel [TWIST]) for detecting virus diversity in wastewater. In addition, we included the comparisons of two extraction kits (Qiagen QIAamp VIRAL RNA Mini Kit and ZymoBIOMICSTM DNA/RNA Minipre Kit) and four virus identification tools (Diamond blast, Kraken2, VirSorter2 and geNomad) for a systematic study. Performances of Qiagen and Zymo extraction kits in recovering viruses and human viruses in wastewater were comparable. By the three untargeted methods we detected 12,808 contigs with lengths longer than 10,000 bp. No contig longer than 10,000 bp was detected by the targeted method. Presence of human viruses were analyzed further by comparing the viral contigs against a custom Swiss-Prot human virus database. There were 45 viruses that are potentially associated with human health found in wastewater, 8 of them were unique to the targeted method and 7 of them were unique to the three untargeted methods. Four enteric viruses Mamastrovirus, Norovirus, Rotavirus and Sapovirus were detected with high abundance in samples prepared with the targeted method. Dimensional scaling analysis demonstrated the divergent virus and human virus communities from the untargeted and targeted methods. Patterns of virus and human virus populations identified by Kraken2 and geNomad were similar. Presence of selected viruses (SARS-CoV-2 [N1&N2], SC2, RSV, Norovirus GI and GII) were confirmed with ddPCR. This work indicates integration of untargeted and targeted sequencing methods, and complementary ddPCR can ensure the accurate detection of known and novel viruses using wastewater surveillance.