Viral wastewater epidemiology by targeted and untargeted genomic sequencing
Wastewater-based epidemiology (WBE) is a powerful tool for monitoring the emergence and spread viral pathogens at population-scale. Typical Polymerase Chain Reaction (PCR)-based methods of quantitative and genomic monitoring of viruses in wastewater provide high sensitivity and specificity. However, these methods are limited to the surveillance of target viruses in a single assay and require prior knowledge of the target genome(s). Metagenomic sequencing methods may represent a target-agnostic approach to viral wastewater monitoring, allowing for the detection of a broad range of target viruses including potentially novel and emerging pathogens. In this study, targeted and untargeted metagenomic sequencing methods were compared with tiled-PCR sequencing for detection and genotyping of viral pathogens in wastewater samples. Deep shotgun metagenomic sequencing was unable to generate sufficient genome coverage of human pathogenic viruses for robust genomic epidemiology, with samples dominated by bacteria. Hybrid-capture enrichment of shotgun libraries for respiratory viruses led to significant increases in genome coverage for a range of targets. Tiled-PCR sequencing led to further improvements in genome coverage compared to hybrid-capture for Severe Acute Respiratory Syndrome Coronavirus 2, enterovirus D68, norovirus GII and human adenovirus-F41 in wastewater samples. In conclusion, untargeted shotgun sequencing was unsuitable for genomic monitoring of the low virus concentrations in wastewater samples analysed in this study. Hybrid-capture enrichment represented a viable method for simultaneous genomic epidemiology of a range of viral pathogens, while tiled-PCR sequencing provided the optimal genome coverage for individual viruses with the minimum sequencing depth.