Sequencing the wastewater virome: Current strategies and bioinformatic pitfalls

Wastewater-based surveillance (WBS) is a powerful tool for monitoring viral pathogens at the population level, complementing clinical surveillance through anonymous and unbiased insights into viral circulation. Wastewater sequencing allows to move beyond detection toward detailed characterization of circulating pathogens. This review outlines the two main sequencing strategies used in WBS: targeted and untargeted approaches. Targeted methods, including amplicon- and probe-based sequencing, provide sensitive detection and recovery of known viruses but are limited in identifying novel pathogens. Untargeted shotgun metagenomic sequencing offers a broader, unbiased view of the wastewater virome, although its performance is challenged by low human viral abundance, complex background signals, and genome assembly challenges. We also discuss key challenges complicating bioinformatic analysis, including limited human viral sequences, co-circulating viral lineages, viral dark matter, reference database limitations, large data volumes, and methodological biases, as well as emerging computational approaches aimed at improving wastewater sequencing analysis and interpretation.