Prospective metagenomic sequencing of wastewater across the United States yields robust viral enrichment and concordance with digital PCR measurements.

Metagenomic sequencing is increasingly applied to wastewater to characterize the diversity, dynamics, and relative abundance of human and animal viruses. Among these sequencing approaches are those that enrich viral nucleic acids from the wastewater matrix, aiming to increase the viral read fraction for analysis. However, the feasibility of scaling targeted viral sequencing to diverse sewersheds across large geographic scales is currently unknown. In this study, we apply hybrid capture metagenomic sequencing to nearly 450 weekly wastewater samples collected during the respiratory virus season in the United States and evaluate sequencing performance for generating public health-relevant data. Analysis of data from 15 wastewater treatment plants demonstrates that our approach enabled efficient capture of pathogens of interest, achieving a median viral read fraction over 19%. Importantly, relative abundance estimates of common pathogens correlated with direct quantification of viral targets using reverse transcription digital droplet PCR. Together, our results demonstrate that hybrid capture sequencing of wastewater is a viable tool to monitor both common and rare pathogens across geographically diverse sewersheds.IMPORTANCEWastewater testing is commonly used to identify and quantify human pathogens at a community scale. However, the most commonly used approaches rely on targeted, PCR-based methods that are highly specific to a single virus. Metagenomic sequencing provides an opportunity to detect and quantify the relative abundance of a wide range of viruses that are important for human health from wastewater, and hybrid capture approaches work by first enriching samples for these extremely rare targets to increase sensitivity. We demonstrate that hybrid capture metagenomic sequencing successfully enriches wastewater samples from diverse locations across the United States and that data derived from sequences are associated with detections of 11 key viruses using standard RT-ddPCR methods. This work suggests that scaling hybrid capture metagenomics for viruses in wastewater is a feasible way to generate data critical for public health to support infectious disease outbreak response.