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 RT-ddPCR. 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.

D.D., B.S., M.G., M.R., P.T., P.K., and V.C.-H. are employees of Verily Life Sciences.

This work was funded by gifts to M.K.W. and A.B.B. from the Sergey Brin Family Foundation.

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Deduplicated sequencing reads are available through the NCBI under BioProject PRJNA1438722. Accessions are also listed in Table S1.