Abstract

Wastewater-based epidemiology (WBE) has emerged as a scalable and cost-effective strategy for monitoring SARS-CoV-2 throughout the COVID-19 pandemic. In the post-COVID era, however, reliance on conventional molecular techniques, such as reverse transcription quantitative PCR (RT-qPCR) and amplicon-based targeted next-generation sequencing (tNGS), limits comprehensive pathogen surveillance due to restricted genomic coverage and throughput, hindering early detection of emerging public health threats. In this study, we evaluated a hybrid capture-based sequencing (HybCapSeq) workflow using five probe panels, including three commercial and two custom panels, to compare SARS-CoV-2 detection performance and per-sample reagent cost with those of RT-qPCR and tNGS. We then applied the comprehensive viral capture panel for longitudinal wastewater surveillance (N = 343) across seven types of sampling sites in two Chinese cities. Temporal trends in SARS-CoV-2 genomic coverage derived from capture sequencing preceded reported clinical case trends by 1–2 weeks, demonstrating its potential as an early-warning indicator for wastewater monitoring. Compared with RT-qPCR, tNGS, and metatranscriptomic sequencing (MetaT), the capture-based approach showed comparable or superior performance in SARS-CoV-2 detection and variant tracking, while also enabling simultaneous detection of diverse human-pathogenic viruses, identification of putative novel viral taxa, and characterization of environmental virome composition. In addition, bacterial reads accounted for 95.05% of total reads in hybrid capture libraries (95% CI: 94.42–95.67%), highlighting the potential of this approach for profiling of bacterial and antimicrobial resistance genes in wastewater. These findings endorse HybCapSeq, combined with strategic probe design and site selection, as a robust WBE platform for pathogen surveillance, epidemic preparedness, and public health response in the post-COVID era.