Considerations and Opportunities for Probe Capture Enrichment Sequencing of Emerging Viruses from Wastewater.

Until recently, wastewater-based monitoring for pathogens of public health concern primarily used PCR-based quantification methods and targeted sequencing for specific pathogens (e.g., SARS-CoV-2). In the past three years, researchers have expanded sequencing to monitor a broad range of pathogens, applying probe capture enrichment to wastewater. The goals of those studies included (1) monitoring and expanding fundamental knowledge of disease dynamics for known pathogens and (2) evaluating the potential for early detection of emerging diseases resulting from zoonotic spillover or novel viral variants. Several studies using off-the-shelf probe panels designed for clinical and environmental surveillance reported that enrichment increased virus relative abundance but did not recover complete genomes for most nonenteric viruses. Based on our experience and recent results reported by others using these panels for wastewater, clinical, and synthetic samples, we discuss challenges and technical factors that affect the rates of false positive and false negative results. We identify trade-offs and opportunities throughout the workflow, including in wastewater sample processing, probe panel design, and bioinformatic analysis. We suggest tailored methods of virus concentration and background removal, carefully designed probe panels, and multithresholded bioinformatics analysis.