Read-level mutation linkage in wastewater sequencing reveals cryptic virus evolution and persistence

Wastewater sequencing has become widely adopted as a complement to clinical genomic surveillance, providing population-level monitoring capable of detecting “cryptic variants”, viruses that are rarely, if ever, observed clinically. However, current wastewater analyses are limited by a lack of integration with up-to-date surveillance data and systematic frameworks for tracking virus evolution. Here, we develop an end-to-end workflow for longitudinal cryptic variant detection and tracking that leverages global SARS-CoV-2 genomic surveillance. Applying this approach for a two-year period, we identified more than 2,000 cryptic variants and characterized their emergence and persistence. Using graph analysis, we inferred relationships between cryptic variants and their likely ancestors, uncovering multistep evolutionary trajectories. Our workflow provides a scalable, accessible, and real-time solution for monitoring cryptic variants and community-level virus diversity.