Wastewater genomic surveillance of SARS-CoV-2 by nanopore sequencing uncovers JN.1 prevalence and novel spike mutational hotspots in Athens during winter 2024-2025.

Wastewater-based epidemiology (WBE) provides an efficient, population-wide tool for monitoring SARS-CoV-2 transmission and evolution, overcoming limitations of clinical testing. Here, we designed and applied an upgraded Spike-Seq workflow integrating third-generation nanopore sequencing on the PromethION platform to achieve complete spike gene coverage from wastewater samples collected in Athens (December 2024-February 2025). This workflow enabled simultaneous quantification of lineage-defining markers and detection of previously uncharacterized mutations in wastewater samples. Our results identified the sustained dominance of the JN.1 lineage (>80% frequency), with only transient signals from XFG, NB.1.8.1, and LP.8.1variants. Notably, we revealed recurrent substitutions in the N-terminal domain and S2 region, including L24F and I1130K, which indicate continued diversification of the dominant JN.1-associated viral population. Integration of sequencing with qPCR viral load data revealed a peak in community infection pressure in January 2025, which decoupled from the rising frequency of specific mutational hotspots. Our findings demonstrate the utility of amplicon-based wastewater nanopore sequencing for monitoring SARS-CoV-2 genetic diversity in wastewater.