Wastewater-based genomic surveillance uncovers broader SARS-CoV-2 diversity and spatial transmission dynamics in Georgia, January 2023–July 2024

Wastewater surveillance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using next-generation sequencing (NGS) enables real-time monitoring of circulating and emerging variants, often providing earlier detection than clinical reporting while capturing presymptomatic, asymptomatic, and symptomatic infections. However, the correlation between lineage abundance in wastewater and clinical specimens from corresponding sewersheds in Georgia remains understudied. From January 2023 through July 2024, the Georgia Public Health Laboratory (GPHL) sequenced SARS-CoV-2 from 669 wastewater samples collected across 17 treatment facilities in 15 counties. These data were compared with 325 clinical specimens from corresponding sewersheds to assess early detection potential. Nucleic acids were extracted using the KingFisher Apex platform, followed by genomic library preparation and paired-end whole-genome sequencing on the Illumina NextSeq 2000. Variant calling and lineage abundance estimation using Freyja identified 2,218 SARS-CoV-2 lineages and sub-lineages, 1,769 in 2023, and 1,255 in 2024, with 806 co-circulating across both years. Clinical sequences spanned 135 lineages/sub-lineages across 15 clades, with >70% in clades 24A (27.4%), 23A (19.4%), 23F (13.5%), and 22E (10.2%). Predominant lineages included XBB.1.5 (11.4%), JN.1 (6.8%), and JN.1.4 (6.2%), reflecting broader clinical trends in Georgia. Clinical data also reflected distinct transmission patterns, including viral introduction and spread within and between counties. Overall, this study provides important insights into the introduction and dissemination of SARS-CoV-2 lineages in Georgia. Expanding wastewater surveillance to include other emerging and reemerging pathogens of public health importance will be particularly valuable during periods of low disease incidence, serving as an early warning system when clinical testing is limited and enabling timely public health responses.IMPORTANCEWastewater-based surveillance of SARS-CoV-2 provides a noninvasive, cost-effective, population-level method for detecting viral genetic material independent of clinical testing. Next-generation sequencing enables timely monitoring of circulating lineages by public health laboratories. The Georgia National Wastewater Surveillance System (GA-NWSS) established in 2021 through collaboration between the Georgia Department of Public Health (DPH) Epidemiology and the Georgia Public Health Laboratory (GPHL) collects weekly samples from 17 wastewater treatment facilities across 15 counties. From January 2023 through July 2024, GPHL sequenced 669 wastewater samples, identifying 2,218 SARS-CoV-2 lineages and sub-lineages, compared with 135 lineages detected in 325 clinical specimens from corresponding sewersheds. Spatial analysis highlights viral spread across state boundaries and within Georgia catchment areas, illustrating evolving transmission patterns. These results demonstrate that wastewater sequencing captures broader genomic diversity than clinical sampling alone, emphasizing its critical role in pathogen genomic surveillance and supporting data-driven public health response to SARS-CoV-2.