Longitudinal sequencing and variant detection of SARS-CoV-2 across Southern California wastewater from April 2020 - August 2021

Wastewater based epidemiology (WBE) is a useful method to detect pathogen prevalence and may serve to effectively monitor diseases at a broad scale. WBE has been used throughout the COVID-19 pandemic to track localized and population-level disease burden through the quantification of SARS-CoV-2 RNA present in wastewater. Aside from case load estimation, WBE is being used to assay viral genomic diversity and the emergence of potential SARS-CoV-2 variants. Here, we present a study in which we sequenced RNA extracted from sewage influent samples obtained from eight wastewater treatment plants representing 16 million people in Southern California over April 2020 - August 2021. We sequenced SARS-CoV-2 with two methods: Illumina Respiratory Virus Enrichment and metatranscriptomic sequencing (N = 269), and QIAseq SARS-CoV-2 tiled amplicon sequencing (N = 95). We were able to classify SARS-CoV-2 reads into lineages and sublineages that approximated several named variants across a full year, and we identified a diversity of single nucleotide variants (SNVs) of which many are putatively novel SNVs, and SNVs of unknown potential function and prevalence. Through our retrospective study, we also show that several sublineages of SARS-CoV-2 were detected in wastewater up to several months before clinical detection, which may assist in the prediction of future Variants of Concern. Lastly, we show that sublineage diversity was similar between wastewater treatment plants across Southern California, and that diversity changed by sampling month indicating that WBE is effective across megaregions. As the COVID-19 pandemic moves to new phases, and additional SARS-CoV-2 variants emerge, the ongoing monitoring of wastewater is important to understand local and population-level dynamics of the virus. Our study shows the potential of WBE to detect SARS-CoV-2 variants throughout Southern Californias wastewater and track the diversity of viral SNVs and strains in urban and suburban locations. These results will aid in our ability to monitor the evolutionary potential of SARS-CoV-2 and help understand circulating SNVs to further combat COVID-19.