Optimization of the Primary Sludge Processing Method for Wastewater Genomic Surveillance of SARS-CoV‑2

Wastewater genomic surveillance (WWGS) of SARS-CoV-2 is typically performed using influent wastewater, but the approach is challenging due to degradation as well as low target concentrations in wastewater. This could be alleviated by utilizing primary sludge; however, this matrix is prone to sequencing library failures. Our study focuses on developing a robust primary sludge-based SARS-CoV-2 genome sequencing method. The study was conducted using 30 parallel influent wastewater and primary sludge samples collected during three different time periods, under three clinically predominant SARS-CoV-2 Omicron lineages in Ottawa, Canada. Results showed that our approach consistently recovered near-complete (≥90%) SARS-CoV-2 genomes from both influent wastewater and primary sludge samples. Prevalent lineage and single nucleotide variant (SNV) profiles were identical (p > 0.05) between influent wastewater and primary sludge. Further analysis indicated that a similar (p > 0.05) number of rare SNVs were detected between influent wastewater and primary sludge. Overall, our approach enables the sequencing of the most concentrated sources of genetic material within the wastewater matrix, providing valuable insights for public health forecasting of infectious disease prevalence beyond the COVID-19 pandemic.