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

Abstract

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.

Keywords: genome sequencing; lineage prevalence; rare SNVs; single nucleotide variants; wastewater and environmental surveillance.

1

Longitudinal comparison of SARS-CoV-2 genome coverage in influent wastewater (Inf) and primary sludge (PS) across three different periods under the three dominant lineages of Omicron VOC. (A) The percentage of genome coverage (left axis) was plotted against the N1 Ct value (right axis) and (B) The mean depth of coverage (×) was plotted against the N1 Ct value. The percentage of genome coverage and mean depth of coverage on the leftmost graph were shown with error bars, as two technical replicates of each paired influent wastewater and primary sludge samples were sequenced during the first period of sampling.

2

Prevalence (%) of different Omicron VOC lineages in influent wastewater (top) and primary sludge (bottom) was accessed using the Freyja bioinformatics tool under the three dominant lineages BA.2 (leftmost), EG.5 (middle), and EG.5.1 (rightmost). The topmost section of the figures shows the legends for each lineage associated with each sampling period.

3

SARS-CoV-2 genetic diversity in influent wastewater (Inf) and primary sludge (PS) samples. (A) Total and unique (detected exclusively in either sample type) SNVs counts in influent wastewater and primary sludge samples across the three sampling periods. (B) Boxplots of Shannon entropy (leftmost of each lineage type) and richness (rightmost of each lineage type) during the periods dominated by lineages BA.2, EG.5, and EG.5.1. The edges of the box represent the first (bottom) and third (top) quartiles, the solid line indicates the median, and the whiskers show the maximum and minimum values.

4

Evaluation of nondetected SNVs in influent wastewater and primary sludge during the sampling periods against the Canadian and global clinical sequences to determine their prevalence. The figure denotes rare SNVs using olive green-colored cells, while commonly reported SNVs in Canadian and global clinical sequences are represented by ash-colored cells.