Grab wastewater samples were collected once a week on a Monday from the inlet of the 21 wastewater treatment plants (WWTPs) in the City of Cape Town during the Easter period of 2021 and Easter 2022, for nine weeks per year (Figure S1 ). The characteristics of the WWTPs are provided in supplementary material (Table S1) . About 500 mL of the influent sample was collected and transported on ice to the laboratory for analysis.
In the laboratory, the total RNA was extracted with the Qiagen RNeasy® PowerSoil® Kit, as per the manufacturer′s instructions (Qiagen, Hilden, Germany) following the method described earlier [26 (link),27 (link)]. In brief, 100 mL of influent wastewater was centrifuged at 2500× g for 20 min after which 2.5 mL of the pellet was used for the analysis. The quality of the total RNA was determined with a NanoDrop® ND-1000 instrument (Nanodrop Technologies, Wilmington, NC, USA). A clinical SARS-CoV-2 nasopharyngeal swab sample with known viral copies was spiked into the wastewater sample. The extraction method was previously tested for efficiency [28 (link)].
In the laboratory, the total RNA was extracted with the Qiagen RNeasy® PowerSoil® Kit, as per the manufacturer′s instructions (Qiagen, Hilden, Germany) following the method described earlier [26 (link),27 (link)]. In brief, 100 mL of influent wastewater was centrifuged at 2500× g for 20 min after which 2.5 mL of the pellet was used for the analysis. The quality of the total RNA was determined with a NanoDrop® ND-1000 instrument (Nanodrop Technologies, Wilmington, NC, USA). A clinical SARS-CoV-2 nasopharyngeal swab sample with known viral copies was spiked into the wastewater sample. The extraction method was previously tested for efficiency [28 (link)].
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