Comparison of the sensitivity of targeted and untargeted (metagenomic) methods for the detection of viral pathogens in wastewater

Timely and accurate pathogen detection is critical for the successful implementation of wastewater surveillance and has broad implications for public health. A wide range of surveillance tools are currently available, offering both quantitative and qualitative insights into the wastewater virome. Careful consideration of molecular methodology is required to successfully implement an effective wastewater surveillance scheme. Using SARS-CoV-2 as a model organism, we compared detection success across multiple approaches, including targeted (RT-PCR, qPCR, random priming RT-PCR) and target-agnostic (Rapid SMART-9N metagenomics) methods. We also estimated the copy number required for reliable detection, examined how the ratio of target to off-target genomes in wastewater affects detection and genome coverage using metagenomics, and assessed the efficacy of hybrid capture enrichment of target genomes in improving metagenomic detection. Our results show significant differences between methods, targeted RT-PCR and qPCR were more likely (68 % and 65 % respectively) to detect SARS-CoV-2 than target agnostic approaches. The inclusion of carrier RNA during extraction significantly increased the likelihood of target detection. Our target-agnostic metagenomic approach was consistently unable to detect our target, and, even in the presence of high concentrations that are atypical for wastewater, detection was limited. Target enrichment increased SARS-CoV-2 detection and maximum coverage by metagenomics (SMART-9N), though was outperformed by targeted amplicon sequencing. Overall, our findings support the use of targeted approaches for the routine surveillance of viral pathogens in wastewater. Whilst metagenomics provides broad insights into the virome, enrichment strategies are essential when using it to detect specific viruses, particularly in complex wastewater matrices. • This study compares targeted and untargeted molecular methods for SARS-CoV-2 detection in wastewater. • RT-PCR and qPCR outperformed target-agnostic metagenomics in sensitivity and detection success. • Inclusion of carrier RNA during RNA extraction significantly enhanced detection reliability. • Metagenomic detection was limited without enrichment, even at high SARS-CoV-2 concentrations. • Hybrid capture enrichment improved metagenomic detection but was less effective than targeted sequencing.