Suitability of concentration and extraction protocols for genomic applications in wastewater-based epidemiology

Abstract

Wastewater-based epidemiology (WBE) has emerged as a powerful tool for community monitoring, offering a non-invasive, cost-effective means of assessing public health. PCR-based methods, including digital droplet PCR (ddPCR), combined with reverse transcription (RT) for RNA viruses, are widely used for detection of known pathogens in wastewater. Recently, sequencing approaches have gained importance. Targeted amplicon sequencing enables high-sensitivity detection of specific pathogens, while metagenomic sequencing provides untargeted profiling of microbial and viral communities, supporting surveillance of novel pathogens and antimicrobial resistance (AMR). A major challenge across all genomic approaches remains the efficient concentration and extraction of nucleic acids, particularly for low-abundance targets like respiratory viruses. This study aimed to evaluate the fit-for-purpose of concentration and extraction protocols for wastewater surveillance, focussing on their suitability for genomic downstream applications: (RT-)ddPCR, amplicon sequencing and metagenomics. Both RNA (viral pathogens) and DNA (AMR markers) targets were included and experiments were performed on raw and spiked wastewater. The results revealed substantial variability in method performance. Ultrafiltration with a concentrating pipette (InnovaPrep) consistently yielded lower viral concentrations, whereas precipitation-based methods, magnetic capture and a column-based concentration/extraction kit provided higher yield and better sensitivity. PCR inhibitors reduced targeted sequencing efficiency, while metagenomics produced consistent taxonomic profiles across methods. Overall, the study highlights the importance of protocol optimization to improve sensitivity in WBE. Optimization of protocols, particularly for detecting low abundance pathogens and inhibitor removal, remains essential. Broader validation across diverse wastewater matrices and microbial species will be critical to strengthen the role of WBE in public health surveillance.