Comparative wastewater virome analysis with different enrichment methods

Wastewater-based epidemiology (WBE) has proven its value for public health. Physical concentration of virus particles is a crucial step for WBE to permit a sensitive and unbiased characterization of the catchment virome. Here we evaluate five different virion concentration techniques, including polyethylene glycol precipitation (PEG), vacuum-based direct capture (VDC), ultrafiltration (UF), NanoTrap® (NT), and membrane adsorption (MEM) for their suitability to concentrate a wide variety of viral taxa from raw wastewater for PCR detection and sequencing-based metagenomic readouts. We found that to capture a taxonomically diverse virome from wastewater, PEG and VDC outperform all other methods tested in enrichment rates, reproducibility, species detection, and captured nucleotide diversity. We observed that different methods exhibit variable concentration efficiencies across taxonomic groups in a reproducible manner, though we could not identify common physiochemical attributes driving this difference. We conclude that both PEG and VDC are equally capable at detecting and enriching a broad range of viral taxa, boosting the genomic information potential and reducing blind spots relative to other tested methods. These results advance WBE towards capturing the complex wastewater virome and help guide protocol choices for potential future viral threats.