The Impact of Viral Concentration Method on Quantification and Long Amplicon Nanopore Sequencing of SARS-CoV-2 and Noroviruses in Wastewater

Wastewater-based surveillance has gained attention in the four years following the start of the COVID-19 pandemic. Accurate pathogen detection, quantification and characterisation rely on the selection of appropriate methodologies. Here, we explore the impact of viral concentration method on RT-qPCR inhibition and quantification of norovirus genogroups I and II (GI and GII), crAssphage, phi6 and SARS-CoV-2. Additionally, their impact on long amplicon sequencing for typing noroviruses and whole-genome sequencing (WGS) SARS-CoV-2 was explored. RT-qPCR inhibition for each viral concentration method was significantly different apart from the two ultrafiltration methods, InnovaPrep^® concentrating pipette (IP) and Vivaspin^® (VS) centrifugal concentrators. Using an ultrafiltration method reduced inhibition by 62.0% to 96.0% compared to the ammonium sulphate (AS) and polyethylene glycol (PEG) precipitation-based methods. Viral quantification was significantly impacted by concentration method with the highest concentrations (copies/L) observed for VS with 7.2- to 83.2-fold differences from AS depending on the target. Norovirus long amplicon sequencing showed genotype-dependent differences with IP performing best for GI and VS for GII although IP performance gains for GI were relatively small. VS outperformed AS and IP across all metrics during SARS-CoV-2 WGS. Overall, VS performed the best when considering all the areas of investigation.