Systematic evaluation of tangential-flow ultrafiltration and Nanotrap microbiome particles for sequencing wastewater RNA virome

In this study, we aimed to systematically evaluate Nanotrap microbiome particles (Nanotrap) and tangential-flow ultrafiltration (TFF) method for recovering wastewater RNA virome and develop an insightful understanding of the impact of wastewater processing methods on RNA virus detection. We hypothesized that TFF would capture a more diverse RNA virome than Nanotrap. To investigate this, we employed two sequencing approaches. First, amplicon-based sequencing was used to compare breadth and depth coverage of SARS-CoV-2 genome, diversity at the lineage level, and variant composition from the same wastewater samples. Second, whole-transcriptome shotgun sequencing was conducted to compare the diversity of RNA virome recovered by both methods at the family level. Our findings inform practical recommendations for the selection and optimization of wastewater processing methods for future wastewater RNA virome analysis.