Recovering wastewater RNA for virome sequencing by systematically optimized tangential-flow ultrafiltration and...

Area of Science:

• Environmental virology
• Genomic epidemiology
• Wastewater-based epidemiology

Background:

• Municipal wastewater contains diverse RNA viruses, posing risks for human, animal, and plant health.
• Genomic sequencing of wastewater is vital for tracking viral pathogens and understanding community health.
• Wastewater processing methods significantly impact RNA virome sequencing outcomes.

Purpose of the Study:

• To systematically evaluate tangential-flow ultrafiltration (TFF) and Nanotrap Microbiome A Particles for RNA virus detection and characterization in wastewater.
• To compare the efficacy of TFF and Nanotrap methods in detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) via amplicon sequencing.
• To assess the impact of these methods on whole-transcriptome shotgun sequencing of the RNA virome.

Main Methods:

• Paired comparison of tangential-flow ultrafiltration (TFF) and Nanotrap Microbiome A Particles using wastewater samples.
• Detection of SARS-CoV-2 using amplicon sequencing.
• Characterization of the RNA virome using whole-transcriptome shotgun sequencing.

Main Results:

• Both TFF and Nanotrap methods recovered similar SARS-CoV-2 variants at the lineage level.
• Optimizing concentration factors for TFF and automated procedures for Nanotrap was critical for high-depth genome coverage.
• The two methods enriched distinct RNA viromes, with TFF showing higher relative abundances of Reoviridae and Coronaviridae.

Conclusions:

• Wastewater processing methods differentially enrich RNA viromes due to distinct concentration mechanisms.
• Method selection and optimization are critical for accurate genomic monitoring of viruses in wastewater.
• Findings have implications for broader environmental applications of wastewater genomic surveillance.