Simplified Inhouse Nanoweb Membrane Enrichment Coupled Viral Whole Genome Shotgun Metagenomics Approach for Waste...

Wastewater/sewage represents a highly complex environmental matrix and harbors various viruses including viruses of epidemic and pandemic potential like Influenza A and, SARS-CoV-2. The limitations of available methods for sample concentration are cost, efficiency and time. NGS- metagenomics offer sample to virus genomic characterization for even non-cultivable viruses at highest resolution. In this study, we have collected samples from sewage treatment plants located in Gwalior region of Central India (December 2023 to March 2025). In this study a novel in house nanoweb membrane-based sample enrichment followed by magnetic bead based nucleic acid extraction was optimized in conjunction to shotgun whole genome metagenomics on Nanopore and ion torrent NGS. Both the methods were found comparable with commercially available methods by virus specific TaqMan qPCR. Both the methods were found successful in virus recovery at two log through 10²GC/50 ml feline calicivirus, Influenza A virus, Zika virus, SARS-CoV-2 spiked in sewage matrix alone or in mixture), suggesting the optimized protocol found working for virus characterization at strain level. Here, we have standardized a simple field amenable waste water sample enrichment followed by nucleic acid extraction protocol, that can easily be integrated with latest onsite downstream molecular diagnostic platforms. The developed method is very simple, cost effective and field deployable. This will help to develop a suitable strategic plan for sewage surveillance towards early warning/microbial forensics and future decisions for prevention and therapeutic interventions.