Enabling Whole-Genome Sequencing of Native Influenza A in Wastewater Using Nanotrap Microbiome Particles and NEBNext Influenza A Integrated Indexing Primers

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Wastewater-based epidemiology (WBE) is an important tool for monitoring infectious diseases at the population level, providing early indicators of viral transmission and evolution. Influenza A viruses, known for their seasonal epidemics and pandemic potential, pose unique challenges for wastewater surveillance due to their low abundance and susceptibility to environmental degradation.1,2 Traditional sequencing approaches for influenza A in wastewater often yield incomplete genomes due to low viral titers and matrix inhibitors, limiting their utility for public health surveillance. In this work, we evaluated native wastewater using an optimized workflow, as native wastewater had not previously yielded successful whole-genome recovery in our hands. Key modifications included on-bead DNase treatment, minimizing refrigerated hold time prior to wastewater processing (<1 day), and increasing nucleic acid binding bead input to improve whole-genome recovery.

This study presents an enhanced workflow using Nanotrap Microbiome A and Nanotrap Microbiome B Particles (combined Nanotrap method) for viral enrichment with the NEBNext® Flu A Integrated Indexing Primer Module protocol, leveraging Oxford Nanopore sequencing for rapid whole-genome characterization. By integrating targeted cDNA synthesis and native barcoding, this approach enables same-day sequencing with improved genome coverage, offering a scalable and high-throughput solution for monitoring influenza A in wastewater.