Developing and benchmarking one health genomic surveillance tools for influenza A virus in wastewater

Abstract Influenza A viruses (IAV) remain a persistent One Health threat, and whole-genome sequencing from wastewater offers a promising surveillance tool. However, IAV is at low abundance in wastewater, making it difficult to sequence. We benchmark four targeted enrichment methods suited for multi-subtype whole-genome IAV sequencing including custom and off-the-shelf methods. Our custom HA tiled-amplicon panel is sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes, although its reliance on conserved and intact primer-binding sites limits primer design to fewer subtypes. A published universal amplicon method targets all IAV subtypes but performs poorly in wastewater due to its reliance on intact genome segments. Meanwhile, probe-capture methods are resilient to RNA degradation. In real wastewater samples, our custom probe panel recovers whole-genome signals from circulating H1N1, H3N2, H5N1, including validated H5N1 2.3.4.4b reads in samples where dPCR detection is limited. However, probe-capture is generally more costly and labor-intensive than amplicon methods. Across all four sequencing methods, two wastewater matrices, and most spike-in and endogenous virus targets, ultrafiltration-based virus concentration consistently outperforms large-volume direct extraction. Together, these benchmarking comparisons and custom panels provide needed information for implementing IAV genomic sequencing in routine wastewater surveillance.