# Developing and Benchmarking One Health Genomic Surveillance Tools for Influenza A Virus in Wastewater

## TL;DR

The paper compares methods for sequencing influenza A virus in wastewater to improve surveillance of low-abundance strains.

## Contribution

A custom HA tiled-amplicon panel was developed and benchmarked against other methods for wastewater-based IAV genomic surveillance.

## Key findings

- The custom HA tiled-amplicon panel was sensitive, fast, and cost-effective for monitoring seasonal IAV variants.
- Probe-capture methods showed resilience to RNA degradation but were less sensitive and more costly than amplicon methods.
- Ultrafiltration-based virus concentration outperformed direct extraction methods across all sequencing techniques.

## 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 benchmarked four targeted enrichment methods suited for whole-genome sequencing including custom and off-the-shelf amplicon and probe-based methods. Our custom HA tiled-amplicon panel was sensitive, fast, and cost-effective, making it suitable for monitoring low-abundance seasonal variants of known subtypes. However, its reliance on conserved and intact primer-binding sites limited primer design to fewer subtypes. A previously published universal amplicon method targeted all IAV subtypes, but it performed poorly in wastewater due to its reliance on intact genome segments. Probe-capture methods were resilient to RNA degradation and mismatches, potentially enabling broader surveillance and detection of emerging strains. However, probes were costly, labor-intensive, and less sensitive than tiled-amplicon. When testing compatibility of sequencing methods with upstream virus concentration and extraction methods, ultrafiltration-based virus concentration outperformed large-volume direct extraction with all four sequencing methods. This set of benchmarking comparisons and custom panels provides needed information for the translation of IAV genomic sequencing into a routine component of wastewater surveillance.