Optimising a whole-genome sequencing workflow for One Health surveillance of Influenza A viruses across human, swine, and avian hosts in a diagnostic setting

Influenza A viruses (IAV) are a global health concern, infecting a wide range of hosts, including humans, birds, and pigs. Whole genome sequencing (WGS) is crucial for genomic surveillance under a One Health framework, providing insights into IAV evolution and transmission. We evaluated several laboratory workflows for next generation sequencing (NGS), aiming to optimise and unify the WGS process across IAV samples of human, swine, and avian origins. Multiple combinations of RNA extraction methods, one-tube RT-PCR protocols and primer sets were systematically tested for their efficacy in generating high-quality PCR and NGS products across all viral segments. We assessed performance based on sequencing quality, including read counts, segment coverage and amplification bias across various host types, subtypes, and viral loads. High-quality RNA extraction was critical for achieving reliable sequencing results, particularly in low viral load samples. Uniform amplification across all IAV genome segments was best achieved using an optimised RT-PCR protocol that reduced amplification bias for shorter fragments, including defective interfering particles (DIPs). Although variability in performance remained across host types and sample qualities, the refined workflow showed consistent improvements in sequencing outcomes, offering a promising foundation for a unified genomic surveillance. While further refinements may be needed for specific contexts, this study provides an improved, broadly applicable workflow that enhances genomic surveillance of influenza A viruses under a One Health framework, facilitating better understanding of viral evolution and transmission across species.