{"doi":"10.1101/2025.09.19.676942","title":"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 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.","journal":"bioRxiv (Cold Spring Harbor Laboratory)","year":2025,"id":576073,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9552,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1484667,"name":"Li-Wen Audrey Wang","orcid":"0009-0002-7760-2096","position":1,"is_corresponding":false},{"id":440060,"name":"James B. Thissen","orcid":"0000-0002-4693-5886","position":2,"is_corresponding":false},{"id":423500,"name":"Kara L. Nelson","orcid":"0000-0001-8899-2662","position":3,"is_corresponding":false},{"id":301737,"name":"Lenore Pipes","orcid":"0000-0003-0056-8045","position":4,"is_corresponding":false},{"id":225972,"name":"Rose S. Kantor","orcid":"0000-0002-5402-8979","position":5,"is_corresponding":false},{"id":1484666,"name":"Minxi Jiang","orcid":"0000-0002-0796-2590","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:57:56.636458Z","pmid":"41000925","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}