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.
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BibTeX
@article{jiang2026developing,
title = {{Developing and benchmarking one health genomic surveillance tools for influenza A virus in wastewater}},
author = {Minxi Jiang and Audrey L. W. Wang and James B. Thissen and Kara L. Nelson and Lenore Pipes and Rose S. Kantor},
journal = {Nature Communications},
year = {2026},
month = oct,
doi = {10.1038/s41467-026-78683-7},
url = {https://www.nature.com/articles/s41467-026-78683-7},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Developing and benchmarking one health genomic surveillance tools for influenza A virus in wastewater
AU - Minxi Jiang
AU - Audrey L. W. Wang
AU - James B. Thissen
AU - Kara L. Nelson
AU - Lenore Pipes
AU - Rose S. Kantor
PY - 2026
DA - 2026/10/17
JO - Nature Communications
DO - 10.1038/s41467-026-78683-7
UR - https://www.nature.com/articles/s41467-026-78683-7
AB - 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.
SN - 2041-1723
ER -