Researchers Correct Avian Influenza Sequence Upload Errors

In a formal response published in Nature Communications, a team of researchers has acknowledged and corrected widespread sequencing errors that appeared in a global avian influenza study. The original article, released on November 2, 2026, was subject to a “Matters Arising” critique by M. Wille and colleagues, who identified anomalies in the genetic data submitted to public repositories. The responding authors, led by Dhammika Leshan Wannigama and including numerous international collaborators, have detailed the specific technical failure that led to the publication of incorrect sequence data. This correction is significant because it clarifies the integrity of the underlying biological findings while addressing the procedural errors that compromised the publicly available genomic records.

The central issue identified by the correspondents was the appearance of hemagglutinin (HA) and neuraminidase (NA) sequences from disparate geographic regions that shared 100% nucleotide identity over prolonged time periods. Such patterns suggested evolutionary stasis that was biologically implausible for avian influenza viruses. The responding team explained that the primary error occurred during the sequence submission process to the Global Initiative on Sharing Avian Influenza Data (GISAID). Following GISAID’s advice for bulk submissions, the researchers utilized the command-line software “FluCLI” to upload their data. During the reformatting of files for this bulk upload, an analysis file originally prepared for phylogenetic tree construction was inadvertently included in the submission package.

This analysis file contained a mixture of the study’s own sequences and reference sequences from other sources. Furthermore, the sample identifiers were reformatted incorrectly, leading to the duplication and mixing of study and reference sequences in the public database. The authors emphasized that these anomalies were not present in their original internal analyses. The erroneous sequences were the result of a submission mistake rather than a flaw in the biological sampling or initial data generation. The team has apologized for the confusion caused by these erroneous sequences and has worked with GISAID to withdraw the affected entries and replace them with the corrected sequence data under the same accession numbers.

To address the specific concerns regarding evolutionary rates, the researchers provided a detailed analysis of expected mutation rates for avian influenza viruses. Citing previous work by Chen and Holmes, the team noted that substitution rates for avian influenza viruses vary between 1.8 and 8.4 × 10−3 per site per year. For the HA gene, the mean substitution rate was 3.92 × 10−3 per site per year, with a 95% highest probability density range of 2.43–5.40 × 10−3. For the NA gene, the mean substitution rate was 3.61 × 10−3 per site per year, with a range of 2.29–4.92 × 10−3. Based on these values, the rough estimate for the mutation rate in the HA gene is 6.7 mutations per year, while the mutation rate for the NA gene is approximately 5.1 mutations per year.

The authors also referenced a 2022 study by Kim et al., which evaluated the effects of host species on avian influenza mutation rates. That study found that mutation rates were higher for viruses infecting wild shorebirds and chickens compared to wild and domestic ducks. Although the exact host of the fecal samples collected in this study or the precise date of deposition is unavailable, the corrected sequences are consistent with the expected rate of influenza virus evolution. The team provided supplementary figures and tables to demonstrate that the corrected data aligns with these established evolutionary benchmarks, thereby refuting the claim of implausible evolutionary stasis.

In response to the TempEst analysis conducted by the correspondents, the responding authors performed their own TempEst analysis using the corrected sequence data. They downloaded available full sequences of the NA gene from Asia, collected between January 1, 1996, and December 31, 2024, and uploaded to GISAID. In total, 817 sequences were downloaded for this verification process. The phylogeny was inferred using the Maximum Likelihood method and the General Time Reversible model of nucleotide substitutions. The resulting tree constructed with the correct sequences did not contain any outliers belonging to the study’s sequences, confirming that the original conclusions of the manuscript were based on correctly generated data.

The correspondence also addressed concerns regarding Australia’s avian influenza status. The authors clarified that their study does not question Australia’s status as free from avian influenza. They noted that they never mentioned Australia in their study or discussed its status. The team agreed with the correspondents that Australia has a strong surveillance and infection-control system and a well-documented history of rapid detection and eradication of avian influenza outbreaks. The phylogenetic tree constructed based on the correct sequence data clearly shows that the study’s sequences are not 100% identical to the two sequences reported in Australia in 2020. The authors emphasized that their research is not sufficient to support any theory about the origin of influenza variants or to identify any specific region as the primary source of such variants.

Regarding the discussion of flyways and global circulation, the authors apologized for the sequence uploading error that made the HA and NA sequences obtained from Papua New Guinea appear 100% identical to previously submitted sequences. When the correct sequences are used, no 100% similarities were observed. The team highlighted that Papua New Guinea lies within the East Asian–Australasian Flyway, which intersects with several other major migratory pathways, including the West Asian–East African Flyway and the Black Sea/Mediterranean Flyway. These flyways are interconnected through northern migratory routes and overlap with the Pacific and Mississippi Americas Flyways. The complex network of migratory routes makes it biologically plausible that avian influenza viruses circulating in one region may be detected in distant geographic areas.

The study region contains more than 100,000 small islands or islets, approximately 98% of which are remote or rural and not subject to testing or monitoring. This creates significant gaps in available data. The authors noted that climate change is rapidly altering these ecosystems, complicating the understanding of virus circulation. During the study period, which spanned from late 2021 to early 2023, the North and South Indian Ocean experienced 14 cyclonic storms. Severe weather events, including record-breaking Cyclone Freddy in 2023, affected regions such as Madagascar and Mozambique. These environmental stressors can disrupt ecosystems, alter bird migratory pathways, and displace human populations, increasing the complexity of disease ecology in these regions.

The authors also addressed the limitations of global influenza surveillance. They noted that systematic surveillance is limited or absent in northern regions such as Siberia and northern Mongolia, as well as across much of Central Asia, Africa, and South and East Asia. Only a handful of countries routinely conduct surveillance studies and release sequence data from wild birds or poultry. The study period overlapped with the global COVID-19 pandemic, during which much existing surveillance capacity was diverted to pandemic management. This diversion left influenza and other pathogens under-monitored, and global restrictions on movement hampered disease surveillance. Large parts of Asia, the Pacific, and Africa are characterized by millions of backyard and smallholder poultry farms, which are rarely subject to surveillance for avian influenza.

The team acknowledged an issue regarding the H3 hemagglutinin clade assignment. This error arose from the automated NextClade platform, which occasionally assigns avian H3 sequences to human-specific clades instead of reporting them as “unassigned.” The authors recognized that it was their responsibility to validate these assignments before including them in the manuscript. They have corrected the record by updating these assignments as “unassigned” clades in the paper, as an author’s correction. This step ensures that the taxonomic classification of the viruses is accurate and does not mislead readers about the host specificity of the lineages.

Finally, the authors provided clarification on ethical and regulatory approvals. The study did not involve direct sampling from live or protected animals, nor did it involve the collection of material from birds’ nests or colonies. All materials consisted of bird feces collected from the environment, representing environmental waste samples. These samples do not fall under the scope of the Convention on International Trade in Endangered Species (CITES) or the Nagoya Protocol. The samples were collected directly into a DNA/RNA preservation buffer, which renders the material non-infectious and non-culturable. The analyses and sequencing were conducted in Thailand, and the study received ethical exemption approval from the institute in Japan. The authors have updated their acknowledgements and ethical statements to reflect their commitment to transparency and adherence to established standards of integrity.

Subject of Research: Correction of sequencing errors in global avian influenza genomic data

Article Title: Reply to: Evidence of widespread sequencing errors in global avian influenza study

Article References: Wannigama, D. L., Amarasiri, M., Phattharapornjaroen, P., Hurst, C., Modchang, C., Besa, J. J. V., Miyanaga, K., Cui, L., Fernandez, S., Huang, A. T., Ounjai, P., Werawatte, W. K. C. P., Rad S. M, A. H., Vatanaprasan, P., Jay, D. J., Saethang, T., Luk-in, S., Kanthawee, P., Thuptimdang, W., … Abe, S. (2026). Reply to: Evidence of widespread sequencing errors in global avian influenza study. Nature Communications, 17(1), Article 11334. https://doi.org/10.1038/s41467-026-78540-7

Image Credits: AI Generated

DOI: 10.1038/s41467-026-78540-7

Keywords: Avian Influenza, Genomic Sequencing, Data Correction, GISAID, Viral Evolution, Phylogenetics, Public Health, Surveillance, Migratory Birds, Scientific Integrity, Reply, Evidence