Fig. 4

Laboratory-based AIV subtype characterisation workflow. (1) Swabs were collected from mallard ducks at several locations around New Zealand. (2) Swabs were sent to the laboratory for AIV testing. Nucleic acid from the collected swabs or AIV isolates was extracted using the KingFisher MagMAX CORE Total Nucleic Acid Purification Kit (Thermo Fisher Scientific, MA, USA). (3–4) The presence of Influenza A virus was tested using the Influenza A RT-qPCR assay. Swabs that were tested positive by RT-qPCR were inoculated into embryonated chicken eggs. (5) Whole-genome amplification was performed by multi-segment reverse transcription PCR (RT-PCR) using AIV-specific primers. (6–7) The size of purified whole-genome amplified AIV amplicons were assessed on a 4200 TapeStation using Genomic DNA (gDNA) ScreenTape Assay Kit (Agilent Technologies, Santa Clara, CA, USA), and the concentration of the amplicons were quantified using the Invitrogen Qubit™ 1 x dsDNA High Sensitivity Quantification Assay kit (Thermo Fisher Scientific, Waltham, MA, USA) on an Invitrogen Qubit™ 4.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA). (8) cDNA was prepared for sequencing using the Oxford Nanopore Technologies (ONT) Rapid PCR Barcoding Kit V14 (Oxford Nanopore Technologies, London, UK) or Rapid Barcoding Kit V14 (Oxford Nanopore Technologies, London, UK) and sequenced using R10.4.1 PromethION or MinION flow cells (Oxford Nanopore Technologies, London, UK). (9) AIV subtypes were identified by manually mapping sequenced reads to a non-redundant Global Initiative on Sharing All Influenza Data (GISAID) dataset. Created in BioRender. Wilson, A. (2025) https://BioRender.com/7erudlf.