Sequencing methods for HA and NA genes of avian influenza viruses from wild bird feces using Oxford Nanopore sequencing

• Published in: Comparative immunology, microbiology and infectious diseases Vol. 102; p. 102076
• Main Authors: Nabeshima, Kei, Asakura, Shingo, Iwata, Ritsuko, Honjo, Hisako, Haga, Atsushi, Goka, Koichi, Onuma, Manabu
• Format: Journal Article
• Language: English
• Published: Elsevier Ltd 01.11.2023
• Subjects: Amplicon sequencing; Avian flu; Nanopore sequencing; Wild bird; Wild bird; Nanopore sequencing; Avian flu; Amplicon sequencing
• ISSN: 0147-9571; 1878-1667
• DOI: 10.1016/j.cimid.2023.102076

We developed a method to determine the sequences of hemagglutinin (HA) and neuraminidase (NA) from RNA extracted directly from wild bird fecal samples, using Nanopore Flongle. We determined the nucleotide sequences and subtypes of HA and NA in 16 and 15 samples respectively, using Flongle. The results of HA and NA subtyping determined by the conventional method were consistent with their subtypes determined by our method, thereby the applicability of this method in the identification of HA and NA subtypes. In addition, the homology between the HA fragments in this and the Sanger methods ranged from 98.5 % to 100 %. Compared with conventional PCR with the Sanger method, this method can easily determine HA and NA subtypes and sequences directly from the fecal samples. It is easier to implement and has lower running costs (USD100$) than other NGS-based methods, making it a useful tool for avian influenza surveillance in wild birds. •We developed a method of sequencing AI from wild bird feces using Nanopore Flongle.•The sequencing accuracy of our method was comparable to the Sanger method.•Our method is a low-cost and scalable sequencing method.•The method is expected to be used for AI surveillance and other virus species.