Abstract Title
Validation of Pre-Barcoded Primer Sets for High-Throughput Nanopore Sequencing of Avian Influenza Viruses
Abstract
The highly pathogenic avian influenza virus (AIV) H5 clade 2.3.4.4b has continuously evolved, leading to the emergence of diverse genotypes. Sharing genetic data and conducting international surveillance of these viruses in wild birds and poultry is essential. This study validated a high-throughput AIV genetic analysis strategy using pre-barcoded primer sets and Nanopore sequencing technology. Previously published primer sets for multi-segment amplification of all eight AIV genes were modified with barcodes and tested on the Nanopore flow cell (R10.4.1) to evaluate gene amplification performance and demultiplexing. The primer set with the highest amplification efficiency, particularly focusing on polymerase and hemagglutinin genes associated with mammalian host susceptibility, was selected. This optimized primer set, along with the Nanopore ligation sequencing kit (LSK-114), was applied to clinical samples (brain, lung, liver, throat swab, and total cloacal swab) from ducks experimentally infected with two highly pathogenic AIV strains (H5N1 and H5N6, clade 2.3.4.4b). The assembled sequences were consistent with results from the Native Barcoding Kit and primer sets used by Zhou et al. (2009) and Van Poelvoorde et al. (2021). Additionally, the whole genome analysis showed efficient sequencing features; improved minimum coverage on key sites, such as PB2 E627 and D701, and reduced unclassified reads. These high-throughput and balanced primer sets are suitable for timely and resource-efficient genetic surveillance of emerging zoonotic avian influenza
Co-Author(s)
Gyeong-Beom Heo, Yeongbu Kim,Se-Hee An, Youn-Jeong Lee, and Kwang-Nyeong Lee
Animal and Plant Quarantine Agency, Gimcheon, Republic of Korea
Abstract Category
Diagnostics, vaccination, or other mitigation strategies for poultry and wildlife