Abstract Title
Optimization of Influenza A Whole-Genome Sequencing Protocol for short- and long-read NGS platforms
Abstract
Avian influenza is widespread in wild birds and is causing outbreaks in poultry and US dairy cows, with several recent human cases, creating a growing need for reliable SNP analysis to track mutations that may facilitate viral replication in different hosts. Accurate SNP analysis requires whole-genome sequencing (WGS) with sufficient read depth across all eight segments. However, coverage in the longer polymerase segments often exhibits a U-shaped pattern, with reduced coverage in the middle regions. This is likely due to the amplification of defective interfering particles (DIPs), which can complicate genome assembly.
This study evaluated an alternative approach to the commonly used amplicon-based protocol for influenza A WGS, which also underlies the Oxford Nanopore influenza ligation WGS V14 protocol. We tested alternative primer sets and RT-PCR kits to improve read depth, particularly in the polymerase segments. Additionally, four purification methods were assessed to optimize the removal of DIPs and other untargeted reads. Automation of the purification step was explored using the KingFisher system and compared to manual workflows. Libraries were prepared using the Native Barcoding Kit V14 and sequenced on a MinION Mk1C with an R10.4.1 flow cell. Sequencing reads were basecalled, demultiplexed, and aligned using MinKNOW and minimap2 in Galaxy.
The alternative primer set and RT-PCR kit significantly increased total read counts and read depth. The alternative purification kit effectively removed short reads, enabling superior coverage depth across polymerase segments. Our refined protocol supports accurate SNP analysis and can be adapted for various short- and long-read NGS platforms.
This study evaluated an alternative approach to the commonly used amplicon-based protocol for influenza A WGS, which also underlies the Oxford Nanopore influenza ligation WGS V14 protocol. We tested alternative primer sets and RT-PCR kits to improve read depth, particularly in the polymerase segments. Additionally, four purification methods were assessed to optimize the removal of DIPs and other untargeted reads. Automation of the purification step was explored using the KingFisher system and compared to manual workflows. Libraries were prepared using the Native Barcoding Kit V14 and sequenced on a MinION Mk1C with an R10.4.1 flow cell. Sequencing reads were basecalled, demultiplexed, and aligned using MinKNOW and minimap2 in Galaxy.
The alternative primer set and RT-PCR kit significantly increased total read counts and read depth. The alternative purification kit effectively removed short reads, enabling superior coverage depth across polymerase segments. Our refined protocol supports accurate SNP analysis and can be adapted for various short- and long-read NGS platforms.
Co-Author(s)
Iryna V. Goraichuk, Jacquline Risalvato, Mary Pantin-Jackwood, David L. Suarez
Southeast Poultry Research Laboratory, U.S. National Poultry Research Center, Agriculture Research Service, U.S Department of Agriculture, Athens, GA, USA
Abstract Category
Diagnostics, vaccination, or other mitigation strategies for poultry and wildlife