Custom barcoded primers for influenza A nanopore sequencing: enhanced performance with reduced preparation time

Authors: Goraichuk, Iryna; Suarez, David

Submitted to: Frontiers in Cellular and Infection Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/10/2025
Publication Date: 4/15/2025
Citation: Goraichuk, I., Suarez, D.L. 2025. Custom barcoded primers for influenza A nanopore sequencing: enhanced performance with reduced preparation time. Frontiers in Cellular and Infection Microbiology. https://doi.org/10.3389/fcimb.2025.1545032.
DOI: https://doi.org/10.3389/fcimb.2025.1545032

Interpretive Summary: Highly pathogenic avian influenza virus is an important disease in both poultry and mammals. Having the sequence of the virus can provide important information about the virus including how virulent it will be in birds and whether antiviral drugs are likely to be effective. Sequence information can also be used to understand where the virus likely came from which can be helpful in blocking it's spread. The more quickly the sequence information can be determined can also be helpful in trying to control the viral outbreak. This paper describes the use of special primers that have an identifiable code (barcodes) that when used with the MinIon sequencer can reduce the processing time to generate sequence and still maintain high sensitivity. Having barcodes allows you to sequence multiple samples at the same time, which allows lower per sample sequence costs. The manuscript provides the data for a faster and cheaper method of sequencing influenza viruses that retains high sensitivity that will help in the control of outbreaks.

Technical Abstract: Highly pathogenic avian influenza is endemic and widespread in wild birds and is causing major outbreaks in poultry world-wide and in U.S. dairy cows, with several recent human cases, highlighting the need for reliable and rapid sequencing to track mutations that may facilitate viral replication in different hosts. SNP analysis is a useful molecular epidemiology tool to track outbreaks, but it requires accurate whole-genome sequencing (WGS) with sufficient read depth across all eight segments. In outbreak situations, where timely data is critical for controlling the spread of the virus, reducing sequencing preparation time while maintaining high-quality standards is particularly important. In this study, we optimized a custom barcoded primer strategy for influenza A whole-genome sequencing on the Nanopore platform, combining the high performance of the Native barcoding kit with the rapid preparation time of the Rapid barcoding kit. Custom barcoded primers were designed to perform barcode attachment during RT-PCR amplification, eliminating the need for separate barcoding and clean-up steps, thus reducing library preparation time. We compared the performance of the custom barcoded primer method with the Native and Rapid barcoding kits in terms of read quality, read depth, and sequencing output. The results show that the custom barcoded primers provided performance comparable to the Native barcoding kit while reducing library preparation time by 2.3X compared to the Native kit and being only 15 minutes longer than the Rapid kit with better depth of sequencing. This approach offers a promising solution for influenza A sequencing, providing both high throughput and time efficiency, which significantly improves the time-to-result turnaround, making sequencing more accessible for real-time surveillance.