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An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Shakiba Kazemian·Stephen Pedroza·Alinne L R Santana-Pereira·Peter J Neasham·James F North·Laura Huber·Miria F Criado·Constantinos S Kyriakis
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Abstract

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

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Influenza A virusLong-read sequencingMultisegment RT-PCROxford Nanopore TechnologiesWhole-genome sequencingJournal Article

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Shakiba Kazemian, Stephen Pedroza, Alinne L R Santana-Pereira, Peter J Neasham, James F North, Laura Huber, Miria F Criado, Constantinos S Kyriakis. 2026-09-08. An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.. https://doi.org/10.1016/j.jviromet.2026.115453

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