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Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses
Jacqueline King1, Timm Harder1, Martin Beer1
1Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493, Greifswald, Insel Riems, Germany.
BMC Infectious Diseases
|September 5, 2020
Summary
A new rapid multiplex workflow using MinION sequencing offers quick whole-genome sequencing for influenza A virus, crucial for diagnostics and surveillance. This method achieves high accuracy and coverage in under 14 hours.
Area of Science:
- Virology
- Genomics
- Bioinformatics
Background:
- Influenza A viruses frequently reassort and possess zoonotic potential, necessitating rapid sequence information acquisition.
- The MinION (Oxford Nanopore Technologies) is emerging as a viable tool for routine whole-genome sequencing, complementing existing next-generation sequencing methods.
- A novel, rapid, and high-throughput MinION multiplexing workflow was developed, utilizing a universal reverse-transcription polymerase chain reaction (RT-PCR).
Purpose of the Study:
- To establish and validate a novel, rapid, and high-throughput MinION multiplexing workflow for influenza A virus whole-genome sequencing.
- To compare the performance of the MinION workflow against the IonTorrent platform for influenza A virus sequencing.
- To demonstrate the applicability of the MinION-MinIT method in human and veterinary diagnostics through sequencing of diverse influenza strains.
Main Methods:
- Twelve influenza A virus samples were universally amplified using a one-step RT-PCR.
- Samples were sequenced on the MinION instrument with a barcoding library preparation kit and processed using the MinIT for live base-calling.
- Identical PCR products were sequenced on an IonTorrent platform, with subsequent data comparison for validation.
Main Results:
- The MinION-MinIT workflow generated over two million reads from twelve samples in a six-hour run.
- 72% of reads were quality screened, trimmed, and mapped to produce full genome sequences with >99.9% identity and 100% segment coverage.
- The entire process from RNA extraction to finished sequences required only 14 hours.
Conclusions:
- A novel and rapid multiplex workflow for influenza A virus sequencing has been successfully developed and validated.
- The protocol is suitable for both clinical and academic settings, supporting real-time diagnostics and passive surveillance.
- This workflow enhances the speed and efficiency of influenza A virus genomic analysis.
