Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses

Abstract

Background: Due to the frequent reassortment and zoonotic potential of influenza A viruses, rapid gain of sequence information is crucial. Alongside established next-generation sequencing protocols, the MinION sequencing device (Oxford Nanopore Technologies) has become a serious competitor for routine whole-genome sequencing. Here, we established a novel, rapid and high-throughput MinION multiplexing workflow based on a universal RT-PCR. Methods: Twelve representative influenza A virus samples of multiple subtypes were universally amplified in a onestep RT-PCR and subsequently sequenced on the MinION instrument in conjunction with a barcoding library preparation kit from the rapid family and the MinIT performing live base-calling. The identical PCR products were sequenced on an IonTorrent platform and, after final consensus assembly, all data was compared for validation. To prove the practicability of the MinION-MinIT method in human and veterinary diagnostics, we sequenced recent and historical influenza strains for further benchmarking. Results: The MinION-MinIT combination generated over two million reads for twelve samples in a six-hour sequencing run, from which a total of 72% classified as quality screened, trimmed and mapped influenza reads to produce full genome sequences. Identities between the datasets of > 99.9% were achieved, with 100% coverage of all segments alongside a sufficient confidence and 4492fold mean depth. From RNA extraction to finished sequences, only 14 h were required. Conclusions: Overall, we developed and validated a novel and rapid multiplex workflow for influenza A virus sequencing. This protocol suits both clinical and academic settings, aiding in real time diagnostics and passive surveillance.

Highly Pathogenic Avian Influenza Viruses at the Wild–Domestic Bird Interface in Europe: Future Directions for Research and Surveillance

“…Next-generation sequencing (NGS) techniques are used to characterize whole genome sequences, but these are time consuming, expensive, and require extensive protocols and large stationary equipment. Recent developments in NGS—i.e., third-generation sequencers, including the portable MinION third-generation nanopore sequencing device—facilitate faster, less expensive, easier to use and more mobile analyses of whole viral genomes [ 188 ]. A broader application of third-generation sequencing will significantly increase surveillance resolution and efficiency by reducing costs and time, while further optimisation of sequencing techniques and analyses is needed to include minority single nucleotide variant analyses [ 189 ].…”

Evaluation of Oxford Nanopore MinION RNA-Seq Performance for Human Primary Cells

“…The ability to sequence long DNA fragments provided by the ONT MinION system offers easier assembly of complex genomes than short-read methods do, but its high error rate is still an obstacle [6,7]. Thanks to the decreased turnaround time at a low cost compared to short-read sequencing, and the continuous performance improvements, also including the option to sequence multiple samples in a single run [19,23,24], this pocket-…”

“…The ability to sequence long DNA fragments provided by the ONT MinION system offers easier assembly of complex genomes than short-read methods do, but its high error rate is still an obstacle [6,7]. Thanks to the decreased turnaround time at a low cost compared to short-read sequencing, and the continuous performance improvements, also including the option to sequence multiple samples in a single run [19,23,24], this pocket-sized device is widely used both for scientific research and clinical applications [36]. Different transcriptomic studies exploited the ability of this technology to uncover the diversity of alternative splicing isoforms and their expression levels [12,14,15].…”

“…The turnaround time and cost for ONT RNA-Seq can be further reduced by sequencing multiple samples on a single run [19,[23][24][25]. Wick et al [23] sequenced 12 bacterial DNA isolates simultaneously on a single MinION flow cell using the ONT native barcoding kit.…”

“…The long reads were then combined in a hybrid assembly with Illumina data to fully resolve the bacterial genome. Recently, King et al [24] proposed a rapid workflow for multiplexed sequencing of influenza A viruses using the ONT technology for real-time analysis, in combination with a one-step RT-PCR and the Rapid Barcoding Kit.…”