Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses
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TECHNICAL ADVANCE
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Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses Jacqueline King, Timm Harder, Martin Beer and Anne Pohlmann*
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. Keywords: Nanopore sequencing, MinION, Influenza A viruses, Full genome sequencing, Next-generation sequencing, Avian influenza viruses, Multiplexing
Background Next-generation sequencing (NGS) methods, especially second-generation sequencers, have shown their capability of whole-genome sequencing (WGS) over the past decade for a wide spectrum of pathogens including influenza A viruses (IAV) [1, 2]. Due to widespread avian influenza * Correspondence: [email protected] Institute of Diagnostic Virology, Friedrich-Loeffler-Institut, Südufer 10, 17493 Greifswald, Insel Riems, Germany
virus (AIV) outbreaks with high mortality among poultry and wild birds in combination with the unceasing risk of zoonosis, avian origin IAV has devastating economic and anthropological impacts [3, 4]. Frequent reassortment events and vast genetic diversity of these viruses show the necessity for fast and accurate WGS [5]. While the characterisation of IAV has greatly benefitted from WGS utilising first- and second-generation sequencers, limiting factors such as high costs, process
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