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Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses

Authors
  • King, Jacqueline1
  • Harder, Timm1
  • Beer, Martin1
  • Pohlmann, Anne1
  • 1 Friedrich-Loeffler-Institut, Südufer 10, Greifswald, Insel Riems, 17493, Germany , Greifswald (Germany)
Type
Published Article
Journal
BMC Infectious Diseases
Publisher
Springer (Biomed Central Ltd.)
Publication Date
Sep 03, 2020
Volume
20
Issue
1
Identifiers
DOI: 10.1186/s12879-020-05367-y
Source
Springer Nature
Keywords
License
Green

Abstract

BackgroundDue 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.MethodsTwelve representative influenza A virus samples of multiple subtypes were universally amplified in a one-step 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.ResultsThe 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.ConclusionsOverall, 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.

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