Details

Autor(en) / Beteiligte

• King, Jacqueline
• Harder, Timm
• Beer, Martin
• Pohlmann, Anne

Titel

Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses

Ist Teil von

• BMC infectious diseases, 2020-09, Vol.20 (1), p.1-648, Article 648

Ort / Verlag

London: BioMed Central Ltd

Erscheinungsjahr

2020

Link zum Volltext

Quelle

Free E-Journal (出版社公開部分のみ）

Beschreibungen/Notizen

• 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. Twelve 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. 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. 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.