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 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.

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: Avian influenza viruses; Full genome sequencing; Influenza A viruses; MinION; Multiplexing; Nanopore sequencing; Next-generation sequencing.

Fig. 1

Comparison of IonTorrent S5XL and MinION workflow from RNA extraction to finished consensus sequences for twelve samples. Overall time is split into hands on and passive time needed for each protocol step