Rapid multiplex MinION nanopore sequencing workflow for Influenza A viruses

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