Evaluation of Rapid Amplicon-Based Nanopore Sequencing Using The Latest Chemistry for Accurate Whole Genome Analysis of Influenza A Virus in Clinical Samples.

MinION sequencing is widely used to sequence influenza A virus (IAV) genomes. However, the accuracy and utility of this approach, which uses the latest chemistry to obtain whole viral genome sequences directly from clinical samples, remain insufficiently investigated. We evaluated the sequencing accuracy of combining simultaneous multi-segment one-step RT-PCR and MinION sequencing using various subtypes of 13 IAV isolates. The latest R10.4.1 chemistry significantly improved sequencing accuracy, achieving ≥99.993% identity with the results of Illumina MiSeq and reducing single nucleotide deletion in homopolymer regions. Applying this method to 11 clinical samples enabled rapid subtype identification and acquisition of eight full-length IAV genomes. In four of these samples, subtype identification of hemagglutinin and neuraminidase was achieved within 20 min of starting the sequencing, and a full-length IAV genome was obtained within 7 h of RNA extraction. However, cross-barcode misassignment during demultiplexing might have affected data interpretation, particularly for samples with low viral genome copy numbers. Although careful data analysis is required for multiplex sequencing of clinical samples with low viral genome copy numbers, this approach can be used for the rapid identification of IAV subtypes and accurate acquisition of full IAV genome sequences from clinical samples.