Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

Influenza is a major global public health threat as a result of its highly pathogenic variants, large zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential of a diagnostic test for influenza which also provides insights on transmission, evolution and drug resistance, and simultaneously detects other viruses. We therefore set out to apply Oxford Nanopore Technology to metagenomic sequencing of respiratory samples. We generated influenza reads down to a limit of detection of 10 2 -10 3 genome copies/ml in pooled samples, observing a strong relationship between the viral titre and the proportion of influenza reads (p = 4.7x10 -5 ). Applying our methods to clinical throat swabs, we generated influenza reads for 27/27 samples with high-to-mid viral titres (Cycle threshold (Ct) values 99% complete sequence for all eight gene segments. We also detected Human Coronavirus and generated a near complete Human Metapneumovirus genome from clinical samples. While further optimisation is required to improve sensitivity, this approach shows promise for the Nanopore platform to be used in the diagnosis and genetic analysis of influenza and other respiratory viruses.