Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples.
Lewandowski K., Xu Y., Pullan ST., Lumley SF., Foster D., Sanderson N., Vaughan A., Morgan M., Bright N., Kavanagh J., Vipond R., Carroll M., Marriott AC., Gooch KE., Andersson M., Jeffery K., Peto TEA., Crook DW., Walker AS., Matthews PC.
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 for 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 102-103 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 <30) and 6/13 samples with low viral titres (Ct values 30-40). No false positive reads were generated from 10 influenza-negative samples. Thus Nanopore sequencing operated with 83% sensitivity (95% CI 67-93%) and 100% specificity (95% CI 69-100%) compared to the current diagnostic standard. Coverage of full length virus was dependent on sample composition, being negatively influenced by increased host and bacterial reads. However, at high influenza titres, we were able to reconstruct >99% complete sequence for all eight gene segments. We also detected a Human Coronavirus co-infection in one clinical sample. 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.