Metagenomic Nanopore sequencing of influenza virus direct from clinical respiratory samples

Kuiama Lewandowski; Yifei Xu; Steven T. Pullan; Sheila F. Lumley; Dona Foster; Nicholas Sanderson; Alison Vaughan; Marcus Morgan; Nicole Bright; James Kavanagh; Richard Vipond; Miles Carroll; Anthony C. Marriott; Karen E Gooch; Monique Andersson; Katie Jeffery; Timothy EA Peto; Derrick W. Crook; A Sarah Walker; Philippa C. Matthews

Influenza is a major global public health threat as a result of its highly pathogenic variants, large 27 zoonotic reservoir, and pandemic potential. Metagenomic viral sequencing offers the potential of 28 a diagnostic test for influenza which also provides insights on transmission, evolution and drug 29 resistance, and simultaneously detects other viruses. We therefore set out to apply Oxford 30 Nanopore Technology to metagenomic sequencing of respiratory samples. We generated 31 influenza reads down to a limit of detection of 10 2 -10 3 genome copies/ml in pooled samples, 32 observing a strong relationship between the viral titre and the proportion of influenza reads (p = 33 4.7x10 -5 ). Applying our methods to clinical throat swabs, we generated influenza reads for 27/27 34 samples with high-to-mid viral titres (Cycle threshold (Ct) values <30) and 6/13 samples with low 35 viral titres (Ct values 30-40). No false positive reads were generated from 10 influenza-negative 36 samples. Thus Nanopore sequencing operated with 83% sensitivity (95% CI 67-93%) and 100% 37 specificity (95% CI 69-100%) compared to the current diagnostic standard. Coverage of full 38 length virus was dependent on sample composition, being negatively influenced by increased 39 host and bacterial reads. However, at high influenza titres, we were able to reconstruct >99% 40 complete sequence for all eight gene segments. We also detected Human Coronavirus and 41 generated a near complete Human Metapneumovirus genome from clinical samples. While 42 further optimisation is required to improve sensitivity, this approach shows promise for the 43 The application of Oxford Nanopore Technologies (ONT; https://nanoporetech.com/) to 75 generate full-length influenza sequences from clinical respiratory samples can address these 76 challenges. ONT offers a 'third-generation', portable, real-time approach to generating long-read 77 sequence data, with demonstrated success across a range of viruses [21, [23] [24] [25] . To date, 78 Nanopore sequencing of influenza has been reported using high titre virus from an in vitro 79 culture system, producing full length genome sequences through direct RNA sequencing [26] or 80 targeted enrichment by either hybridisation of cDNA [27] or influenza-specific PCR amplification 81 [28] . 82 83 We therefore aimed to optimise a metagenomic protocol for detecting influenza viruses directly 84 from clinical samples using Nanopore sequencing. We determine its sensitivity compared to 85 existing diagnostic methods and its accuracy compared to short-read (Illumina) sequencing, 86 using clinical samples from hospital patients during an influenza season, and samples from a 87 laboratory controlled infection in ferrets. 88