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

We consistently retrieved Hazara virus reads from all three dilution series by Nanopore 124 sequencing, independently of influenza virus titre in the sample (Fig 2B) . Sequencing from 125 dilution series 1 and 2 gave a consistent proportion of total reads mapping to the Hazara 126 genome, across dilutions and between the first two pools with mean values per pool of 1.4x10 3 127 ±660 RPM (reads per million (of total reads) ± standard deviation) and 1.2x10 3 ±350 RPM 128 respectively. The pool-3 dilution series generated 260 ±340 RPM Hazara reads across samples, 129 and showed a decreasing trend associated with increased dilution factor, as increasingly more 130 non-viral RNA was introduced from this high-background pool. 131 132 Limit of influenza virus detection by Nanopore from pooled samples 133 Nanopore sequencing of the triplicate SISPA preparations of the influenza A positive pool 134 produced mean 5.3x10 4 ±3.6x10 4 RPM mapping to the influenza A genome (Fig 2B) . Across the 135 dilution series, the proportion of influenza reads was strongly associated with influenza virus 136 titre (p-value = 4.7x10 -5 ), but was also influenced by which negative pool was used for dilution, 137 consistent with the pattern observed for the Hazara control. Sequencing the negative controls 138 (pools with no influenza virus spike) generated no reads mapping to influenza. At influenza virus 139 titres <10 3 copies/ml, influenza reads were inconsistently detected across the samples (Fig 2B) , 140 suggesting the limit of detection is between 10 2 -10 3 influenza copies/ml. 141 142