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 first sequenced five influenza A positive and five influenza-negative throat swabs, each 91 spiked with Hazara virus control at 10 4 genome copies/ml. Using the SISPA approach [21] 92 followed by Nanopore sequencing, we produced metagenomic data dominated by reads that 93 were bacterial in origin, with extremely few viral reads detected. Passing the sample through a 94 0.4 µm filter prior to nucleic acid extraction increased the detection of viral reads by several 95 orders of magnitude ( Fig S1) . Filtration is relatively expensive, so we also assessed the 96 alternative approach of adding a rapid centrifugation step to pellet bacterial and human cells, 97 followed by nucleic acid extraction from the supernatant. We used a pooled set of influenza A 98 positive samples (concentration 10 6 genome copies/ml), to provide a large enough sample to 99 . CC-BY-NC 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/676155 doi: bioRxiv preprint assess reproducibility, with the Hazara control spiked in at 10 4 genome copies/ml. Enrichment 100 for influenza and Hazara was similar for filtration vs centrifugation, based on reads mapping to 101 the viral genome ( Fig S2) . As centrifugation is simpler and cheaper, we selected this approach 102 for all further testing. 103 104 Method optimisation to reduce time for cDNA synthesis 105