Amplicon based MinION sequencing of SARS-CoV-2 and metagenomic characterisation of nasopharyngeal swabs from patients with COVID-19

Shona C Moore; Rebekah Penrice-Randal; Muhannad Alruwaili; Xiaofeng Dong; Steven T Pullan; Daniel Carter; Kevin Bewley; Qin Zhao; Yani Sun; Catherine Hartley; En-min Zhou; Tom Solomon; Michael B. J. Beadsworth; James Cruise; Debby Bogaert; Derrick W T Crook; David A Matthews; Andrew D. Davidson; Zana Mahmood; Waleed Aljabr; Julian Druce; Richard T Vipond; Lisa Ng; Laurent Renia; Peter Openshaw; J Kenneth Baillie; Miles W Carroll; Calum Semple; Lance Turtle; Julian Alexander Hiscox

First, an amplicon-based system and the second, a metagenomic approach, that can also be used to assess the background microbiome. For the amplicon-based system, a series of primers were designed such that the SARS-CoV-2 genome could be amplified in appropriately 1000 base paired sequential fragments, with an approximately 200 base pair overlap to allow sequence assembly from the amplicon data (Fig. 1A) . The primers were selected on the basis of conserved regions in the SARS-CoV-2 genome based upon an initial deposition of 17 genomes. The rationale to generate short amplicons was also to allow the selection of primer pairs that could amplify longer segments should one set of primers fail on a particular sample. To test whether the primers could generate amplicons, RNA was purified from Vero cells that had been infected with an isolate of SARS-CoV-2 (MT007544.1 GenBank). This RNA was used as a template for cDNA synthesis followed by PCR using the conserved primers. This generated 30 amplicons covering the SARS-CoV-2 genomes and these were sequenced using MinION (Fig. 1B) . The amplicon-based approach was then evaluated on nasopharyngeal swabs collected from two patients with COVID-19.