Direct RNA nanopore sequencing of SARS-CoV-2 extracted from critical material from swabs

Abstract

ABSTRACTBackgroundIn consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily.MethodsHere, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retro-transcription.ResultsUsing an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapisid (N) gene, which have been reported previously in studies conducted in other countries.ConclusionTo the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs.Despite these limitations, this approach provides the advantages of true native RNA sequencing, and does not include amplification steps that could introduce systematic errors.This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.We deposited the gene sequence in the NCBI database under the following URL:https://www.ncbi.nlm.nih.gov/nuccore/MT457389