Abstract
In 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 (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to 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.
Highlights
The characterization of the SARS-CoV-2 genome has become a priority for global healthcare in order to identify more suitable vaccines and therapeutic drugs [1].The use of third-generation sequencing technologies has significantly increased in recent years, because these methods yield reliably long reads even from biological samples with noise [2]
We investigated the suitability of Nanopore Oxford MinION Mk1B [12], a third-generation nanopore-based platform, for the identification of a critical target such as SARS-CoV-2 native RNA from a swab sample and, at the same time, tested its sensitivity in the characterization of the viral mutational landscape
To characterize the SARS-CoV-2 mutational landscape, we used RNA extracted from samples collected from five male and five female patients who tested positive for SARS-CoV2 in the swab test, with the corresponding cycle threshold (Ct) values ranging from 18 to 24
Summary
The characterization of the SARS-CoV-2 genome has become a priority for global healthcare in order to identify more suitable vaccines and therapeutic drugs [1].The use of third-generation sequencing technologies has significantly increased in recent years, because these methods yield reliably long reads even from biological samples with noise [2]. The use of swab samples for RNA extraction shows critical issues, regarding both the fragmentation level and concentration. Such limitations affect the read coverage of sequencing, which is decreased in a considerable manner. Protocols used for RNA library construction often require at least 100 ng total RNA. Under certain conditions, it is not possible to obtain that much RNA to satisfy this requirement. In these cases, in order to increase RNA amount, several protocols suggest RNA enrichment by reverse transcription and amplification of the cDNA, prior to library preparation [3]
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