Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

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.

SARS-CoV-2 Variant Surveillance in Genomic Medicine Era

“…Direct RNA sequencing is relatively recent approach in sequencing technologies that do not require RNA revers-transcription and allow the direct determination of the sequence of single nucleic acid molecules, without amplification [34]. This technology provides longer reads than regular NGS methods, but with higher error rates [35].…”

Lessons Learned and Yet-to-Be Learned on the Importance of RNA Structure in SARS-CoV-2 Replication

“…These findings provide important insights into SARS-CoV-2 base modification; however, additional studies utilizing direct RNA sequencing (described in more detail below) to determine RNA base modifications will be required to fully elucidate these patterns of modification and potentially other modifications yet to be detected. Currently, there are multiple studies that have performed direct RNA-seq on SARS-CoV-2 ( 49 , 254 , 255 ); however, these investigations primarily focused on the transcriptome and largely omit details on epitranscriptomics and thus could be valuable sources of data on epitranscriptomics for subsequent analysis. The value in revisiting these data sets has already been demonstrated by Fleming et al whereby in silico analysis of the direct RNA-seq data sets of SARS-CoV-2 provided by Kim et al ( 49 ) and Miladi et al ( 254 ) suggested the presence of multiple pseudouridine residues, which were not observed previously and were conserved across the sgRNA ( 256 ).…”

Transcriptomics and RNA-Based Therapeutics as Potential Approaches to Manage SARS-CoV-2 Infection

“…In contrast, direct RNA-seq (DRS) is a powerful tool that allows for the characterization of the transcriptional landscape of viruses with complex genomes such as SARS-CoV-2 [ 91 ]. When reading the RNA genome directly, the complete sequence is obtained using long reads that do not require an assembly step or obtaining the precise identity of the nucleotide bases since it is not required to synthesize cDNA or perform PCR [ 92 , 93 ]. DRS is performed efficiently using the third-generation platforms Oxford Nanopore and PacBio [ 94 ].…”