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.

Keywords: COVID-19; MinION; SARS-CoV-2; direct RNA nanopore sequencing; swab.

Figure 1

Representative scheme of the direct RNA seq. protocol (SQK-RNA002, Nanopore Technologies). dsRNA-DNA hybrids are synthesized during the library preparation. Next, the adapters needed for sequencing through nanopores are added at the 3′ poly A end, with the motor protein bound in 3′ to 5′ direction. Thereby, only the RNA strand will be sequenced, whereas the cDNA strand will be excluded.

Figure 2

The MinKNOW duty time plots depict the sum of total channel activity within a particular period of time. The number of sequencing pores decrease over time. In the categorized plot, the imbalance of adapters is apparent.

Figure 3

Genome IGV analysis that shows the frequency of the identified NC_045512:c.28881_28882_28883delinsAAC mutation in the reads from sequenced samples.

Figure 4

The sequences obtained by Sanger sequencing of RNA extracted from swabs that tested positive for SARS-CoV-2 infection. The rectangle covers coordinates from 28,881 to 28,883 for underlines, as all samples contained the NC_045512:c.28881_28882_28883delinsAAC mutation.

Figure 5

Identification of the reported mutation in the CNCB database. (A) Strains isolated from 18.3% of COVID-19 cases (over 21,000 complete genomes sequenced from isolated strains) contained the reported mutation. (B) Plot depicting the distribution of these cases from the end of February to the beginning of June.