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

  1. Davide Vacca
  2. Antonino Fiannaca
  3. Fabio Tramuto
  4. Valeria Cancila
  5. Laura La Paglia
  6. Walter Mazzucco
  7. Alessandro Gulino
  8. Massimo La Rosa
  9. Carmelo Massimo Maida
  10. Gaia Morello
  11. Beatrice Belmonte
  12. Alessandra Casuccio
  13. Rosario Maugeri
  14. Gerardo Iacopino
  15. Carmela Rita Balistreri
  16. Francesco Vitale
  17. Claudio Tripodo
  18. Alfonso Urso

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

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.

Article activity feed

  1. Version published to 10.3390/life12010069
  2. ScreenIT

    SciScore for 10.1101/2020.12.21.20191346: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    No key resources detected.

    Results from OddPub: Thank you for sharing your data.

    Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.

    Results from TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    Results from JetFighter: We did not find any issues relating to colormaps.

    Results from rtransparent:
    • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2020.12.21.20191346 on medRxiv