Sequencing Using a Two-Step Strategy Reveals High Genetic Diversity in the S Gene of SARS-CoV-2 after a High-Transmission Period in Tunis, Tunisia

  1. Wasfi Fares
  2. Kais Ghedira
  3. Mariem Gdoura
  4. Anissa Chouikha
  5. Sondes Haddad-Boubaker
  6. Marwa Khedhiri
  7. Kaouthar Ayouni
  8. Asma Lamari
  9. Henda Touzi
  10. Walid Hammemi
  11. Zina Medeb
  12. Amel Sadraoui
  13. Nahed Hogga
  14. Nissaf ben Alaya
  15. Henda Triki

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Abstract

The method of choice for SARS-CoV-2 variant detection is whole-genome sequencing using next-generation sequencing (NGS) technologies. Resources for this technology remain limited in many low- and middle-income countries, where it is not possible to perform whole-genome sequencing for representative numbers of SARS-CoV-2-positive cases.

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  1. Version published to 10.1128/spectrum.00639-21
  2. ScreenIT

    SciScore for 10.1101/2021.06.18.449083: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: This study was approved by the Bio-Medical Ethics Committee of the Pasteur Institute of Tunis, Tunisia réf. 2020/14/I/LR16IPT/V1 Primer design: Primers were designed using PrimerDesign-M online software, available through https://www.hiv.lanl.gov/content/sequence/PRIMER_DESIGN/primer_design.html (26, 27), based on an alignment of 13451 SARS-CoV-2 complete genome sequences.
    Sex as a biological variableThe study population included 91 males and 110 females, their age ranged from 5 to 98 years.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    They were submitted to the NCBI database under accession number MZ150010 - MZ150210.
    NCBI
    suggested: (NCBI, RRID:SCR_006472)
    Sequences’ raw data have been processed using fastqc version 0.11.9 for quality control (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
    https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
    suggested: (FastQC, RRID:SCR_014583)
    Low quality reads and adapters have been filtered using trimmomatic version 0.39 (28) with a Phred quality score of 30 as threshold.
    trimmomatic
    suggested: (Trimmomatic, RRID:SCR_011848)
    Phred
    suggested: (Phred, RRID:SCR_001017)
    Genome consensus sequences were assembled by mapping on the SARS-CoV-2 reference genome of GenBank accession number NC045512 (Wuhan-Hu-1 isolate) using Spades assembler version 3.15.0 (29), with thresholds of 80% for nucleotide sequence coverage and 90% for nucleotide similarity.
    Spades
    suggested: (SPAdes, RRID:SCR_000131)
    Phylogenetic analysis: The obtained partial S gene sequences and selective whole genome sequences were aligned together with representative SARS-CoV-2 reference sequences of the nine recognized GISAID clades publically available in the GISAID database using MUSCLE multiple sequence alignment algorithms (32) implemented in MEGAX (33).
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)

    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:
    • No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
    • No funding statement was detected.
    • No protocol registration statement was detected.

    Results from scite Reference Check: We found no unreliable references.

    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 10.1101/2021.06.18.449083v1 on bioRxiv