P1 variants and key amino acid mutations at the Spike gene identified using Sanger protocols

  1. Gabriela Bastos Cabral
  2. Cintia Mayumi Ahagon
  3. Giselle Ibette Silva Lopez-Lopes
  4. Igor Mohamed Hussein
  5. Paula Morena Guimaraes
  6. Audrey Cilli
  7. Valeria Oliveira Silva
  8. Maria do Carmo CT Timenetsky
  9. Ivy de Jesus Alves
  10. Andrea GC Bombonatte
  11. Fabiana C Pereira dos Santos
  12. Luís Fernando de Macedo Brígido

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Abstract

SARS-CoV-2 variants, along with vaccination, mark the second year of the pandemic. The spike region is a focal point in COVID-19 pathogenesis, with different amino acid changes potentially modulating vaccine response and some being part of variant signatures. NGS is the standard tool to sequence the virus but limitations of different sources hinders expansion of genomic surveillance in many places. To improve surveillance capability we developed a Sanger based sequencing protocol to obtain coverage of most (>95%) spike gene. Eleven nasopharyngeal swabs collections had RNA extracted for real time PCR diagnosis and leftover RNA had up to 3785 bp sequenced at an ABI3500 using dye termination chemistry of nested PCR products of two reactions of one-step RT-PCR. P1 amino acid mutations signatures were present in 18% (2/11), with 82% (9/11) with three or more additional amino acid changes (GISAID CoVsurver list). Most sequences (86%, 6/7) from 2021 have the E484K, whereas the mutation was not present in samples collected in 2020 (0/4, p=0.015).The swiftness that favorable mutations to the virus may prevail and their potential impact in vaccines and other current interventions need broader surveillance and more public health attention.

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  1. ScreenIT

    SciScore for 10.1101/2021.03.21.21253158: (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

    Experimental Models: Organisms/Strains
    SentencesResources
    Primers design: In order to design primer suitable PCR sets to partial SARS-CoV-2 Spike (S1 and S2 region) protein, 3 sequences (NC_045512.2); SARS-CoV-2 Wuhan-Hu-1, and two 2 sequences from the first case reported in Brazil, MT350282.1 and MT126808.1; (Araujo, 2020) were obtained from NCBI and imported into BioEdit sequence alignment editor (version 7.0.5.2) program.
    Wuhan-Hu-1
    suggested: None
    Software and Algorithms
    SentencesResources
    Primers design: In order to design primer suitable PCR sets to partial SARS-CoV-2 Spike (S1 and S2 region) protein, 3 sequences (NC_045512.2); SARS-CoV-2 Wuhan-Hu-1, and two 2 sequences from the first case reported in Brazil, MT350282.1 and MT126808.1; (Araujo, 2020) were obtained from NCBI and imported into BioEdit sequence alignment editor (version 7.0.5.2) program.
    BioEdit
    suggested: (BioEdit, RRID:SCR_007361)

    Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).

    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.
    • Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
    • 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.

  2. Version published to 10.1101/2021.03.21.21253158v1 on medRxiv