Real-time SARS-CoV-2 diagnostic and variants tracking over multiple candidates using nanopore DNA sequencing

  1. François Stüder
  2. Jean-Louis Petit
  3. Stefan Engelen
  4. Marco Antonio Mendoza-Parra

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Abstract

Since December 2019, a novel coronavirus responsible for a severe acute respiratory syndrome (SARS-CoV-2) is accountable for a major pandemic situation. The emergence of the B.1.1.7 strain, as a highly transmissible variant has accelerated the world-wide interest in tracking SARS-CoV-2 variants’ occurrence. Similarly, other extremely infectious variants, were described and further others are expected to be discovered due to the long period of time on which the pandemic situation is lasting. All described SARS-CoV-2 variants present several mutations within the gene encoding the Spike protein, involved in host receptor recognition and entry into the cell. Hence, instead of sequencing the whole viral genome for variants’ tracking, herein we propose to focus on the SPIKE region to increase the number of candidate samples to screen at once; an essential aspect to accelerate diagnostics, but also variants’ emergence/progression surveillance. This proof of concept study accomplishes both at once, population-scale diagnostics and variants' tracking. This strategy relies on (1) the use of the portable MinION DNA sequencer; (2) a DNA barcoding and a SPIKE gene-centered variant’s tracking, increasing the number of candidates per assay; and (3) a real-time diagnostics and variant’s tracking monitoring thanks to our software RETIVAD. This strategy represents an optimal solution for addressing the current needs on SARS-CoV-2 progression surveillance, notably due to its affordable implementation, allowing its implantation even in remote places over the world.

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  1. Version published to 10.1038/s41598-021-95563-w
  2. ScreenIT

    SciScore for 10.1101/2021.05.18.21257281: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    DNA libraries were sequenced on Nanopore FLO-MIN106D flowcells (R9) with the MinION Mk1C instrument (72h sequencing; high-accuracy base calling).
    MinION
    suggested: (MinION, RRID:SCR_017985)
    BWA aligner following nanopore parameters).
    BWA
    suggested: (BWA, RRID:SCR_010910)
    E-Sarbeco amplicon sequence, or SPIKE gene; defined as entry parameters) is visualized within scatterplot view (png file) updated during the processing (RETIVAD produces a png file per detected PCR barcode monomers; thus allowing to generate scatterplots displaying as many lines as detected cDNA barcode monomers; Figure S4).
    SPIKE
    suggested: (SPIKE, RRID:SCR_010466)
    For it, the racon package (version 1.4.20) is used for raw de novo DNA assembly, followed by the alignment with Minimap2 (version 2.17).
    Minimap2
    suggested: (Minimap2, RRID:SCR_018550)

    Results from OddPub: Thank you for sharing your code.

    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.
    • Thank you for including a protocol registration statement.

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

    About SciScore

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  3. Version published to 10.1101/2021.05.18.21257281v1 on medRxiv