Guidelines for accurate genotyping of SARS-CoV-2 using amplicon-based sequencing of clinical samples

  1. Slawomir Kubik
  2. Ana Claudia Marques
  3. Xiaobin Xing
  4. Janine Silvery
  5. Claire Bertelli
  6. Flavio De Maio
  7. Spyros Pournaras
  8. Tom Burr
  9. Yannis Duffourd
  10. Helena Siemens
  11. Chakib Alloui
  12. Lin Song
  13. Yvan Wenger
  14. Alexandra Saitta
  15. Morgane Macheret
  16. Ewan W. Smith
  17. Philippe Menu
  18. Marion Brayer
  19. Lars M. Steinmetz
  20. Ali Si-Mohammed
  21. Josiane Chuisseu
  22. Richard Stevens
  23. Pantelis Constantoulakis
  24. Michela Sali
  25. Gilbert Greub
  26. Carsten Tiemann
  27. Vicent Pelechano
  28. Adrian Willig
  29. Zhenyu Xu

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Abstract

Background

SARS-CoV-2 genotyping has been instrumental to monitor virus evolution and transmission during the pandemic. The reliability of the information extracted from the genotyping efforts depends on a number of aspects, including the quality of the input material, applied technology and potential laboratory-specific biases. These variables must be monitored to ensure genotype reliability. The current lack of guidelines for SARS-CoV-2 genotyping leads to inclusion of error-containing genome sequences in studies of viral spread and evolution.

Results

We used clinical samples and synthetic viral genomes to evaluate the impact of experimental factors, including viral load and sequencing depth, on correct sequence determination using an amplicon-based approach. We found that at least 1000 viral genomes are necessary to confidently detect variants in the genome at frequencies of 10% or higher. The broad applicability of our recommendations was validated in >200 clinical samples from six independent laboratories. The genotypes of clinical isolates with viral load above the recommended threshold cluster by sampling location and period. Our analysis also supports the rise in frequency of 20A.EU1 and 20A.EU2, two recently reported European strains whose dissemination was favoured by travelling during the summer 2020.

Conclusions

We present much-needed recommendations for reliable determination of SARS-CoV-2 genome sequence and demonstrate their broad applicability in a large cohort of clinical samples.

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    SciScore for 10.1101/2020.12.01.405738: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Mapping to human transcriptome and genome were performed using STAR (ver 2.7) (41) and bwa mem (version 0.7.12-r1039) (42), respectively.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Read coverage was estimated using bedtools coverage from the bedtools suite (version v2.29.2) (43) for the portion of the viral genome targeted by the amplicons (positions 36-29,844 of the reference genome).
    bedtools
    suggested: (BEDTools, RRID:SCR_006646)
    Read subsampling was performed with samtools v1.9 (44).
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Statistical analysis: Statistical data analyses were performed using the R software environment for statistical computing (46) and graphics with the ggplot2 package (47).
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    The datasets generated with synthetic SARS-CoV-2 genome and analysed during the current study are available in the Sequence Read Archive (SRA) repository under accession number SUB8654793.
    Sequence Read Archive
    suggested: (DDBJ Sequence Read Archive, RRID:SCR_001370)

    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.

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  2. Version published to 10.1101/2020.12.01.405738 on bioRxiv