SARS-CoV-2 Testing in the Community: Testing Positive Samples with the TaqMan SARS-CoV-2 Mutation Panel To Find Variants in Real Time

  1. Fiona Ashford
  2. Angus Best
  3. Steven J. Dunn
  4. Zahra Ahmed
  5. Henna Siddiqui
  6. Jordan Melville
  7. Samuel Wilkinson
  8. Jeremy Mirza
  9. Nicola Cumley
  10. Joanne Stockton
  11. Jack Ferguson
  12. Lucy Wheatley
  13. Elizabeth Ratcliffe
  14. Anna Casey
  15. Tim Plant
  16. The COVID-19 Genomics UK (COG-UK) Consortium, Aggarwal Dinesh University of Cambridge, Department of Medicine, Cambridge, UK, Blane Beth University of Cambridge, Department of Medicine, Cambridge, UK, Brooks Ellena University of Cambridge, Department of Medicine, Cambridge, UK, Carabelli Alessandro M. University of Cambridge, Department of Medicine, Cambridge, UK, Churcher Carol M. University of Cambridge, Department of Medicine, Cambridge, UK, Galai Katerina University of Cambridge, Department of Medicine
  17. Joshua Quick
  18. Alex Richter
  19. Nicholas Loman
  20. Alan McNally

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Abstract

Genome sequencing is a powerful tool for identifying SARS-CoV-2 variant lineages; however, there can be limitations due to sequence dropout when used to identify specific key mutations. Recently, ThermoFisher Scientific has developed genotyping assays to help bridge the gap between testing capacity and sequencing capability to generate real-time genotyping results based on specific variants.

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  1. Version published to 10.1128/jcm.02408-21
  2. ScreenIT

    SciScore for 10.1101/2021.11.17.21266297: (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.
    RandomizationFrom our pool of positive samples 185 were randomly selected for the mutation panel assay following the assay workflow (Figure 1).
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    RNA extraction: RNA was extracted from 200 µl of patient sample using the Thermo Fisher MagMax Viral/Pathogen II Nucleic Acid isolation kits with MagMax magnetic beads and MS-2 phage internal control, using the automated Thermo Fisher Kingfisher Flex Magnetic Particle Processor (11).
    Thermo Fisher MagMax
    suggested: None
    Thermo Fisher Kingfisher
    suggested: None
    Designing a Genotyping Panel for the Mutation Assay: TaqMan probes specific to SNPs found in variants known to be circulating widely within the United Kingdom around March-May 2021 were used in this study.
    Mutation Assay
    suggested: None

    Results from OddPub: Thank you for sharing your code and data.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    However, previous studies have discussed there are limitations due to sequence drop-out when used to identify specific key mutations (18, 19, 20, 21). Nanopore sequenced long-reads can be susceptible to errors where identifying specific mutations may be problematic. These allelic drop-outs potentially affect PCR-based (tile amplicon) targeted sequencing, thus resulting in incomplete genome coverage, especially at lower amounts, resulting in the loss of both 5’ and 3’ regions that fall outside primer binding positions. The presence of SNPs in the forward and / or reverse primer binding sites may lead to complete or partial lack of amplification. For the study presented here, we demonstrated that genotyping has two major functions. 1) Genotyping is a powerful additional, more in-depth assay for identifying specific mutations and has real clinical health value allowing laboratories to report and action VOC much quicker than genome sequencing. 2) Genotyping is an excellent additional complement to the already powerful tool of genome sequencing already proven for assigning lineages via phylogenetic trees. Our data confirms that SARS-CoV-2 genotyping is essential for real-time identification of VOC here now and tracking those that emerge for informing public health strategy.

    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.

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

    About SciScore

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  3. Version published to 10.1101/2021.11.17.21266297v1 on medRxiv