Multiplex SARS-CoV-2 Genotyping Reverse Transcriptase PCR for Population-Level Variant Screening and Epidemiologic Surveillance

  1. Hannah Wang
  2. Jacob A. Miller
  3. Michelle Verghese
  4. Mamdouh Sibai
  5. Daniel Solis
  6. Kenji O. Mfuh
  7. Becky Jiang
  8. Naomi Iwai
  9. Marilyn Mar
  10. ChunHong Huang
  11. Fumiko Yamamoto
  12. Malaya K. Sahoo
  13. James Zehnder
  14. Benjamin A. Pinsky

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Abstract

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with concerning phenotypic mutations is of public health interest. Genomic surveillance is an important tool for a pandemic response, but many laboratories do not have the resources to support population-level sequencing.

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  1. Version published to 10.1128/jcm.00859-21
  2. Version published to 10.1101/2021.04.20.21255480v2 on medRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.04.20.21255480: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: This study was conducted with Stanford institutional review board approval (protocol 48973), and individual consent was waived.
    Consent: This study was conducted with Stanford institutional review board approval (protocol 48973), and individual consent was waived.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationAuthentication: controls, oligonucleotides, cycling conditions, fluorescence thresholds, analytical validation, and assay interpretation for reporting are provided in the Supplemental Methods, Tables S1-S6, and Fig. S1.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    We therefore designed a one-step multiplex allele-specific reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) to detect these three non-synonymous single nucleotide variants in the spike gene (N501Y: RefSeq NC_045512 23063A>T; E484K: 23012G>A, L452R: 22917 T>G).
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    All tests were conducted at the Stanford Clinical Virology Laboratory, which serves tertiary-care academic hospitals and affiliated outpatient facilities in the San Francisco Bay Area.
    Stanford Clinical Virology Laboratory
    suggested: None
    We adapted an existing WGS pipeline for poliovirus genotyping to conduct SARS-CoV-2 whole-genome amplicon-based sequencing (Supplemental Methods).
    WGS
    suggested: None

    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: We detected the following sentences addressing limitations in the study:
    Limitations of this assay, as described by the authors, are N1-FAM autofluorescence leading to false B.1.1.7 drop-out and absence of the ORF1ab 3675-3677 deletion in select B.1.351 isolates. While these deletions are surrogates for B.1.1.7, B.1.351, and P.1, screening for N501Y and E484K may be more specific to variants of concern/interest and appear to be causally-associated with concerning phenotypes. Another genotyping NAAT utilized a two-step approach combining the CDC-based laboratory-developed RT-qPCR and the ThermoFisher TaqPath COVID-19 RT-PCR.(36) With the intent to identify B.1.1.7 variants, the authors screened 1,035 samples for TaqPath S gene dropout and genotyped two specimens by multiplex droplet digital PCR amplifying HV69-70del, N501Y, del145, and S982A in two reactions, which were positive for these four mutations. Next-generation sequencing confirmed B.1.1.7 lineage. Limitations of this genotyping approach include dependence upon S gene dropout followed by more labor-intensive ddPCR. The largest NAAT-based genotyping series thus far was a national effort in France recently reported by Haim-Boukobza et al.(37) The authors used two separate assays to screen for the HVdel69-70 and N501Y mutations in 35,208 samples, with an uninterpretable assay rate of 19.0% and sequencing concordance Kappa coefficient of 0.87-0.88. To our knowledge, the present study is the largest NAAT-based SARS-CoV-2 genotyping initiative in the United States. While large multi-institutiona...

    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.

  4. Version published to 10.1101/2021.04.20.21255480v1 on medRxiv