SARS-CoV-2 Variants in Paraguay: Detection and Surveillance with an Economical and Scalable Molecular Protocol

  1. Magaly Martinez
  2. Phuong-Vi Nguyen
  3. Maxwell Su
  4. Fátima Cardozo
  5. Adriana Valenzuela
  6. Laura Franco
  7. María Eugenia Galeano
  8. Leticia Elizabeth Rojas
  9. Chyntia Carolina Díaz Acosta
  10. Jonás Fernández
  11. Joel Ortiz
  12. Florencia del Puerto
  13. Laura Mendoza
  14. Eva Nara
  15. Alejandra Rojas
  16. Jesse J. Waggoner

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Abstract

SARS-CoV-2 variant detection relies on resource-intensive whole-genome sequencing methods. We sought to develop a scalable protocol for variant detection and surveillance in Paraguay, pairing rRT-PCR for spike mutations with Nanopore sequencing. A total of 201 acute-phase nasopharyngeal samples were included. Samples were positive for the SARS-CoV-2 N2 target and tested with the Spike SNP assay to detect mutations associated with the following variants: alpha (501Y), beta/gamma (417variant/484K/501Y), delta (452R/478K), and lambda (452Q/490S). Spike SNP calls were confirmed using amplicon (Sanger) sequencing and whole-genome (Nanopore) sequencing on a subset of samples with confirmed variant lineages. Samples had a mean N2 Ct of 20.8 (SD 5.6); 198/201 samples (98.5%) tested positive in the Spike SNP assay. The most common genotype was 417variant/484K/501Y, detected in 102/198 samples (51.5%), which was consistent with the P.1 lineage (gamma variant) in Paraguay. No mutations (K417 only) were found in 64/198 (32.3%), and K417/484K was identified in 22/198 (11.1%), consistent with P.2 (zeta). Seven samples (3.5%) tested positive for 452R without 478K, and one sample with genotype K417/501Y was confirmed as B.1.1.7 (alpha). The results were confirmed using Sanger sequencing in 181/181 samples, and variant calls were consistent with Nanopore sequencing in 29/29 samples. The Spike SNP assay could improve population-level surveillance for mutations associated with SARS-CoV-2 variants and inform the judicious use of sequencing resources.

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  1. Version published to 10.3390/v14050873
  2. ScreenIT

    SciScore for 10.1101/2021.09.15.21263618: (What is this?)

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

    Table 1: Rigor

    EthicsIRB: This study was reviewed and approved by the IICS Scientific and Ethics Committee (P38/2020) and the Emory Institutional Review Board (study 00110736)
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Sequencing: All rRT-PCR products from the first Spike SNP reaction (348-base-pair amplicon) were stored at 4°C and later shipped to GeneWiz (South Plainfield, NJ) for Sanger sequencing.
    GeneWiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Amplicon sequence was considered to be high quality and included for analysis if it achieved a quality score ≥ 40 and had base calls for all probe targets in at least one direction.
    Amplicon
    suggested: (Amplicon, RRID:SCR_003294)
    The genomic libraries were prepared and then loaded on a R9.4.1 flow cell (ONT FLO-MIN106) for sequencing with MinION device (MIN-101B).
    MinION
    suggested: (MinION, RRID:SCR_017985)
    Basecalling, demultiplexing and trimming was carried out by Guppy toolkit integrated in the MinKNOW Nanopore software (Oxford Nanopore Technologies, UK)
    MinKNOW Nanopore
    suggested: None
    Genome assembly and consensus sequences were obtained with Nanopore EPI2ME (using Flye and Medaka respectively).
    Flye
    suggested: (Flye, RRID:SCR_017016)
    Statistics: Basic statistical analyses were performed using GraphPad Prism version 9.0.2 (GraphPad Software, San Diego, CA).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    A limitation to the current study is the utilization of retrospective convenience samples to evaluate the testing protocol. Although new data on SARS-CoV-2 variants in Paraguay were generated, we cannot calculate variant prevalence from these data. In conclusion, the Spike SNP assay provides accurate detection of mutations that are associated with VOCs/VOIs in Paraguay. This can be implemented in SARS-CoV-2 testing protocols to triage samples for whole genome sequencing in the MinION platform or direct amplicon sequencing, which provides additional information. The assay can be implemented in any laboratory performing rRT-PCR to improve surveillance for these mutations and inform the judicious use of scarce sequencing resources.

    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

    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.

  3. Version published to 10.1101/2021.09.15.21263618v1 on medRxiv