DNA spike-ins enable confident interpretation of SARS-CoV-2 genomic data from amplicon-based sequencing

  1. Kim A. Lagerborg
  2. Erica Normandin
  3. Matthew R. Bauer
  4. Gordon Adams
  5. Katherine Figueroa
  6. Christine Loreth
  7. Adrianne Gladden-Young
  8. Bennett Shaw
  9. Leah Pearlman
  10. Erica S. Shenoy
  11. David Hooper
  12. Virginia M. Pierce
  13. Kimon C. Zachary
  14. Daniel J. Park
  15. Bronwyn L. MacInnis
  16. Jacob E. Lemieux
  17. Pardis C. Sabeti
  18. Steven K Reilly
  19. Katherine J. Siddle

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Abstract

The rapid global spread and continued evolution of SARS-CoV-2 has highlighted an unprecedented need for viral genomic surveillance and clinical viral sequencing. Amplicon-based sequencing methods provide a sensitive, low-cost and rapid approach but suffer a high potential for contamination, which can undermine lab processes and results. This challenge will only increase with expanding global production of sequences by diverse research groups for epidemiological and clinical interpretation. We present an approach which uses synthetic DNA spike-ins (SDSIs) to track samples and detect inter-sample contamination through a sequencing workflow. Applying this approach to the ARTIC Consortium’s amplicon design, we define a series of best practices for Illumina-based sequencing and provide a detailed characterization of approaches to increase sensitivity for low-viral load samples incorporating the SDSIs. We demonstrate the utility and efficiency of the SDSI method amidst a real-time investigation of a suspected hospital cluster of SARS-CoV-2 cases.

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    SciScore for 10.1101/2021.03.16.435654: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Sample collection and study design: This study was approved by the Partners Institutional Review Board under protocol 2019P003305 and we obtained samples under a waiver of consent for viral sequencing.
    Consent: Sample collection and study design: This study was approved by the Partners Institutional Review Board under protocol 2019P003305 and we obtained samples under a waiver of consent for viral sequencing.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    When we performed the permissive BLAST search described above on the 140 bp core SDSI sequences and filtered results to homo sapiens and SARS-CoV-2 with >50 percent identity and >50 query cover, there were no significant hits.
    BLAST
    suggested: (BLASTX, RRID:SCR_001653)
    Metagenomic sequencing and comparison: Metagenomic sequencing data and genome assemblies used for the comparison of amplicon-based sequencing were previously prepared, sequenced, analyzed as described previously,1 and the data are publicly available at NCBI’s GenBank and SRA databases under BioProject PRJNA622837.
    BioProject
    suggested: (NCBI BioProject, RRID:SCR_004801)
    Data has been made available in both the Short Read Archive and NCBI GenBank under Bioproject PRJNA622837.
    Short Read Archive
    suggested: None
    We used iVar version 1.2.1 for primer trimming on all samples followed by assembly with minimap2 set to a minimum coverage of either 3, 10, or 20, skipping deduplication procedures.
    iVar
    suggested: None
    We constructed a maximum likelihood tree using iqtree with a GTR substitution model and edited and interpreted the tree in Figtree v1.4.4.
    Figtree
    suggested: (FigTree, RRID:SCR_008515)

    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.
    • 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.

  2. Version published to 10.1101/2021.03.16.435654v1 on bioRxiv