HiSpike Method for High-Throughput Cost Effective Sequencing of the SARS-CoV-2 Spike Gene

  1. Ephraim Fass
  2. Gal Zizelski Valenci
  3. Mor Rubinstein
  4. Paul J. Freidlin
  5. Shira Rosencwaig
  6. Inna Kutikov
  7. Robert Werner
  8. Nofar Ben-Tovim
  9. Efrat Bucris
  10. Oran Erster
  11. Neta S. Zuckerman
  12. Orna Mor
  13. Ella Mendelson
  14. Zeev Dveyrin
  15. Efrat Rorman
  16. Israel Nissan

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Abstract

The changing nature of the SARS-CoV-2 pandemic poses unprecedented challenges to the world's health systems. Emerging spike gene variants jeopardize global efforts to produce immunity and reduce morbidity and mortality. These challenges require effective real-time genomic surveillance solutions that the medical community can quickly adopt. The SARS-CoV-2 spike protein mediates host receptor recognition and entry into the cell and is susceptible to generation of variants with increased transmissibility and pathogenicity. The spike protein is the primary target of neutralizing antibodies in COVID-19 patients and the most common antigen for induction of effective vaccine immunity. Tight monitoring of spike protein gene variants is key to mitigating COVID-19 spread and generation of vaccine escape mutants. Currently, SARS-CoV-2 sequencing methods are labor intensive and expensive. When sequence demands are high sequencing resources are quickly exhausted. Consequently, most SARS-CoV-2 strains are sequenced in only a few developed countries and rarely in developing regions. This poses the risk that undetected, dangerous variants will emerge. In this work, we present HiSpike, a method for high-throughput cost effective targeted next generation sequencing of the spike gene. This simple three-step method can be completed in < 30 h, can sequence 10-fold more samples compared to conventional methods and at a fraction of their cost. HiSpike has been validated in Israel, and has identified multiple spike variants from real-time field samples including Alpha, Beta, Delta and the emerging Omicron variants. HiSpike provides affordable sequencing options to help laboratories conserve resources for widespread high-throughput, near real-time monitoring of spike gene variants.

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  1. Version published to 10.3389/fmed.2021.798130
  2. Version published to 10.21203/rs.3.rs-333890/v1 on Research Square
  3. ScreenIT

    SciScore for 10.1101/2021.03.02.21252290: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Point coverage depth was determined with samtools depth with base quality threshold of 15 and mapping quality threshold of 15.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)

    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:
    Nevertheless, HiSpike does have some limitations, chief among them being that it does not sequence genomic regions outside the spike gene, which are covered by other protocols for whole genome sequencing [33, 35-37]. Rarely, mutations outside the spike gene can influence transmissibility. However, the most important aspect of this limitation is that molecular epidemiology including its uses for clade definition and outbreak profiling to aid contact tracing, is mostly effective at the whole genome level. To the best of our knowledge, HiSpike is currently the only documented sequencing tool for specific monitoring of spike mutations. To ease transitions to the HiSpike method, we included a detailed user instruction manual (Supplementary file S2). We propose using HiSpike for routine sequencing of positive SARS-CoV-2 samples for near real-time monitoring of emerging spike mutations. In this manner, public health resources can help contain potential super spreading or vaccine-escape variants, even before these variants cause frequency shifts at the local, regional, or global levels.

    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 10.1101/2021.03.02.21252290 on medRxiv