Cell-free DNA tissues of origin by methylation profiling reveals significant cell, tissue, and organ-specific injury related to COVID-19 severity

  1. Alexandre Pellan Cheng
  2. Matthew Pellan Cheng
  3. Wei Gu
  4. Joan Sesing Lenz
  5. Elaine Hsu
  6. Erwin Schurr
  7. Guillaume Bourque
  8. Mathieu Bourgey
  9. Jerome Ritz
  10. Francisco M. Marty
  11. Charles Y. Chiu
  12. Donald C. Vinh
  13. Iwijn De Vlaminck

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Abstract

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  1. Version published to 10.1016/j.medj.2021.01.001
  2. ScreenIT

    SciScore for 10.1101/2020.07.27.20163188: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: High frequency sampling: Clinical samples from UCSF were processed through protocols approved by the UCSF Institutional Review Board (protocol number 10-00476, 18-25287).
    RandomizationRandomized clinical trial: Individuals diagnosed with COVID-19 were recruited to a randomized, controlled clinical trial at the McGill University Health Center, where they received either Lopinavir/ritonavir, or standard-of-care (https://clinicaltrials.gov/ct2/show/NCT04330690).
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Following treatment with bisulfite, whole-genome sequencing (WGS) libraries were prepared according to manufacturer’s protocols (Zymo EZ Methylation-Gold kit, #D5005 and Swift Biosciences Accel-NGS Methyl-Seq DNA Library Kit #30024) using a dual indexing barcode strategy (Swift biosciences #38096
    WGS
    suggested: None
    Swift Biosciences
    suggested: None
    Human genome alignment: Adapter sequences were trimmed using BBDUK (BBTools software suite [47])
    BBTools
    suggested: (Bestus Bioinformaticus Tools, RRID:SCR_016968)
    Resulting sequences were aligned to the human genome (version hg19) and deduplicated using Bismark [48]
    Bismark
    suggested: (Bismark, RRID:SCR_005604)
    Alignment files were filtered with a minimum mapping quality of 10 using SAMtools [49]
    SAMtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    Bisulfite conversion efficiency: The bisulfite conversion efficiency achieved in experiments was estimated using MethPipe [51] by calculating the reported methylation density of cytosines present at C[A/T/G] dinucleotides, which are rarely methylated in mammalian genomes.
    MethPipe
    suggested: (MethPipe, RRID:SCR_005168)

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

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    This study has several limitations. First, we assayed samples from only hospitalized patients, and we have not evaluated cfDNA profiles for mild COVID-19 cases. Second, while this study spans two independent cohorts, with patient groups that are genetically and geographically unrelated, the overall sample size and patient numbers may not be sufficient to generalize our findings to the entire spectrum of COVID-19 cases. Nonetheless, our analysis of cfDNA tissues of origin can provide immediate insights into the dynamics and pathogenesis of COVID-19. Last, the resolution of our measurements is limited by the availability of isolated cells and tissue methylation patterns. Our current reference dataset does not include all known human cell types and tissue types. Therefore, we are not sensitive to those rarer tissues that may play a role in the pathogenesis of COVID-19. More comprehensive investigations are therefore needed to confirm and further refine the observations reported here. In summary, we report the application of cfDNA profiling to quantify cellular and tissue specific injury due to COVID-19.

    Results from TrialIdentifier: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04330690RecruitingTreatments for COVID-19: Canadian Arm of the SOLIDARITY Tria…

    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.

  3. ScreenIT

    SciScore for 10.1101/2020.07.27.20163188: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementClinical samples from UCSF were processed through protocols approved by the UCSF Institutional Review Board (protocol number 10-00476, 18-25287).RandomizationRandomized clinical trial cohort To test the robustness of these initial observations, we assayed an additional 52 samples collected from 28 patients that were recruited into a randomized control trial at the McGill University Health Centre in Montreal, Canada.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableIncreased erythroid turnover may be due to erythroid destruction as the primary driver, followed by compensatory production, and is supported by anemia (Hgb <13.5 g/dL for men and Hgb < 12 for women) found in 26 of 33 COVID-19 patients across both studies.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    Following treatment with bisulfite, whole-genome sequencing (WGS) libraries were prepared according to manufacturer’s protocols (Zymo EZ Methylation-Gold kit, #D5005 and Swift Biosciences AccelNGS Methyl-Seq DNA Library Kit #30024) using a dual indexing barcode strategy (Swift biosciences #38096, NEBNext Multiplex Oligos for Illumina E7500L, or custom primers).
    WGS
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>Swift Biosciences</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Adapter sequences were trimmed using BBDUK (BBTools software suite [47]).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BBTools</b></div>
        <div>suggested: (Bestus Bioinformaticus Tools, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016968">SCR_016968</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Alignment files were filtered with a minimum mapping quality of 10 using SAMtools [49].</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SAMtools</b></div>
        <div>suggested: (Samtools, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002105">SCR_002105</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">cfDNA methylation densities were extracted using Bismark [48] and averaged over the DMRs.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Bismark</b></div>
        <div>suggested: (Bismark, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005604">SCR_005604</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The bisulfite conversion efficiency achieved in experiments was estimated using MethPipe [51] by calculating the reported methylation density of cytosines present at C[A/T/G] dinucleotides, which are rarely methylated in mammalian genomes.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MethPipe</b></div>
        <div>suggested: (MethPipe, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005168">SCR_005168</a>)</div>
      </div>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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 is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  4. Version published to 10.1101/2020.07.27.20163188v1 on medRxiv