Identification of driver genes for critical forms of COVID-19 in a deeply phenotyped young patient cohort

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1. Version published to 10.1126/scitranslmed.abj7521

   Jan 19, 2022
2. 

   ScreenIT

   Jul 1, 2021

   SciScore for 10.1101/2021.06.21.21257822: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Autoantibodies against type I IFNs: The blocking activity of anti-IFNα and anti-IFNω autoantibodies was determined by assessing a reporter luciferase activity as previously described (Bastard et al., 2021).	anti-IFNα suggested: (Elabscience Cat# E-EL-M3054, RRID:AB_2891274) anti-IFNω suggested: None
   The membranes were blocked for 1 h in 5% skimmed milk/TBS 0.05%/Tween 20 and then incubated with the anti-ADAM9 antibody (ab218242; Abcam, Cambridge, UK) for 2 h at 4°C in 5% BSA/TBS 0.1% Tween at 1/1000 dilution.	anti-ADAM9 suggested: None
   An anti-GAPDH antibody (MAB374, Merck Millipore, Burlington, MA, USA) …

   SciScore for 10.1101/2021.06.21.21257822: (What is this?)

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

   Table 1: Rigor

   NIH rigor criteria are not applicable to paper type.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Autoantibodies against type I IFNs: The blocking activity of anti-IFNα and anti-IFNω autoantibodies was determined by assessing a reporter luciferase activity as previously described (Bastard et al., 2021).	anti-IFNα suggested: (Elabscience Cat# E-EL-M3054, RRID:AB_2891274) anti-IFNω suggested: None
   The membranes were blocked for 1 h in 5% skimmed milk/TBS 0.05%/Tween 20 and then incubated with the anti-ADAM9 antibody (ab218242; Abcam, Cambridge, UK) for 2 h at 4°C in 5% BSA/TBS 0.1% Tween at 1/1000 dilution.	anti-ADAM9 suggested: None
   An anti-GAPDH antibody (MAB374, Merck Millipore, Burlington, MA, USA) was used for loading control.	anti-GAPDH suggested: None
   Flow cytometry staining: The cells were fixed for 20 min in 3.6% paraformaldehyde at 4°C, washed in 5% FCS in PBS and stained with anti-nucleocapsid antibody (GTX135357, Genetex, Irvine, CA, USA) at a 1/200 dilution in Perm/WashTM (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) for 45 min at room temperature.	anti-nucleocapsid suggested: (GeneTex Cat# GTX135357, RRID:AB_2868464) GTX135357 suggested: (GeneTex Cat# GTX135357, RRID:AB_2868464)
   The antibody was then revealed by incubation with an Alexa 647-labeled goat anti-rabbit monoclonal antibody (Ab150083, Abcam, Cambridge, UK) diluted 1/200 in 5% FCS in PBS for 45 min at room temperature.	anti-rabbit suggested: (Abcam Cat# ab150083, RRID:AB_2714032)
   Experimental Models: Cell Lines
   Sentences	Resources
   The three viral antigens included in our multiplex bead array comprise two representations of the Spike protein (a soluble trimeric form of the spike glycoprotein stabilized in the pre-fusion conformation expressed in HEK, and the Spike S1 domain expressed in CHO cells), and the C-terminal domain of the Nucleocapsid protein (expressed in E.coli).	CHO suggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)
   A monolayer of Vero cells seeded at 60000 cells/well in black-well plates was infected with the pseudotype virus (virus only) or virus and serum mixture overnight at 37°C - 5% CO2.	Vero suggested: None
   Briefly, HEK293T cells were transfected with the firefly luciferase plasmids under the control of human ISRE promoters in the pGL4.45 backbone, and a constitutively expressing Renilla luciferase plasmid for normalization (pRL-SV40).	HEK293T suggested: None
   Cell culture: Vero 76 cell lines were grown at 37 °C under 5% CO2 and maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 100 units/ml penicillin and supplemented with 10% fetal bovine serum (Pan Biotech, Aidenbach, Germany)	Vero 76 suggested: None
   ACE2-expressing A549 cells (A549-ACE2) were grown at 37 °C under 5% CO2 and maintained in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) containing 10 µg/ml of blasticidine S (Invitrogen, Carlsbad, CA, USA).	A549 suggested: None
   In vitro viral infections: Vero 76 and A549-ACE2 cell lines were infected with wild-type SARS-CoV-2 virus at Multiplicities Of Infections (MOIs) of 10 and 400, respectively.	A549-ACE2 suggested: None
   Recombinant DNA
   Sentences	Resources
   Briefly, HEK293T cells were transfected with the firefly luciferase plasmids under the control of human ISRE promoters in the pGL4.45 backbone, and a constitutively expressing Renilla luciferase plasmid for normalization (pRL-SV40).	pGL4.45 suggested: None Renilla luciferase suggested: RRID:Addgene_118016) pRL-SV40 suggested: None
   Software and Algorithms
   Sentences	Resources
   The mass cytometry standard files produced with the Helios instrument were analyzed using Maxpar® Pathsetter software v.	Maxpar® Pathsetter suggested: None
   The FCS files from each group (healthy, critical, non-critical) were then concatenated using CyTOF® software v.	CyTOF® suggested: (Fluidigm CyTOF, RRID:SCR_021055)
   7.0.8493.0 for viSNE analysis (Cytobank Inc, Mountain View, CA, USA).	Cytobank suggested: (Cytobank, RRID:SCR_014043)
   Differential protein expression analysis was performed using the limma bioconductor package in R (Ritchie et al., 2015).	limma suggested: (LIMMA, RRID:SCR_010943) bioconductor suggested: (Bioconductor, RRID:SCR_006442)
   Differential protein expression analysis: Summed peptides normalized label-free quantification (LFQ values from MaxQuant software) values were used for differential protein expression analysis.	MaxQuant suggested: (MaxQuant, RRID:SCR_014485)
   The following day, cells were lysed using passive lysis buffer (Promega, Madison, WI, USA) and luciferase activity was measured upon addition of substrate (Promega® CellTiterGlo®, Madison, WI, USA) using Biotek plate reader and Gen5 software.	Gen5 suggested: (Gen5, RRID:SCR_017317)
   To determine neutralizing titers, the concentration of the antibody resulting in 50% neutralization (NT50) was determined using XLFit and graphed using Prism (GraphPad, San Diego, CA, USA)	Prism suggested: (PRISM, RRID:SCR_005375) GraphPad suggested: (GraphPad Prism, RRID:SCR_002798)
   Whole-genome sequencing (WGS): WGS data was generated from DNA isolated from whole blood.	WGS suggested: None
   Functional annotation of the variants was performed using Variant Effect Predictor from Ensembl (version 101).	Variant Effect Predictor suggested: None Ensembl suggested: (Ensembl, RRID:SCR_002344)
   GATK version 4 (Van der Auwera et al., 2013; DePristo et al., 2011) was used for the joint genotyping process and variant quality score recalibration (VQSR).	GATK suggested: (GATK, RRID:SCR_001876)
   The raw sequencing data were aligned to a reference human genome build 38 (GRCh38) using the short reads aligner STAR (Dobin et al., 2013).	STAR suggested: (STAR, RRID:SCR_004463)
   Quantification of gene expression was performed using RSEM (Li and Dewey, 2011) with GENCODE annotation v25 (http://www.gencodegenes.org).	GENCODE suggested: (GENCODE, RRID:SCR_014966) http://www.gencodegenes.org suggested: (HapMap 3 and ENCODE 3, RRID:SCR_004563)
   We then performed DGE analysis on all 69 samples using the trimmed mean of M-values method (TMM) from the edgeR R package (Robinson and Oshlack, 2010; Robinson et al., 2010).	edgeR suggested: (edgeR, RRID:SCR_012802)
   We used the TensorFlow (Abadi et al., 2016) Python package to construct the DANNs.	Python suggested: (IPython, RRID:SCR_001658)
   The membrane was then incubated with the secondary antibody coupled to HRP (Bio-Rad Laboratories, Hercules, CA, USA)	Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

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   Results from OddPub: Thank you for sharing your data.

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   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.

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   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.
   • Thank you for including a protocol registration statement.

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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.06.21.21257822v1 on medRxiv

   Jun 25, 2021