Acute COVID-19 gene-expression profiles show multiple etiologies of long-term sequelae

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   ScreenIT

   Oct 8, 2021

   SciScore for 10.1101/2021.10.04.21264434: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: As the research laboratory processing needed to begin proximal to sample collection, a portion of the data was generated prior to obtaining informed consent.
   Sex as a biological variable	For each RNA-seq sample, we defined expressed sex based on the relative abundance of the sex-specific genes UTY (male) and Xist (female).
   Randomization	This is often controlled by constructing batches using a randomization procedure that balances key outcome variables (e.g., case/control status) across batches.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Next, a 1:3,000 dilution of goat anti-human IgG/A/M F(ab)– horseradish peroxidase conjugated …

   SciScore for 10.1101/2021.10.04.21264434: (What is this?)

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

   Table 1: Rigor

   Ethics	Consent: As the research laboratory processing needed to begin proximal to sample collection, a portion of the data was generated prior to obtaining informed consent.
   Sex as a biological variable	For each RNA-seq sample, we defined expressed sex based on the relative abundance of the sex-specific genes UTY (male) and Xist (female).
   Randomization	This is often controlled by constructing batches using a randomization procedure that balances key outcome variables (e.g., case/control status) across batches.
   Blinding	not detected.
   Power Analysis	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Next, a 1:3,000 dilution of goat anti-human IgG/A/M F(ab)– horseradish peroxidase conjugated secondary antibody was prepared in PBS and 100 μl of this secondary antibody was added to each well for 1 hour (SouthernBiotech #2020-04 Lot No. L4206-Q408B, #2050-04 Lot No. C5213-RI66P, #2040-04 Lot No. B3919-NE80C).	anti-human IgG/A/M suggested: None
   Lastly, we tested for differences in gene expression that are independent of the antibody response to the spike protein by fitting all models a second time with 3 additional coefficients controlling for the log2 titers of anti-spike-protein IgG, IgA, and IgM.	anti-spike-protein IgG suggested: None IgM suggested: None
   We controlled for multiple testing using Holm’s method for family-wise error rate control39 among all comparisons performed within a given symptom or cell type between DE signatures with at least 100 DEGs either before or after controlling for anti-spike antibody titers.	anti-spike suggested: None
   Software and Algorithms
   Sentences	Resources
   Given the monumental hurdles of consenting sick and infectious patients in isolation rooms, the Human Research Protection Program allowed for sample collection, which occurred at the time of clinical collection and included at most an extra 5-10 cc of blood, prior to obtaining research consent.	Human Research Protection Program suggested: None
   Blood used for Olink and enzyme-linked immunosorbent assays (ELISA) were collected in SST tubes (Becton Dickinson #367985) and blood used for whole genome sequencing (WGS) in CPT Vacutainer tubes (Becton Dickinson #362761).	WGS suggested: None
   All plates were then put into the KingFisher to start DNA extraction.	KingFisher suggested: (Hamilton NIMBUS Presto and ID NIMBUS Presto Assay Ready Workstation, RRID:SCR_019998)
   This configuration enabled 2×150 bp paired end reads into resulting FASTQ files in 2-5 days per batch that were sent through primary data quality control using MultiQC to assess read depth and quality metrics17.	MultiQC suggested: (MultiQC, RRID:SCR_014982)
   Quality-filtered raw data was converted into FASTQ files using bcl2fastq (Illumina).	bcl2fastq suggested: (bcl2fastq , RRID:SCR_015058)
   RNA-seq reads were aligned to the GRCh38 primary assembly18 with Gencode gene annotation v3019 by STAR (v2.7.3a)20 using per-sample 2-pass mapping (--twopassMode Basic) and chimeric alignment options (--chimOutType Junctions SeparateSAMold -chimSegmentMin 15 -chimJunctionOverhangMin 15).	Gencode suggested: (GENCODE, RRID:SCR_014966)
   MultiQC17 was used to compile and summarize per-sample statistics from STAR, Picard Tools and featureCounts (i.e. gene-level counts, mtRNA counts, globinRNA counts, etc.) into an interactive HTML report.	STAR suggested: (STAR, RRID:SCR_004463)
   We therefore excluded samples with DV200 below 80% as well as samples with fewer than 10 million mapped reads counted by featureCounts.	featureCounts suggested: (featureCounts, RRID:SCR_012919)
   Gene expression was normalized for composition bias using the trimmed mean of M-values method, implemented by calcNormFactors in the edgeR package25,26 and transformed to normalized log2 CPM with observation weights computed by voomWithDreamWeights from the variancePartition package27,28.	edgeR suggested: (edgeR, RRID:SCR_012802) variancePartition suggested: (variancePartition, RRID:SCR_019204)
   The last 3 fixed effects listed are sequencing quality metrics computed by Picard Tools: PCT_R2_TRANSCRIPT_STRAND_READS is “the fraction of reads that support the model where R2 is on the strand of transcription and R1 is on the opposite strand”; PCT_INTRONIC_BASES is the “fraction of PF_ALIGNED_BASES that correspond to gene introns”; and WIDTH_OF_95_PERCENT is difference between the 2.5 percentile and the 97.5 percentile of the insert size distribution.	Picard suggested: (Picard, RRID:SCR_006525)
   Gene Ontology term enrichment analyses for DE signatures: For each DE test, the downregulated and upregulated DEGs were separately tested for Gene Ontology (GO) term enrichment for all GO terms annotated to at least 10 expressed genes, using the Bioconductor packages goseq, topGO, and org.	topGO suggested: (topGO, RRID:SCR_014798)
   Study data were collected and managed using REDCap electronic data capture tools hosted by Scientific Computing at the Icahn School of Medicine at Mount Sinai48,49.	REDCap suggested: (REDCap, RRID:SCR_003445)
   Canonical correlations between all technical, clinical, and demographic variables were calculated using the canCorPairs function and visualized using the plotCorrMatrix function from the Bioconductor package variancePartition29,55.	Bioconductor suggested: (Bioconductor, RRID:SCR_006442)

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   Results from OddPub: Thank you for sharing your code and 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.
   • No protocol registration statement was detected.

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

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   Version published to 10.1101/2021.10.04.21264434v1 on medRxiv

   Oct 5, 2021