Identification and quantification of SARS-CoV-2 leader subgenomic mRNA gene junctions in nasopharyngeal samples shows phasic transcription in animal models of COVID-19 and dysregulation at later time points that can also be identified in humans

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   ScreenIT

   Mar 15, 2021

   SciScore for 10.1101/2021.03.03.433753: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   The LeTRS was also tested with a combined Nanopore cDNA ARTIC v3 amplicon dataset of 7 published viral cell culture samples (barcode01-barcode07) 16, and a dataset from a published direct RNA Nanopore sequencing analysis Vero cells infected with SARS-CoV-2 or an uninfected negative control 2.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Software and Algorithms
   Sentences	Resources
   The generated raw FastQ files (2 x 250 bp) were …

   SciScore for 10.1101/2021.03.03.433753: (What is this?)

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

   Table 1: Rigor

   Institutional Review Board Statement	not detected.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Sex as a biological variable	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Experimental Models: Cell Lines
   Sentences	Resources
   The LeTRS was also tested with a combined Nanopore cDNA ARTIC v3 amplicon dataset of 7 published viral cell culture samples (barcode01-barcode07) 16, and a dataset from a published direct RNA Nanopore sequencing analysis Vero cells infected with SARS-CoV-2 or an uninfected negative control 2.	Vero suggested: CLS Cat# 605372/p622_VERO, RRID:CVCL_0059)
   Software and Algorithms
   Sentences	Resources
   The generated raw FastQ files (2 x 250 bp) were trimmed to remove Illumina adapter sequences using Cutadapt v1.2.1 26.	Cutadapt suggested: (cutadapt, RRID:SCR_011841)
   The reads were further trimmed to remove low quality bases, using Sickle v1.200 27 with a minimum window quality score of 20.	Sickle suggested: (Sickle, RRID:SCR_006800)
   The LeTRS was also tested with a combined Nanopore cDNA ARTIC v3 amplicon dataset of 7 published viral cell culture samples (barcode01-barcode07) 16, and a dataset from a published direct RNA Nanopore sequencing analysis Vero cells infected with SARS-CoV-2 or an uninfected negative control 2.	SARS-CoV-2 suggested: (Active Motif Cat# 91351, RRID:AB_2847848)
   Sequencing data alignment and basic filtering: LeTRS controlled Hisat2 v2.1.0 28 to map the paired-end Illumina reads against the SARS-CoV-2 reference genome (NC_045512.2) with the default setting, and Minimap2 v2.1 29 to align the Nanopore cDNA reads and direct RNA-seq reads on the viral genome using Minimap2 with “–ax splice” and “-ax splice -uf -k14” parameters, respectively.	Minimap2 suggested: (Minimap2, RRID:SCR_018550)
   LeTRS provided 10 known leader-TRS junctions to improve alignment accuracy by using “--known-splicesite-infile” function in Hisat2 and “--junc-bed” function in Minimap2, but this application could be optionally switched off by users.	Hisat2 suggested: (HISAT2, RRID:SCR_015530)
   In order to remove low mapping quality and mis-mapped reads before searching the leader-TRS junction sites, LeTRS used Samtools v1.9 30 to have basic filtering for the reads in the output Sam/Bam files according to their alignment states as shown (Table 9 - basic filtering).	Samtools suggested: (SAMTOOLS, RRID:SCR_002105)
   Leader-TRS junction plotting: LeTRS-plot was developed as an automatic plotting tool that interfaces with the R package ggplot2 v3.3.3 to view the leader-TRS junctions in the tables generated by LeTRS (Figure 3-5).	ggplot2 suggested: (ggplot2, RRID:SCR_014601)

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   Results from OddPub: Thank you for sharing your code.

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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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

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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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   Version published to 10.1101/2021.03.03.433753 on bioRxiv

   Mar 3, 2021