• Introduction: Quick Start, Tutorial, Flowchart, Outputs structure
• Install: Dependencies, Containers, References, Test datasets
• Inputs: Data, Design, Parameters
• 1. Preprocessing: ATAC reads, ATAC peaks, mRNA
• 2. Differential Analysis: ATAC, mRNA, Split
• 3. Enrichment Analysis: Enrichment, Figures, Tables

List of processes

• DA_mRNA__doing_differential_analysis
• DA_mRNA__plotting_differential_analysis_results
• DA_mRNA__saving_detailed_results_tables

DA_mRNA__doing_differential_analysis

Description

Differential gene expression analysis is carried out with sleuth.

Outputs

• Sleuth R object: 1_Preprocessing/2_Differential_Analysis/mRNA__all_genes__rsleuth/${comparison}__mRNA_DEG_rsleuth.rds
• Promoter of all detected genes: 1_Preprocessing/2_Differential_Analysis/mRNA__all_genes__bed_promoters/${comparison}__all_genes_prom.rds

DA_mRNA__plotting_differential_analysis_results

Description

This process makes standardized (i.e. similar types of plots are produced for mRNA-Seq data) PCA and volcano plots, and some other plots produced directly by sleuth.

Parameters

• params.sleuth_plots__fdr_threshold: Peaks with FDR less than or equal to this value are colored in red in the volcano plot. Default: 0.05.
• params.sleuth_plots__top_n_labels: The top n peaks with lowest FDR will have their annotated gene displayed on the volcano plot. Default: 15.

Outputs

• Volcano plots:

  • Figures_Individual/2_Differential_Analysis/mRNA__volcano/${comparison}__mRNA_volcano.pdf
  • Figures_Merged/2_Differential_Analysis/mRNA__volcano.pdf.

• PCA plots (PC 1 and 2):

  • Figures_Individual/2_Differential_Analysis/mRNA__PCA_1_2/${comparison}__mRNA_PCA_1_2.pdf.
  • Figures_Merged/2_Differential_Analysis/mRNA__PCA_1_2.pdf.
    • top left panel: percentage of variance explained by the top 5 first principal components
    • top right panel: PCA plot for principal components 1 and 2
    • bottom panels: genes annotated to peaks that contribute the most to principal components 1 (left) and 2 (right). Color code: red or -1 indicates that the peak is a positive contributor. Blue or +1 indicates that the peak is a negative contributor.

• PCA plots (PC 3 and 4):

  • Figures_Individual/2_Differential_Analysis/mRNA__PCA_3_4/${comparison}__mRNA_PCA_3_4.pdf.
  • Figures_Merged/2_Differential_Analysis/mRNA__PCA_3_4.pdf.
    • Same as above but for principal components 3 and 4.

• Other plots;

  • Figures_Individual/2_Differential_Analysis/mRNA__other_plots/${comparison}__mRNA_other_plots.pdf
  • Figures_Individual/2_Differential_Analysis/mRNA__other_plots/${comparison}__mRNA_other_plots.pdf

    • MA plot: Make an 'MA plot' for a given test. MA plots display, for each transcript, the mean of abundances across samples on the x-axis and fold change on the y-axis.
      

    • Density plot: Plot the density of a grouping.
      

DA_mRNA__saving_detailed_results_tables

Description

The detailed ATAC-seq results table is created in R, which gene id, name, coordinates, significance, log2 fold change and other sleuth columns.

Outputs

• Tables_Individual/2_Differential_Analysis/mRNA_detailed/${comparison}__res_detailed_mRNA.{csv,xlsx}
• Tables_Merged/2_Differential_Analysis/mRNA_detailed.{csv,xlsx}.