RNA-seq analysis pipeline, featureCounts input, edgeR, functional enrichment, visualization

• Analysis_STAR.Rmd - RNA-seq analysis pipeline for STAR counts. Prerequisites:

  • A path to data folder. This folder should have 3 subfolders:
    • 02_STAR-align - gzipped count files with .tab extension outputted by STAR aligner
    • results - folder where the results will be stored
    • data - Must have sample_annotation.csv file, example below

• enrichR_analysis.Rmd - Analyze gene lists using enrichR. Analyze all genes, and up- and downregulated genes separately. Uses DEGs.xlsx produced by Analysis*.Rmd.

• enrichR_plot.Rmd - barplot of selected enrichment results, similar to Example. WIP

• GSEA.Rmd - GSEA analysis using MSigDb.

• Pathview.Rmd - visualization of top KEGG pathways. Uses DEGs.xlsx produced by Analysis*.Rmd. Example

Outdated scripts

• Analysis_featurecounts.Rmd - RNA-seq analysis pipeline for featureCount counts. Prerequisites:
  • A path to data folder. This folder should have 3 subfolders:
    • 03_featureCount - gzipped count files outputted by featureCount
    • results - folder where the results will be stored
    • data - Must have sample_annotation.csv file. Annotation file should have "Sample" column with sample names, and any other annotation columns. Include "Group" column containing covariate of interest. Example:

# Sample,Group
VLI10_AA_S61_L006_R1_001.txt.gz,AA
VLI10_AA_S61_L007_R1_001.txt.gz,AA
VLI10_AA_S61_L008_R1_001.txt.gz,AA
VLI11_C_S62_L006_R1_001.txt.gz,C
VLI11_C_S62_L007_R1_001.txt.gz,C
VLI11_C_S62_L008_R1_001.txt.gz,C

• Figure_clusterProfiler_nes.Rmd - Takes the results of edgeR analysis from an Excel file, performs GO and KEGG GSEA and plots the results as horizontal barplots, sorted by normalized enrichment score (NES). Example

• Figure_clusterProfiler_asis.Rmd - Takes the results of edgeR analysis from an Excel file, performs GO and KEGG GSEA and plots the results as horizontal barplots, sorted by p-value, as they come out of the enrichment analysis.

• Figure_heatmap.Rmd - make heatmap for selected genes. Uses TMP.xlsx produced by Analysis*.Rmd and a custom signature of gene names

Supplemental scripts

• calcTPM.R - a function to calculate TPMs from gene counts

• utils.R - helper functions

scripts folder

Scripts for running RNA-seq preprocessing steps on a cluster using PBS job submission system. subread-featurecounts scripts are in the dcaf/ngs.rna-seq repository

• submit00_fastqc.sh - FASTQC on raw FASTQ files
• MultiQC commands to summarize QC reports generated by TrimGalore and STAR

multiqc --filename multiqc_01_trimmed.html --outdir multiqc_01_trimmed 01_trimmed/
multiqc --filename multiqc_02_STAR-align.html --outdir multiqc_02_STAR-align 02_STAR-align/

• submit01_trimgalore.sh - Adapter trimming using TrimGalore
• submit02_STAR-index.sh - Index the genome for the STAR aligner
• submit02_STAR.sh - Align samples using STAR. Requires input01_toStarAlign.list text file with the list of input files, each string contains (comma-separated) file name(s), space separates first and second read pairs

CaSpER pipeline detecting CNVs from RNA-seq data

Dedicated repository with detailed instructions: mdozmorov/CaSpER_pipeline

• submit05_BAFExtract-index.sh - indexing the genome for BAFExtract
• submit05_BAFExtract.sh - BAFExtract run

Misc

• DESeq results to pathways in 60 Seconds with the fgsea package, https://stephenturner.github.io/deseq-to-fgsea/