Differential expression analysis and Report page overview
RNA-seq differential expression analysis using DESeq2 starts with the gene count matrix files produced by our RNA-seq Expression Count alignment and QC pipelinecalls differentially expressed genes between conditions, performs PCA analysis and pairwise correlations of the input samples, as well as gene set enrichment analyses, and an embedded genome browser to directly view all samples used in the analysis.
Results
Under the "Report" tab, a series of interactive and downloadable plots allow for in-depth data exploration
Volcano plot and Heatmap
-interactive volcano plot allowing adjustment of the p-value and fold-change threshold, downloadable gene lists using these custom thresholds, and annotation of specific genes of interest
Genome Browser
-under the "Genome Browser" tab, an interactive, embedded IGV browser session displaying the normalized signal bigwig tracks of all samples used in the analysis
*multiple genomic loci can be visualized by specifying them with spaces in-between:
chr15:61,977,809-61,997,892 chr13:5,848,131-5,883,750 chr8:72,315,172-72,325,543
Diff expr genes
-an interactive and downloadable table containing the differential expression results
PCA
-PCA results of the samples included in the analysis
Correlation Plot
-pairwise correlation heatmap of all samples used in the analysis
GSEA GO
-gene set enrichment results of gene ontology (GO) terms
GSEA Pathway
-gene set enrichment results of Reactome pathways
Here is a video overview of running our RNA-Seq Alignment/QC (STAR) and
DESeq2 Differential Expression Analysis pipelines with a few clicks
Output Files
deseq
deseq/<ANALYSIS_NAME>.cls
-class file for downstream GSEA analyses
deseq/corr_plot.png
-pairwise correlation heatmap
deseq/<ANALYSIS_NAME>.diffexpr.w_symbols.txt
-differential expression analysis results table
deseq/<ANALYSIS_NAME>.norm.gct
-normalized gene count file; can be used in further downstream GSEA analyses
deseq/<ANALYSIS_NAME>.pca-loadings.txt
-computed PCA loadings
deseq/<ANALYSIS_NAME>.pca-rotation.txt
-transformed gene count data for PCA analysis
deseq/<ANALYSIS_NAME>.vst.txt
-normalized gene count data using a variance-stabilized transformation
Create Lists
lists/Diffexpr.min_count_10.pval_0.05.all_ids.txt
-list of all gene ID's use in the analysis
lists/Diffexpr.min_count_10.pval_0.05.down.txt
-table of significantly down-regulated genes and their results from DESeq2
lists/Diffexpr.min_count_10.pval_0.05.up.txt
-table of significantly up-regulated genes and their results from DESeq2