There are 3 parts - tabs in the Navigation Bar:
Single gene look-up
Selecting a gene by symbol or Ensembl ID generates (top) a bar plot of TPMs in control (NBH) 10 weeks timepoint samples, and (bottom) a bar plot of fold enrichment between RML and control, for each cell type, for both timepoints. You can toggle between invidual (free) scaling and fixed scaling for y-axis.
The dot plot shows each individual gene as a single point. Data is separated into cell types and timepoints. Points can be selected by clicking or dragging a rectangular selection while holding left mouse button. Selected genes are highlighted, labeled, and listed in the table below the plot, which can be printed or exported in multiple formats using the widget above the table. 'Reset' button in the side panel de-selects all highlighted genes. Genes can also be de-selected by clicking and dragging (dragging across a mix of selected and not-selected points will invert the selection). The slider in the side panel restricts the displayed genes based on FDR threshold (BH).
Gene Set Enrichment Analysis
The analysis or gene set enrichement based on two tools (selected using a toogle widget in the side panel):
1. Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles (Subramanian et al., 2005).
2. clusterProfiler: an R Package for Comparing Biological Themes Among Gene Clusters (Guangchuang et al., 2012).
The main difference between these tools is that GSEA uses a ranked gene lists, and the analysis results are separated into gene sets regulated positively and negatively (a toogle is used to select this). The ranking is based on a formula:
rank = Log2FC ∗ (-log10FDR)
Cell type-specificity threshold - slider-selection of input genes, based on fold enrichment of RiboTag IPs compared to total brain samples; the slider value defines the quantile of genes used as input for the analysis (e.g., choosing 0.8 will use only top 20% of cell type-specific genes, etc.). To select all genes, set the silder to zero.
Q-value threshold - slider restricting the enrichment results on the basis of q-value (higher value will preserve only the results with more significant enrichement score).
LFC threshold - slider-selection of input genes based on their Log2FC (input genes FDR threshold (BH) is set to 0.1 for all data in this case).
In the main enrichment results plot (bottom of the page), enrichment results are displayed for each cell type and timepoint. Specific gene sets can be selected by mouse click, to explore the detailed information in the tab panel (top right) containing:
Volcano plot - use a slider to adjust the y-axis. Hovering mouse cursor on the points will display more details of the hovered genes. In case of GSEA analysis, all genes pertaining to the selected gene set are dispayed; in case of GO enrichement, only the genes fulfilling the input criteria (FDR, L2FC) are displayed.
GSEA enrichement plot (only in case of GSEA analysis)
Summary - summary of the data for the selected gene set.