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Gene Set Enrichment Analysis (GSEA) Guide ​

Gene Set Enrichment Analysis (GSEA) examines whether predefined sets of genes are statistically over-represented at the top or bottom of a ranked list. This method complements Over-Representation Analysis (ORA) by using continuous ranked expression values rather than discrete significance thresholds. The Omics Studio interface provides a guided workflow for performing GSEA and interpreting the results.

Starting a GSEA ​

  1. Open your study in Explorer. From the left navigation menu, select Enrichment Analysis (the percent-sign icon). A small pop-out menu appears—choose Gene Set Enrichment Analysis. The page displays a description of GSEA and a button labelled Start your GSEA Analysis.
  2. Click Start your GSEA Analysis to launch the wizard.

Step 1 – Define Analysis Criteria ​

This step determines which genes enter the analysis and which comparison(s) to evaluate:

  • Thresholds: Select at least one significance criterion. Options include P-value ≤ 0.05, Adjusted P-value ≤ 0.05, and fold-change cut-offs for up- or down-regulated genes. You can also specify log2 fold-change thresholds.
  • Select your comparisons: Choose one or more contrasts from your study (e.g., Group A vs Group B). Selected comparisons appear as tags beneath the field.
  • My Lists (optional): Restrict the analysis to genes from a predefined list.

Once at least one threshold and comparison are chosen, the Continue button becomes active.

Step 2 – Choose Databases ​

GSEA requires gene-set annotations. Choose one or more databases that match your organism and data type:

  • Click the Choose your database(s) dropdown to view available options.
  • Select a database; it appears as a tag beneath the dropdown. For example, Homo sapiens | Biological Process | Gene Ontology.
  • You can also select lists from My Lists to provide custom gene-set definitions.

After selecting at least one database, click Process. Omics Studio processes the ranked gene list in the background.

Step 3 – Choose Your Plots ​

While processing, select one or more visualization types to generate:

  • Bar Chart and Dot Plot are available. Click each card to select it; selected plots are highlighted.
  • A progress bar indicates processing status. When the message Your data is ready appears, the Continue button becomes active.
  • Click Continue to open the results.

Exploring GSEA Results ​

The results page presents three tabs—Bar Chart, Dot Plot, and Table—along with a Focus panel for filters.

Focus Panel (Filters and Selection) ​

The Focus panel provides controls that affect all result views:

  • Comparison selection: Displays selected comparisons; toggle them on/off to focus on subsets.
  • Filters: Options include Top 15 (lowest p-values), P-value, Adjusted P-value, Size (number of genes in a set), NES – Up-regulated, and NES – Down-regulated.
  • Include only: Specify a custom list of gene sets.
  • Current selection and Related identifiers: Show selected gene sets and the contributing genes.

Table Tab ​

The Table tab lists enriched gene sets with these columns:

  • Group – Comparison (e.g., Group A vs Group B)
  • ID – Identifier (e.g., GO term)
  • Description – Biological process or pathway name
  • NES – Normalized Enrichment Score (positive = up-regulated, negative = down-regulated)
  • ES – Raw enrichment score
  • p-value – Statistical significance
  • Size – Number of genes in the set

Use the Focus panel to filter, then select rows to examine contributing genes.

Bar Chart Tab ​

The Bar Chart displays gene sets as horizontal bars.

  • Positive vs negative enrichment: Right-extending bars (NES > 0) show up-regulated sets; left-extending bars (NES < 0) show down-regulated sets.
  • Height control: Adjust the chart height to show more or fewer gene sets.
  • Search and zoom: Locate gene sets or zoom into the chart.
  • Subtabs: Enrichment shows a single comparison, while Grouped Comparisons stacks multiple comparisons.
  • Interaction: Click a bar to select and highlight a gene set across all views.

Dot Plot Tab ​

The Dot Plot represents gene sets as points.

  • Axes: X-axis = NES, Y-axis = gene-set descriptions.
  • Colour and size: Encode NES direction and set size or significance.
  • Height control and search: Adjust view or find specific sets.
  • Subtabs: Enrichment shows a single comparison; Grouped Comparisons shows multiple.
  • Interaction: Click a dot to highlight the gene set in all views and inspect contributing genes.

Summary ​

To perform a Gene Set Enrichment Analysis in Omics Studio:

  1. Launch the GSEA wizard and set thresholds and comparisons.
  2. Select one or more gene-set databases (e.g., Gene Ontology, Reactome).
  3. Choose plots (bar chart and/or dot plot) and wait for processing.
  4. Explore results with the Table, Bar Chart, and Dot Plot tabs. Apply filters, adjust chart settings, and inspect contributing genes.

Following this workflow helps uncover biological processes and pathways that are coordinately up- or down-regulated in your dataset, providing deeper functional insights than unranked enrichment methods.