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Over-Representation Analysis Guide ​

This guide explains how to perform an Over-Representation Analysis (ORA) in Omics Studio and how to interpret the results. ORA helps you identify enriched biological pathways or processes in a list of genes, proteins, or metabolites. The workflow involves running a wizard to specify analysis criteria, choosing annotation databases and plot types, and then exploring the resulting data using tables and interactive plots.

Starting the ORA Wizard ​

  1. Open the study in Explorer. In the left navigation of Explorer, expand Enrichment Analysis and select Over-Representation Analysis. A description of ORA appears with a button labelled Start your ORA analysis.
  2. Click Start your ORA analysis. The wizard launches and walks you through three steps.

Step 1 – Define Analysis Criteria ​

In this step you specify which features (genes/proteins/metabolites) should be considered significant and select the comparisons to include. Key fields include:

  • Thresholds: Check one or more criteria to limit the feature list. Options include P-value ≤ 0.05, Adjusted P-value ≤ 0.05, and log2 fold-change thresholds (for up-regulated and down-regulated features).
  • Select your comparisons: Choose one or more statistical contrasts from your study (e.g., Group A vs Group B). The dropdown lists all comparisons and shows the associated organism and data type. After you pick a comparison, it appears as a tag under the field.
  • My Lists (optional): Select a predefined list (such as a gene list or custom set) to restrict analysis to specific identifiers.

When at least one threshold and one comparison are selected, the Continue button becomes active. Click Continue to proceed.

Step 2 – Choose Databases ​

The second step asks you to select one or more annotation databases. The available databases depend on your organism and data type. Examples include Reactome pathways, ChEBI metabolite pathways, Gene Ontology (GO) categories, and others.

Use the dropdown to pick appropriate databases; each choice appears as a tag. For example, Homo sapiens | Pathways | Reactome might be selected. Once at least one database is chosen, click Process. Omics Studio processes the data in the background.

Step 3 – Choose Your Plots ​

While processing, the wizard allows you to select one or more plot types. Options typically include Bar Chart and Dot Plot.

Click the icons to select your desired plots; the selected plots are highlighted. When processing is finished, a message appears stating Your data is ready and the Continue button becomes active. Click Continue to view your results.

Exploring ORA Results ​

The results page contains three tabs—Table, Bar Chart, and Dot Plot—and a Focus panel on the right. Each component offers interactive settings to refine and interpret your results.

Focus Panel ​

The Focus panel appears on the right side of all result tabs. It provides filters and options that affect what is displayed:

  • Comparison selection: A list of selected comparisons; you can check/uncheck to view a subset of comparisons.
  • Filters: Options include Top N (e.g., Top 15), P-value, Adjusted P-value, Gene Ratio, Count, and Include only up/down-regulated.
  • Current selection summary: Shows how many pathways/terms are currently displayed. An Exclude toggle removes selected terms.
  • Related identifiers: When you select a term in the table or chart, this section lists the identifiers contributing to that enrichment.

Table Tab ​

The Table tab shows a list of enriched terms with several columns: Group, ID, Description, Gene Ratio, and Background Ratio. You can:

  • Sort or filter columns: Click column headers to sort. Use Focus panel filters to limit results.
  • Select terms: Each row has a checkbox. Selecting terms highlights them across other plots and populates the Related identifiers section.
  • Navigate pages: Use page controls at the bottom to move between pages of results.

Bar Chart Tab ​

The Bar Chart tab displays enrichment results as horizontal bars. Longer bars correspond to higher gene ratios or counts. Features include:

  • Enrichment vs Cross-Group Comparisons: Two subtabs appear above the chart. Enrichment shows bars for a single comparison, while Cross-Group Comparisons compares multiple comparisons.
  • Height and search controls: Adjust how many bars are displayed. Zoom and search allow focusing on subsets of pathways.
  • Preview metrics: The Focus panel preview shows whether bars encode Gene Ratio or Count.
  • Selecting bars: Click a bar to select a term; it highlights across the table and dot plot and updates Related identifiers.

Dot Plot Tab ​

The Dot Plot tab shows a scatter-like plot where each dot represents an enriched term. Dot size and colour typically encode Count or Gene Ratio.

  • Enrichment vs Cross-Group Comparisons: Subtabs let you view single or cross-group enrichment.
  • Height and search controls: Adjust height, zoom, or search for pathways.
  • Selecting dots: Click a dot to select a term, highlight it across other views, and see associated identifiers.

Summary ​

By following this workflow you can perform an Over-Representation Analysis in Omics Studio:

  1. Launch the ORA wizard and set thresholds and comparisons.
  2. Choose annotation databases such as Reactome or GO categories.
  3. Pick the plot types to generate and wait for processing.
  4. Explore the results using the Table, Bar Chart, and Dot Plot tabs. Apply filters in the Focus panel, adjust chart settings, and select rows/bars/dots to see related identifiers.

This comprehensive interface allows you to identify enriched pathways, compare them across experimental conditions, and focus on the most significant biological processes.