Making sense of gene and proteins lists with functional enrichment analysis

14 Reactome (web)

14.1

Reactome is an open-source database of curated biological pathways across species, offering pathway maps and enrichment tools to analyse gene lists in a pathway-focused context. It’s ideal for visualising data within established biochemical and cellular processes.

14.1.1 Steps to perform ORA in Reactome:

• Hit the Analysis Tools tab

- Choose Analyse gene list from the left panel

• Upload list of features on the box or choose a file, hit continue

• Select preferred options:

  - Project to Human: This option will convert identifiers from non-human species into human equivalents, allowing you to analyse data across species.

  - Include interactors: This option integrates interactors from IntAct, a protein interaction database. Including interactors broadens the background network, potentially offering deeper insights.

• Hit Analyse!

14.1.2 Steps to perform GSA in Reactome:

Note: GSA appears to be non-functional at reactome.org as of August 2026.

Skip to “Browse the Reactome Results” below.

• If Analyse gene expression was chosen instead, Reactome offers the following gene set analysis:

• Let’s try CAMERA as it represents the camera() function of limma package in R for a gene set analysis.

• Choose TMM normalisation to ensure consistency with the input data used in other tools within our workshop.

• Select data type and provide input data

• Annotate columns by adding extra info as follows:

• Save dataset and Continue

• You can now browse the results

Question

When running FEA in Reactome, how do you prefer the analysis methods?

- PADOG (Pathway Analysis with Down-weighting of Overlapping Genes)

- CAMERA (Correlation Adjusted Mean Rank)

- ssGSEA (Single Sample Gene Set Enrichment Analysis)

 

14.1.3 Browse the Reactome results

Results can be interactively browsed using the reactome pathway or voronoi visualisation modes:

User can explore the pathway names listed in the table within the Analysis tab and they are displayed as popups on the pathway diagrams.

One can also select a pathway of interest by navigating through the left panel or by simply searching for the term in the search box.

The enriched table can be downloaded as shown below:

The diagram can be downloaded using this icons:

Here is a sample pathway diagram from Reactome GSA.

In case ssGSEA was selected, an overall output would look like below: