There are several methods of functional enrichment. From the web tool PANTHER we will perform several approaches to know the functions in which our list of genes of interest are participating.

To functionally characterize a group of genes of interest associated with retinal dystrophies.

We have 73 genes in which we have detected variants in different subjects with retinal dystrophies. They are included in the file named dystrophies.

Open the data file “dystrophies.txt” with a notepad or similar and inspect its contents.

• Step 1. From the home of the tool go to the “Gene List Analysis” tab. Upload this txt file or copy the ids of the genes in the window indicated for it.
• Step 2. Select the organism. In this case “homo sapiens”.
• Step 3. Choose the type of analysis. Next we review all the available options:

• We will start with a description of the functions annotated (GO terms, signaling pathways) to these genes and for this we will use these two options: “Functional classification viewed in gene list” and “Functional classification viewed in graphics charts” which will provide us respectively the list and the graphical summary of functions associated to this group of genes.

• We need:
  1. After obtaining the functional classification list of these genes, we will save it in a file (“Send list to file”).
  2. From the option “Functional classification viewed in graphic charts” get a bar chart for each Gene Ontology ontology. The same information, please represent it from a “pie chart”.

• This method provides us with the functions that are overrepresented in our gene list versus the rest of the reference genome.
• The functional results will characterize the genes included in our list of interest.
• We would like to:
  1. Perform an analysis using the PANTHER GO-Slim Biological Processes. The results we obtain will be visualized in a “multiple pie chart”. Are there functional differences between the reference genome and our set of genes?
  2. Repeat the analysis but this time we will use all Biological Processes. Comment on the results.

1. What do you think about the functions detected in the functional description? General or specific?
2. When you perform the overrepresentation analysis, how are the functions with respect to the previous functional description? Do they inform us more specifically about the functional activity of these genes?

• ClusterProfiler package implements methods to analyze and visualize functional profiles GO, KEGG, DisGeNET, Reactome…) of gene and gene clusters.
• We are going to work the same previous exercises, but from R using clusterProfiler package. Are you ready? This script + data could help us.
• More detailed information: Annotation and visualization, clusterProfiler-book