43.2.1.1 Step 1

Open http://biit.cs.ut.ee/gprofiler/, paste the gene list, and select Bos taurus as the organism (species of interest) and the output type as Excel spreadsheet (Figures 43.4 and 43.5)

43.2.1.2 Step 2

Download the Excel file to check for the annotations enriched in the differentially expressed genes (see Figure 43.6):

The output shows the significance of terms and the genes associated with the query (Q) in the term (T). The first term – response to abiotic stimulus (Biological process – BP (t type)) has a term ID of GO:0009628, with a p‐value of 1.79E‐07. The term has 625 genes associated with it, of which only 210 are enriched in the gene list, out of a total of 4133 genes considered.

43.2.1.3 Step 3: Representing the functional terms graphically

The most common way of representing the functional terms is by choosing the top ten terms (by sorting on the basis of p‐value) in each category (Biological processes – BP; Molecular process – MP and cellular component – CC), and representing the term on the y‐axis and the significance (–log₁₀P) on the x‐axis, as shown below for the biological processes. The same can be done for all the categories (Figure 43.7).

43.2.1.4 Step 4

Interpretation of the data is completely the researcher’s purview.

43.2.2.1 Step 1

Open https://david.ncifcrf.gov and upload a multi‐list file if you have > 3000 genes to be annotated. The multi‐list file should be a list1 and list2, separated by a tab (shown in the figure below). Upload this list into DAVID, select the official gene symbol from the drop‐down menu (as an identifier), check the radio button against the gene list and submit (see Figure 43.8).

43.2.2.2 Step 2

Select an appropriate background (here it is Bos taurus) against which you wish to test your gene list. Create a combined list by clicking “combine” after selecting both the lists, and select the combined list to get the functional annotations (see Figure 43.8).

43.2.2.3 Step 3

Click on the functional annotation tool in the window to get the annotation summary results (see Figure 43.8).

43.2.2.4 Step 4

The + button can be clicked in the window to get the results. To get the gene ontology terms, click the + button by the side of the gene ontology and then proceed to any particular category – BP, MP or CC. Clicking on the chart option opens up a window with all the specific terms. Here we click on BP to get all the gene ontology terms enriched for biological processes in our differentially expressed genes. The details containing the genes associated with each gene can be downloaded and opened in Excel for further use. The same can be done to visualize pathways enriched in the DEGs (see Figure 43.9).

43.2.3.1 Step 1

Open the ClueGO app in Cytoscape, paste genes in the window (see Figure 43.10).

43.2.3.2 Step 2

Select a gene ontology or pathway and start. Here we selected immune system processes, as shown in Figure 43.10.

43.2.3.3 Step 3

Represent a network with GO term as node label, percentage associated genes as node color, and P‐Value Corrected with Bonferroni step‐down as node size (these parameters are selected as per the requirements of the researcher) (see Figures 43.11, 43.12 and 43.13).

43.2.3.4 Step 4

The attributes of the network can be exported in a table format (see Figure 43.14).