Viewing GSA results and creating a gene list

Once we have performed GSA to identify differentially expressed genes, we can create a list of significantly differentially expressed genes using cutoff thresholds.

Double click the Feature list data node to open the task report

The task report spreadsheet will open showing genes on rows and the results of the GSA on columns (Figure 1).

Figure 1. Viewing the GSA task report spreadsheet

To get a sense of what filtering thresholds to set, we can view a volcano plot for a comparison.

Select next to the 5uM vs. 0uM comparison

A volcano plot will open showing p-value on the y-axis and fold-change on the x-axis (Figure 2).

Figure 2. Viewing GSA results with a volcano plot

Thresholds for the cutoff lines are set using the text boxes on the left-hand side. The default thresholds are 2 and 0.05.

Select Gene-specific analysis report to return to the task report spreadsheet
Select FDR step up
Select the triangle next to FDR step up to open the FDR step up options
Select All contrasts
Set the cutoff value to 0.05

This will include genes that have a FDR step up value of less than or equal to 0.05 for all three contrasts, 5μM vs. 0μM, 10μM vs. 0μM and 5μM:10μM vs. 0μM. FDR step up is the false discovery rate adjusted p-value used by convention in microarray and next generation sequencing data sets in place of unadjusted p-value.

Select Fold-change
Select the triangle next to Fold-change to open the Fold-change options
Leave All contrasts selected
Set to From -2 to 2 with Exclude range selected

Note that the number of genes that pass the filter is listed at the top of the filter menu next to Results: and will update to reflect any changes to the filter. Here, 21 genes pass the filter (Figure 3).