To identify transcripts that are both significantly differentially expressed AND alternatively-spliced among the four tissue samples, we can use the Create Gene List function.
- Select Create Gene List from the Analyze Known Genes panel of the RNA-Seq workflow to invoke the List Manager dialog
- Select the Advanced tab (Figure 1)
- Select Specify New Criteria to invoke the Configure Criteria dialog (Figure 2)
- In the Configure Criteria dialog box (Figure 2), provide a name for the list (Diff Exp)
- Select 1/transcripts (RNA-Seq_results.transcripts) from theSpreadsheet drop-down menu
- Select 8. p-value(DiffExp) from the Column drop-down menu
- Set Include p-values to significant with FDR with a value of 0.05
A list of 30,305 transcripts that pass this criteria will be generated according to the # pass score on the right-hand side of the dialog. If the settings are changed, this number will automatically update.
- Select OK
- Repeat the same steps to create a list of transcripts that are likely alternatively spliced, named Alt Splice, using the same p-value cutoff and Column set to 10. p-value (AltSplice) (Figure 3)
- Select OK to generate Alt Splice
- Select both lists in the right-hand panel under the Criteria panel while holding the Ctrl key on your keyboard
- Select Intersection from the left-hand panel of the List Manager dialog (Figure 4)
- Enter a name for the criteria (Diff Exp and Alt Splice)
- Select OK to close the naming dialog and OK again to close the list creation hint dialog
- Select Save List from the Manage criteria section of the List Manager dialog (Figure 5)
- Select Diff Exp and Alt Splice in the List Creator dialog (Figure 6)
- Select OK to save the list
- Select Close to exit the List Manager dialog and view the Diff_Exp_and_Alt_Splice spreadsheet (Figure 7)
This list of differentially expressed and alternatively spliced transcripts will be used in the next section of the tutorial.