The basic method of creating a gene list from ANOVA results based on fold-change and p-value cut-offs is detailed in Creating gene lists from ANOVA results. Advanced options enable the creation of lists based on more complex criteria. For example, we can use the Create Gene List function to identify transcripts that are both significantly differentially expressed AND alternatively-spliced among the four tissue samples.
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)
Figure 1. Creating a gene list using advanced options
Select Specify New Criteria to invoke the Configure Criteria dialog (Figure 2)
Figure 2. Configuring criteria for transcripts with a p-value < 0.05
In the Configure Criteria dialog box (Figure 2), provide a name for the list (Diff Exp)
Select 1/transcripts (RNA-Seq_results.transcripts) from the_Spreadsheet_ 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 38,285 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)
Figure 3. Configuring criteria for a list of alternatively spliced genes
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)
Figure 4. Creating a gene list at the intersection of two criteria
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)
Figure 5. Saving a created list criteria
Select Diff Exp and Alt Splice in the List Creator dialog (Figure 6)
Figure 6. Selecting list to save in List Creator dialog
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)
Figure 7. A list of the differentially expressed and alternatively spliced genes is now available for downstream analysis
This list of differentially expressed and alternatively spliced transcripts will be used later in the tutorial.
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