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- Select Differential Expression Analysis from the Analyze Known Genes section of the RNA-Seq workflowThe
The Differential Expression Analysis dialog offers the choice of analyzing at Gene-,Transcript-, or Exon-level.
- Select Gene-level
- Specify the 1/gene_rpkm (RNA-Seq_results.gene.rpkm) spreadsheet from the Spreadsheet drop-down menu (Figure 1)
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If the ANOVA were now performed (without contrasts), a p-value for differential expression would be calculated, but it would only indicate if there are differences within the factor Tissue; it would not inform you which groups are different or give any information on the magnitude of the change difference between groups (fold-change or ratio). To get this more specific information, you need to define linear contrasts.
- Select Contrasts... to open the Configure dialog
- For Select Factor/Interaction, Tissue will be the only factor available as it was the only factor included in the ANOVA model in the previous step; if multiple factors were included, they could be selected in the Select Factor/Interaction: drop-down menu. The levels in this factor are listed on the Candidate Level(s) panel on the left side of the dialog
- For this data set, verify that No is selected for Data is already log transformed?
- Left click to select muscle from muscle from the Candidate Level(s) panel and move it to the Group 1 panel (renamed muscle) by selecting Add Contrast Level > in the top half of the dialog. Label 1 will be changed to the subgroup name automatically, but you can also manually specify the label name
- Select not muscle from the Candidate Level(s) panel and move it to the Group 2 panel (renamed not muscle)
- The Add Contrast button can now be selected (Figure 3)
- Select OK to return to the ANOVA dialog
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