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| exclude | Additional Assistance |
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To perform gene set and pathway analysis, we need to create a list of genes that overlap with differentially methylated CpG loci.
- Select Find Overlapping Genes... from the Tools section of the main toolbar (Figure 1)
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| SubtitleText | Selecting Finding Overlapping Genes form the main toolbar |
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| AnchorName | Selecting Finding Overlapping Genes |
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- Select Create a new spreadsheet with genes that overlap with the regions in the Find Overlapping Genes dialog
- Select OK
- Select an annotation - this tutorial uses RefSeq Transcripts - 2014-01-03
- You can select a name for the new list, we have named it gene-list
- Select OK
A new spreadsheet will be created as a child spreadsheet (Figure 2)
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| SubtitleText | Annotating the differentially methylated CpG loci with genes |
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| AnchorName | Overlapping Genes List |
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Partek Genomics Suite offers several tools to help interpret this list of genes. First, let's look at Gene Set Analysis.
- Select Gene Set Analysis from the Biological Interpretation section of the Illumina BeadArray Methylation workflow
- Select GO Enrichment for Select the method of analysis
- Select Next >
- Select female_only/LCLs_vs_B_cells/gene-list (gene-list.txt) for the source spreadsheet
- Select Next >
- Select Invoke gene ontology browser on the result and leave the rest of the options set to defaults for Configure the parameters of the test (Figure 3)
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| SubtitleText | Configuring the parameters of the test |
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| AnchorName | Configuring Gene Set Analysis |
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- Select Next >
- Select Default Mapping File for Select the method of mapping genes to genes sets
- Select Next >
A new spreadsheet will be created with categories ranked by enrichment score and the Gene Ontology Browser will launch to graphically display the results of the spreadsheet (Figure 4). The results show which gene sets are over represented in the list of genes overlapped by differentially regulated CpG loci between the experimental and control groups.
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| SubtitleText | GO enrichment browser showing gene groups overrepresented in the list of genes which overlap with differentially methylated CpG loci |
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| AnchorName | GO Enrichment spreadsheet |
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Next, we can perform Pathway Analysis to see which pathways are over represented in the gene overlapped by differentially regulated CpG loci.
- Select Pathway Analysis from the Biological Interpretation section of the Illumina BeadArray Methylation workflow
- Select Pathway Enrichment for Select the method of analysis
- Select Next >
- Select female_only/LCLs_vs_B_cells/gene-list (gene-list.txt) for the source spreadsheet
- Select Next >
- Leave the default selections for the Configure parameters of the test panel
- Select Next >
- Leave the default selections for the Result File and Select the parameters panels
- Select Next > to run the analysis
ThePathway-Enrichment spreadsheet will be added to the spreadsheet tree in Partek Genomics Suite and the Partek® Pathway™ software will open to provide visualization of the most significantly enriched pathway as a pathway diagram (Figure 4). The color of the gene boxes reflects p-values of the associated differentially methylated CpG loci (bright orange is insignificant, purple is highly significant). The Color by option can be changed another column from the gene-list.txt spreadsheet.
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| SubtitleText | : Partek Pathway illustrating one of the pathways overrepresented in the list of genes overlapping the differentially methylated CpG sites. |
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| AnchorName | Pathway Diagram |
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The Pathway-Enrichment spreadsheet can also be viewed in Partek Pathway by switching to the Pathway-Enrichment section of the menu tree on the left-hand side of the window. From the spreadsheet view, you can select a pathway name to visualize that pathway. Alternatively, you can open a pathway visualization in Partek Pathway from the Pathway-Enrichment spreadsheet in Partek Genomics Suite by right-clicking on a row and selecting Show pathway... from the pop-up menu. Please note that if you have closed Partek Pathway and have reopened it, you will need to import a gene list if you want to color the visualization by attributes form the gene list. For more information about using Partek Pathway, consult the User's Guide section on Partek Pathway.