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To detect differential methylation between CpG loci in different HSCP populations, go to Analysis > Detect Differential Methylation. In the ANOVA dialog (Figure 1), select Add Factor to move the factor 2. HPSC from Experimental Factor(s) to the ANOVA Factor(s) box. experimental groups, we can perform an ANOVA test. For this tutorial, we will perform a simple two-way ANOVA to compare the methylation states of the two experimental groups.
- Select Detect Differential Methylation from the Analysis section of the Illumina BeadArray Methylation workflow
A new child spreadsheet, mvalue, is created when Detect Differential Methylation is selected. M-values are an alternative metric for measuring methylation. β-values can be easily converted to M-values using the following equation: M-value = log2( β / (1 - β)).
An M-value close to 0 for a CpG site indicates a similar intensity between the methylated and unmethylated probes, which means the CpG site is about half-methylated. Positive M-values mean that more molecules are methylated than unmethylated, while negative M-values mean that more molecules are unmethylated than methylated. As discussed by Du and colleagues, the β-value has a more intuitive biological interpretation, but the M-value is more statistically valid for the differential analysis of methylation levels.
Because we are performing differential methylation analysis, Partek Genomics Suite automatically creates an M-values spreadsheet to use for statistical analysis.
- Select 2. Cell Type and 3. Gender from the Experimental Factor(s) panel
- Select Add Factor > to move 2. Cell Type and 3. Gender to the ANOVA Factor(s) panel (Figure 1)
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- Select Contrasts...
- Leave Data is already log transformed? set to No
- Leave Report comparisons as set to Difference
For methylation data, fold-change comparisons are not appropriate. Instead, comparisons should be reported as the difference between groups.
- Select 2. Cell Type from the Select Factor/Interaction drop-down menu
- Select LCLs
- Select Add Contrast Level > for the upper group
- Select B cells
- Select Add Contrast Level > for the lower group
- Select Add Contrast (Figure 2)
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For this exercise, we shall compare primed HPSC with suppression of OCT4 (shPOU5F1) and primed HPSC with suppression of NANOG (shNANOG) with the baseline primed cells. To start with, select the Primed group in the Candidate Level(s) box and push Add Contrast Level > to move the Primed group to Group 2 (lower box). Then Ctrl & select both shPOU5F1 and shNANOG in the Candidate Level(s) box and push Add Contrast Level > to move them to Group 1 (upper box). Then click Add Combinations and confirm that two contrast have been created as seen in Figure 3.
and Select Add Contrast to confirm and then OK to go back to the ANOVA dialog. Note that the Contrasts button has now changed to Contrasts Included. Select OK to start the calculation. Select Contrast and use the Add Contrast Level> buttons to move Tumor to Group 1 and Normal to Group 2 (Figure 10).
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- Select OK to close the Configuration dialog
The Contrasts... button of the ANOVA dialog now reads Contrasts Included
- Select OK to close the ANOVA dialog and run the ANOVA
If this is the first time you have analyzed a MethylationEPIC array using the Partek Genomics Suite software, the manifest file may need to be configured. If it needs configuration, the Configure Annotation dialog will appear (Figure 3).
- Select Chromosome is in one column and the physical location is in another column for Choose the column configuration
- Select Ilmn ID for Marker ID
- Select CHR for Chromosome i
- Select MAPINFO for Physical Position
- Select Close
This enables Partek Genomics Suite to parse out probe annotations from the manifest file.
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