Detecting Differentially Methylated CpG Islands

The approach described in previous sections relies on ANOVA to detect differentially methylated CpG sites and takes individual sites as a starting point for interpretation. Since ANOVA compares M values at each site independently, this strategy is robust to type I/type II probe bias.

An alternative could be to first summarize all the probes belonging to a CpG island region (i.e. island, N-shore, N-shelf, S-shore, S-shelf) and then use ANOVA to compare regions across the groups. Since the summarization will include both type I and type II probes, you may want to split the analysis in two branches and analyze type I and type II probes independently. To do this, we need to annotate each probe as type I or type II.

Select the female_only spreadsheet
Select Transform from the main toolbar
Select Create Transposed Spreadsheet... from the Transform drop-down menu (Figure 1)

Figure 1. Creating a transposed spreadsheet

Select Sample ID for Column: and numeric for Data Type:
Select OK

A new temporary spreadsheet will be created with a row for each probe and columns for each sample.

Right-click on column 1. ID to bring up the pop-up menu
Select Insert Annotation
Select Add as categorical
Select Infinium_Design_Type and UCSC_CpG_Islands_Name from the Column Configuration options
Select OK to add the Inifinium design type and CpG island name as categorical columns on the spreadsheet

Now we can use the interactive filter to create two separate spreadsheets for type I and type II probes.

Select () to launch the interactive filter
Select 2. Infinium_Design_Type from the drop-down menu if not selected by default
Left-click the type I column to exclude it
Right-click the temporary spreadsheet in the spreadsheet tree to bring up the pop-up dialog
Select Clone...
Name the new spreadsheet female_only_typeII_probes
Select OK
Repeat process to create a spreadsheet for type I probes

We can now use these spreadsheets to generate lists of M values at CpG island regions

Select spreadsheet female_only_typeII_probes
Select Stat from the main toolbar
Select Column Statistics... under Descriptive
Add Mean to the Selected Measure(s) panel
Select Group By and set it to 3. UCSC_CpG_Islands_Name (Figure )
Select OK

The new spreadsheet (Figure ) has one CpG island region per row (listed in column #2, Level), samples on columns, and the values in the cells represent the mean of M values of all the CpG probes in the region.

Note the first row, with label “– Mean”. It corresponds to all the probes that map outside of USCS CpG islands. As it is not needed for the downstream analysis, we will remove it.

Right-click on the row header for Mean
Select Delete to remove the row

The final step is to transpose the spreadsheet back to its original orientation.

Select the female_only_typeII_probes spreadsheet
Select Transform from the main toolbar
Select Create Transposed Spreadsheet... from the Transform drop-down menu
Select Labelfor Column: and numeric for Data Type:
Select OK

The layout now is as follows: one sample per row with CpG island regions on columns; cell entries correspond to mean methylation status of the region (Figure ). This spreadsheet can then be used as a starting point for ANOVA and other analyses.

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