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

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

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• 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 (Figure 2)
  
  

• Select OK to add the Inifinium design type and UCSC 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 (Image Added) 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... (Figure 3)
  
  

• Name the new spreadsheet female_only_typeII_probes
• Select OK 
• Save the created spreadsheet, we chose the file name female_only_typeII_probes
• Repeat process to create a spreadsheet for type I probes

The temporary spreadsheet is no longer needed so we can close it.

• Close the temporary spreadsheet by selecting it in the file tree and selecting (Image Added)

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 (Figure 4)
  
  

• Add Mean to the Selected Measure(s) panel 
• Select Group By and set it to 3to 3. UCSC_CpG_Islands_Name Name (Figure 5)
  
  

• Select OK 

The new temporary spreadsheet (Figure ) has one CpG island region per row (listed in column #2, LevelFigure 6), 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 UCSC CpG islands. As it is not needed for the downstream analysis, we will remove it.

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The final step is to transpose the spreadsheet data 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 Label 2. Level for Column: and numeric for Data Type:
• Select OK

The layout now of the new transposed spreadsheet 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 7). The column with a blank value for the column header is the average of all probes not associated with CpG island regions. You can delete this column if you like. 

• Right-click the transposed spreadsheet, 2_transpose
• Select Save as... from the pop-up menu
• Name it mvalues_typeII_probes_CpG_islands 
• Close the source temporary spreadsheet by selecting it in the spreadsheet tree and selecting (Image Added)

The mvalues_typeII_probes_CpG_islands spreadsheet can be used as a starting point for ANOVA and other analyses. You can also repeat the steps above to create an equivalent spreadsheet for type I probes. 

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