# Detect 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 **mvalue** spreadsheet * Select **Transform** from the main toolbar * Select **Create Transposed Spreadsheet...** from the *Transform* drop-down menu (Figure 1) ![](/files/xhu6bKiPqiIMIabkI133) 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 (Figure 2) ![](/files/yL5mr8reoAAoSkKfk7Ew) Figure 2. Adding Infinium design type and CpG island annotations * 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 separate spreadsheets for type I and type II probes. * Select (![](/files/FMRDfrNB5wLvfxnRcctA)) 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) ![](/files/TvLb1bQaqct3njTLah7z) Figure 3. Creating a probe list with only Infinium type II probes * 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 (![](/files/It9h1gyJIE3c0yemgRhI)) We can 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) ![](/files/KIk8y26RiSI8Z5EVfGr1) Figure 4. Selecting column statistics * Add **Mean** to the *Selected Measure(s)* panel * Select **Group By** and set it to **3. UCSC\_CpG\_Islands\_Name** (Figure 5) ![](/files/375QcEh09ojal3SYIsgo) Figure 5. Configuring column statistics * Select **OK** The new temporary spreadsheet has one CpG island region per row (Figure 6), samples on columns, and the values in the cells represent the mean of M values of all the CpG probes in the region. ![](/files/yn3TeGgbkfdVJYou17OU) Figure 6. New spreadsheet with average M values for probes at each CpG island; probes not at CpG islands are collected into the first row "- Mean" Note the first row, with label “*– Mean*”. It corresponds to all the probes that map outside of UCSC 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 data back to its original orientation. * Select **Transform** from the main toolbar * Select **Create Transposed Spreadsheet...** from the *Transform* drop-down menu * Select **2. Level** for *Column:* and **numeric** for *Data Type:* * Select OK The layout 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 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. ![](/files/0qqi09cVTu6OS2sL6IHt) Figure 7. Spreadsheet with average M values of probes in each CpG island for each sample * 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 (![](/files/EC3TWoQo2sxOKbQuJmkn)) The *mvalues\_typeII\_probes\_CpG\_islands* spreadsheet can be used as a starting point for ANOVA and other analyses. 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