Annotate samples

Each row of the spreadsheet (Figure 1) corresponds to a single sample. The first column is the names of the .idat files and the remaining columns are the array probes. The table values are β-values, which correspond to the percentage methylation at each site. A β-value is calculated as the ratio of methylated probe intensity over the overall intensity at each site (the overall intensity is the sum of methylated and unmethylated probe intensities).

Figure 1. Spreadsheet after .idat file import: samples on rows (Sample IDs are based on file names), probes on columns, cell values are functionally normalized beta values (default settings)

Before we can perform any analysis, the study samples need to be organized into their experimental groups.

• Select Add Sample Attributes from the Import section of the Illumina BeadArray Methylation workflow

• Select Add a Categorical Attribute from the Add Sample Attributes dialog (Figure 2)

Figure 2. Adding sample attributes. Adding Attributes from an Existing Column can be used to split file names into sections, based on delimiters (e.g. _, -, space etc.). Adding a Numeric or Categorical Attribute enables the user to manually specify sample attributes

• Select OK

The Create categorical attribute dialog allows us to create groups for a categorical attribute. By default, two groups are created, but additional groups can be added.

• Set Attribute name: to Cell Type

• Rename the groups B cells and LCLs

• Drag and drop the samples from the Unassigned list to their groups as listed in the table below

There should now be two groups with eight samples in each group (Figure 3).

Figure 3. Adding Cell Type attribute as a categorical group

• Select OK

• Select Yes from the Add another categorical attribute dialog

• Set Attribute name: to Gender

• Rename the groups Male and Female

• Drag and drop the samples from the Unassigned list to their groups as listed in the table below

There should now be two groups with four samples in Male and twelve samples in Female (Figure 4).

Figure 4. Adding Gender attribute as a categorical group

• Select OK

• Select No from the Add another categorical attribute dialog

• Select Yes to save the spreadsheet

Two new columns have been added to spreadsheet 1 (Methylation) with the cell type and gender of each sample (Figure 5).

Figure 5. Annotated beta values spreadsheet

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