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An alternative metric for measurement of methlyation levels are M-values. 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, we need to convert our data to from β-values to M-values.
- Select Convert Beta Value to M Value from the Import section of the Illumina BeadArray Methylation workflow
The original data (β-values) will be overwritten.
- Select () from the icon bar to save the current spreadsheet
Before we can perform any analysis, the study samples need to be organized into their experimental groups.
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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 Experimental Group
- Select New Group twice to add two additional groups
- Rename the groups Primed+shCTRL, Naive+shCTRL, Naive+shPOU5F1, and Naive+shNANOGCell 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
Sample ID | Group NameCell Type |
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GSM2515899GSM2452106_200526580002_R01C01 | Primed shCTRL |
GSM2515900_200526580002_R02C01 | Primed shCTRL |
GSM2515901_200526580002_R03C01 | Naive shCTRL |
GSM2515902_200526580002_R04C01 | Naive shCTRL |
GSM2515903_200526580002_R05C01 | Naive shPOU5F1 |
GSM2515904_200526580002_R06C01 | Naive shPOU5F1 |
GSM2515905_200526580002_R07C01 | Naive shNANOG |
GSM2515906_200526580002_R08C01 | Naive shNANOG |
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200483200025_R04C01 | B cells |
GSM2452107_200483200021_R01C01 | B cells |
GSM2452108_200483200021_R02C01 | B cells |
GSM2452109_200483200025_R06C01 | B cells |
GSM2452110_200483200025_R07C01 | B cells |
GSM2452111_200483200021_R08C01 | B cells |
GSM2452112_200483200021_R06C01 | B cells |
GSM2452113_200483200021_R04C01 | B cells |
GSM2452114_200483200025_R01C01 | LCLs |
GSM2452115_200483200025_R03C01 | LCLs |
GSM2452116_200483200021_R03C01 | LCLs |
GSM2452117_200483200025_R05C01 | LCLs |
GSM2452118_200483200025_R02C01 | LCLs |
GSM2452119_200483200021_R07C01 | LCLs |
GSM2452120_200483200021_R05C01 | LCLs |
GSM2452121_200483200025_R08C01 | LCLs |
There should now be two groups with eight samples in each group (Figure 3).
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- 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
Sample ID | Gender |
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GSM2452106_200483200025_R04C01 | Female |
GSM2452107_200483200021_R01C01 | Female |
GSM2452108_200483200021_R02C01 | Male |
GSM2452109_200483200025_R06C01 | Female |
GSM2452110_200483200025_R07C01 | Female |
GSM2452111_200483200021_R08C01 | Female |
GSM2452112_200483200021_R06C01 | Female |
GSM2452113_200483200021_R04C01 | Male |
GSM2452114_200483200025_R01C01 | Female |
GSM2452115_200483200025_R03C01 | Female |
GSM2452116_200483200021_R03C01 | Male |
GSM2452117_200483200025_R05C01 | Female |
GSM2452118_200483200025_R02C01 | Female |
GSM2452119_200483200021_R07C01 | Female |
GSM2452120_200483200021_R05C01 | Female |
GSM2452121_200483200025_R08C01 | Male |
There should now be two groups with four samples in Male and twelve samples in Female (Figure 4).
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- Select OK
- Select No from the Add another categorical attribute dialog
- Select Yes to save the spreadsheet
A Two new column has columns have been added to spreadsheet 1 (Differential Methylation Analysis) with the experimental group cell type and gender of each sample (Figure 45).
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