Differential Methylation

Introduction: Differential Methylation of Sample Groups

Differential methylation analysis was conducted on site and region level according to the sample groups specified in the analysis.

Comparisons

The following comparisons were made:

P-values

In the following anlyses, p-values on the site level were computed using the limma method. I.e. hierarchical linear models from the limma package were employed and fitted using an empirical Bayes approach on derived M-values.

Site Level

Differential methylation on the site level was computed based on a variety of metrics. Of particular interest for the following plots and analyses are the following quantities for each site: a) the difference in mean methylation levels of the two groups being compared, b) the quotient in mean methylation and c) a statistical test (t-test or limma depending on the settings) assessing whether the methylation values in the two groups originate from distinct distributions. Additionally each site was assigned a rank based on each of these three criteria. A combined rank is computed as the maximum (i.e. worst) rank among the three ranks. The smaller the combined rank for a site, the more evidence for differential methylation it exhibits. This section includes scatterplots of the site group means as well as volcano plots of each pairwise comparison colored according to the combined ranks or p-values of a given site.

The following rank cutfoffs have been automatically selected for the analysis of differentially methylated sites:

Rank Cutoff
EWS_FLI1_high vs. EWS_FLI1_low (based on Cell_type) 1227880
comparison
differential methylation measure

Figure 1

Figure 1

Scatterplot for differential methylation (sites). If the selected criterion is not rankGradient: The transparency corresponds to point density. If the number of points exceeds 2000000 then the number of points for density estimation is reduced to that number by random sampling.The1% of the points in the sparsest populated plot regions are drawn explicitly (up to a maximum of 10000 points).Additionally, the colored points represent differentially methylated sites (according to the selected criterion). If the selected criterion is rankGradient: median combined ranks accross hexagonal bins are shown as a gradient according to the color legend.

comparison
difference metric
significance metric

Figure 2

Figure 2

Volcano plot for differential methylation quantified by various metrics. Color scale according to combined ranking.

Differential Methylation Tables

A tabular overview of measures for differential methylation on the site level for the individual comparisons are provided in this section. Below, a brief explanation of the different columns can be found:

The tables for the individual comparisons can be found here:

Region Level

Differential methylation on the region level was computed based on a variety of metrics. Of particular interest for the following plots and analyses are the following quantities for each region: the mean difference in means across all sites in a region of the two groups being compared and the mean of quotients in mean methylation as well as a combined p-value calculated from all site p-values in the region [1]. Additionally each region was assigned a rank based on each of these three criteria. A combined rank is computed as the maximum (i.e. worst) value among the three ranks. The smaller the combined rank for a region, the more evidence for differential methylation it exhibits. Regions were defined based on the region types specified in the analysis. This section includes scatterplots of the region group means as well as volcano plots of each pairwise comparison colored according to the combined rank of a given region.

The following rank cutfoffs have been automatically selected for the analysis of differentially methylated regions:

tiling genes promoters cpgislands
EWS_FLI1_high vs. EWS_FLI1_low (based on Cell_type) 36294 17436 10870 3553
comparison
regions
differential methylation measure

Figure 3

Figure 3

Scatterplot for differential methylation (regions). If the selected criterion is not rankGradient: The transparency corresponds to point density. The 1% of the points in the sparsest populated plot regions are drawn explicitly. Additionally, the colored points represent differentially methylated regions (according to the selected criterion). If the selected criterion is rankGradient: median combined ranks accross hexagonal bins are shown as a gradient according to the color legend.

comparison
regions
difference metric
significance metric

Figure 4

Figure 4

Volcano plot for differential methylation quantified by various metrics. Color scale according to combined ranking.

Differential Methylation Tables

A tabular overview of measures for differential methylation on the region level for the individual comparisons are provided in this section.

The tables for the individual comparisons can be found here:

tiling genes promoters cpgislands
EWS_FLI1_high vs. EWS_FLI1_low (based on Cell_type) csv csv csv csv

Enrichment Analysis

No Enrichment Analysis was conducted

References

  1. Makambi, K. (2003) Weighted inverse chi-square method for correlated significance tests. Journal of Applied Statistics, 30(2), 225234