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 (limma or t-test 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
450k vs. non.450k (based on Platform) 25099
EPIC vs. non.EPIC (based on Platform) 17301
WGBS vs. non.WGBS (based on Platform) 143304
infant blood vs. non.infant blood (based on tissue) 121866
non-malignant tissue associated fibroblasts (NAF) vs. non.non-malignant tissue associated fibroblasts (NAF) (based on tissue) 91577
cancer associated fibroblasts (CAF) vs. non.cancer associated fibroblasts (CAF) (based on tissue) 94986
primary prostate epithelial cells (PrEC) vs. non.primary prostate epithelial cells (PrEC) (based on tissue) 72278
prostate cancer cell line (LNCaP) vs. non.prostate cancer cell line (LNCaP) (based on tissue) 113868
Rep1 vs. Rep2 (based on PrEC_comparison) 893
LNCaP vs. PrEC (based on LNCaP_PrEC_comp) 137907
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 2e+06 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
450k vs. non.450k (based on Platform) 1821 112 452 347
EPIC vs. non.EPIC (based on Platform) 2377 129 117 132
WGBS vs. non.WGBS (based on Platform) 10884 1899 1241 5746
infant blood vs. non.infant blood (based on tissue) 5009 1703 3391 2557
non-malignant tissue associated fibroblasts (NAF) vs. non.non-malignant tissue associated fibroblasts (NAF) (based on tissue) 7220 1820 3154 789
cancer associated fibroblasts (CAF) vs. non.cancer associated fibroblasts (CAF) (based on tissue) 10552 1663 2262 1272
primary prostate epithelial cells (PrEC) vs. non.primary prostate epithelial cells (PrEC) (based on tissue) 4402 1305 1295 2483
prostate cancer cell line (LNCaP) vs. non.prostate cancer cell line (LNCaP) (based on tissue) 7590 2076 1312 4330
Rep1 vs. Rep2 (based on PrEC_comparison) 452 0 0 263
LNCaP vs. PrEC (based on LNCaP_PrEC_comp) 11615 1987 3029 2197
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
450k vs. non.450k (based on Platform) csv csv csv csv
EPIC vs. non.EPIC (based on Platform) csv csv csv csv
WGBS vs. non.WGBS (based on Platform) csv csv csv csv
infant blood vs. non.infant blood (based on tissue) csv csv csv csv
non-malignant tissue associated fibroblasts (NAF) vs. non.non-malignant tissue associated fibroblasts (NAF) (based on tissue) csv csv csv csv
cancer associated fibroblasts (CAF) vs. non.cancer associated fibroblasts (CAF) (based on tissue) csv csv csv csv
primary prostate epithelial cells (PrEC) vs. non.primary prostate epithelial cells (PrEC) (based on tissue) csv csv csv csv
prostate cancer cell line (LNCaP) vs. non.prostate cancer cell line (LNCaP) (based on tissue) csv csv csv csv
Rep1 vs. Rep2 (based on PrEC_comparison) csv csv csv csv
LNCaP vs. PrEC (based on LNCaP_PrEC_comp) csv csv csv csv

GO Enrichment Analysis

GO Enrichment Analysis was conducted. The wordclouds and tables below contains significant GO terms as determined by a hypergeometric test.

comparison
Hypermethylation/hypomethylation
ontology
regions
differential methylation measure

Figure 5

Figure 5

Wordclouds for GO enrichment terms.

comparison
Hypermethylation/hypomethylation
ontology
regions
differential methylation measure

GOMFID Pvalue OddsRatio ExpCount Count Size Term
GO:1902511 0.0024 Inf 0.0024 1 1 negative regulation of apoptotic DNA fragmentation
GO:0032076 0.0048 427.3421 0.0048 1 2 negative regulation of deoxyribonuclease activity
GO:0050976 0.0048 427.3421 0.0048 1 2 detection of mechanical stimulus involved in sensory perception of touch
GO:0051083 0.0048 427.3421 0.0048 1 2 'de novo' cotranslational protein folding
GO:0060133 0.0048 427.3421 0.0048 1 2 somatotropin secreting cell development
GO:0035803 0.0072 213.6579 0.0072 1 3 egg coat formation
GO:0044571 0.0072 213.6579 0.0072 1 3 [2Fe-2S] cluster assembly

LOLA Enrichment Analysis

No LOLA 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