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:

The table below summarizes information on the comparisons.

comparison adjustment covariateTable
1 1 vs. 5 (based on cmp_cellnum) sex,donorId,flowcell csv
2 02I vs. GNN (based on cmp_donor_02I_vs_GNN) flowcell,nominalCellNumber csv
3 02I vs. GPJ (based on cmp_donor_02I_vs_GPJ) flowcell,nominalCellNumber csv
4 02I vs. 2I_ (based on cmp_donor_02I_vs_2I_) flowcell,nominalCellNumber csv
5 GNN vs. GPJ (based on cmp_donor_GNN_vs_GPJ) flowcell csv
6 2I_ vs. GNN (based on cmp_donor_GNN_vs_2I_) flowcell csv
7 2I_ vs. GPJ (based on cmp_donor_GPJ_vs_2I_) flowcell csv
8 2N vs. 4N (based on cmp_ploidy_2N_vs_4N) sex,donorId,flowcell,nominalCellNumber csv
9 2N vs. 8N (based on cmp_ploidy_2N_vs_8N) sex,donorId,flowcell,nominalCellNumber csv
10 16N vs. 2N (based on cmp_ploidy_2N_vs_16N) sex,donorId,flowcell,nominalCellNumber csv
11 2N vs. 32N (based on cmp_ploidy_2N_vs_32N) sex,donorId,flowcell,nominalCellNumber csv
12 4N vs. 8N (based on cmp_ploidy_4N_vs_8N) sex,donorId,flowcell,nominalCellNumber csv
13 16N vs. 8N (based on cmp_ploidy_8N_vs_16N) sex,donorId,flowcell,nominalCellNumber csv
14 16N vs. 32N (based on cmp_ploidy_16N_vs_32N) sex,donorId,flowcell,nominalCellNumber csv

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.

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 methylation difference in a region of the two groups being and of quotient of mean methylation levelsas well as a p-value obtained from statistical testing (limma or t-test; depending on parameter settings). 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 cpgislands genes promoters ensembleRegBuildBPall ensembleRegBuildBPallMerged
1 vs. 5 (based on cmp_cellnum) 0 0 0 0 0 0
02I vs. GNN (based on cmp_donor_02I_vs_GNN) 0 0 0 0 0 0
02I vs. GPJ (based on cmp_donor_02I_vs_GPJ) 0 0 0 0 0 0
02I vs. 2I_ (based on cmp_donor_02I_vs_2I_) 0 0 0 0 0 0
GNN vs. GPJ (based on cmp_donor_GNN_vs_GPJ) 0 0 0 0 0 0
2I_ vs. GNN (based on cmp_donor_GNN_vs_2I_) 0 0 0 0 0 0
2I_ vs. GPJ (based on cmp_donor_GPJ_vs_2I_) 0 0 0 0 0 0
2N vs. 4N (based on cmp_ploidy_2N_vs_4N) 0 0 0 0 0 0
2N vs. 8N (based on cmp_ploidy_2N_vs_8N) 0 0 0 0 0 0
16N vs. 2N (based on cmp_ploidy_2N_vs_16N) 0 0 0 0 0 0
2N vs. 32N (based on cmp_ploidy_2N_vs_32N) 0 0 0 0 0 0
4N vs. 8N (based on cmp_ploidy_4N_vs_8N) 0 0 0 0 0 0
16N vs. 8N (based on cmp_ploidy_8N_vs_16N) 0 0 0 0 0 0
16N vs. 32N (based on cmp_ploidy_16N_vs_32N) 0 0 0 0 0 0
comparison
regions
differential methylation measure

Figure 1

Figure 1

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 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 region level for the individual comparisons are provided in this section.

The tables for the individual comparisons can be found here:

tiling cpgislands genes promoters ensembleRegBuildBPall ensembleRegBuildBPallMerged
1 vs. 5 (based on cmp_cellnum) csv csv csv csv csv csv
02I vs. GNN (based on cmp_donor_02I_vs_GNN) csv csv csv csv csv csv
02I vs. GPJ (based on cmp_donor_02I_vs_GPJ) csv csv csv csv csv csv
02I vs. 2I_ (based on cmp_donor_02I_vs_2I_) csv csv csv csv csv csv
GNN vs. GPJ (based on cmp_donor_GNN_vs_GPJ) csv csv csv csv csv csv
2I_ vs. GNN (based on cmp_donor_GNN_vs_2I_) csv csv csv csv csv csv
2I_ vs. GPJ (based on cmp_donor_GPJ_vs_2I_) csv csv csv csv csv csv
2N vs. 4N (based on cmp_ploidy_2N_vs_4N) csv csv csv csv csv csv
2N vs. 8N (based on cmp_ploidy_2N_vs_8N) csv csv csv csv csv csv
16N vs. 2N (based on cmp_ploidy_2N_vs_16N) csv csv csv csv csv csv
2N vs. 32N (based on cmp_ploidy_2N_vs_32N) csv csv csv csv csv csv
4N vs. 8N (based on cmp_ploidy_4N_vs_8N) csv csv csv csv csv csv
16N vs. 8N (based on cmp_ploidy_8N_vs_16N) csv csv csv csv csv csv
16N vs. 32N (based on cmp_ploidy_16N_vs_32N) csv csv csv csv csv csv

Enrichment Analysis

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 3

Figure 3

Wordclouds for GO enrichment terms.

comparison
Hypermethylation/hypomethylation
ontology
regions
differential methylation measure

GOMFID Pvalue OddsRatio ExpCount Count Size Term
GO:0006370 2e-04 126.2097 0.021 2 33 7-methylguanosine mRNA capping
GO:0036260 2e-04 111.7571 0.0236 2 37 RNA capping
GO:0097310 6e-04 Inf 6e-04 1 1 cap2 mRNA methylation
GO:1901676 6e-04 Inf 6e-04 1 1 positive regulation of histone H3-K27 acetylation
GO:0048861 0.0025 580.6667 0.0025 1 4 leukemia inhibitory factor signaling pathway
GO:0072108 0.0025 580.6667 0.0025 1 4 positive regulation of mesenchymal to epithelial transition involved in metanephros morphogenesis
GO:0051461 0.0032 435.4722 0.0032 1 5 positive regulation of corticotropin secretion
GO:0060708 0.0032 435.4722 0.0032 1 5 spongiotrophoblast differentiation
GO:1903025 0.0032 435.4722 0.0032 1 5 regulation of RNA polymerase II regulatory region sequence-specific DNA binding
GO:0009249 0.0038 348.3556 0.0038 1 6 protein lipoylation
GO:0080009 0.0038 348.3556 0.0038 1 6 mRNA methylation
GO:0019827 0.0042 25.0419 0.1001 2 157 stem cell population maintenance
GO:0072307 0.0045 290.2778 0.0045 1 7 regulation of metanephric nephron tubule epithelial cell differentiation
GO:0006123 0.0051 248.7937 0.0051 1 8 mitochondrial electron transport, cytochrome c to oxygen
GO:0008635 0.0051 248.7937 0.0051 1 8 activation of cysteine-type endopeptidase activity involved in apoptotic process by cytochrome c
GO:0060426 0.0051 248.7937 0.0051 1 8 lung vasculature development
GO:0060463 0.0051 248.7937 0.0051 1 8 lung lobe morphogenesis
GO:0006108 0.0057 217.6806 0.0057 1 9 malate metabolic process
GO:0006122 0.0057 217.6806 0.0057 1 9 mitochondrial electron transport, ubiquinol to cytochrome c
GO:0060290 0.0057 217.6806 0.0057 1 9 transdifferentiation
GO:0045333 0.0059 21.0557 0.1185 2 186 cellular respiration
GO:0060180 0.0064 193.4815 0.0064 1 10 female mating behavior
GO:0006107 0.007 174.1222 0.007 1 11 oxaloacetate metabolic process
GO:0042511 0.007 174.1222 0.007 1 11 positive regulation of tyrosine phosphorylation of Stat1 protein
GO:0045835 0.007 174.1222 0.007 1 11 negative regulation of meiotic nuclear division
GO:0048711 0.0076 158.2828 0.0076 1 12 positive regulation of astrocyte differentiation
GO:0045651 0.0083 145.0833 0.0083 1 13 positive regulation of macrophage differentiation
GO:0060707 0.0083 145.0833 0.0083 1 13 trophoblast giant cell differentiation
GO:2000696 0.0089 133.9145 0.0089 1 14 regulation of epithelial cell differentiation involved in kidney development
GO:0015671 0.0095 124.3413 0.0096 1 15 oxygen transport
GO:0072160 0.0095 124.3413 0.0096 1 15 nephron tubule epithelial cell differentiation
GO:0072283 0.0095 124.3413 0.0096 1 15 metanephric renal vesicle morphogenesis
GO:0044706 0.0097 16.2216 0.153 2 240 multi-multicellular organism process