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 CMP vs. MEP (based on cmp_ct_CMPvMEP) sex,donorId,flowcell,nominalCellNumber csv
2 CMP vs. GMP (based on cmp_ct_CMPvGMP) donorId,flowcell,nominalCellNumber csv
3 CLP vs. CMP (based on cmp_ct_CMPvCLP) sex,donorId,flowcell,nominalCellNumber csv
4 GMP vs. MLP3 (based on cmp_ct_MLP3vGMP) donorId,flowcell,nominalCellNumber csv
5 CLP vs. MLP0 (based on cmp_ct_CLPvMLP0) donorId,flowcell,nominalCellNumber csv
6 CLP vs. MLP1 (based on cmp_ct_CLPvMLP1) sex,donorId,nominalCellNumber csv
7 CLP vs. MLP2 (based on cmp_ct_CLPvMLP2) sex,donorId,flowcell,nominalCellNumber csv
8 CLP vs. MLP3 (based on cmp_ct_CLPvMLP3) sex,donorId,flowcell,nominalCellNumber csv
9 MLP1 vs. MLP2 (based on cmp_ct_MLP1vMLP2) sex,donorId,flowcell,nominalCellNumber csv
10 MLP2 vs. MLP3 (based on cmp_ct_MLP2vMLP3) sex,donorId,flowcell,nominalCellNumber csv
11 MLP1 vs. MLP3 (based on cmp_ct_MLP1vMLP3) sex,donorId,flowcell,nominalCellNumber csv
12 MLP0 vs. MLP1 (based on cmp_ct_MLP0vMLP1) donorId,flowcell,nominalCellNumber csv
13 MLP0 vs. MLP2 (based on cmp_ct_MLP0vMLP2) donorId,flowcell,nominalCellNumber csv
14 MLP0 vs. MLP3 (based on cmp_ct_MLP0vMLP3) donorId,flowcell,nominalCellNumber csv
15 HSC vs. MPP (based on cmp_ct_HSCvMPP) flowcell,cellSourceCurated,nominalCellNumber csv
16 CMP vs. MPP (based on cmp_ct_MPPvCMP) flowcell,cellSourceCurated,nominalCellNumber csv
17 HSC vs. ML (based on cmp_ct_HSCvML) flowcell,cellSourceCurated,nominalCellNumber csv
18 LYM vs. MYE (based on cmp_ct_MYEvLYM) donorId,flowcell,nominalCellNumber csv
19 BM vs. CB (based on cmp_src_HSC_BMvCB) flowcell,nominalCellNumber csv
20 BM vs. FL (based on cmp_src_HSC_BMvFL) flowcell,nominalCellNumber csv
21 BM vs. PB (based on cmp_src_HSC_BMvPB) flowcell,nominalCellNumber csv
22 CB vs. FL (based on cmp_src_HSC_CBvFL) donorId,flowcell,nominalCellNumber csv
23 CB vs. PB (based on cmp_src_HSC_CBvPB) sex,donorId,nominalCellNumber csv
24 FL vs. PB (based on cmp_src_HSC_FLvPB) donorId,flowcell,nominalCellNumber csv
25 BM vs. CB (based on cmp_src_MPP_BMvCB) nominalCellNumber csv
26 BM vs. PB (based on cmp_src_MPP_BMvPB) flowcell,nominalCellNumber csv
27 CB vs. PB (based on cmp_src_MPP_CBvPB) donorId,flowcell,nominalCellNumber csv
28 HSC vs. MPP (based on cmp_HSCvMPP_PB) donorId,flowcell,nominalCellNumber csv
29 HSC vs. MPP (based on cmp_HSCvMPP_CB) sex,donorId,nominalCellNumber csv
30 HSC vs. MPP (based on cmp_HSCvMPP_BM) 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
CMP vs. MEP (based on cmp_ct_CMPvMEP) 0 0 0 0 0 0
CMP vs. GMP (based on cmp_ct_CMPvGMP) 0 0 0 0 0 0
CLP vs. CMP (based on cmp_ct_CMPvCLP) 0 0 0 0 0 0
GMP vs. MLP3 (based on cmp_ct_MLP3vGMP) 0 0 0 0 0 0
CLP vs. MLP0 (based on cmp_ct_CLPvMLP0) 0 0 0 0 0 0
CLP vs. MLP1 (based on cmp_ct_CLPvMLP1) 0 0 0 0 0 0
CLP vs. MLP2 (based on cmp_ct_CLPvMLP2) 0 0 0 0 0 0
CLP vs. MLP3 (based on cmp_ct_CLPvMLP3) 0 0 0 0 0 0
MLP1 vs. MLP2 (based on cmp_ct_MLP1vMLP2) 0 0 0 0 0 0
MLP2 vs. MLP3 (based on cmp_ct_MLP2vMLP3) 0 0 0 0 0 0
MLP1 vs. MLP3 (based on cmp_ct_MLP1vMLP3) 0 0 0 0 0 0
MLP0 vs. MLP1 (based on cmp_ct_MLP0vMLP1) 0 0 0 0 0 0
MLP0 vs. MLP2 (based on cmp_ct_MLP0vMLP2) 0 0 0 0 0 0
MLP0 vs. MLP3 (based on cmp_ct_MLP0vMLP3) 0 0 0 0 0 0
HSC vs. MPP (based on cmp_ct_HSCvMPP) 0 0 0 0 0 0
CMP vs. MPP (based on cmp_ct_MPPvCMP) 0 0 0 0 0 0
HSC vs. ML (based on cmp_ct_HSCvML) 0 0 0 0 0 0
LYM vs. MYE (based on cmp_ct_MYEvLYM) 0 0 0 0 0 0
BM vs. CB (based on cmp_src_HSC_BMvCB) 0 0 0 0 0 0
BM vs. FL (based on cmp_src_HSC_BMvFL) 0 0 0 0 0 0
BM vs. PB (based on cmp_src_HSC_BMvPB) 0 0 0 0 0 0
CB vs. FL (based on cmp_src_HSC_CBvFL) 0 0 0 0 0 0
CB vs. PB (based on cmp_src_HSC_CBvPB) 0 0 0 0 0 0
FL vs. PB (based on cmp_src_HSC_FLvPB) 0 0 0 0 0 0
BM vs. CB (based on cmp_src_MPP_BMvCB) 0 0 0 0 0 0
BM vs. PB (based on cmp_src_MPP_BMvPB) 0 0 0 0 0 0
CB vs. PB (based on cmp_src_MPP_CBvPB) 0 0 0 0 0 0
HSC vs. MPP (based on cmp_HSCvMPP_PB) 0 0 0 0 0 0
HSC vs. MPP (based on cmp_HSCvMPP_CB) 0 0 0 0 0 0
HSC vs. MPP (based on cmp_HSCvMPP_BM) 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
CMP vs. MEP (based on cmp_ct_CMPvMEP) csv csv csv csv csv csv
CMP vs. GMP (based on cmp_ct_CMPvGMP) csv csv csv csv csv csv
CLP vs. CMP (based on cmp_ct_CMPvCLP) csv csv csv csv csv csv
GMP vs. MLP3 (based on cmp_ct_MLP3vGMP) csv csv csv csv csv csv
CLP vs. MLP0 (based on cmp_ct_CLPvMLP0) csv csv csv csv csv csv
CLP vs. MLP1 (based on cmp_ct_CLPvMLP1) csv csv csv csv csv csv
CLP vs. MLP2 (based on cmp_ct_CLPvMLP2) csv csv csv csv csv csv
CLP vs. MLP3 (based on cmp_ct_CLPvMLP3) csv csv csv csv csv csv
MLP1 vs. MLP2 (based on cmp_ct_MLP1vMLP2) csv csv csv csv csv csv
MLP2 vs. MLP3 (based on cmp_ct_MLP2vMLP3) csv csv csv csv csv csv
MLP1 vs. MLP3 (based on cmp_ct_MLP1vMLP3) csv csv csv csv csv csv
MLP0 vs. MLP1 (based on cmp_ct_MLP0vMLP1) csv csv csv csv csv csv
MLP0 vs. MLP2 (based on cmp_ct_MLP0vMLP2) csv csv csv csv csv csv
MLP0 vs. MLP3 (based on cmp_ct_MLP0vMLP3) csv csv csv csv csv csv
HSC vs. MPP (based on cmp_ct_HSCvMPP) csv csv csv csv csv csv
CMP vs. MPP (based on cmp_ct_MPPvCMP) csv csv csv csv csv csv
HSC vs. ML (based on cmp_ct_HSCvML) csv csv csv csv csv csv
LYM vs. MYE (based on cmp_ct_MYEvLYM) csv csv csv csv csv csv
BM vs. CB (based on cmp_src_HSC_BMvCB) csv csv csv csv csv csv
BM vs. FL (based on cmp_src_HSC_BMvFL) csv csv csv csv csv csv
BM vs. PB (based on cmp_src_HSC_BMvPB) csv csv csv csv csv csv
CB vs. FL (based on cmp_src_HSC_CBvFL) csv csv csv csv csv csv
CB vs. PB (based on cmp_src_HSC_CBvPB) csv csv csv csv csv csv
FL vs. PB (based on cmp_src_HSC_FLvPB) csv csv csv csv csv csv
BM vs. CB (based on cmp_src_MPP_BMvCB) csv csv csv csv csv csv
BM vs. PB (based on cmp_src_MPP_BMvPB) csv csv csv csv csv csv
CB vs. PB (based on cmp_src_MPP_CBvPB) csv csv csv csv csv csv
HSC vs. MPP (based on cmp_HSCvMPP_PB) csv csv csv csv csv csv
HSC vs. MPP (based on cmp_HSCvMPP_CB) csv csv csv csv csv csv
HSC vs. MPP (based on cmp_HSCvMPP_BM) 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:0060324 0.0018 36.0623 0.0641 2 53 face development
GO:0071049 0.0024 871.2778 0.0024 1 2 nuclear retention of pre-mRNA with aberrant 3'-ends at the site of transcription
GO:0002824 0.003 27.8396 0.0823 2 68 positive regulation of adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains
GO:0019318 0.0032 11.7184 0.3025 3 250 hexose metabolic process
GO:0033091 0.0036 435.6111 0.0036 1 3 positive regulation of immature T cell proliferation
GO:0033133 0.0036 435.6111 0.0036 1 3 positive regulation of glucokinase activity
GO:0044375 0.0036 435.6111 0.0036 1 3 regulation of peroxisome size
GO:0006094 0.0041 23.5385 0.0968 2 80 gluconeogenesis
GO:0015746 0.0048 290.3889 0.0048 1 4 citrate transport
GO:0016557 0.0048 290.3889 0.0048 1 4 peroxisome membrane biogenesis
GO:0060528 0.0048 290.3889 0.0048 1 4 secretory columnal luminar epithelial cell differentiation involved in prostate glandular acinus development
GO:0070427 0.0048 290.3889 0.0048 1 4 nucleotide-binding oligomerization domain containing 1 signaling pathway
GO:0070673 0.0048 290.3889 0.0048 1 4 response to interleukin-18
GO:0071034 0.0048 290.3889 0.0048 1 4 CUT catabolic process
GO:0071224 0.0048 290.3889 0.0048 1 4 cellular response to peptidoglycan
GO:1903142 0.0048 290.3889 0.0048 1 4 positive regulation of establishment of endothelial barrier
GO:0046364 0.005 21.3379 0.1065 2 88 monosaccharide biosynthetic process
GO:0000467 0.006 217.7778 0.006 1 5 exonucleolytic trimming to generate mature 3'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA)
GO:0070671 0.006 217.7778 0.006 1 5 response to interleukin-12
GO:0071038 0.006 217.7778 0.006 1 5 nuclear polyadenylation-dependent tRNA catabolic process
GO:0071051 0.006 217.7778 0.006 1 5 polyadenylation-dependent snoRNA 3'-end processing
GO:0071225 0.006 217.7778 0.006 1 5 cellular response to muramyl dipeptide
GO:0010906 0.0066 18.3341 0.1234 2 102 regulation of glucose metabolic process
GO:0043144 0.0072 174.2111 0.0073 1 6 snoRNA processing
GO:0043634 0.0072 174.2111 0.0073 1 6 polyadenylation-dependent ncRNA catabolic process
GO:0045627 0.0072 174.2111 0.0073 1 6 positive regulation of T-helper 1 cell differentiation
GO:0071029 0.0072 174.2111 0.0073 1 6 nuclear ncRNA surveillance
GO:0071035 0.0072 174.2111 0.0073 1 6 nuclear polyadenylation-dependent rRNA catabolic process
GO:0060770 0.0084 145.1667 0.0085 1 7 negative regulation of epithelial cell proliferation involved in prostate gland development
GO:0070131 0.0084 145.1667 0.0085 1 7 positive regulation of mitochondrial translation
GO:0002819 0.0087 15.9274 0.1416 2 117 regulation of adaptive immune response
GO:0034475 0.0096 124.4206 0.0097 1 8 U4 snRNA 3'-end processing