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
B-cell vs. non.B-cell (based on description) 516598
Common lymphoid progenitor vs. non.Common lymphoid progenitor (based on description) 539963
Common myeloid progenitor vs. non.Common myeloid progenitor (based on description) 540402
Erythrocyte vs. non.Erythrocyte (based on description) 622247
Hematopoietic stem cell vs. non.Hematopoietic stem cell (based on description) 606222
Monocyte vs. non.Monocyte (based on description) 327297
T helper cell (CD4+) vs. non.T helper cell (CD4+) (based on description) 494457
differentiated vs. non.differentiated (based on differentiation_level) 526419
progenitor vs. non.progenitor (based on differentiation_level) 526949
stemCell vs. non.stemCell (based on differentiation_level) 606222
lymphoid vs. myeloid (based on blood_lineage) 603864
CLP vs. CMP (based on cmp_blood_CLP_CMP) 638615
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
B-cell vs. non.B-cell (based on description) 2086 431 613 686
Common lymphoid progenitor vs. non.Common lymphoid progenitor (based on description) 9034 1297 1924 919
Common myeloid progenitor vs. non.Common myeloid progenitor (based on description) 7167 953 1266 810
Erythrocyte vs. non.Erythrocyte (based on description) 20260 1808 2223 876
Hematopoietic stem cell vs. non.Hematopoietic stem cell (based on description) 33610 6281 5851 3072
Monocyte vs. non.Monocyte (based on description) 32447 3498 3083 1183
T helper cell (CD4+) vs. non.T helper cell (CD4+) (based on description) 1592 227 257 307
differentiated vs. non.differentiated (based on differentiation_level) 2059 358 486 488
progenitor vs. non.progenitor (based on differentiation_level) 4422 569 922 722
stemCell vs. non.stemCell (based on differentiation_level) 33610 6281 5851 3072
lymphoid vs. myeloid (based on blood_lineage) 2926 630 747 602
CLP vs. CMP (based on cmp_blood_CLP_CMP) 21667 2687 2995 1867
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
B-cell vs. non.B-cell (based on description) csv csv csv csv
Common lymphoid progenitor vs. non.Common lymphoid progenitor (based on description) csv csv csv csv
Common myeloid progenitor vs. non.Common myeloid progenitor (based on description) csv csv csv csv
Erythrocyte vs. non.Erythrocyte (based on description) csv csv csv csv
Hematopoietic stem cell vs. non.Hematopoietic stem cell (based on description) csv csv csv csv
Monocyte vs. non.Monocyte (based on description) csv csv csv csv
T helper cell (CD4+) vs. non.T helper cell (CD4+) (based on description) csv csv csv csv
differentiated vs. non.differentiated (based on differentiation_level) csv csv csv csv
progenitor vs. non.progenitor (based on differentiation_level) csv csv csv csv
stemCell vs. non.stemCell (based on differentiation_level) csv csv csv csv
lymphoid vs. myeloid (based on blood_lineage) csv csv csv csv
CLP vs. CMP (based on cmp_blood_CLP_CMP) 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