RnBeads: Example 3

Option	Value
analyze.sites	yes
differential.report.sites	yes
region.types	default
differential.permutations	0
differential.comparison.columns	Sample_ID, Sentrix_ID, Sentrix_Position, Sample_Plate, Sample_Well, Sample_Group, Passage_No, Treatment, barcode, Predicted Male Probability, Predicted Gender
differential.comparison.columns.all.pairwise	default
columns.pairing	default
differential.site.test.method	limma
differential.variability	no
differential.variability.method	diffVar
covariate.adjustment.columns	default
differential.adjustment.sva	yes
differential.adjustment.celltype	yes
differential.enrichment.go	no
differential.enrichment.lola	no
differential.enrichment.lola.dbs	${LOLACore}

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:

hESC vs. hiPSC (based on Sample_Group) [3 vs. 5]
KOSR vs. TeSR (based on Treatment) [2 vs. 2]
female vs. male (based on Predicted Gender) [5 vs. 3]

The table below summarizes information on the comparisons.

	comparison	adjustment	covariateTable
1	hESC vs. hiPSC (based on Sample_Group)	ct_1,ct_2	csv
2	KOSR vs. TeSR (based on Treatment)	ct_1,ct_2	csv
3	female vs. male (based on Predicted Gender)	ct_1,ct_2	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.

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
hESC vs. hiPSC (based on Sample_Group)	4615
KOSR vs. TeSR (based on Treatment)	0
female vs. male (based on Predicted Gender)	12118

comparison
differential methylation measure

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

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:

id: site id
Chromosome: chromosome of the site
Start: start coordinate of the site
Strand: strand of the site
mean.g1,mean.g2: (where g1 and g2 is replaced by the respective group names in the table) mean methylation in each of the two groups
mean.diff: difference in methylation means between the two groups: mean.g1-mean.g2. In case of paired analysis, it is the mean of the pairwise differences.
mean.quot.log2: log2 of the quotient in methylation: log2((mean.g1+epsilon)/(mean.g2+epsilon)), where epsilon:=0.01. In case of paired analysis, it is the mean of the pairwise quotients.
diffmeth.p.val: p-value obtained from linear models employed in the limma package (or alternatively from a two-sided Welch t-test; which type of p-value is computed is specified in the differential.site.test.method option).
max.g1,max.g2: maximum methylation level in group 1 and 2 respectively
min.g1,min.g2: minimum methylation level in group 1 and 2 respectively
sd.g1,sd.g2: standard deviation of methylation levels
min.diff: Minimum of 0 and the smallest pairwise difference between samples of the two groups
diffmeth.p.adj.fdr: FDR adjusted p-value of all sites
combinedRank: mean.diff, mean.quot.log2 and diffmeth.p.val are ranked for all sites. This aggregates them using the maximum, i.e. worst rank of a site among the three measures
num.na.g1,num.na.g2: number of NA methylation values for groups 1 and 2 respectively
mean.covg.g1,mean.covg.g2: mean coverage of groups 1 and 2 respectively (In case of Infinium array methylation data, coverage is defined as combined beadcount.)
min.covg.g1,min.covg.g2: minimum coverage of groups 1 and 2 respectively
max.covg.g1,max.covg.g2: maximum coverage of groups 1 and 2 respectively
covg.thresh.nsamples.g1,covg.thresh.nsamples.g2: number of samples in group 1 and 2 respectively exceeding the coverage threshold (5) for this site.

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
hESC vs. hiPSC (based on Sample_Group)	199	31	66	220
KOSR vs. TeSR (based on Treatment)	0	0	0	0
female vs. male (based on Predicted Gender)	2477	759	756	827

comparison
regions
differential methylation measure

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

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.

id: region id
Chromosome: chromosome of the region
Start: start coordinate of the region
End: end coordinate of the region
[symbol]: associated gene symbol to the given region [only valid for gene associated regions]
[entrezID]: Entrez ID of the gene associated with the region [only valid for gene associated regions]
mean.mean.g1,mean.mean.g2: (where g1 and g2 is replaced by the respective group names in the table) mean of mean methylation levels for group 1 and 2 across all sites in a region
mean.mean.diff: Mean difference in means across all sites in a region
mean.mean.quot.log2: log2 of the mean quotient in means across all sites in a region
comb.p.val: Combined p-value aggregating p-values of all sites in the region using a generalization of Fisher's method [1]
comb.p.adj.fdr: FDR adjusted combined p-value
combinedRank: mean.mean.diff, mean.mean.quot.log2 and comb.p.val are ranked for all regions. This column aggregates them using the maximum, i.e. worst rank of a site among the three measures
num.sites: number of sites associated with the region
mean.num.na.g1,mean.num.na.g2: Mean number of NA methylation values accross all sites in group 1 and group 2 respectively
mean.mean.covg.g1,mean.mean.covg.g2: Mean value of mean coverage values (across all samples in a group) across all sites in a region
mean.nsamples.covg.thresh.g1,mean.nsamples.covg.thresh.g2: mean number of samples (accross all considered sites) that have a coverage larger than 5 for the site in group 1 and group 2 respectively

The tables for the individual comparisons can be found here:

	tiling	genes	promoters	cpgislands
hESC vs. hiPSC (based on Sample_Group)	csv	csv	csv	csv
KOSR vs. TeSR (based on Treatment)	csv	csv	csv	csv
female vs. male (based on Predicted Gender)	csv	csv	csv	csv

GO Enrichment Analysis

No GO Enrichment Analysis was conducted

LOLA Enrichment Analysis

No LOLA Enrichment Analysis was conducted

Differential Methylation

Parameter Overview

Introduction: Differential Methylation of Sample Groups

Comparisons

P-values

Site Level

Differential Methylation Tables

Region Level

Differential Methylation Tables

GO Enrichment Analysis

LOLA Enrichment Analysis

References