Differential analysis

DMC or DMR/DMG

DMtools dmDMR integrates several commonly used methods for detecting differentially methylated Cytosines (DMCs) and DMR base on DM format, for example, the beta-binomial distribution model for data with replicates and Fisher’s exact test for data without replicates. In addition, DMtools can not only scan the whole genome for DMRs but also operate on predefined windows, such as gene bodies, transposable elements (TEs), untranslated regions (UTRs), and CpG islands.

For each sliding window or predefined window, differential analysis can be performed if it meets the following criteria: 1 the region contains at least m valid CpG (or non-CpG) sites (e.g., m = 5) in both samples; 2 each valid CpG site is covered by at least n bisulfite sequencing reads (e.g., n = 5). Users can choose a suitable statistical method to perform hypothesis tests. Each predefined window or sliding window acquires one p value from the selected statistical testing method. Finally, the p values are adjusted with the false discovery rate (FDR) method for multiple hypothesis testing, proposed by Benjamini and Hochberg. If the adjusted p value of DMCs is less than the predefined threshold, the number of DMCs in the window is greater than the preset threshold, and the difference of DNA ML between the two samples is greater than the preset threshold, the window is defined as a DMR.

You can get dmc and dmr result with:

Usage

DMC and DMR detect

$ dmtools dmDMR -p mutant.output.dm \
-1 mutant.methratio.dm -2 WT.methratio.dm \
--methdiff 0.2 --minstep 100 --mindmc 5 --pval 0.01

Obtained hyper、hypo dmc/dmr from dmc/dmr results

$ awk -v OFS="\t" 'gsub(/\,/,"\t",$NF)' mutant.output.dmr | \
awk '$(NF-2)>4 && $NF<=1'  > mutant.output.hyper.dmr
$ awk -v OFS="\t" 'gsub(/\,/,"\t",$NF)' mutant.output.dmr | \
awk '!($(NF-2)>4 && $NF<=1)'  > mutant.output.hypo.dmr
$ awk '$NF>0' mutant.output.dmc | awk '{print $1"\t"$2"\t"$2}' \
> mutant.output.hyper.dmc
$ awk '$NF<0' mutant.output.dmc | awk '{print $1"\t"$2"\t"$2}' \
> mutant.output.hypo.dmc

Parameters

[ Main paramaters ]
-p	output file prefix
-1	sample1 methy dm files, sperate by comma.
-2	sample2 methy dm files, sperate by comma.
--mindmc	min dmc sites in dmr region. [default : 4]
--minstep	min step in bp [default : 100]
--maxdis	max length of dmr [default : 0]
--pvalue	pvalue cutoff, default: 0.01
--FDR	adjust pvalue cutoff default : 1.0
--methdiff	the cutoff of methylation differention. default: 0.25 [CpG]
--element	caculate predefinded region, input file with id.
--context	Context for DM. CG/CHG/CHH/C, [C]
-h|--help

1. Auto define DMR region according the dmc

dmDMR -p dm.output -1 [sample1.methC.dm,replicates ..] \
-2 [sample2.methC.dm,replicates ..]

2. Pre-definded regions (Gene/TE/UTR/CDS or other regions, not suggest)

dmDMR -L -o_dm dm.output -1 [sample1.methC.dm,replicates ..] \
-2 [sample2.methC.dm,replicates ..]

Output file

1. DMC

# format
Chrom position starnd context pvalue adjust_pvalue combine_pvalue corrected_pvalue \
cover_sample1 meth_sample1 cover_sample2 cover_sample2 meth.diff

2. DMR

# format
Chrom start end methlevelInSample1 methlevelInSample2 NdmcInRegion hypermdc,hypodmc

Tip

For feature requests or bug reports please open an issue on github.