Question: ChIP-seq differential binding with multiple replicates using MACS2 bdgdiff.
gravatar for shawn.w.foley
11 months ago by
shawn.w.foley190 wrote:


I'm trying to perform a differential binding ChIP-seq experiment and am struggling with the best way to incorporate my replicates using MACS2. I have two sets of samples, control rep 1-3 and control input, as well as treated rep 1-3 and treated input. If I run macs2 callpeak as recommended I run:

macs2 callpeak -B -t ctrl.rep1.bam ctrl.rep2.bam ctrl.rep3.bam -c ctrl.input.bam -f BAM -g hs -n ctrl.repAll.peaks --nomodel --extsize 146 --buffer-size 1000000

macs2 callpeak -B -t treat.rep1.bam treat.rep2.bam treat.rep3.bam -c treat.input.bam -f BAM -g hs -n treat.repAll.peaks --nomodel --extsize 146 --buffer-size 1000000

This will generate output files in the bedgraph format that I can then use to run macs2 bdgdiff:

macs2 bdgdiff --t1 treat.repAll_treat_pileup.bdg --t2 ctrl.repAll_treat_pileup.bdg --c1 treat.repAll_control_lambda.bdg --c2 ctrl.repAll_control_lambda.bdg -l 146 -d1 spikeIn_treat -d2 spikeIn_ctrl --o-prefix treat_vs_ctrl

However, these bedgraph files are the POOLED replicates. I want to be able to utilize my individual replicates in order to get some statistical power.

1) Does anyone have experience running macs2 bdgdiff across multiple replicates for treated/untreated samples?

2) How would I combine the log odds ratio in the macs2 bdgdiff output files to get a meaningful measurement of the difference in binding pre- and post-treatment?

I've been searching online and can't find anything. Any help would be appreciated.

chip-seq macs2 bdgdiff • 808 views
ADD COMMENTlink written 11 months ago by shawn.w.foley190

bdgdiff is not very useful, imo. I'd recommend calling peaks on each replicate individually and using something like DiffBind for more robust, believable results that are actually based on signal at your peaks. The stats are a lot more useful as well, and it integrates nicely with their QC package (ChIPQC) as well.

ADD REPLYlink written 11 months ago by jared.andrews071.8k
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