Question: Peak Calling with MACS, Analysis with DiffBind, Danio Rerio!
gravatar for kyle.s.shank
3.6 years ago by
kyle.s.shank0 wrote:

Hello all,

This is a broad question - so forgive the length and thanks in advance for any/all help!

Our lab is doing its first ATAC-seq analysis on Dario Rerio. We received six total FASTQ batches (3 treatment, 3 control). Performed alignment with bowtie, some transforming with samtools, and performed peak calling in MACS. Here is the basic MACS call:

#macs2 callpeak --name macs2 -t <treatment reads>.bam -c <control reads>.bam --outdir <outdir> -f BAM --name <treatment> --nomodel -p 1e-5 -g 1464443456  --shift -100 --extsize 200 --bdg

following advice seen elsewhere on this site/the MACS2 Github account for the --shift -100 --extsize 200 arguments.

Everything looks great in IGV: fairly consistent peaks across all three replicates. Went to perform the analysis in DiffBind, and I'm running into a wall. sampleSheet looks like this:

SampleID / Tissue / Condition /Replicate / bamReads /ControlID /bamControl / Peaks / PeakCaller

V1 /V / Treated / 1 / V1_GT16_02784_S1sfm.bam / V0 / V0_GT16_02781_S4sfm.bam / V_Peaks.xls / macs
W1  /W / Treated / 2 / W1_GT16_02785_S2sfm.bam / W0 / W0_GT16_02782_S5sfm.bam / W_Peaks.xls / macs
X1 / X /Treated / 3 / X1_GT16_02786_S3sfm.bam / X0 / X0_GT16_02783_S6sfm.bam / X_Peaks.xls / macs

Reading in works fine: dba() and dba.count() pose no issues. However, when trying to run dba.contrast(), I repeatedly receive the same error: Warning message: No contrasts added. Perhaps try more categories, or lower value for minMembers. I understand that this may very well be due to there actually being no contrasts to find in the data, but is there a chance that I should also be reading in the Peak files for the control sets as well to allow for contrasts to be found? I thought that would be the incorrect process, but I admit that I'm unsure.

Thanks again for any and all help!

atac-seq diffbind • 2.7k views
ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by kyle.s.shank0


Did you manage to finish the analysis with Diffbind? Was it the best tool to use? I'm analysing ATAC-seq data for the first time here at our institute and have also just finished MACS2 peak calling.

ADD REPLYlink written 3.0 years ago by YaGalbi1.5k
gravatar for Sinji
3.6 years ago by
UT Southwestern Medical Center
Sinji3.0k wrote:

I'm unfortunately not familiar with the DiffBind package, but I do work quite a bit with the DiffReps stand alone command line program so that might be something to try instead since it's easier to use. It's built for ChIP-seq, but it should work ATAC-seq I believe.

ADD COMMENTlink modified 3.6 years ago • written 3.6 years ago by Sinji3.0k
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