Question: Peak Calling with MACS, Analysis with DiffBind, Danio Rerio!
0
gravatar for kyle.s.shank
2.6 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 • 2.1k views
ADD COMMENTlink modified 2.6 years ago • written 2.6 years ago by kyle.s.shank0

Hi,

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 2.1 years ago by YaGalbi1.4k
0
gravatar for Sinji
2.6 years ago by
Sinji2.8k
UT Southwestern Medical Center
Sinji2.8k 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 2.6 years ago • written 2.6 years ago by Sinji2.8k
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