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.count() pose no issues. However, when trying to run
dba.contrast(), I repeatedly receive the same error:
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!