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!
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.