I used DiffBind for a collaborator who noticed that, contrary to his expectations, a particular region (which overlaps highly significant (pvalue << 0.001) peaks from MACS2 in each replicate of condition Y) got a very low FDR when compared to condition X (in which there are no peaks from any of the 4 replicates). Could you please help me to figure out if there's anything wrong in my code? Unfortunately, I cannot share the real data (it's still unpublished), but here's the anonymised experimental setup and the commands I used to compare conditions Y vs X, as well as Z vs X:
> samples SampleID Factor Condition Replicate bamReads bamControl Peaks PeakCaller 1 condX_repA TF condX 1 A-TF.bam A-I.bam A-condX.bed bed 2 condX_repB TF condX 2 B-TF.bam B-I.bam B-condX.bed bed 3 condX_repC TF condX 3 C-TF.bam C-I.bam C-condX.bed bed 4 condX_repD TF condX 4 D-TF.bam D-I.bam D-condX.bed bed 5 condY_repE TF condY 1 E-TF.bam E-I.bam E-condY.bed bed 6 condY_repF TF condY 2 F-TF.bam F-I.bam F-condY.bed bed 7 condY_repG TF condY 3 G-TF.bam G-I.bam G-condY.bed bed 8 condY_repH TF condY 4 H-TF.bam H-I.bam H-condY.bed bed 9 condZ_repJ TF condZ 1 J-TF.bam J-I.bam J-condZ.bed bed 10 condZ_repK TF condZ 2 K-TF.bam K-I.bam J-condZ.bed bed 11 condZ_repL TF condZ 3 L-TF.bam L-I.bam L-condZ.bed bed 12 condZ_repM TF condZ 4 M-TF.bam M-I.bam M-condZ.bed bed > db_analysis = dba(sampleSheet="sampleInfo.csv", minOverlap=4, bCorPlot=FALSE) > db_analysis = dba.count(db_analysis, minOverlap=4, bCorPlot=FALSE) > db_analysis = dba.contrast(db_analysis, minMembers=4, categories=DBA_CONDITION) > db_analysis = dba.analyze(db_analysis, bCorPlot=FALSE) > dba.report(db_analysis, contrast=1, th=1, file="sites_Y_vs_X", ext="txt") > dba.report(db_analysis, contrast=2, th=1, file="sites_Z_vs_X", ext="txt")
Thanks a lot for your help!