Hello! I am trying to annotate H3K9me2 called peaks(macs2) to genes. I am using the homer's annotatePeaks.pl script for the annotation and from the resulting tsv file I am filtering for promoters and genes as the scrip tries to annotate the peaks to all the genomic features of the input gtf file. My problem is that after the filtering I get more than 18.000 annotated genes(almost the whole mouse genome) and that seems to be wrong.
Is it something I am missing?
Peak calling is done with macs2(0.05 cutoff)
I only have one replicate and I use idr analysis for self consistency(reject peaks with idr >0.05)
Macs2 at the strand column outputs a dot (.) whereas annotatePeaks.pl at the help page needs a + or - strand information.
Have you run some exploratory analysis, how many peaks you get and how big they are? In particular, if you have no replicates and only a treatment vs. input call, you might already have a lot of noise in your peak calls. You might also want to try SICER instead for peak calling, which is better suited for histone ChIP-seq.
PS: There is no strand specificity in ChIP-seq. Some tools output a . , others just put + as default, but the meaning is the same. So you can just replace the dots with plus signs if needed by the downstream tool.
Thanks a lot Mathias. Your answer is very informative to me since a didnt know neither sicer nor the strand output. Thanks a lot!
You are very welcome.