Hi, I am trying to analyze ChIP seq data for a factor which do not behave as a typical transcription factor. When I am using MACS2 i hardly get any significant peaks. Around 20. And when I perform Homer I get 20000. I am not able to understand which peak caller to go for. Macs is not even picking up peaks which I can visually see in IGV. Can somebody help me to understand the reason behind it. I am using broad peak calling option. If I decide to go ahead with Homer then on what basis I should filter my peaks. I am planning correlated binding profile with transcriptome data further.
I encountered this exact situation in the past and I made the decision to abandon both HOMER and MACS2. I spent, literally, months working with both programs using every possible parameter configuration but they were simply unable to detect the peaks correctly. As they couldn't identify peaks correctly, neither could any of the statistics from them be trusted.
Two programs that worked better for me were:
- SICER (http://home.gwu.edu/~wpeng/Software.htm)
- deepTools (http://deeptools.readthedocs.io/en/latest/index.html)
SICER takes a while to understand, but you'll just have to read the manual. You could take a look at this presentation from a colleague in Boston: http://cistrome.org/~czang/data/SICERtest/ChIPseq_SICER_CZ.pdf
With deepTools, which I would say is the superior ChIP-seq analysis suite, the
bamCoverage function will help you to find peak regions, precisely, and then there are other functions that can allow you to compare. I think that producing a bedgraph peaks file with deepTools/bamCoverage and then feeding this into SICER for differential analysis is a possibility, but that's a pipeline that I've yet to try.