Hi everyone,
I apologies for posting yet another beginners question about ChIP-seq. I don't have a bioinformatician to hand to help me so I really rely on you guys and this site to teach me how to analyse my data. Thank you in advance!
I have some ChIP-seq data (input control and IP sample) that I'm running with MACS2 (version 2.1.0) in Galaxy. I've managed to generate peaks and all the usual output files (peak.xls, peak.bed, treatment.bedgraph, control.bedgraph, narrow peaks.tab, summits.bed etc etc). From reading around here, it seems a good thing to do next is to take a look at my peaks in a genome browser. When I look at my two bedgraph files in IGB, I see peaks in my IP which is fine. However, if I then look at the peaks.xls file of my called peaks, they don't seem to reflect what I'm seeing in the IGB browser at all. For example, if I zoom into a co-ordinate that's in my peak.xls file in my bedgraph files on IGB I don't see a peak and vice versa, when I see what looks like a valid peak in IGB, it isn't called in my peak.xls file.
Can someone please point out to me what I'm misunderstanding here? I understand that sometimes called peaks aren't obvious to the eye as there are various normalizations to background and statistical modelling going on behind the scenes to call them, but still, mine aren't convincing me at all.
Help!! Please!!
Have I missed a step, am I looking at the wrong files, or completely misinterpreting something (or all 3)?
silly question: are you using the same genome built to 1) align and 2) visualize? i.e. hg19? also, did you use a control for your MACS data?
Hi TriS
Thanks for replying to me.
Yep I'm using hg19 for everything. And yes I used an input control for each of my IP samples. Am I looking at the correct files? Should the bedGRAPH files (so the treatment pile up.bdg and the control lamda.bdg files) reflect the called peaks outputted in the peaks.xls file??
Yes, you should see a bar for the bed file and a peak for the bedgraph. Do you have bedgraph for the input? I would almost tell you to try to plot the BAM files you used to call the peaks, this would tell you if there is any problem with the analysis in MACS.
Yup I have a bedgraph for the control too and I'm viewing them both (so the control and the treatment bdg files) in the genome browser at the same time. The control file looks pretty flat compared to my treatment track which I'm guessing/hoping is a good thing? I will look at loading the original BAM files into IGB as you suggest. I'm not sure if it is worth trying to run my BAM files on an older version of MACS? From what I understand, MACS1 and MACS2 and very comparable anyway??
Yes, for the control you do want a flat line :)
I don't think you will get very different results using MACS1 or MACS2. try the BAM files first. if you know a specific region you want to check (i.e. the one that gives you problems), you can extract that from the BAM file using samtools so that you will have a much smaller file to work with
Thanks TriS I'll try it out.