Normalizing Peaks in ChIP seq

Question

[mropri]

Hi,

I have run peak calling using MACS2 on 3 samples and get a narrowPeaks file for each sample. First, does MACS do local normalizing when calling peaks, meaning is each sample peak intensity normalized? Also, can I now compare the peak intensities between sample or do I have to do global normalizing meaning normalizing the peak intensities of the 3 samples together after peak calling ? Lastly, the narrowPeaks file should output 10 columns from MACS2 according to this https://hbctraining.github.io/Intro-to-ChIPseq/lessons/05_peak_calling_macs.html. However, I get a file with 5 columns like the image below. Is the 5th column the normalized peak intensities? Appreciate the help and time to answer my questions.

[ATpoint]

The only reliable comparison I could think of would be a differential analysis that requires groups of samples and experimental replication. What exactly do you want to show?

[mropri]

Hi,

I have more samples in the pipeline and will have 6 total files with 3 replicates for 2 conditions. I just wanted to be sure that 5th column is showing normalized peak intensities and that I can compare them as is between control and treatment or do I need to do additional normalization. Appreciate your help and comment.

[Ram]

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