1) Doubt about DiffBind's MA plots in general Hello, I am doing ChIP-seq analysis of H3K4me3 in human samples across different conditions. For that, my pipeline includes DiffBind to make volcano, MA plots , PCA and heatmaps. The DiffBind manual says that MA plots are good to see the effect of normalization and puts the following example (please, see picture below). The problem is that, when I consult with other people, they say I should expect that the blue density plot is symmetrical around the X axis (when my data is properly normalized). If I see this image in the manual I don't think it's symmetrical. Therefore, I was wondering that maybe the DiffBind variant of an MA plot is done differently and requires a slightly different interpretation. What's your view in the topic?
2) This problem applied to my data. A second question is: I expect that the blue density plot doesn't have to be always perfect around the X axis, but where should we "draw the line"? I am uploading one of my MA plots to illustrate this. Across my conditions (let's say incremental conditions A B C against condition H that's healthy control), the plot A vs H is symmetrical, BvsH as well, but CvsH is less (please see the second picture below, the one with many pink points). To my eyes, this is a nice result showing a transition in my biological process (because of the loss of H3K4me3 binding that you can see), but I am afraid that, because the blue density plot is too much shifted down, the result is actually not legit because of a normalization problem or something like that. I asked in Biostars and some people suggested to do the analysis in CSAW and compare MA plots. I am doing it, but I wanted to ask here these doubts about the DiffBind inner workings anyways.
3) In the second panel with the three pictures, in A vs H you can see that the majority of the sites are symmetrical to the X axis except for a blob in the upper left quadrant. What is happening there?