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I have paired end ChIP-seq data with 101 bp and 2 biological replicates for each one. I have done peak calling with macs2 but I have some questions about it.

I also faced with an warning:

WARNING @ Thu, 07 Jun 2018 17:06:05: #2 Since the d (197) calculated from paired-peaks are smaller than 2*tag length, it may be influenced by unknown sequencing problem!
WARNING @ Thu, 07 Jun 2018 17:06:05: #2 You may need to consider one of the other alternative d(s): 197
WARNING @ Thu, 07 Jun 2018 17:06:05: #2 You can restart the process with --nomodel --extsize XXX with your choice or an arbitrary number. Nontheless, MACS will continute computing.

  1. I have added --nomodel --extsize 197 ; --nomodel --extsize 147 and --nomodel --extsize 202 (separately to macs2 command) and got the results without any warning? Which one is more correct?

  2. Are broad peaks extended of narrow peaks? If I apply intersect between them I should expect find 100% overlap between narrow peaks and broad ones?

  3. Which kind of peak (narrow/broad) is proper for H3k27ac, H3k4me1, H3k4me3, H3k27me3 study?

  4. If there is no control group for using as background, can I use default parameters?

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  1. Your original command without --nomodel --extsize ... is probably the most accurate. This warning stems from a time when reads were much much shorter and likely never made that much sense to begin with.
  2. Broad peak calling in MACS2 basically works by finding a bunch of nearish narrow peaks and merging them. If you have really broad signals then use something like histoneHMM instead.
  3. H3K27ac, H3K4me1 and H3K4me3 are narrow. H3K27me3 is broad.
  4. Yeah, but you'll need to spend a bit more time with the data to ensure that the peaks are reasonable (you'll have to tweak the parameters if not).
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    $\begingroup$ Adding to Devon Ryan's answer, see this image from ENCODE. $\endgroup$
    – EagleEye
    Jun 14 '18 at 14:11

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