<ol>
<li>Your original command without <code>--nomodel --extsize ...</code> 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.</li>
<li>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 <code>histoneHMM</code> instead.</li>
<li>H3K27ac, H3K4me1 and H3K4me3 are narrow. H3K27me3 is broad.</li>
<li>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).</li>
</ol>

CC BY-SA 4.0
2018-06-14T14:11:23.530Z
Adding to Devon Ryan's answer, see [this image](https://i.sstatic.net/olCjj.png) from [ENCODE](https://www.encodeproject.org/chip-seq/histone/).
EagleEye
CC BY-SA 4.0
2018-06-07T18:52:02.940Z
2018-06-07T18:52:02.940Z
Devon Ryan
<p>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.</p>

<p>I also faced with an warning:</p>

<blockquote>
  <p>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!<br>
  WARNING @ Thu, 07 Jun 2018 17:06:05: #2 You may need to consider one of the other alternative d(s): 197<br>
  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. </p>
</blockquote>

<ol>
<li><p>I have added <code>--nomodel --extsize 197 ; --nomodel --extsize 147</code> and <code>--nomodel --extsize 202</code> (separately to macs2 command) and got the results without any warning? Which one is more correct?</p></li>
<li><p>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?</p></li>
<li><p>Which kind of peak (narrow/broad) is proper for H3k27ac, H3k4me1, H3k4me3, H3k27me3 study?</p></li>
<li><p>If there is no control group for using as background, can I use default parameters?</p></li>
</ol>

CC BY-SA 4.0
2018-06-07T19:10:19.570Z
cross posted: https://www.biostars.org/p/319418/
Pierre
<chip-seq>
Macs2 peak calling?
# Macs2 peak calling?

(Asked by: username_0 on 2018-06-07)

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?

**Tags:** chip-seq
> **Comments:**
> **username_1**: cross posted: https://www.biostars.org/p/319418/  

---

## 1 answers

### Answer by username_2 on 2018-06-07. Score: 4 | Accepted answer

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).

> **Comments:**
> **username_3**: Adding to Devon Ryan's answer, see [this image](https://i.sstatic.net/olCjj.png) from [ENCODE](https://www.encodeproject.org/chip-seq/histone/).  

---

