Question: Dear all,

I am confused with the --broad-cutoff option in macs2.
In the manual, it said that --broad-cutoff is cutoff for broad region. This option is not available unless --broad is set. If -p is set, this is a pvalue cutoff, otherwise, it's a qvalue cutoff. DEFAULT: 0.1

If I understand it correctly, if I only set the --broad-cutoff 0.01, the cutoff will filter out broad peak with q-value > 0.01.
However, when I processing a histone modification ChIP-seq data, I got some result with q-value > 0.01 [-log10(qvalue) <2].

Here is the result file.

# This file is generated by MACS version 2.1.0.20150731
# Command line: callpeak -t /mouseAnalysis/data/align/CYJ-1_dedup.bam -c /mouseAnalysis/data/align/CYJ-1i_dedup.bam -f BAM -n CYJ-1_CYJ-1i_macs.tmp -g mm -B --broad --broad-cutoff 0.01
--fix-bimodal --nomodel --extsize=270 --outdir /mouseAnalysis/data/peakCall/
# ARGUMENTS LIST:
# name = CYJ-1_CYJ-1i_macs.tmp
# format = BAM
# ChIP-seq file = ['/mouseAnalysis/data/align/CYJ-1_dedup.bam']
# control file = ['/mouseAnalysis/data/align/CYJ-1i_dedup.bam']
# effective genome size = 1.87e+09
# band width = 300
# model fold = [5, 50]
# qvalue cutoff for narrow/strong regions = 5.00e-02
# qvalue cutoff for broad/weak regions = 1.00e-02
# Larger dataset will be scaled towards smaller dataset.
# Range for calculating regional lambda is: 1000 bps and 10000 bps
# Broad region calling is on

# tag size is determined as 49 bps
# total tags in treatment: 8279763
# tags after filtering in treatment: 8279763
# maximum duplicate tags at the same position in treatment = 1
# Redundant rate in treatment: 0.00
# total tags in control: 9357359
# tags after filtering in control: 9357359
# maximum duplicate tags at the same position in control = 1
# Redundant rate in control: 0.00
# d = 270
chr start end length pileup -log10(pvalue) fold_enrichment -log10(qvalue) name
chr11 3132147 3132916 770 46.55 5.11039 1.29032 1.91284 CYJ-1_CYJ-1i_macs.tmp_peak_1
chr11 3168653 3168940 288 39.21 4.37530 1.18763 1.48268 CYJ-1_CYJ-1i_macs.tmp_peak_2
chr11 3193021 3193581 561 46.44 5.97673 1.34145 2.72753 CYJ-1_CYJ-1i_macs.tmp_peak_3

**My questions are:**

Do I understand the --broad-cutoff correctly?
Why the filter doesn't work?

Thanks.

Answer: Yes you are correct the broad cut-off will not work unless you point out the exact pvalue or qvalue threshold. Unless you do that you will see all the reads for the broad-cutoff you set , you see a lot of data since your FDR cut-off is set at 10% and then the broad finding is applied on them. If you run both the narrow call and broad call with the q-value 10% and for broad you ad extra paramteer of --broad-cutoff 0.01 then you can fetch the length of the peaks and plot a histogram to understand the peak length distribution to understand how broad are the peaks. So to properly apply the --broad-cutoff you have to play with p-value or q-value score along with the broad-cutoff option. You can take a look [here][1] you can see for borad calls you need to set a stringent p or q value cut-off parameters along with broad-cutoff to get proper output. This is not very clearly mentioned in the document so you need to check it yourself using varied runs to see what you get in terms of the peaks.

[1]: http://crazyhottommy.blogspot.it/2015/09/macs2-parallel-peak-calling.html