Question: Empty output from MACS2 when using control
0
gravatar for lm687
2.7 years ago by
lm68750
lm68750 wrote:

Hello,

I have been trying to perform peak calling with MACS2. I get an output when I don't use a control file, but blank files when I do. I'm reproducing someone else's results so there shouldn't be a problem with the input (I have only aligned the reads to the genome using BWA). I am running it with default parameters

macs2 callpeak -t SRR001992.bam -c SRR001999.bam

and changing the cutoff -q still yields blank files. Does anyone know where the problem could be? Thank you!

chip-seq macs2 peak calling • 1.9k views
ADD COMMENTlink modified 3 months ago by oriolebaltimore60 • written 2.7 years ago by lm68750
1

Can you maybe post the output of macs2?

Default params include -mfold 5 50, try lowering it to 2 50 maybe. In several datasets I cannot get any peaks with the default mfold. 

ADD REPLYlink written 2.7 years ago by rioualen280

Thank you, I have modified the parameters but I still get the same blank output. The files peaks.narrowPeak and summits.bed are empty. peaks.xls is:

# This file is generated by MACS version 2.1.0.20151222
# Command line: callpeak -t bwaSRR001992_sorted.bam -c bwaSRR001998_sorted.bam -m 2 50 -n macs2_with_control/bwaSRR001992_sorted.bam
# ARGUMENTS LIST:
# name = macs2_with_control/bwaSRR001992_sorted.bam
# format = AUTO
# ChIP-seq file = ['bwaSRR001992_sorted.bam']
# control file = ['bwaSRR001998_sorted.bam']
# effective genome size = 2.70e+09
# band width = 300
# model fold = [2, 50]
# qvalue cutoff = 5.00e-02
# Larger dataset will be scaled towards smaller dataset.
# Range for calculating regional lambda is: 1000 bps and 10000 bps
# Broad region calling is off

# tag size is determined as 26 bps
# total tags in treatment: 4374226
# tags after filtering in treatment: 3824323
# maximum duplicate tags at the same position in treatment = 1
# Redundant rate in treatment: 0.13
# total tags in control: 4090488
# tags after filtering in control: 3608175
# maximum duplicate tags at the same position in control = 1
# Redundant rate in control: 0.12
# d = 131
# alternative fragment length(s) may be 131 bps

model.r contains vectors p, m, xcorr and x.

Thank you

ADD REPLYlink written 2.7 years ago by lm68750

Another try would be to load these files in the browser and to see how similar the profiles are, that could be an another reason.

ADD REPLYlink written 2.7 years ago by Sukhdeep Singh9.5k

thank you, I will do this. Does the amount of total tags in the control seem reasonable?

ADD REPLYlink modified 2.7 years ago • written 2.7 years ago by lm68750

This I think has no limits, depending upon how much was sequenced and how the data was pre-processed. But macs integrates a scaling option, so for most cases, you don't need to worry about that.

# Larger dataset will be scaled towards smaller dataset.

ADD REPLYlink modified 2.7 years ago • written 2.7 years ago by Sukhdeep Singh9.5k

thank you for the information!

ADD REPLYlink written 2.7 years ago by lm68750

If you like someone's answer and they satisfy you, you thank people by upvoting them on Q/A platforms and forums.
Good Luck!

ADD REPLYlink written 2.7 years ago by Sukhdeep Singh9.5k

How to solve this issue? I am having a similar problem.

ADD REPLYlink written 20 months ago by xiang-jiao.yang0
1
gravatar for Sukhdeep Singh
2.7 years ago by
Sukhdeep Singh9.5k
Netherlands
Sukhdeep Singh9.5k wrote:

Another user had this problem, from the Tao's answer

Can’t see any problem. Some guesses:

  1. qvalue cutoff is too high. Try lower it such as -q 0.1
  2. disk full
  3. chromosome names in ChIP file and control file are not consistent. e.g. ‘chr1’ in ChIP but ‘chromosome1’ in control
ADD COMMENTlink modified 2.7 years ago • written 2.7 years ago by Sukhdeep Singh9.5k

Thank you very much! Unfortunately, I don't think it can be either of these

ADD REPLYlink written 2.7 years ago by lm68750
0
gravatar for darsal2
2.5 years ago by
darsal20
Chicago, IL, USA. University of Illinois at Chicago
darsal20 wrote:

Did you ever figure out a solution? I am also having the same problem. All of my output files are empty... I'm running it using these parameters:

macs2 callpeak -t 4_D.bam -c 1_D.bam -g 2.5e8 -nomodel -n 4v1rep41_test -q 0.01

ADD COMMENTlink written 2.5 years ago by darsal20
0
gravatar for macmath
13 months ago by
macmath130
France
macmath130 wrote:

Could you check the parameter specifically based on the differences between regular peak vs broad peak

Example for regular peak calling: macs2 callpeak -t ChIP.bam -c Control.bam -f BAM -g hs -n test -B -q 0.01

Example for broad peak calling: macs2 callpeak -t ChIP.bam -c Control.bam --broad -g hs --broad-cutoff 0.1

ADD COMMENTlink written 13 months ago by macmath130
0
gravatar for oriolebaltimore
3 months ago by
United States
oriolebaltimore60 wrote:

Any solution to this problem. I ran with IP and Input as control. I have 0 peaks list in xls file and also in narrowPeaks file.

Appreciate any solutions. I tried all changing q to 0.01, 0.1 , 0.5 etc. Also checked chromosome numbers formats etc.

Thanks Adrian

ADD COMMENTlink written 3 months ago by oriolebaltimore60

I think it's better to make this a reply or a separate question. But to answer your question I doubt there's much you can adjust to get more peaks. Sometimes there's just not much signal in the IP relative to control. Maybe the answer is to optimize the antibody or use another one.

ADD REPLYlink written 3 months ago by goodez260

Thanks. I checked and I suspect the control - input FASTQ file is questionable. This was obtained from SRA.

ADD REPLYlink written 3 months ago by oriolebaltimore60
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