Last updated: 2018-06-27

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    File Version Author Date Message
    Rmd 15c0dd0 Briana Mittleman 2018-06-27 try no shift
    html 8120471 Briana Mittleman 2018-06-27 Build site.
    Rmd 5e81c8e Briana Mittleman 2018-06-27 add code for macs2 peak calling
    html ee777df Briana Mittleman 2018-06-26 Build site.
    Rmd 789d8ef Briana Mittleman 2018-06-26 start test macs analysis. download package

In this analysis I want to test macs2 as a potential peak caller in the 3’ seq data. This is a widely used peak caller for chip seq data.

I have to create a specific environment to install macs2 because you need to use python 2.7. I call it macs-env. To access this environment I use source activate macs-env.

First, I will merge all of my files into 1 bam file. Using samtools merge. I will do all of my work for this in data/macs2


#!/bin/bash

#SBATCH --job-name=merge
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END

module load samtools

samtools merge macs2/allBamFiles.bam bam/*.bam

I will create a script in the code directory to call the peaks:

#!/bin/bash

#SBATCH --job-name=macs2nomod
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=macs2nomod.out
#SBATCH --error=macs2nomod.err
#SBATCH --partition=broadwl
#SBATCH --mem=8G
#SBATCH --mail-type=END


module load Anaconda3 

source activate macs-env

macs2 callpeak -t /project2/gilad/briana/threeprimeseq/data/macs2/allBamFiles.bam  -f "BAM" -g 'hs' --outdir /project2/gilad/briana/threeprimeseq/data/macs2/ -n threeprimebatch1 --slocal 1000 --nomodel

This method called 102988 peaks. This is likely more than the true PAS.

Update the -m (MFOLD) term to change the fold enrichment. They must be lower than the upper limmit and higher than the lower limit. The default is 5 50. I will try to make this 20 100.

#!/bin/bash

#SBATCH --job-name=macs2nomod20.100
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=macs2nomod.20.100.out
#SBATCH --error=macs2nomod.20.100.err
#SBATCH --partition=broadwl
#SBATCH --mem=8G
#SBATCH --mail-type=END


module load Anaconda3 

source activate macs-env

macs2 callpeak -t /project2/gilad/briana/threeprimeseq/data/macs2/allBamFiles.bam  -f "BAM" -g 'hs' --outdir /project2/gilad/briana/threeprimeseq/data/macs2/ -n threeprimebatch1.20.100 --slocal 1000 --nomodel -m 20 100

This did not change anything. I am going to try a higher cutoff.

#!/bin/bash

#SBATCH --job-name=macs2nomod40.400
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=macs2nomod.40.200.out
#SBATCH --error=macs2nomod.40.200.err
#SBATCH --partition=broadwl
#SBATCH --mem=8G
#SBATCH --mail-type=END


module load Anaconda3 

source activate macs-env

macs2 callpeak -t /project2/gilad/briana/threeprimeseq/data/macs2/allBamFiles.bam  -f "BAM" -g 'hs' --outdir /project2/gilad/briana/threeprimeseq/data/macs2/ -n threeprimebatch1.40.200 --slocal 1000 --nomodel -m 40 200

Try to not have a shift.

#!/bin/bash

#SBATCH --job-name=macs2noshift
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=macs2nomod.noshift.out
#SBATCH --error=macs2nomod.noshift.err
#SBATCH --partition=broadwl
#SBATCH --mem=8G
#SBATCH --mail-type=END


module load Anaconda3 

source activate macs-env

macs2 callpeak -t /project2/gilad/briana/threeprimeseq/data/macs2/allBamFiles.bam  -f "BAM" -g 'hs' --outdir /project2/gilad/briana/threeprimeseq/data/macs2/ -n threeprimebatch1.noshift --slocal 1000 --nomodel --shift 0 --extsize 200

Does not look like this changed anything.

I also want to run this using seperate files for the total and nuclear fractions.

I wil first merge the total and nuclear bam files seperatly.


#!/bin/bash

#SBATCH --job-name=mergeTN
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --partition=broadwl
#SBATCH --mem=20G
#SBATCH --mail-type=END

module load samtools

samtools merge macs2/TotalBamFiles.bam bam/*T*.bam


samtools merge macs2/NuclearBamFiles.bam bam/*N*.bam

Now I can run the original call peaks on each seperatly with macs2_nomod_TN.sh.

#!/bin/bash

#SBATCH --job-name=macs2nomodTN
#SBATCH --account=pi-yangili1
#SBATCH --time=8:00:00
#SBATCH --output=macs2nomodTN.out
#SBATCH --error=macs2nomodTN.err
#SBATCH --partition=broadwl
#SBATCH --mem=8G
#SBATCH --mail-type=END


module load Anaconda3 

source activate macs-env

macs2 callpeak -t /project2/gilad/briana/threeprimeseq/data/macs2/TotalBamFiles.bam  -f "BAM" -g 'hs' --outdir /project2/gilad/briana/threeprimeseq/data/macs2/ -n Totalthreeprimebatch1 --slocal 1000 --nomodel 

macs2 callpeak -t /project2/gilad/briana/threeprimeseq/data/macs2/NuclearBamFiles.bam  -f "BAM" -g 'hs' --outdir /project2/gilad/briana/threeprimeseq/data/macs2/ -n Nuclearthreeprimebatch1 --slocal 1000 --nomodel

Session information

sessionInfo()
R version 3.4.2 (2017-09-28)
Platform: x86_64-apple-darwin15.6.0 (64-bit)
Running under: macOS Sierra 10.12.6

Matrix products: default
BLAS: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.4/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

attached base packages:
[1] stats     graphics  grDevices utils     datasets  methods   base     

loaded via a namespace (and not attached):
 [1] workflowr_1.0.1   Rcpp_0.12.17      digest_0.6.15    
 [4] rprojroot_1.3-2   R.methodsS3_1.7.1 backports_1.1.2  
 [7] git2r_0.21.0      magrittr_1.5      evaluate_0.10.1  
[10] stringi_1.2.2     whisker_0.3-2     R.oo_1.22.0      
[13] R.utils_2.6.0     rmarkdown_1.8.5   tools_3.4.2      
[16] stringr_1.3.1     yaml_2.1.19       compiler_3.4.2   
[19] htmltools_0.3.6   knitr_1.18

This reproducible R Markdown analysis was created with workflowr 1.0.1