Last updated: 2018-07-30

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    File Version Author Date Message
    Rmd 422a428 Briana Mittleman 2018-07-30 add peak cove pipeline and combined lane qc

I need to create a processing pipeline that I can run each time I get more individuals that will do the following:

  • combine all total and nuclear libraries (as a bigwig/genome coverage)

  • call peaks with Yang’s script

  • filter peaks with Yang’s script

  • clean peaks

  • run feature counts on these peaks for all fo the individuals

Create bedgraph and bigwig:

I can do this step in my snakefile. First, I added the following to my environemnt.

  • ucsc-bedgraphtobigwig
  • ucsc-bigwigmerge
  • ucsc-wigtobigwig
  • ucsc-bigwigtobedgraph

I want to create bedgraph for each file. I will add a rule to my snakefile that does this and puts them in the bedgraph directory.

#add to directory
dir_bedgraph= dir_data + "bedgraph/"

#add to rule_all  

expand(dir_bedgraph + "{samples}.bg", samples=samples)

#rule
rule bedgraph: 
  input:
    bam = dir_sort + "{samples}-sort.bam"
  output: dir_bedgraph + "{samples}.bg"
  shell: "bedtools genomecov -ibam {input.bam} -bg -5 > {output}"

I want to add more memory for this rule in the cluster.json

"bedgraph" :
    {
            "mem": 16000
    }

I will use the bedgraphtobigwig tool.

#add to directory
dir_bigwig= dir_data + "bigwig/"
dir_sortbg= dir_data + "bedgraph_sort/"

#add to rule_all  
expand(dir_sortbg + "{samples}.sort.bg", samples=samples)
expand(dir_bigwig + "{samples}.bw", samples=samples)

rule sort_bg:
    input: dir_bedgraph + "{samples}.bg"
    output: dir_sortbg + "{samples}.sort.bg"
    shell: "sort -k1,1 -k2,2n {input} > {output}"

rule bg_to_bw:
    input: 
        bg=dir_sortbg + "{samples}.sort.bg"
        len= chrom_length 
    output: dir_bigwig + "{samples}.bw"
    shell: "bedGraphToBigWig {input.bg} {input.len} {output}""

Merge BW

This next step will take all of the files in the bigwig directory and merge them. To do this I will create a script that creates a list of all of the files then uses this list in the merge script.

mergeBW.sh

#!/bin/bash

#SBATCH --job-name=mergeBW
#SBATCH --account=pi-yangili1
#SBATCH --time=24:00:00
#SBATCH --output=mergeBW.out
#SBATCH --error=mergeBW.err
#SBATCH --partition=broadwl
#SBATCH --mem=40G
#SBATCH --mail-type=END

module load Anaconda3
source activate three-prime-env

ls -d -1 $PWD/* /project2/gilad/briana/threeprimeseq/data/bigwig | tail -n +2 > /project2/gilad/briana/threeprimeseq/data/list_bw/list_of_bigwig.txt

bigWigMerge -inList /project2/gilad/briana/threeprimeseq/data/list_bw/list_of_bigwig.txt /project2/gilad/briana/threeprimeseq/data/mergedBW/merged_combined_YL-SP-threeprimeseq.bg

The result of this script will be a merged bedgraph of all of the files.

Session information

sessionInfo()
R version 3.5.1 (2018-07-02)
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/3.5/Resources/lib/libRblas.0.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/3.5/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_1.1.1   Rcpp_0.12.18      digest_0.6.15    
 [4] rprojroot_1.3-2   R.methodsS3_1.7.1 backports_1.1.2  
 [7] git2r_0.23.0      magrittr_1.5      evaluate_0.11    
[10] stringi_1.2.4     whisker_0.3-2     R.oo_1.22.0      
[13] R.utils_2.6.0     rmarkdown_1.10    tools_3.5.1      
[16] stringr_1.3.1     yaml_2.1.19       compiler_3.5.1   
[19] htmltools_0.3.6   knitr_1.20

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