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import argparse
import sys
import os
import glob
#this is built for mapping single end nascent data!!! This program expects a indir and a outdir. They must both end in a slash.
#First choose which steps do you want to run
inputfiletype=".fastq"
#Flip reads is required for some protocols such as PRO-seq and the NEB random prime kit
flipreads = True
#Check read quality?
checkquality=True
#Trim adaptors?
trimgalore = True
fastqtosam = True
samtobam = True
bamtosortedbam = True
sortedbamtobai = True
sortedbamtobedgraph = True
readcountcorrectbedgraph = True
igvcreate = True
millionsmappedcount = True
submit=True
#What bowtie options do you want to use
bowtieoptions = "--very-sensitive"
processers="1"
if fastqtosam==True:
processers="32"
queue="long"
memamount="200"
memtype="gb" #gb, mb, kb
time="48:00:00" #hours:min:seconds
igvdir="/opt/igvtools/2.3.75/"
#-------- these are variables you probably don't need to check
bowtieindexdic = {"hg19":"/scratch/Shares/dowell/pubgro/genomefiles/bowtiebwaindexs/hg19_Bowtie2_index", "mm10":"/scratch/Shares/dowell/pubgro/genomefiles/bowtiebwaindexs/mm10_Bowtie2_index", "dm3":"/scratch/Shares/dowell/pubgro/genomefiles/bowtiebwaindexs/dm3.fa.Bowtie2"}
memdic = {"gb":"G", "kb":"K", "mb":"M"}
samtoolsmem = memamount+memdic[memtype]
sbatchmem = memamount+memtype
#--------------all scripts below this line
def flipfile(passfile, outdir, wfname):
wf = open(wfname, "a")
rootname = passfile.split("/")[-1]
rootname = rootname.strip(".fastq")
outfastqdir = outdir+"flipped/"
ensure_dir(outfastqdir)
outfastq = outfastqdir+rootname+".flip.fastq"
wf.write("date\n")
wf.write("\n")
wf.write("module load fastx-toolkit/0.0.13\n")
wf.write("/opt/fastx-toolkit/0.0.13/bin/fastx_reverse_complement -Q33 -i "+passfile+" -o "+outfastq+" \n")
wf.write("echo flipped\n")
wf.write("date\n")
wf.close()
return outfastq
def checkqualityfile(passfile, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
wf.write("module load fastqc/0.11.5\n")
qualoutdir = outdir+"qual/"
ensure_dir(qualoutdir)
wf.write("fastqc "+passfile+" -o "+qualoutdir+"\n")
wf.write("echo qual\n")
wf.write("date\n")
wf.close()
def trimgalorefile(passfile, outdir, wfname):
rootname = passfile.split("/")[-1]
rootname = rootname.strip(".fastq")
cutoutdir = outdir+"cutadapt/"
ensure_dir(cutoutdir)
#MA_DMSO_Groseq_S7_R1_001.flip_trimmed.fq
#MA_DMSO_Groseq_S7_R1_001.flip.fastq
trimmedfastq = cutoutdir+rootname+"_trimmed.fq"
trimmedfastq2 = cutoutdir+rootname+".trimmed.fastq"
wf = open(wfname, "a")
wf.write("date\n")
wf.write("\n")
wf.write("module load python/2.7.14/cutadapt/1.12\n")
wf.write("module load trim_galore/0.4.3\n")
wf.write("echo $PATH\n")
wf.write("echo $PYTHONPATH\n")
wf.write("/opt/trim_galore/0.4.3/trim_galore --path_to_cutadapt /opt/cutadapt/python-2.7.14/1.12/bin/cutadapt -o "+cutoutdir+" "+passfile+"\n")
wf.write("mv "+trimmedfastq+" "+trimmedfastq2+"\n")
wf.write("echo trimmed\n")
wf.write("date\n\n\n")
wf.close()
return trimmedfastq2
def bowtie2file(passfile, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
wf.write("module load samtools/1.3.1\n")
wf.write("module load bowtie/2.2.9\n\n")
samdir = outdir+"sams/"
ensure_dir(samdir)
rootname = passfile.split("/")[-1]
rootname = rootname.strip(".fastq")
samfile = samdir+rootname+".sam"
stderrfile = samdir+rootname+".stderr"
bowtieindex = bowtieindexdic[genome]
wf.write("bowtie2 -p "+processers+" "+bowtieoptions+" -x "+bowtieindex+" -U "+passfile+" >"+samfile+" 2>"+stderrfile+" \n")
wf.write("echo mapped\n")
wf.write("date\n")
wf.close()
return samfile
def samtobamfile(passfile, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
wf.write("wc -l "+passfile+" > "+passfile+".wc\n")
rootname=passfile.split("/")[-1]
rootname=rootname.strip(".sam")
bamoutdir = outdir+"bams/"
ensure_dir(bamoutdir)
bamfile = bamoutdir+rootname+".bam"
wf.write("samtools view -S -b -o "+bamfile+" "+passfile+" 2>"+bamfile+".err\n")
wf.write("samtools flagstat "+bamfile+" > "+bamfile+".flagstat 2>"+bamfile+".flagstat.err\n")
wf.write("echo bam\n")
wf.write("date\n")
wf.close()
return bamfile
def bamtosortedbamfile(bamfile, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
sortedbamfile = bamfile.strip(".bam")+".sorted.bam"
sortedbamdir = outdir+"sortedbams/"
ensure_dir(sortedbamdir)
sortedbamfile = sortedbamdir+sortedbamfile.split("/")[-1]
wf.write("samtools sort -m "+samtoolsmem+" "+bamfile+" >"+sortedbamfile+"\n")
wf.write("samtools flagstat "+sortedbamfile+" >"+sortedbamfile+".flagstat 2>"+sortedbamfile+".flagstat.err\n")
wf.write("echo sorted.bam\n")
wf.write("date\n")
return sortedbamfile
def sortedbamtobai(sortedbam, wfname):
wf = open(wfname, "a")
wf.write("date\n")
wf.write("samtools index "+sortedbam+"\n")
wf.write("echo indexed.bam\n")
wf.write("date\n")
def sortedbamtobedgraphfile(sortedbam, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
wf.write("module load bedtools/2.25.0\n")
wf.write("echo bedgraph\n")
rootname = sortedbam.split("/")[-1]
rootname = rootname.strip(".sorted.bam")
Bedgraphoutdir = outdir+"/bedgraphs/"
ensure_dir(Bedgraphoutdir)
wf.write("genomeCoverageBed -bg -strand + -ibam "+sortedbam+" -g "+genome+" > "+Bedgraphoutdir+rootname+".pos.BedGraph\n")
wf.write("genomeCoverageBed -bg -strand - -ibam "+sortedbam+" -g "+genome+" | awk -F '\t' -v OFS='\t' '{ $4 = - $4 ; print $0 }' > "+Bedgraphoutdir+rootname+".neg.BedGraph\n")
wf.write("cat "+Bedgraphoutdir+rootname+".pos.BedGraph "+Bedgraphoutdir+rootname+".neg.BedGraph > "+Bedgraphoutdir+rootname+".unsorted.BedGraph\n")
wf.write("sortBed -i "+Bedgraphoutdir+rootname+".unsorted.BedGraph >"+Bedgraphoutdir+rootname+".BedGraph\n")
wf.write("date\n")
Bedgraphfile = Bedgraphoutdir+rootname+".BedGraph"
return Bedgraphfile
def readcountcorrectbedgraphfile(Bedgraphfile, sortedbam, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
wf.write("echo readcountcorrectedbedgraph\n")
wf.write("module load python/2.7.14\n")
#need to copy readcountcorrectBG.py to out dir
rootname = Bedgraphfile.strip(".BedGraph")
flagstatfile = sortedbam+".flagstat"
readcountcorrBedgraphfile = rootname +".mp.BedGraph"
wf.write("python "+outdir+"readcountcorrectBG.py "+Bedgraphfile+" "+flagstatfile+" "+readcountcorrBedgraphfile+"\n")
wf.write("date\n")
return readcountcorrBedgraphfile
def igvcreatefile(readcountcorrBedgraphfile, outdir, wfname):
wf = open(wfname, "a")
wf.write("date\n")
tdffiledir=outdir+"tdfs/"
ensure_dir(tdffiledir)
rootname = readcountcorrBedgraphfile.split("/")[-1]
rootname = rootname.strip(".mp.BedGraph")
tdffile = tdffiledir+rootname+".tdf"
igvgenomefile = igvdir+"/genomes/"+genome+".chrom.sizes"
wf.write(igvdir+"/igvtools toTDF "+readcountcorrBedgraphfile+" "+tdffile+" "+igvgenomefile+"\n")
wf.write("echo tdf\n")
wf.write("date\n")
def ensure_dir(file_path):
directory = os.path.dirname(file_path)
if not os.path.exists(directory):
os.makedirs(directory)
def main(indir, outdir):
#make outdir
ensure_dir(outdir)
#make e_and_o
os.system("cp --no-clobber readcountcorrectBG.py "+outdir+"\n")
ensure_dir(outdir+"e_and_o/")
indirfiles=[infile for infile in glob.glob(os.path.join(indir, '*'+inputfiletype))]
wf2 = open(outdir+"rsync_out.sh", "w")
for filename in indirfiles:
passfile = filename
rootname = filename.split("/")[-1]
rootname = rootname.strip(inputfiletype)
wfname = outdir+rootname+"_map.slurm"
wf = open(wfname, "w")
wf.write("#!/bin/bash\n")
wf.write("#SBATCH --job-name="+rootname+" # Job name\n")
wf.write("#SBATCH --mail-type=ALL # Mail events (NONE, BEGIN, END, FAIL, ALL)\n")
wf.write("#SBATCH --mail-user="+email+"# Where to send mail\n")
wf.write("#SBATCH --nodes=1\n")
wf.write("#SBATCH --ntasks="+processers+"# Number of CPU (processer cores i.e. tasks) In this example I use 32 for bowtie2. I only need one, since none of the commands I run are parallelized.\n")
wf.write("#SBATCH --time="+time+" # Time limit hrs:min:sec\n")
wf.write("#SBATCH -p "+queue+"\n")
wf.write("#SBATCH --mem="+sbatchmem+" # Memory limit\n")
wf.write("#SBATCH --output="+outdir+"e_and_o/"+rootname+".%j.out\n")
wf.write("#SBATCH --error="+outdir+"e_and_o/"+rootname+".%j.err\n")
wf.write("\n\n\npwd; hostname; date\n")
wf.close()
if flipreads==True:
passfile = flipfile(passfile, outdir, wfname)
if checkquality==True:
checkqualityfile(passfile, outdir, wfname)
if trimgalore==True:
passfile = trimgalorefile(passfile, outdir, wfname)
checkqualityfile(passfile, outdir, wfname)
if fastqtosam==True:
passfile = bowtie2file(passfile, outdir, wfname)
if samtobam==True:
passfile = samtobamfile(passfile, outdir, wfname)
if bamtosortedbam==True:
sortedbam = bamtosortedbamfile(passfile, outdir, wfname)
if sortedbamtobai==True:
sortedbamtobai(sortedbam, wfname)
if sortedbamtobedgraph==True:
Bedgraphfile = sortedbamtobedgraphfile(sortedbam, outdir, wfname)
if readcountcorrectbedgraph==True:
readcountcorrBedgraphfile = readcountcorrectbedgraphfile(Bedgraphfile, sortedbam, outdir, wfname)
if igvcreate==True:
igvcreatefile(readcountcorrBedgraphfile, outdir, wfname)
wf = open(wfname, "a")
wf.write("date\n")
wf.close()
if submit==True:
os.system('sbatch '+wfname)
if submit==True:
print "To check if your scripts are still runing, type squeue or qstat on the command line."
print "If your scripts error they are in "+outdir
print "Once your jobs have ended check these files for quality reports in "+outdir+"qual/."
print "Once jobs are complete you can use the files in rsync_out.sh to move the imporant files to projects."
else:
print "instructions"
wf2.close()
if __name__=="__main__":
parser = argparse.ArgumentParser(description='Make a slurm script for processing GRO-seq data and submit it to the queue on fiji.')
parser.add_argument("indir", help="Directory with fastq or bam files. Must be located on /scratch/.", type=str)
parser.add_argument("outdir", help="Directory with fastq or bam files. Must be located on /scratch/.", type=str)
args = parser.parse_args()
print args.indir
# if len(sys.argv)<2:
# print "to run"
# print "python create_scipts_to_map_on_fiji.py <indir> <outdir> <genome> <your_email>"
# print "\n\n"
# print "genome options are hg19, mm10, dm3"
# print "Remember that the indir(in direcory) and outdir(out directory) must be on /scratch/!!!"
# print "But you should ALWAYs keep a copy of raw data on /projects/ becuase /projects/ is backed up and scratch is not."
# else:
# indir = sys.argv[1]
# outdir = sys.argv[2]
# genome = sys.argv[3]
# email=sys.argv[4]
# if indir.startswith("/scratch/") and outdir.startswith("/scratch/"):
#
# main(indir, outdir)
# else:
# print "Your in directory and out directory must be on /scratch/", indir, outdir
#test
#python create_scipts_to_map_on_fiji.py /scratch/Users/allenma/171025_NB501447_0179_fastq/Allen_Dowell-371/ /scratch/Users/allenma/pro/ hg19 allenma@colorado.edu