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wmd.0.3.py
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#!/usr/local/bin/python2.7
import sys
import subprocess
import argparse
import re
import cogent
from cogent.db.ncbi import EFetch
import cogent.parse.genbank
import matplotlib
import pysam
from Bio import SeqIO
import os
import time
#import ggplot
import math
import smtplib
import jinja2
def main():
global args
global refName
global refNamePrefix
global mappingStatsTable
readStatsTable = []
filteredStatsTable = []
mappingStatsTable = {}
gconfStatsTable = {}
templateLoader = jinja2.FileSystemLoader( searchpath="/opt/wmd/" )
templateEnv = jinja2.Environment( loader=templateLoader )
TEMPLATE_FILE = 'wmd-template.html'
template = templateEnv.get_template(TEMPLATE_FILE)
# recognize zipped files
# zip filtered fastq files
# correct % read bases mapped
# calculate percent valid unique
# output results to table
# add MrFast, crack, soap
# enable output of each subroutine to be passed to whatever subsequent subroutine
# find longest common substring for paired end
# get valid pairs
parser = argparse.ArgumentParser(description='Execute modular SNV analysis pipeline.')
parser.add_argument('--name', '-n', metavar='name', nargs='+', help='Sample names (exculding .fastq)', required=True)
parser.add_argument('--mappers', '-m', metavar='map1', nargs='+',help='Mapping algorithms to execute (default: novoalign)', default='novoalign', choices=['bowtie2','novoalign','bwa'])
parser.add_argument('--variant_callers', '-v', metavar='vc1', nargs='+', help='Variant calling algorithms to execute (default gconf)', default='gconf',\
choices=['samtools','gconf','gatk','varscan'])
parser.add_argument('--reference','-r', metavar='ref', nargs=1, help='Fasta reference (gi, accession number, or fasta file).', required=True)
parser.add_argument('--threads', '-t', metavar='t', nargs=1, help='Number of threads to run for each program (default: 8)', default=8, type=int)
parser.add_argument('--all', '-a', help='Run all mappers and variant callers', action='store_true')
parser.add_argument('--mincov','-c',help='Minimum coverage to call variant',default=4, type=int)
parser.add_argument('--paired','-p',help='Samples are paired-end/mate-pair. Prefix MUST be the same between both samples with corresponding *.1 *.2 appendices',action='store_true')
parser.add_argument('--minqual','-q',help='Minimum mean quality score to filter reads (default: 12)', default=12, type=int)
parser.add_argument('--illumina', help='Illumina quality encoding', action='store_true')
parser.add_argument('--percent',help='Percent of reads to use per fastq file', type=float, default=100)
parser.add_argument('--delete_indices', help='Delete reference indices. (default:off)', action='store_true')
parser.add_argument('--email', help='Send email to addresses when run has completed.', type=str, nargs='+', metavar='[email protected]')
parser.add_argument('--verbose', '-V' ,help='Verbose output', action='store_true')
args = parser.parse_args()
# use glob to find paired end files
if args.all:
args.mappers = ['bowtie2','novoalign','bwa']
if os.path.isfile('wmd-gconf-bam-files.txt'):
os.remove('wmd-gconf-bam-files.txt')
if '.fasta' in args.reference[0] or '.fa' in args.reference[0]:
refName = args.reference[0]
else:
refName = args.reference[0]+'.fasta'
print 'Downloading reference from NCBI'
ef = EFetch(id=args.reference[0], rettype='fasta')
lines = ef.read().splitlines()
with open(refName, 'w') as f:
for line in lines:
f.write(line+'\n')
print 'Download successful'
print 'Indexing reference'
refNamePrefix = refName.split('.fa')
bowtie2index = ['bowtie2-build','-q',refName,refName]
novoindex = ['novoindex',refNamePrefix[0]+'.ndx',refName]
bwaindex = ['bwa','index','-a','bwtsw',refName]
samtoolsindex = ['samtools','faidx',refName]
indexCommands = []
if 'bowtie2' in args.mappers\
and not os.path.isfile(refName+'.1.bt2')\
and not os.path.isfile(refName+'.2.bt2')\
and not os.path.isfile(refName+'.3.bt2')\
and not os.path.isfile(refName+'.4.bt2')\
and not os.path.isfile(refName+'.rev.1.bt2')\
and not os.path.isfile(refName+'.rev.2.bt2'): indexCommands.append(bowtie2index)
if 'novoalign' in args.mappers\
and not os.path.isfile(refNamePrefix[0]+'.ndx'): indexCommands.append(novoindex)
if 'bwa' in args.mappers\
and not os.path.isfile(refName+'.pac')\
and not os.path.isfile(refName+'.ann')\
and not os.path.isfile(refName+'.bwt')\
and not os.path.isfile(refName+'.amb')\
and not os.path.isfile(refName+'.pac'): indexCommands.append(bwaindex)
if not os.path.isfile(refName+'.fai'): indexCommands.append(samtoolsindex)
processes=[]
for index in indexCommands:
processes.append(subprocess.Popen(index))
exit_codes =[p.wait() for p in processes]
print 'Done indexing reference'
if not os.path.isfile(refNamePrefix[0]+'.dict'):
print 'Making sequence dictionary'
subprocess.call(['java','-jar','/opt/wmd/picard-tools-1.104/CreateSequenceDictionary.jar','R=',refName,'O=',refNamePrefix[0]+'.dict','QUIET=','true'])
#remove trailing .fastq
temp = [x.split('.fastq')[0] for x in args.name]
args.name = temp
for sampleName in args.name:
# echo "echo blah" | qsub
#qsub -hold_jid job1, job2 ... to wait for all of the jobs to be completed
#Popen.wait = checks to see if the Popen process is done
# save all bam files and run gconf on them at once
#prefix = sampleName.split('.fastq')
#sampleName = prefix[0]
print 'Starting\t',sampleName
print 'Quality filtereing reads'
prinseq = ['prinseq-lite.pl']
if args.paired:
prinseq.extend(['-fastq',sampleName+'.1.fastq','-fastq2',sampleName+'.2.fastq'])
else:
prinseq.extend(['-fastq',sampleName+'.fastq'])
prinseq.extend(['-min_len','50','-min_qual_mean',str(args.minqual),'-out_good',sampleName+'.filtered','-out_bad','null'])
#if args.illumina:
#prinseq.insert(1,'-phred64')
#with open(sampleName+'.filtered.prinseq-stats.tsv','w') as stats:
prinseqProc = subprocess.Popen(prinseq,stderr=subprocess.PIPE)
prinseqOut = prinseqProc.communicate()
data = prinseqOut[1].split('\n\t')
data.pop(0)
#data.remove('Sequences filtered by specified parameters:')
#print data
filtered = False
for entry in data:
row = entry.split(':')
if 'Sequences filtered by specified parameters:' in entry:
filtered = True
elif filtered:
filteredStatsTable.append([row[0].strip(),row[1].strip()])
else:
readStatsTable.append([row[0].strip(),row[1].strip()])
#print readStatsTable
if args.paired:
subprocess.call(['mv',sampleName+'.filtered_1.fastq',sampleName+'.1.filtered.fastq'])
subprocess.call(['mv',sampleName+'.filtered_2.fastq',sampleName+'.2.filtered.fastq'])
#if args.paired:
#return
#subprocess.call(['prinseq-lite.pl','-fastq',sampleName+'.1.fastq','fastq2',sampleName+'.2.fastq','-min_len','50','-min_qual_mean',str(args.minqual),\
#'-out_good',sampleName+'.filtered','-out_bad','null'],stderr=stats)
#else:
#subprocess.call(['prinseq-lite.pl','-fastq',sampleName+'.fastq','-min_len','50','-min_qual_mean',str(args.minqual),'-out_good',sampleName+'.filtered',\
#'-out_bad','null'],stderr=stats)
#subprocess.call(['fastq_quality_filter','-Q33', '-q 20', '-p 75', '-i',i+'.fastq', '-o',i+'.filtered.fastq'])
print 'Done filtering'
if not args.paired:
print 'Calculating read quality statistics'
if not os.path.exists(sampleName+'_filtered_fastqc'):
os.makedirs(sampleName+'_filtered_fastqc')
subprocess.call(['fastqc','-t', str(args.threads), '-o', sampleName+'_filtered_fastqc', sampleName+'.filtered.fastq'])
print 'Done calculating read quality statistics'
#sampleName = sampleName+'.filtered'
#subprocess.call(['ln','-s',refName,sampleName+'.ref.fasta'])
#subprocess.call(['ln','-s',refName+'.fai',sampleName+'.ref.fasta.fai'])
with open(sampleName+'.log','w') as log:
log.write('Starting\t'+sampleName+'\n')
sample = WMD(sampleName)
if 'bowtie2' in args.mappers:
#bowtie2(i)
sample.runBowtie2()
#gatkPreProcessing(sampleName,'bowtie2')
#gconf(i,'bowtie2')
#MapStats(sampleName,'bowtie2')
if 'novoalign' in args.mappers:
#novoalign(i)
sample.runNovoalign()
#gatkPreProcessing(sampleName,'novoalign')
#gconf(i,'novoalign')
#MapStats(sampleName,'novoalign')
if 'bwa' in args.mappers:
#bwa(i)
sample.runBwa()
#gatkPreProcessing(sampleName,'bwa')
#gconf(i,'bwa')
#MapStats(sampleName,'bwa')
#if '.fasta' in args.reference[0] or '.fa' in args.reference[0]:
# subprocess.call(['unlink',sampleName+'.ref.fasta'])
gconf('wmd-gconf-bam-files.txt')
templateVars = {'statsTitle' : 'WMD Read Statistics',
'readStatsTable' : readStatsTable,
'filteredTitle' : 'Sequences filtered by specified parameters',
'filteredStatsTable' : filteredStatsTable
}
outputHTML = template.render(templateVars)
with open('wmd-stats.html','w') as html:
html.write(outputHTML)
if args.email:
message = """From: WMD <[email protected]>
Subject: WMD Run Finished
Your WMD run has completed. DO NOT REPLY.
"""
s = smtplib.SMTP('localhost')
s.sendmail('[email protected]',args.email,message)
s.quit()
return
class WMD(object):
def __init__(self, name):
self.name = name+'.filtered'
self.first = name+'.1.filtered'
self.second = name+'.2.filtered'
#index commands
#self.bowtie2index = ['bowtie2-build','-q',self.name+'.ref.fasta',self.name+'.ref.fasta']
#self.novoindex = ['novoindex',self.name+'.ref.ndx',self.name+'.ref.fasta']
#self.bwaindex = ['bwa','index','-a','bwtsw',name+'.ref.fasta']
#mapping commands
self.bowtie2 = ['bowtie2','-p',str(args.threads),'--very-sensitive-local',refName,self.name+'.fastq','-S',self.name+'.bowtie2.sam']
self.bowtie2pe = ['bowtie2','-p',str(args.threads),'--very-sensitive-local',refName,'-1',self.first+'.fastq','-2',self.second+'.fastq','-S',self.name+'.bowtie2.sam']
#self.novoalign = ['novoalign','-o','SAM','-d',self.name+'.ref.ndx','-f',self.name+'.fastq']
#self.novoalignpe = ['novoalign','-o','SAM','-d',self.name+'.ref.ndx','-f',self.name+'.1.fastq',self.name+'.2.fastq']
self.bwa = ['bwa','mem','-t',str(args.threads),refName,name+'.fastq']
self.bwape = ['bwa','mem','-t',str(args.threads),refName,self.first+'.fastq',self.second+'.fastq']
#variant calling commands
self.varscan = ['java','-jar','/opt/wmd/varscan/VarScan.v2.3.7.jar','--min-coverage',str(args.mincov),'--output-vcf','1',name+'.'+mapper+'.mpileup.tsv']
self.gatk = ['java','-jar','/opt/wmd/gatk/GenomeAnalysisTK.jar','-T','HaplotypeCaller','--nt',str(args.threads),'-R',refName,'-I',name+'.'+mapper
if args.illumina:
#self.bowtie2.insert(1,'--phred64')
#self.bowtie2pe.insert(1,'--phred64')
self.bowtie2pe.insert(1,'--rf')
#self.novoalign.insert(1,'-F')
#self.novoalign.insert(2,'ILMFQ')
#self.novoalign.insert(3,'-i')
#self.novoalign.insert(4,'MP')
#self.novoalignpe.insert(1,'-F')
#self.novoalignpe.insert(2,'ILMFQ')
#self.novoalignpe.insert(3,'-i')
#self.novoalignpe.insert(4,'MP')
#def indexIt(self,command):
#print 'Indexing '+self.name+' '+command[0]
#with open(self.name+'.log','a') as log:
# subprocess.call(command,stderr=log, stdout=log)
#print 'Finished indexing '+self.name+' '+command[0]
def mapIt(self,command):
print 'Aligning '+self.name+' with '+command[0]
stdoutSam = ('bwa')
if any(sam in command[0] for sam in stdoutSam): # if mapper outputs sam to STDOUT
with open(self.name+'.'+command[0]+'.sam','w') as outfile,open(self.name+'.log','a') as log:
subprocess.call(command,stderr=log, stdout=outfile)
else:
with open(self.name+'.log','a') as log:
subprocess.call(command,stderr=log, stdout=log)
print 'Finished aligning '+self.name+' with '+command[0]
def runBowtie2(self):
name = self.name
#self.indexIt(self.bowtie2index)
if args.paired:
self.mapIt(self.bowtie2pe)
else:
self.mapIt(self.bowtie2)
samToBam(name,'bowtie2')
gatkPreProcessing(name,'bowtie2')
MapStats(name,'bowtie2')
if args.delete_indices: subprocess.call(['rm',name+'.ref.fasta.1.bt2',name+'.ref.fasta.2.bt2',name+'.ref.fasta.3.bt2',name+'.ref.fasta.4.bt2',name+'.ref.fasta.rev.1.bt2',name+'.ref.fasta.rev.2.bt2'])
return
def runNovoalign(self):
name = self.name
first = self.first
second = self.second
#self.indexIt(self.novoindex)
wc = ""
samFiles = []
processes = []
# convert lines to # reads, divide by threads, multiply by 4 (to get # reads)
if args.paired:
wc = subprocess.Popen(['wc','-l',first+'.fastq'],stdout=subprocess.PIPE)
out = wc.communicate()
wc.wait()
reads = int(out[0].split(' ')[0])/4
readsPerFile = math.trunc(math.ceil(reads/int(args.threads)))
linesPerFile = str(readsPerFile * 4)
#cut = str(linesPerFile*4)
#print out[0].split(' ')[0],reads,readsPerFile,linesPerFile
#lines = str(math.trunc(math.ceil(int(out[0].split(' ')[0])/int(args.threads))))
firstFastq = subprocess.Popen(['split','-d','-l',linesPerFile,first+'.fastq',first+'.fastq.'])
secondFastq = subprocess.Popen(['split','-d','-l',linesPerFile,second+'.fastq',second+'.fastq.'])
firstFastq.wait()
secondFastq.wait()
for i in range(0,int(args.threads)):
if int(len(str(i))) == 1:
suffix = '0'+str(i)
else:
suffix = str(i)
with open(name+'.novoalign.'+str(i),'w') as sam:
samFiles.append(name+'.novoalign.'+str(i))
#p = subprocess.Popen(['novoalign','-o','SAM','-d',refNamePrefix[0]+'.ndx','-f',first+'.fastq.'+suffix,second+'.fastq.'+suffix], stdout=sam)
p = subprocess.Popen('novoalign -r Random -e 1 -o SAM -d '+refNamePrefix[0]+'.ndx -f '+first+'.fastq.'+suffix+' '+second+'.fastq.'+suffix+' | perl -pe \'s/\t\t/\t/g\'', stdout=sam, shell=True)
#processes.append(subprocess.Popen(['novoalign','-o','SAM','-d',refNamePrefix[0]+'.ndx','-f',first+'.fastq.'+suffix,second+'.fastq.'+suffix], stdout=sam))
processes.append(p)
#self.mapIt(self.novoalignpe)
else:
wc = subprocess.Popen(['wc','-l',name+'.fastq'],stdout=subprocess.PIPE)
out = wc.communicate()
wc.wait()
reads = int(out[0].split(' ')[0])/4
readsPerFile = math.trunc(math.ceil(reads/int(args.threads)))
linesPerFile = str(readsPerFile * 4)
#lines = str(math.trunc(math.ceil(int(out[0].split(' ')[0])/int(args.threads))))
subprocess.call(['split','-d','-l',linesPerFile,name+'.fastq',name+'.fastq.'])
for i in range(0,int(args.threads)):
if int(len(str(i))) == 1:
suffix = '0'+str(i)
else:
suffix = str(i)
with open(name+'.novoalign.'+str(i),'w') as sam:
samFiles.append(name+'.novoalign.'+str(i))
#processes.append(subprocess.Popen(['novoalign','-o','SAM','-d',refNamePrefix[0]+'.ndx','-f',name+'.fastq.'+suffix], stdout=sam))
p = subprocess.Popen('novoalign -r Random -e 1 -o SAM -d '+refNamePrefix[0]+'.ndx -f '+name+'.fastq.'+suffix+' | perl -pe \'s/\t\t/\t/g\'', stdout=sam, shell=True)
processes.append(p)
exit_codes = [p.wait() for p in processes]
subprocess.call('rm *fastq.*', shell=True)
mergeCommand = ['java','-jar','/opt/wmd/picard-tools-1.104/MergeSamFiles.jar']
for i in samFiles:
mergeCommand.append('I=')
mergeCommand.append(i)
mergeCommand.extend(['O=',name+'.novoalign.sam','USE_THREADING=','true'])
subprocess.call(mergeCommand)
for sam in samFiles:
subprocess.call(['rm',sam])
#self.mapIt(self.novoalign)
samToBam(name,'novoalign')
gatkPreProcessing(name,'novoalign')
MapStats(name,'novoalign')
if args.delete_indices: subprocess.call(['rm',name+'.ref.ndx'])
print 'Done with novoalign'
return
def runBwa(self):
name = self.name
#self.indexIt(self.bwaindex)
if args.paired:
self.mapIt(self.bwape)
else:
self.mapIt(self.bwa)
samToBam(name,'bwa')
gatkPreProcessing(name,'bwa')
MapStats(name,'bwa')
if args.delete_indices: subprocess.call(['rm',name+'.ref.fasta.amb',name+'.ref.fasta.ann',name+'.ref.fasta.bwt',name+'.ref.fasta.pac',name+'.ref.fasta.sa'])
print 'Done with BWA'
return
def runVarscan(self):
name = self.name
def samToBam(name,mapper):
print 'Converting '+name+'-'+mapper+' SAM to BAM'
with open(name+'.log','a') as log:
log.write('******sam2bam******')
with open(name+'.'+mapper+'.temp', 'w') as outfile:
subprocess.call(['samtools','view','-bS',name+'.'+mapper+'.sam'], stdout=outfile,stderr=log)
subprocess.call(['samtools','sort',name+'.'+mapper+'.temp',name+'.'+mapper])
subprocess.call(['samtools','index',name+'.'+mapper+'.bam'])
subprocess.call(['rm',name+'.'+mapper+'.temp',name+'.'+mapper+'.sam'])
print 'Finished converting '+name+'-'+mapper+' SAM to BAM'
return
def gconf(fof):
print 'Starting GConf'
subprocess.call(['gc.0.12.pl','--fof',fof,'-r',refName,'--samtools','/opt/wmd/samtools-0.1.19/samtools','-w','-z','-l','/opt/wmd/gc_pvalue_lookup.transposed.tsv',\
'-c',str(args.mincov),'-t',str(args.threads)])
print 'Finished GConf'
return
def gatkPreProcessing(name,mapper):
print 'Starting GATK pre-processing'
with open(name+'.log','a') as log, open('wmd-gconf-bam-files.txt','a') as bams:
log.write('******GATK Preprocessing******')
print 'Adding ReadGroups'
subprocess.call(['java','-jar','/opt/wmd/picard-tools-1.104/AddOrReplaceReadGroups.jar','I=',name+'.'+mapper+'.bam','O=',name+'.'+mapper+'.rg.bam','LB=','Ion',\
'PL=','Ion','PU=','Ion','SM=',name],stdout=log,stderr=log)
print 'Indexing bam'
subprocess.call(['samtools','index',name+'.'+mapper+'.rg.bam'])
print 'Finding indels'
subprocess.call(['java','-jar','/opt/wmd/gatk/GenomeAnalysisTK.jar','-T','RealignerTargetCreator','-R',refName,\
'-I',name+'.'+mapper+'.rg.bam','-o',name+'.'+mapper+'.rg.intervals'],stdout=log,stderr=log)
print 'Realigning indels'
subprocess.call(['java','-jar','/opt/wmd/gatk/GenomeAnalysisTK.jar','-T','IndelRealigner','-R',refName,\
'-I',name+'.'+mapper+'.rg.bam','-targetIntervals',name+'.'+mapper+'.rg.intervals','-o',name+'.'+mapper+'.rg.realigned.bam'],stdout=log, stderr=log)
bams.write(name+'.'+mapper+'.rg.realigned.bam\n')
subprocess.call(['rm',name+'.'+mapper+'.bam',name+'.'+mapper+'.bam.bai',name+'.'+mapper+'.rg.intervals',name+'.'+mapper+'.rg.bam',name+'.'+mapper+'.rg.bam.bai'])
return
def MapStats(name,mapper):
print 'Starting MapStats'
try:
bamfile = pysam.Samfile(name+'.'+mapper+'.rg.realigned.bam', 'rb')
except IOError:
print "Bam file didn't open"
mapped = float(bamfile.mapped)
unmapped = float(bamfile.unmapped)
totalreadbases = float()
refs = bamfile.references
reflens = bamfile.lengths
totalrefbases = float()
totalcov = float()
refbasescovered = float()
readbasesmapped = float()
uss = {} # unique start sites hash
usscount = float()
uniqueMappedReads = {}
fastqFiles = []
#temp = args.name
if args.paired:
fastqFiles.append(name+'.1.filtered')
fastqFiles.append(name+'.2.filtered')
else:
fastqFiles.append(name)
for fastq in fastqFiles:
for seq in SeqIO.parse(fastq+".fastq","fastq"):
totalreadbases += len(seq.seq)
for i in range(0,len(reflens)):
totalrefbases += reflens[i]
for p in bamfile.pileup(refs[i],1,reflens[i]):
if p.n > 0:
refbasescovered += 1
totalcov += p.n
for alignedread in bamfile.fetch(refs[i],1,reflens[i]):
#print "tid",alignedread.tid,"\taend",alignedread.aend,"\talen",alignedread.alen,"\tpos",alignedread.pos,"\tpositions",alignedread.positions
uss[refs[i]+str(alignedread.aend)] = 1
uss[refs[i]+str((alignedread.pos - 1))] = 1
for key in uss.keys():
usscount += 1
with open(name+'.'+mapper+'.rg.realigned.map-stats.tsv', 'w') as stats:
#out = "Percent Reads Mapped = %s\nPercent Ref Bases Covered = %s\nPercent Read Bases Mapped = %s\nAverage Coverage = %s\nPercent Unique Start Sites = %s" % \
#(((mapped/(mapped + unmapped)) * 100), ((refbasescovered/totalrefbases)*100),((totalcov/totalreadbases)*100),((totalcov/totalrefbases)*100),\
#((usscount/totalrefbases)*100))
stats.write("Percent Reads Mapped = %.2f\nPercent Ref Bases Covered = %.2f\nPercent Read Bases Mapped = %.2f\nAverage Coverage = %.2f\nPercent Unique Start Sites = %.2f" % \
((mapped/(mapped+unmapped))*100,(refbasescovered/totalrefbases)*100,(totalcov/totalreadbases)*100,totalcov/totalrefbases,(usscount/totalrefbases)*100))
#stats.write(out)
#for p in bamfile.pileup():
# print p.pos,"\t",p.n
bamfile.close()
print "Done calculating MapStats"
return
if __name__ == '__main__':
main()