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outLyzer_V3.2.py
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outLyzer_V3.2.py
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#! /usr/bin/env python
#! -*- coding: utf-8 -*-
import sys
import os
import re
import subprocess
import numpy as np
from scipy.stats import t
import math
import argparse
import time
from multiprocessing import Process
from argparse import RawTextHelpFormatter
from shutil import copyfile
from collections import defaultdict
import shlex
### MultiProcessing
def launchCommandLine(commandLine):
os.system(commandLine)
def ifFolderNotExistCreate(path):
if (os.path.exists(path)) == False:
os.makedirs(path)
def manageCommandListToLaunch(commandList,maxJob):
jobs = []
for commandLine in commandList:
if len(jobs) == maxJob:
while len(jobs) >= maxJob:
for job in jobs:
if not job.is_alive():
jobs.remove(job)
time.sleep(3)
process = Process(target=launchCommandLine, args=(commandLine, ))
process.start()
jobs.append(process)
while len(jobs) > 0 :
for job in jobs:
if not job.is_alive():
jobs.remove(job)
time.sleep(3)
### outLyzer
def dataListCreation(datafile,sep):
"""This function reads csv file containing variant identification
and create a list of lists to analyse it.
Argument 1: datafile = csv file to analyse"""
liste = datafile.readlines()
liste2 =[]
for i in liste:
j =i.split(sep)
liste2.append(j)
return liste2
def createBedList(bedFilePath):
bedFile = open(bedFilePath,'r')
bedList = dataListCreation(bedFile, '\t')
bedFile.close()
for line in bedList:
line[2] = line[2].replace('\n','')
return bedList
def cutBedFile(bedFilePath,cutNb,outputPath):
ifFolderNotExistCreate(outputPath)
bedFile = open(bedFilePath,'r')
bedList = dataListCreation(bedFile, '\t')
bedFile.close()
lineNb = int(math.ceil(len(bedList)/float(cutNb)))
BedIntervals = range(0,len(bedList),lineNb)
fileNb = 1
for interval in BedIntervals[1:]:
fileToWrite = open('%sbedFile_%i.bed'%(outputPath,fileNb),'w')
for line in bedList[interval-lineNb:interval]:
lineToWrite = '\t'.join(line)
fileToWrite.write(lineToWrite)
fileToWrite.close()
fileNb += 1
fileToWrite = open('%sbedFile_%i.bed'%(outputPath,fileNb),'w')
for line in bedList[BedIntervals[-1]:len(bedList)]:
lineToWrite = '\t'.join(line)
fileToWrite.write(lineToWrite)
fileToWrite.close()
bedList = os.listdir(outputPath)
finalBedList = []
for bedFile in bedList:
finalBedList.append(os.path.join(outputPath,bedFile))
return finalBedList
def createOutLyzerCommandList(output,pythonPath,samtoolsPath,progPath,cut,bedList,bamFile,refFile,studentValue,balance,Qscore,SDQ,WinSize,AS,HSM,multiplicativeFactor,verbose,FRcor,WSmin,force):
outLyzerCommandList = []
for bedFile in bedList:
commandLine = '%s %s subprocess -samtools %s -bam %s -cut %i -output %s -ref %s -bed %s -t %f -bal %f -Q %i -SDQ %i -WS %i -x %f -verbose %i -WSmin %i -force %f'%(pythonPath,progPath,samtoolsPath,bamFile,cut,output,refFile,bedFile,studentValue,balance,Qscore,SDQ,WinSize,multiplicativeFactor,verbose,WSmin,force)
if AS:
commandLine = commandLine+' -AS'
if HSM:
commandLine = commandLine+' -HSM %s'%HSM
if FRcor:
commandLine = commandLine+' -FRcor'
outLyzerCommandList.append(commandLine)
return outLyzerCommandList
def launchPileupCommandForBedFile(bamFile,samtoolsPath,referenceFile,bedFile,output):
samtoolsCmd = '%s mpileup -d 100000 -Q 0 -A -R -B -f %s -x -l %s %s'%(samtoolsPath,referenceFile,bedFile,bamFile)
samtoolsResultCmd = subprocess.Popen(shlex.split(samtoolsCmd),stdout=subprocess.PIPE,stderr=subprocess.PIPE, universal_newlines=True)
samtoolsResultCmdOut,samtoolsResultCmdErr = samtoolsResultCmd.communicate()
pileupList = samtoolsResultCmdOut.splitlines()
pileupList.pop()
return pileupList
def launchPileupCommandForPosition(bamFile,samtoolsPath,referenceFile,genomicPosition,windowSize):
name = bamFile.split('.')[0].split('_')[0]
name = name.split('/')[-1]
chromosome = genomicPosition.split(':')[0]
positiontoTest = int(genomicPosition.split(':')[-1])
samtoolsPosition = subprocess.check_output('%s mpileup -d 10000 -Q 0 -A -R -B -f %s -x -r %s:%i-%i %s 2>/dev/null'%(samtoolsPath,referenceFile,chromosome,positiontoTest,positiontoTest,bamFile),shell=True).decode('utf-8')
if samtoolsPosition == '':
print('No reads mapped on position')
else:
genomicInterval = '%s:%i-%i'%(chromosome,round(positiontoTest-(windowSize/2)),round(positiontoTest+(windowSize/2)))
samtoolsOutput = subprocess.check_output('%s mpileup -d 10000 -Q 0 -A -R -B -f %s -x -r %s %s 2>/dev/null'%(samtoolsPath,referenceFile,genomicInterval,bamFile),shell=True).decode('utf-8')
pileupList = samtoolsOutput.split('\n')
pileupList.pop()
return pileupList
def calcThompsonTau(n,studentValue):
tStu = t.ppf(1-(studentValue/2),n-2)
tau = (tStu*(n-1))/(math.sqrt(n)*math.sqrt(n-2+tStu**2))
return tau
def thompsonTest(listToTest,studentValue):
condition = 'continue'
mean = np.mean(listToTest)
stdDev = np.std(listToTest)
deltaMax = abs(listToTest[-1]-mean)
tau = calcThompsonTau(len(listToTest),studentValue)
testValue = tau*stdDev
if deltaMax > testValue:
listToTest.pop()
else:
condition = 'stop'
return listToTest,condition
def reject_outliersReads(altReadList,studentValue):
filteredAltReadList = []
for number in altReadList:
num = int(number)
if num != 0:
filteredAltReadList.append(num)
filteredAltReadList.sort()
outliersReject = 'continue'
if not not filteredAltReadList :
while outliersReject == 'continue':
newAltReadList,condition = thompsonTest(filteredAltReadList,studentValue)
if condition=='stop':
break
else:
newAltReadList = []
return newAltReadList
def calcConfidenceIntervalFromReads(altReadList,studentValue,DP,force):
altReadsOutliers = reject_outliersReads(altReadList,studentValue)
forced = ''
if not len(altReadsOutliers) == 0:
maxOutliers = max(altReadsOutliers)
if force > 0 and maxOutliers > math.ceil(force*DP):
forced = '*'
while maxOutliers != 'NA' and maxOutliers > math.ceil(force*DP):
altReadsOutliers.pop()
altReadsOutliers = reject_outliersReads(altReadsOutliers,studentValue)
if len(altReadsOutliers) != 0:
maxOutliers = max(altReadsOutliers)
else:
maxOutliers = 'NA'
else:
maxOutliers = 'NA'
return maxOutliers,forced
def forwardReverseOrigin(Indel):
if str.isupper(Indel):
return 'Forward'
else:
return 'Reverse'
def countMaxIndel(indelDic):
maxIndel = ''
maxCount = 0
for element in indelDic:
totalReads = int(indelDic[element]['Forward'])+int(indelDic[element]['Reverse'])
if totalReads > maxCount:
maxCount = totalReads
maxIndel = element
return maxIndel
def countDelInPileup(baseCount):
delDic = {}
while bool(re.search('[-]',baseCount)):
indelSearch = re.search('[-]([0-9]+)+([ATCGNatcgn]+)',baseCount)
indelReal = indelSearch.group(0)[0:int(indelSearch.group(1))+len(indelSearch.group(1))+1]
indelContent = indelSearch.group(2)[0:int(indelSearch.group(1))]
orientation = forwardReverseOrigin(indelContent)
if not indelReal.upper() in delDic.keys():
delDic[indelReal.upper()] = {'Forward':0,'Reverse':0}
delDic[indelReal.upper()][orientation] = baseCount.count(indelReal)
else:
delDic[indelReal.upper()][orientation] = baseCount.count(indelReal)
baseCount = baseCount.replace(indelReal,'')
if bool(delDic):
mainDel = countMaxIndel(delDic)
return mainDel,delDic[mainDel]
else:
return 'None',delDic
def countInsInPileup(baseCount):
delDic = {}
while bool(re.search('[+]',baseCount)):
insSearch = re.search('[+]([0-9]+)+([ATCGNatcgn]+)',baseCount)
insReal = insSearch.group(0)[0:int(insSearch.group(1))+len(insSearch.group(1))+1]
insContent = insSearch.group(2)[0:int(insSearch.group(1))]
orientation = forwardReverseOrigin(insContent)
if not insReal.upper() in delDic.keys():
delDic[insReal.upper()] = {'Forward':0,'Reverse':0}
delDic[insReal.upper()][orientation] = baseCount.count(insReal)
else:
delDic[insReal.upper()][orientation] = baseCount.count(insReal)
baseCount = baseCount.replace(insReal,'')
if bool(delDic):
mainDel = countMaxIndel(delDic)
return mainDel,delDic[mainDel]
else:
return 'None',delDic
def countPointMutations(baseCount,pileupInfosDic):
baseCountNoIndel = eraseIndelfromPileup(baseCount)
baseCountDic={'For':baseCountNoIndel.count('.'),'Rev':baseCountNoIndel.count(','),'a':baseCountNoIndel.count('a'),'A':baseCountNoIndel.count('A'),'c':baseCountNoIndel.count('c'),'C':baseCountNoIndel.count('C'),'g':baseCountNoIndel.count('g'),'G':baseCountNoIndel.count('G'),'t':baseCountNoIndel.count('t'),'T':baseCountNoIndel.count('T')}
qualDic={'A':[],'C':[],'G':[],'T':[]}
baseList = ['a','A','c','C','g','G','t','T']
for position,base in enumerate(baseCountNoIndel):
if base in baseList:
phredQual = ord(pileupInfosDic['baseQual'][position])
if base == 'a' or base =='A':
qualDic['A'].append(phredQual)
elif base == 'c' or base =='C':
qualDic['C'].append(phredQual)
elif base == 'g' or base =='G':
qualDic['G'].append(phredQual)
elif base == 't' or base =='T':
qualDic['T'].append(phredQual)
return baseCountDic,qualDic
def eraseIndelfromPileup(baseCount):
while bool(re.search('[+-]',baseCount)):
indelSearch = re.search('[+-]([0-9]+)+[ATCGNatcgn]+',baseCount)
indelReal = indelSearch.group(0)[0:int(indelSearch.group(1))+len(indelSearch.group(1))+1]
baseCount = baseCount.replace(indelReal,'')
return baseCount
def mainPileupLineInfosInDic(pileupLine):
pileupInfosDic = {}
pileupInfosDic['refBase'] = pileupLine[2]
pileupInfosDic['depth'] = pileupLine[3]
pileupInfosDic['baseCount'] = re.sub(r"(\^.)",r"",pileupLine[4].replace('$','').replace('N','.').replace('n',','))
pileupInfosDic['baseQual'] = pileupLine[5]
return pileupInfosDic
def defineMainPointMutation(pileupLine, pileupInfosDic):
baseCountDic,qualDic = countPointMutations(pileupLine, pileupInfosDic)
nucleotideList=['A','C','G','T']
finalAltBase = ''
finalAltBaseFreq = 0
finalBaseQual = 0
finalQualStd = 0
altReads = 0
for base in nucleotideList:
if not base == pileupInfosDic['refBase']:
totalBaseCount = baseCountDic[base]+baseCountDic[base.lower()]
if totalBaseCount == 0:
phredQual = 'NA'
phredQualStd = 'NA'
else:
phredQual = '%.2f'%(float(sum(qualDic[base]))/(baseCountDic[base.lower()]+baseCountDic[base])-33)
phredQualStd = round(np.std(qualDic[base]),2)
altFreqBase = round(((baseCountDic[base]+baseCountDic[base.lower()])/float(pileupInfosDic['depth']))*100,3)
if altFreqBase > finalAltBaseFreq:
finalAltBase = base
finalAltBaseFreq = altFreqBase
finalBaseQual = phredQual
finalQualStd = phredQualStd
altReads = baseCountDic[base]+baseCountDic[base.lower()]
return finalAltBase,finalAltBaseFreq,finalBaseQual,finalQualStd,altReads
def defineLeadingMutationType(delReads,insReads,baseCountDic,finalAltBase):
leadingMutation = 'WT'
leadingMutationReads = 0
if bool(delReads):
totalDelReads = int(delReads['Forward'])+int(delReads['Reverse'])
if totalDelReads > leadingMutationReads:
leadingMutationReads = totalDelReads
leadingMutation = 'Deletion'
else:
totalDelReads = 0
if bool(insReads):
totalInsReads = int(insReads['Forward'])+int(insReads['Reverse'])
if totalInsReads > leadingMutationReads:
leadingMutationReads = totalInsReads
leadingMutation = 'Insertion'
else:
totalInsReads = 0
if finalAltBase != '':
pointAltReads = int(baseCountDic[finalAltBase])+int(baseCountDic[finalAltBase.lower()])
if pointAltReads > leadingMutationReads:
leadingMutationReads = pointAltReads
leadingMutation = 'PointMutation'
else:
pointAltReads = 0
return leadingMutation
def countAltReads(baseCount):
baseCountNoIndel = eraseIndelfromPileup(baseCount)
indelCount = baseCount.count('-')+baseCount.count('+')
altReads = baseCountNoIndel.count('a')+baseCountNoIndel.count('A')+baseCountNoIndel.count('c')+baseCountNoIndel.count('C')+baseCountNoIndel.count('g')+baseCountNoIndel.count('G')+baseCountNoIndel.count('t')+baseCountNoIndel.count('T')
totalAltReads = indelCount + altReads
return totalAltReads
def extractpileupLineInfos(pileupLine):
mutationStats = {}
mutationStats['Chromosome']= pileupLine[0]
mutationStats['GenomicPosition'] = pileupLine[1]
mutationStats['refBase'] = pileupLine[2]
mutationStats['DP'] = pileupLine[3]
baseCount = re.sub(r"(\^.)",r"",pileupLine[4].replace('$',''))
pileupInfosDic = mainPileupLineInfosInDic(pileupLine)
delName,delReads = countDelInPileup(baseCount)
insName,insReads = countInsInPileup(baseCount)
baseCountDic, qualDic = countPointMutations(baseCount, pileupInfosDic)
finalAltBase,finalAltBaseFreq,finalBaseQual,finalQualStd,altReads = defineMainPointMutation(baseCount, pileupInfosDic)
leadingMutation = defineLeadingMutationType(delReads, insReads, baseCountDic, finalAltBase)
totalAlt = countAltReads(baseCount)
mutationStats['totalAlt'] = int(totalAlt)
mutationStats['WT_Bal'] = '%i/%i'%(int(baseCountDic['For']),int(baseCountDic['Rev']))
if leadingMutation == 'Insertion':
mutationStats['altReadsMutation'] = insReads['Forward']+insReads['Reverse']
mutationStats['AF'] = round((float(insReads['Forward'])+float(insReads['Reverse']))*100 / float(pileupInfosDic['depth']),3)
mutationStats['alt'] = insName
mutationStats['Qual'] = '.'
mutationStats['Qual_StdDev'] = '.'
mutationStats['balance'] = '%s/%s'%(insReads['Forward'],insReads['Reverse'])
elif leadingMutation == 'Deletion':
mutationStats['altReadsMutation'] = delReads['Forward']+delReads['Reverse']
mutationStats['AF'] = round((float(delReads['Forward'])+float(delReads['Reverse']))*100 / float(pileupInfosDic['depth']),3)
mutationStats['alt'] = delName
mutationStats['Qual'] = '.'
mutationStats['Qual_StdDev'] = '.'
mutationStats['balance'] = '%s/%s'%(delReads['Forward'],delReads['Reverse'])
elif leadingMutation =='PointMutation':
mutationStats['altReadsMutation'] = altReads
mutationStats['AF'] = finalAltBaseFreq
mutationStats['alt'] = finalAltBase
mutationStats['Qual'] = finalBaseQual
mutationStats['Qual_StdDev'] = finalQualStd
mutationStats['balance'] = '%s/%s'%(baseCountDic[finalAltBase],baseCountDic[finalAltBase.lower()])
else:
mutationStats['altReadsMutation'] = '0'
mutationStats['AF'] = '.'
mutationStats['alt'] = 'WT'
mutationStats['Qual'] = '.'
mutationStats['Qual_StdDev'] = '.'
mutationStats['balance'] = '.'
return mutationStats
def writeVcfHeader(commandLine,vcfFile):
openVcfFile = open(vcfFile,'w')
patientID = vcfFile.split('/')[-1].split('.')[0].split('_')[0]
header="""##fileformat=VCFv4.2
##fileDate=%s
##source=%s
##reference=/data/genome/hg19/hg19_order.fa
##INFO=<ID=DP,Number=1,Type=Integer,Description="Raw Depth">
##INFO=<ID=AF,Number=1,Type=Float,Description="Allele Frequency">
##INFO=<ID=SDQ,Number=1,Type=Float,Description="Standard Deviation of average Phred Score">
##INFO=<ID=OUTLIER,Number=1,Type=Integer,Description="Background Noise Threshold (Number of Reads)">
##FILTER=<ID=noise_background,Description="Mutation's Rate is Higher than Surrounding Background Noise">
##FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">
##FORMAT=<ID=BAL,Number=2,Type=String,Description="Count of alternative Forward / Reverse Reads">
##FORMAT=<ID=WTbal,Number=1,Type=String,Description="Forward / Reverse WT reads at the position">
##FORMAT=<ID=MSS,Number=1,Type=String,Description="Proximity analysis of Stretch and repetition motifs around mutation">
#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\tFORMAT\t%s\n"""%(time.strftime("%Y%m%d"),commandLine,patientID)
openVcfFile.write(header)
openVcfFile.close()
def defineWindowToScan(altReadList,positionInExon,windowSize):
if len(altReadList)< windowSize:
newAltReadList = altReadList
else:
if positionInExon-round(windowSize/2) < 0:
newAltReadList = altReadList[0:windowSize]
elif positionInExon+round(windowSize/2)>len(altReadList):
newAltReadList = altReadList[-windowSize:]
else:
newAltReadList = altReadList[int(positionInExon-round(windowSize/2)):int(positionInExon+round(windowSize/2))]
return newAltReadList
def getsubs(loc, s):
substr = s[loc:]
i = -1
while(substr):
yield substr
substr = s[loc:i]
i -= 1
def testMotifRec(seq):
check=False
for i in range(2,int(len(seq)/2),1):
first = seq[0:i]
second = seq[i:2*i]
if first == second:
if len(first)!=first.count(first[0]):
check=True
break
return check
def stretchValid(seq):
if len(seq)==seq.count(seq[0]):
return True
else:
return False
def motifDistanceFromMutation(motif,seq, mutPos):
subStart = seq.find(motif)
dist1 = subStart-mutPos
dist2 = (subStart+len(motif))-mutPos
distList = [abs(dist1),abs(dist2)]
finalDist = 0
if dist1<=0 and dist2>0:
finalDist = 0
else:
finalDist = min(distList)
return finalDist
def findMotif(seq,minStretch,mutPos):
occ = defaultdict(int)
for i in range(len(seq)):
for sub in getsubs(i,seq):
occ[sub] += 1
maxStretch = ''
maxMotif = ''
for motif in occ:
if occ[motif]>=2 and len(motif)>6 and len(motif)>len(maxMotif) and not stretchValid(motif):
if testMotifRec(motif):
maxMotif = motif
if len(motif)>=minStretch and stretchValid(motif) and len(motif)>len(maxStretch):
if len(motif)!=0:
maxStretch=motif
motDic = {}
if len(maxStretch)>0:
motDic['stretch']=[maxStretch,motifDistanceFromMutation(maxStretch, seq, mutPos)]
if len(maxMotif)>0:
motDic['motif']=[maxMotif,motifDistanceFromMutation(maxMotif, seq, mutPos)]
return motDic
def defineMotifStatus(seq,mutPos):
motifStatus = ''
motifDic = findMotif(seq, 5, mutPos)
if 'stretch' in motifDic.keys():
motifStatus += 'S%id%i'%(len(motifDic['stretch'][0]),motifDic['stretch'][1])
if 'motif' in motifDic.keys():
if len(motifStatus)!=0:
motifStatus += '|'
motifStatus += 'M%id%i'%(len(motifDic['motif'][0]),motifDic['motif'][1])
if len(motifStatus) == 0:
motifStatus = 'None'
return motifStatus
def likelihoodMutationState(mutationStats,balance,quality,SDquality,FRcor):
warning = 0
if mutationStats[4] != 'WT':
if FRcor:
WT_F = float(mutationStats[12].split('/')[0])
WT_R = float(mutationStats[12].split('/')[1])
Alt_F = float(mutationStats[11].split('/')[0])
Alt_R = float(mutationStats[11].split('/')[1])
if len(mutationStats[3])==1 and len(mutationStats[4])==1:
diffF = round((WT_F+Alt_F) / (float(mutationStats[5])),3)-0.5
else:
diffF = round((WT_F) / (float(mutationStats[5])),3)-0.5
if diffF > 0:
balanceProp = round(((Alt_F-(Alt_F*diffF))/(Alt_F+Alt_R)),3)
elif diffF < 0:
balanceProp = round(((Alt_R+(Alt_R*diffF))/(Alt_F+Alt_R)),3)
else:
balanceProp = round((Alt_F/(Alt_F+Alt_R)),3)
else:
balanceProp = float(mutationStats[11].split('/')[0]) /( float(mutationStats[11].split('/')[0])+float(mutationStats[11].split('/')[1]))
else:
balanceProp = 'NA'
if mutationStats[4].startswith('+') or mutationStats[4].startswith('-'):
if balanceProp >= (balance) and balanceProp <= (1-balance):
likelihoodStatus=1
else:
likelihoodStatus=0
elif mutationStats[4] != 'WT':
if float(mutationStats[9]) >= quality and balanceProp >= balance and balanceProp <= 1-balance and float(mutationStats[10])<SDquality:
likelihoodStatus=1
else:
likelihoodStatus=0
else:
likelihoodStatus=0
return likelihoodStatus
def calcExonBackGroundNoise(altReadList,depthList,studentValue,force):
meanDepth = sum(depthList)/len(depthList)
maxOutlier,forced = calcConfidenceIntervalFromReads(altReadList, studentValue,meanDepth,force)
if maxOutlier !='NA':
sensitivityLimit = float(maxOutlier)/meanDepth
else:
sensitivityLimit = 'NA'
return sensitivityLimit
def defineMutationPosInSeq(altReadList,positionInExon,windowSize):
if len(altReadList)< windowSize:
mutationPosition = positionInExon
else:
if positionInExon-round(windowSize/2) < 0:
mutationPosition = positionInExon
elif positionInExon+round(windowSize/2)>len(altReadList):
mutationPosition = round(windowSize/2)+(positionInExon+round(windowSize/2)-len(altReadList))
else:
mutationPosition = round(windowSize/2)
return mutationPosition
def detectMutations(exonMatrix,studentValue,balance,quality,SDquality,windowSize,multiplicativeFactor,fileToWrite,FRcor,force):
altReadList = exonMatrix[:,7].tolist()
refSeqList = exonMatrix[:,3].tolist()
depthList = [int(i) for i in exonMatrix[:,5].tolist()]
exonSensitivityLimit = calcExonBackGroundNoise(altReadList, depthList, studentValue,force)
for rowNum in range(0,len(exonMatrix)):
windowList = defineWindowToScan(altReadList, int(rowNum), windowSize)
maxOutliers,forced = calcConfidenceIntervalFromReads(windowList, studentValue,int(exonMatrix[rowNum,5]),force)
if maxOutliers == 'NA' or int(exonMatrix[rowNum,6])>(maxOutliers*multiplicativeFactor):
isReal = likelihoodMutationState(exonMatrix[rowNum,:],balance,quality,SDquality,FRcor)
if isReal:
refSeqToScan = ''.join(defineWindowToScan(refSeqList, int(rowNum), 60))
mutPos = defineMutationPosInSeq(refSeqList, int(rowNum), 60)
motifStatus = defineMotifStatus(refSeqToScan,mutPos)
if '+' in exonMatrix[rowNum,4] or '-' in exonMatrix[rowNum,4]:
ref,alt = transformIndelAnnotation(exonMatrix[rowNum,3], exonMatrix[rowNum,4])
lineToWrite = '%s\t%s\t.\t%s\t%s\t%s\tPASS\tDP=%s;AF=%s;SDQ=%s;OUTLIER=%s\tGT:BAL:WTbal:MSS\t0/1:%s:%s:%s\n'%(exonMatrix[rowNum,1],exonMatrix[rowNum,2],ref,alt,exonMatrix[rowNum,9],exonMatrix[rowNum,5],exonMatrix[rowNum,8],exonMatrix[rowNum,10],str(maxOutliers)+forced,exonMatrix[rowNum,11],exonMatrix[rowNum,12],motifStatus)
fileToWrite.write(lineToWrite)
else:
lineToWrite = '%s\t%s\t.\t%s\t%s\t%s\tPASS\tDP=%s;AF=%s;SDQ=%s;OUTLIER=%s\tGT:BAL:WTbal:MSS\t0/1:%s:%s:%s\n'%(exonMatrix[rowNum,1],exonMatrix[rowNum,2],exonMatrix[rowNum,3].upper(),exonMatrix[rowNum,4].upper(),exonMatrix[rowNum,9],exonMatrix[rowNum,5],exonMatrix[rowNum,8],exonMatrix[rowNum,10],str(maxOutliers)+forced,exonMatrix[rowNum,11],exonMatrix[rowNum,12],motifStatus)
fileToWrite.write(lineToWrite)
return maxOutliers,exonSensitivityLimit
def transformIndelAnnotation(ref,alt):
regex = re.compile('[\+\-][0-9]+')
if '+' in alt:
newAlt = regex.sub(ref,alt).upper()
newRef = ref.upper()
else:
newRef = regex.sub(ref,alt).upper()
newAlt = ref.upper()
return newRef,newAlt
def hotSpotStartPos(hotSpotBed):
hotSpotBedFile = open(hotSpotBed,'r')
hotSpotList = dataListCreation(hotSpotBedFile, '\t')
hotSpotBedFile.close()
hotSpotDic = {}
for line in hotSpotList:
chrom = line[0]
start = line[1]
infos = line[2].replace('\n','').replace('\r','')
hotSpotDic['%s_%s'%(chrom,start)] = infos
return hotSpotDic
def readPileup(pileup,bedNum,studentValue,balance,quality,SDquality,windowSize,vcfPath,multiplicativeFactor,HSMfile,FRcor,WSmin,force):
previousPos = ''
firstLine = 1
countPosInExon = 0
unCoveredRegions = []
lastLine = '%s_%s'%(pileup[-1].split('\t')[0],pileup[-1].split('\t')[1])
fileToWrite = open('%s/tempCalling_%i.vcf'%(vcfPath,int(bedNum)),'a')
if HSMfile != None:
hotSpotDic = hotSpotStartPos(HSMfile)
metricsFile = open('%s/tempHSM_%s.txt'%(vcfPath,bedNum),'w')
hotSpotMark = {}
exonSensitivity = {}
for pileupLine in pileup:
pileupLine = pileupLine.split('\t')
Chromosome = pileupLine[0]
genomicPos = int(pileupLine[1])
mutationStats = extractpileupLineInfos(pileupLine)
rowToAdd = [countPosInExon,mutationStats['Chromosome'],mutationStats['GenomicPosition'],mutationStats['refBase'],mutationStats['alt'],mutationStats['DP'],mutationStats['altReadsMutation'],int(mutationStats['totalAlt']),mutationStats['AF'],mutationStats['Qual'],mutationStats['Qual_StdDev'],mutationStats['balance'],mutationStats['WT_Bal']]
if HSMfile != None:
if '%s_%i'%(Chromosome,genomicPos) in hotSpotDic.keys():
hotSpotMark['%s_%i'%(Chromosome,genomicPos)] = int(mutationStats['DP'])
if firstLine == 1:
exonMatrix = [rowToAdd]
countPosInExon += 1
firstLine = 0
previousPos = genomicPos
else:
if previousPos+1 == genomicPos:
exonMatrix.append(rowToAdd)
previousPos = genomicPos
countPosInExon += 1
if '%s_%i'%(Chromosome,genomicPos) == lastLine:
exonMatrix = np.asarray(exonMatrix)
if len(exonMatrix)>WSmin:
maxOutlier,exonSensitivityLimit = detectMutations(exonMatrix,studentValue,balance,quality,SDquality,windowSize,multiplicativeFactor,fileToWrite,FRcor,force)
exonSensitivity['%s_%s_%s'%(exonMatrix[0,1],exonMatrix[0,2],exonMatrix[-2,2])]=exonSensitivityLimit
if HSMfile != None:
if not not hotSpotMark:
for position in hotSpotMark:
metricsFile.write('%s\t%f\n'%(hotSpotDic[position],round(float(maxOutlier)/int(hotSpotMark[position])*100*multiplicativeFactor,2)))
else:
uncovRegion = '%s_%s_%s'%(exonMatrix[0,1],exonMatrix[0,2],exonMatrix[-1,2])
unCoveredRegions.append(uncovRegion)
else:
exonMatrix = np.asarray(exonMatrix)
if len(exonMatrix)>WSmin:
maxOutlier,exonSensitivityLimit = detectMutations(exonMatrix,studentValue,balance,quality,SDquality,windowSize,multiplicativeFactor,fileToWrite,FRcor,force)
exonSensitivity['%s_%s_%s'%(exonMatrix[0,1],exonMatrix[0,2],exonMatrix[-2,2])]=exonSensitivityLimit
else:
uncovRegion = '%s_%s_%s'%(exonMatrix[0,1],exonMatrix[0,2],exonMatrix[-1,2])
unCoveredRegions.append(uncovRegion)
countPosInExon = 0
rowToAdd = [countPosInExon,mutationStats['Chromosome'],int(mutationStats['GenomicPosition']),mutationStats['refBase'],mutationStats['alt'],mutationStats['DP'],mutationStats['altReadsMutation'],int(mutationStats['totalAlt']),mutationStats['AF'],mutationStats['Qual'],mutationStats['Qual_StdDev'],mutationStats['balance'],mutationStats['WT_Bal']]
exonMatrix = [rowToAdd]
previousPos = genomicPos
countPosInExon += 1
if HSMfile != None:
if not not hotSpotMark:
for position in hotSpotMark:
if maxOutlier !='NA':
metricsFile.write('%s\t%f\n'%(hotSpotDic[position],round(float(maxOutlier)/int(hotSpotMark[position])*100*multiplicativeFactor,2)))
else:
metricsFile.write('%s\t%f\n'%(hotSpotDic[position],'NA'))
hotSpotMark = {}
fileToWrite.close()
return exonSensitivity,unCoveredRegions
def readPileupForPosition(pileup,studentValue,balance,quality,SDquality,windowSize,genomicPosition,force):
previousPos = ''
firstLine = 1
countPosInExon = 0
for pileupLine in pileup:
pileupLine = pileupLine.split('\t')
Chromosome = pileupLine[0]
genomicPos = int(pileupLine[1])
mutationStats = extractpileupLineInfos(pileupLine)
rowToAdd = [countPosInExon,mutationStats['Chromosome'],int(mutationStats['GenomicPosition']),mutationStats['refBase'],mutationStats['alt'],mutationStats['DP'],mutationStats['altReadsMutation'],int(mutationStats['totalAlt']),mutationStats['AF'],mutationStats['Qual'],mutationStats['Qual_StdDev'],mutationStats['balance'],mutationStats['WT_Bal']]
if firstLine == 1:
exonMatrix = [rowToAdd]
countPosInExon += 1
firstLine = 0
previousPos = genomicPos
else:
if previousPos+1 == genomicPos:
exonMatrix.append(rowToAdd)
previousPos = genomicPos
countPosInExon += 1
exonMatrix = np.asarray(exonMatrix)
detectMutationsAtPosition(exonMatrix, studentValue, balance, quality, SDquality, windowSize, genomicPosition,force)
def detectMutationsAtPosition(exonMatrix,studentValue,balance,quality,SDquality,windowSize,genomicPosition,force):
altReadList = exonMatrix[:,7].tolist()
refSeqList = exonMatrix[:,3].tolist()
altReadPos = ""
for rowNum in range(0,len(exonMatrix)):
positionToFind = '%s:%s'%(exonMatrix[rowNum,1],exonMatrix[rowNum,2])
if positionToFind == genomicPosition:
altReadPos = rowNum
maxOutliers,forced = calcConfidenceIntervalFromReads(altReadList, studentValue,int(exonMatrix[rowNum,5]),force)
Chr = exonMatrix[rowNum,1]
genPos = exonMatrix[rowNum,2]
ref = exonMatrix[rowNum,3]
alt = exonMatrix[rowNum,4]
depth = exonMatrix[rowNum,5]
AF = exonMatrix[rowNum,8]
Qual = exonMatrix[rowNum,9]
Qual_StdDev = exonMatrix[rowNum,10]
balance = exonMatrix[rowNum,11]
WTbalance = exonMatrix[rowNum,12]
WT_F = float(exonMatrix[rowNum,12].split('/')[0])
WT_R = float(exonMatrix[rowNum,12].split('/')[1])
refSeqToScan = ''.join(defineWindowToScan(refSeqList, int(rowNum), 60))
mutPos = defineMutationPosInSeq(refSeqList, int(rowNum), 60)
motifDic = findMotif(refSeqToScan, 5, mutPos)
if 'stretch' in motifDic.keys():
stretchInfos = '%s (l = %i / d = %i bases from mutation)'%(motifDic['stretch'][0],len(motifDic['stretch'][0]),motifDic['stretch'][1])
else:
stretchInfos = 'None'
if 'motif' in motifDic.keys():
motifInfos = '%s (l = %i / d = %i bases from mutation)'%(motifDic['motif'][0],len(motifDic['motif'][0]),motifDic['motif'][1])
else:
motifInfos = 'None'
if exonMatrix[rowNum,4] == 'WT':
forwardPer,reversePer = 0,0
correctedFper,correctedRper = 0,0
overAllBalance = round((WT_F/float(depth))*100,1)
overAllBalanceR = 100-overAllBalance
else:
Alt_F = float(exonMatrix[rowNum,11].split('/')[0])
Alt_R = float(exonMatrix[rowNum,11].split('/')[1])
if len(ref) ==1 and len(alt) ==1:
diffF = round((WT_F+Alt_F) / (float(depth)),3)-0.5
forwardPer = round((Alt_F/(Alt_F+Alt_R))*100,1)
reversePer = 100-forwardPer
overAllBalance = round(((WT_F+Alt_F)/float(depth))*100,1)
overAllBalanceR = 100-overAllBalance
else:
diffF = round((WT_F) / (float(depth)),3)-0.5
forwardPer = round((Alt_F/(Alt_F+Alt_R))*100,1)
reversePer = 100-forwardPer
overAllBalance = round(((WT_F)/float(depth))*100,1)
overAllBalanceR = 100-overAllBalance
if diffF>0:
correctedFper = round(((Alt_F-(Alt_F*diffF))/(Alt_F+Alt_R))*100,1)
correctedRper = 100-correctedFper
else:
correctedRper = round(((Alt_R+(Alt_R*diffF))/(Alt_F+Alt_R))*100,1)
correctedFper = 100-correctedRper
print("AltReads Before mutation: ",altReadList[:rowNum])
print("AltReads Mutation at mutation position: ", altReadList[rowNum])
print("AltReads after Mutation",altReadList[rowNum+1:])
print ("""
Mutation Position: %s:%s
Reference Allele: %s
Alternative Allele: %s
Depth: %s
Allele Frequency (%%): %s
Phred Quality: %s
Phred Standard Deviation: %s
Forward / Reverse alt: %s (%s%% / %s%%)
overAll Balance: %s%% / %s%%
Corrected alt F/R: %s%% / %s%%
Raw background Noise: %s
Stretch nearby: %s
Motif nearby: %s
"""%(Chr,genPos,ref,alt,depth,AF,Qual,Qual_StdDev,balance,forwardPer,reversePer,overAllBalance,overAllBalanceR,correctedFper,correctedRper,str(maxOutliers)+forced,stretchInfos,motifInfos))
if len(exonMatrix)-1 != windowSize:
print('/!\ Warning: insufficient coverage for some positions on analyzed region.')
def writeMetricsFile(sensitivityDic,bamFile,output,bedNum):
metricsFile = open('%s/tempAS_%i.metrics'%(output,int(bedNum)),'w')
for interval in sensitivityDic:
lineToWrite = '%s\t%s\n'%(interval.replace('_','\t'),sensitivityDic[interval])
metricsFile.write(lineToWrite)
metricsFile.close()
def sortVcfResults(vcfOutput,tempVcfList,tempOutput):
vcfToWrite = open(vcfOutput,'a')
vcfDic = {}
for element in tempVcfList:
vcfNum = int(element.split('.')[-2].split('_')[-1])
vcfDic[vcfNum] = element
sortVcfList = sorted(vcfDic.keys())
for number in sortVcfList:
vcfToRead = open('%s%s'%(tempOutput,vcfDic[number]),'r')
for line in vcfToRead:
vcfToWrite.write(line)
vcfToRead.close()
vcfToWrite.close()
def sortASresults(ASoutput,AStempList,tempOutput):
mergedTempList = []
for tempFile in AStempList:
AStoRead = open('%s%s'%(tempOutput,tempFile),'r')
for line in AStoRead:
mergedTempList.append(line.replace('\n',''))
AStoRead.close()
sortTempFile = sorted(mergedTempList)
AStoWrite = open(ASoutput,'w')
for element in sortTempFile:
AStoWrite.write(element+'\n')
AStoWrite.close()
def writeHSMresults(HSMoutput,HSMtempList,tempOutput):
HSMtoWrite = open('%s'%HSMoutput,'w')
HSMtoWrite.write('Position_Informations\tSensitivity_Threshold(%)\n')
for tempFile in HSMtempList:
HSMtoRead = open('%s/%s'%(tempOutput,tempFile),'r')
for line in HSMtoRead:
HSMtoWrite.write(line)
HSMtoRead.close()
HSMtoWrite.close()
def findTempVcf(tempOutLyzerDir):
allFiles = os.listdir(tempOutLyzerDir)
tempoutLyzerResults = []
for element in allFiles:
if re.search('tempCalling',element):
tempoutLyzerResults.append(element)
return tempoutLyzerResults
def findTempAS(tempOutLyzerDir):
allFiles = os.listdir(tempOutLyzerDir)
tempoutLyzerResults = []
for element in allFiles:
if re.search('tempAS',element):
tempoutLyzerResults.append(element)
return tempoutLyzerResults
def findTempHSM(tempOutLyzerDir):
allFiles = os.listdir(tempOutLyzerDir)
tempoutLyzerResults = []
for element in allFiles:
if re.search('tempHSM',element):
tempoutLyzerResults.append(element)
return tempoutLyzerResults
def writeFinalResults(bamName,output,ASargs,HSMargs,commandLine,erase):
tempOutLyzerDir = '%s/outLyzerTemp_%s/'%(args.output,bamName)
vcfTempList = findTempVcf(tempOutLyzerDir)
AStempList = findTempAS(tempOutLyzerDir)
HSMtempList = findTempHSM(tempOutLyzerDir)
vcfOutput = '%s/%s.vcf'%(args.output,bamName)
ASoutput = '%s/%s.metrics'%(args.output,bamName)
HSMoutput = '%s/%s_HSM.txt'%(args.output,bamName)
writeVcfHeader(commandLine, vcfOutput)
sortVcfResults(vcfOutput,vcfTempList,tempOutLyzerDir)
if 'uncoveredRegions.bed' in os.listdir(tempOutLyzerDir):
copyfile('%s/uncoveredRegions.bed'%tempOutLyzerDir, '%s/%s_uncoveredRegions.bed'%(output,bamName))
if ASargs:
sortASresults(ASoutput, AStempList, tempOutLyzerDir)
if HSMargs:
writeHSMresults(HSMoutput, HSMtempList, tempOutLyzerDir)
if erase == 0:
os.system('rm -r %s'%(tempOutLyzerDir))
def launchCallingFunction(args):
if checkInputsCalling(args) == True:
print('starting outLyzer Calling Process')
startScript = time.time()
commandLine = ' '.join(sys.argv)
progPath = sys.argv[0]
bamName = args.bam.split('/')[-1].split('.')[0]
tempOutLyzerDir = '%s/outLyzerTemp_%s/'%(args.output,bamName)
if os.path.exists(tempOutLyzerDir):
os.system('rm -r %s'%(tempOutLyzerDir))
bedList = cutBedFile(args.bed, args.cut, tempOutLyzerDir)
outLyzerCommandList = createOutLyzerCommandList(tempOutLyzerDir, args.pythonPath, args.samtools, progPath, args.cut, bedList, args.bam, args.ref, args.t, args.bal, args.Q, args.SDQ, args.WS, args.AS,args.HSM, args.x,args.verbose,args.FRcor,args.WSmin,args.force)
manageCommandListToLaunch(outLyzerCommandList, args.core)
if args.verbose == 1:
print('Formatting results for %s'%bamName)
writeFinalResults(bamName, args.output, args.AS,args.HSM,commandLine,args.NoEraseTemp)
endScript = time.time()
print('outLyzer analysis performed in %f sec.'%(endScript-startScript))
def launchPositionAnalysisFunction(args):
if checkInputsPositionAnalysis(args) == True:
print('start analysis on position: ',args.position)
pileup = launchPileupCommandForPosition(args.bam, args.samtools, args.ref, args.position, args.WS)
if pileup != None:
if not args.force:
force = 0
else:
force = args.force
readPileupForPosition(pileup, args.t, args.bal, args.Q, args.SDQ, args.WS, args.position,force)
else:
print('End of Analysis')
def launchSubProcess(args):
bamName = args.bam.split('/')[-1]
bamPart = args.bed.split('/')[-1].split('.')[0].split('_')[-1]
if args.verbose == 1:
print("pileup conversion for %s part %s / %s"%(bamName,bamPart,args.cut))
pileup = launchPileupCommandForBedFile(args.bam, args.samtools, args.ref, args.bed,args.output)
bedNum = args.bed.split('.')[-2].split('_')[-1]
if args.verbose == 1:
print('statistical analysis for %s part %s / %s'%(bamName,bamPart,args.cut))
exonSensitivityLimit,uncoveredRegions = readPileup(pileup, bedNum, args.t, args.bal, args.Q, args.SDQ, args.WS, args.output, args.x, args.HSM,args.FRcor,args.WSmin,args.force)
if args.AS:
writeMetricsFile(exonSensitivityLimit, args.bam, args.output, bedNum)
if len(uncoveredRegions)>0:
uncovFile = open('%s/uncoveredRegions.bed'%args.output,'a')
for region in uncoveredRegions:
uncovFile.write('%s\n'%region)
uncovFile.close()
def checkInputsCalling(args):
checkResponse = True
pathToCheckList = [args.bed,args.bam,args.ref,args.output]
for pathToCheck in pathToCheckList:
if os.path.exists(pathToCheck) == False:
print("%s doesn't exists"%pathToCheck)
checkResponse = False
if not args.output.endswith('/'):
print("""'/' is missing to the end of %s"""%args.output)
checkResponse = False
if args.HSM:
if os.path.exists('%s'%args.HSM) == False:
print (os.path.exists('%s'%args.HSM))
print("%s doesn't exists"%args.HSM)
checkResponse = False
return checkResponse
def checkInputsPositionAnalysis(args):
checkResponse = True
pathToCheckList = [args.bam,args.ref]
for pathToCheck in pathToCheckList:
if os.path.exists(pathToCheck) == False:
print("%s doesn't exists"%pathToCheck)
checkResponse = False
return checkResponse
def printLicense(args):
print("""Copyright Etienne Muller (2016)
outLyzer is a computer program whose purpose is to detect low allele
frequency mutations, designed to analyze tumor samples in a clinical
context.
This software is governed by the CeCILL license under French law and
abiding by the rules of distribution of free software. You can use,
modify and/ or redistribute the software under the terms of the CeCILL
license as circulated by CEA, CNRS and INRIA at the following URL
"http://www.cecill.info".
As a counterpart to the access to the source code and rights to copy,
modify and redistribute granted by the license, users are provided only
with a limited warranty and the software's author, the holder of the
economic rights, and the successive licensors have only limited
liability.
In this respect, the user's attention is drawn to the risks associated
with loading, using, modifying and/or developing or reproducing the
software by the user in light of its specific status of free software,
that may mean that it is complicated to manipulate, and that also
therefore means that it is reserved for developers and experienced
professionals having in-depth computer knowledge. Users are therefore
encouraged to load and test the software's suitability as regards their
requirements in conditions enabling the security of their systems and/or
data to be ensured and, more generally, to use and operate it in the
same conditions as regards security.
The fact that you are presently reading this means that you have had
knowledge of the CeCILL license and that you accept its terms.""")
if __name__ == "__main__":
parser = argparse.ArgumentParser(description="""
# _ __
# ___ _ _| |_ / / _ _ _______ _ __
# / _ \| | | | __|/ / | | | |_ / _ \ '__|
# | (_) | |_| | |_/ /__| |_| |/ / __/ |
# \___/ \__,_|\__\____/\__, /___\___|_|
# |___/
OutLyzer is a variant-caller conceived for low allele-ratio mutations detection,
based on sequencing background noise evaluation.
It evaluates if the mutation is significantly different from background noise,
using modified Thompson tau technique.
Version: 3.2""",formatter_class=RawTextHelpFormatter)
subparsers = parser.add_subparsers(metavar='{calling,positionAnalysis,LICENSE}',help="outLyzer functions")
# create the parser for the "calling Command" command
parser_calling = subparsers.add_parser('calling',description='outLyzer calling function, analyze the whole BAM file and displays results in a VCF (Variant Calling Format) File')
parser_calling.add_argument('-samtools',help='Complete Samtools path if not specified in environment variable', default='samtools')
parser_calling.add_argument('-pythonPath',help='Complete python path if different from default python version', default='python')
parser_calling.add_argument('-core',help='define number of cores used to process analysis [1]',type = int, default = 1)
parser_calling.add_argument('-cut',help='defines into how many parts bed file is divide [3]',type=int,default=3)
parser_calling.add_argument('-bed',help='bed file required for analysis [REQUIRED]',required=True)
parser_calling.add_argument('-bam',help='bam File to analyze [REQUIRED]',required=True)
parser_calling.add_argument('-ref',help='faidx indexed reference sequence file (fasta) [REQUIRED]',required=True)
parser_calling.add_argument('-output',help='output Path To write results [REQUIRED]',required=True)
parser_calling.add_argument('-t',help='Student t value used in modified Thompson tau technique [0.001]',default=0.001,type=float)
parser_calling.add_argument('-bal',help='minimum Forward / Reverse read proportion [0.3]',default=0.3,type=float)
parser_calling.add_argument('-Q',help='minimum average Phred Score to be considered as a real mutation (only relevant for SNP) [20]',default=20,type=int)
parser_calling.add_argument('-SDQ',help='maximum Standard deviation authorized for average Phred Score [7]',default=7,type=int)
parser_calling.add_argument('-WS',help='Window Size: region (number of bp) around the mutation on which background noise have to be determined [200]',default=200,type=int)
parser_calling.add_argument('-WSmin',help='Window Size Minimum Size: minimum region size (number of bp) required for analysis [10]',default=10,type=int)
parser_calling.add_argument('-x',help='Multiplicative factor that specifies how often the mutation must be above background noise [2]',default=2,type=float)
parser_calling.add_argument('-AS',help='Analysis sensitivity: Returns an additional file containing analysis average sensitivity for each line of bed file',action='store_true')
parser_calling.add_argument('-FRcor',help='Forward Reverse Correction: take into account any imbalance in the Forward-Reverse reads distribution in the Forward / Reverse alternative Read Proportion (-bal option)',action='store_true')
parser_calling.add_argument('-force',help='Force noise background determination to go below a fixed proportion of the Depth [default = disabled; 0 -> 1 ex: "-force 0.2"] ',type = float, default=0)
parser_calling.add_argument('-NoEraseTemp',help='Do not erase temporary files [default = disabled; enabled = 1 ',type = float, default=0)
parser_calling.add_argument('-HSM',help='HotSpot Metrics: Produce sensitivity Threshold for HotSpot positions, in an additional file. Requires formated HotSpot File in argument (see documentation for more details).')
parser_calling.add_argument('-verbose',help='If verbose mode is set to 1, details analysis process steps [0]',type = int, default=0)
parser_calling.set_defaults(func=launchCallingFunction)
#create the parser for subprocess command
parser_subprocess = subparsers.add_parser('subprocess')
parser_subprocess.add_argument('-samtools',help='Complete Samtools path if not specified in environment variable', default='samtools')
parser_subprocess.add_argument('-pythonPath',help='Complete python path if different from default python version', default='python')