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eKLIPse_sc.py
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eKLIPse_sc.py
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import json
import os
import sys
import pybam
from string import split
from eKLIPse_fct import *
#=================================================================================================================================================#
#=================================================================================================================================================#
#=================================================================================================================================================#
def Read_alignment(titleBam,dicoInit,lstError):
try:
pathSCjson = os.path.join(dicoInit['pathTmpDir'],titleBam+"_SC.json")
pathSCfasta = os.path.join(dicoInit['pathTmpDir'],titleBam+".fasta")
FASTA = open(pathSCfasta,'w')
# Init dicoBam
dicoBam = {}
for pos in range(1,dicoInit["dicoGbk"]['refLength']+1,1):
dicoBam[pos] = { 'nb_reads_F':0, 'nb_reads_R':0,\
'nb_sc_reads_F':0, 'nb_sc_reads_R':0,\
'nb_sc_fasta_F':0, 'nb_sc_fasta_R':0 }
#***** BROWSE READS & SEARCH SCR *****#
# Switch to downsampled BAM if exist
if dicoInit['dicoBam'][titleBam]['path_downsampling']!="" : dicoInit['dicoBam'][titleBam]['path'] = dicoInit['dicoBam'][titleBam]['path_downsampling']
for alignment in pybam.read(dicoInit['dicoBam'][titleBam]['path']):
if alignment.file_chromosomes[alignment.sam_refID]==dicoInit["dicoBam"][titleBam]['refName'] and alignment.sam_mapq>=dicoInit['minQ'] and not alignment.sam_cigar_string.__contains__("H") and len(alignment.sam_seq)>=dicoInit['minlen']:# and not explain_sam_flags(alignment.sam_flag).__contains__("second in pair") and not explain_sam_flags(alignment.sam_flag).__contains__("supplementary"):
#***** RETRIEVE positions tuple & lastMapped infos *****#
positionsLstTuple,lastMappedPos,lastMappedPosRead = cigar_list_to_tuple(alignment.sam_cigar_list,alignment.sam_pos0)
#***** FORWARD reads *****#
if explain_sam_flags(alignment.sam_flag)=="" or explain_sam_flags(alignment.sam_flag).__contains__("mate reverse strand"):
# Count reads
for posTuple in positionsLstTuple:
try : dicoBam[posTuple[1]+1]['nb_reads_F']+=1
except: pass # None case
# Count softclipped (right soft-clipping)
length, operation = alignment.sam_cigar_list[len(alignment.sam_cigar_list)-1]
if alignment.sam_qname=="MG1U0:00377:02144": print
if operation=="S":
# dicoBam[lastMappedPos]['nb_sc_reads_F']+=1
# Write to Fasta (apply filter) / not consider 'N'
if length-alignment.sam_seq[len(alignment.sam_seq)-length:].count("N")>=dicoInit['SCsize'] and lastMappedPos+length<=dicoInit["dicoGbk"]['refLength']:
dicoBam[lastMappedPos]['nb_sc_reads_F']+=1
nb_mapped_part = min(dicoInit["MappedPart"],lastMappedPosRead)
FASTA.write(">"+str(lastMappedPos)+"_"+str(nb_mapped_part)+"_scrF_"+alignment.sam_qname+"\n"+alignment.sam_seq[len(alignment.sam_seq)-length-nb_mapped_part:]+"\n")
dicoBam[lastMappedPos]['nb_sc_fasta_F']+=1
#***** REVERSE reads *****#
else:
# Count reads
for posTuple in positionsLstTuple:
try : dicoBam[posTuple[1]+1]['nb_reads_R']+=1
except: pass
# Count softclipped (left soft-clipping)
length, operation = alignment.sam_cigar_list[0]
if operation=="S":
# dicoBam[alignment.sam_pos0+1]['nb_sc_reads_R']+=1
# Write to Fasta (apply filter)
if length-alignment.sam_seq[0:length].count("N")>=dicoInit['SCsize'] and alignment.sam_pos0+1-length>=0:
dicoBam[alignment.sam_pos0+1]['nb_sc_reads_R']+=1
nb_mapped_part = min(dicoInit["MappedPart"],length)
FASTA.write(">"+str(alignment.sam_pos0+1)+"_"+str(nb_mapped_part)+"_scrR_"+alignment.sam_qname+"\n"+alignment.sam_seq[0:length+nb_mapped_part]+"\n")
dicoBam[alignment.sam_pos0+1]['nb_sc_fasta_R']+=1
# CLOSE files
FASTA.close()
# WRITE .json results
JSON = open(pathSCjson,'wb')
JSON.write(json.dumps(dicoBam))
JSON.close()
except:
exc_type, exc_value, exc_traceback = sys.exc_info()
lstError.append("ReadThread \""+titleBam+"\": "+str(exc_value)+" (line "+str(exc_traceback.tb_lineno)+")")
#=================================================================================================================================================#
#=================================================================================================================================================#
#=================================================================================================================================================#
def SC_blast(titleBam,dicoInit,lstError):
try:
pathBLASTjson = os.path.join(dicoInit['pathTmpDir'],titleBam+"_BLAST.json")
pathSCfasta = os.path.join(dicoInit['pathTmpDir'],titleBam+".fasta")
pathSCDELBLAST = os.path.join(dicoInit['pathTmpDir'],titleBam+"_blast.out")
pathSCjson = os.path.join(dicoInit['pathTmpDir'],titleBam+"_SC.json")
dicoDel = {}
# Require sc json files to modify positionsRef
dicoBam = load_json(pathSCjson)
#***** LAUNCH BlastN *****# (if fasta not empty)
if os.path.getsize(pathSCfasta)!=0:
cmd = dicoInit['pathBlastN']+" -task blastn-short -query "+pathSCfasta+" -db "+os.path.join(dicoInit['pathTmpDir'],"ref.fa")+" -outfmt \"10 qseqid qstart qend sstart send qlen length\" -out "+pathSCDELBLAST+" -perc_identity "+str(dicoInit['blastIdThreshold'])+" -max_hsps 1 -gapopen "+str(dicoInit['blastGapOpen'])+" -gapextend "+str(dicoInit['blastGapExt'])
os.system(cmd)
#***** Read Blast output file *****#
IN = open(pathSCDELBLAST,'r')
lst_lines = split(IN.read(),"\n")
IN.close()
for line in lst_lines:
if line!="":
# Blast line parser
splitLine = split(line,",")
splitQread = split(splitLine[0],"_")
SCposRead = int(splitQread[0])
SCmappedpart = int(splitQread[1])
readOrient = splitQread[2]
qstart = int(splitLine[1])
qend = int(splitLine[2])
sstart = int(splitLine[3])
send = int(splitLine[4])
qlen = int(splitLine[5])
alignLength = int(splitLine[6])
covPercent = min((float(alignLength+SCmappedpart)*100.0)/float(qlen),100.0)
# Find correspunding positions
if covPercent>=dicoInit['blastCovThreshold'] and sstart<send:
if readOrient=="scrF":
delStart = min(SCposRead-SCmappedpart+qstart-1,dicoInit["dicoGbk"]['refLength']-1)
delEnd = min(sstart,dicoInit["dicoGbk"]['refLength']-1)
delName = str(delStart)+"-"+str(delEnd)
# Filter deletions
if (delStart<delEnd and delEnd-delStart>=dicoInit['SCsize']) or (delStart>delEnd and delStart-delEnd>=dicoInit["mitosize"]):
if delName in dicoDel:
dicoDel[delName]['scrF']['nbBlast']+=1
dicoDel[delName]['scrF']['initial_SCposRead'].append(SCposRead)
if send>dicoDel[delName]['scrF']['limit']: dicoDel[delName]['scrF']['limit'] = send
else: dicoDel[delName] = { 'scrF':{'nbBlast':1,'limit':send,'initial_SCposRead':[SCposRead]}, 'scrR':{'nbBlast':0,'limit':99999,'initial_SCposRead':[]}, 'freqF':0.0, 'freqR':0.0 }
else:
delStart = min(send-(SCmappedpart-qlen+alignLength)+1,dicoInit["dicoGbk"]['refLength']-1)
delEnd = min(SCposRead,dicoInit["dicoGbk"]['refLength']-1)
delName = str(delStart)+"-"+str(delEnd)
# Filter deletions
if (delStart<delEnd and delEnd-delStart>=dicoInit['SCsize']) or (delStart>delEnd and delStart-delEnd>=dicoInit["mitosize"]):
if delName in dicoDel:
dicoDel[delName]['scrR']['nbBlast']+=1
dicoDel[delName]['scrR']['initial_SCposRead'].append(SCposRead)
if sstart<dicoDel[delName]['scrR']['limit']: dicoDel[delName]['scrR']['limit'] = sstart
else: dicoDel[delName] = { 'scrF':{'nbBlast':0,'limit':0,'initial_SCposRead':[]}, 'scrR':{'nbBlast':1,'limit':sstart,'initial_SCposRead':[SCposRead]}, 'freqF':0.0, 'freqR':0.0 }
#***** WRITE .json results file *****#
JSON = open(pathBLASTjson,'wb')
JSON.write(json.JSONEncoder().encode(dicoDel))
JSON.close()
except:
exc_type, exc_value, exc_traceback = sys.exc_info()
lstError.append("SCBlastThread \""+titleBam+"\": "+str(exc_value)+" (line "+str(exc_traceback.tb_lineno)+")")
#=================================================================================================================================================#
#=================================================================================================================================================#
#=================================================================================================================================================#
def deletionPrediction(titleBam,dicoInit,lstError):
try:
pathSCjson = os.path.join(dicoInit['pathTmpDir'],titleBam+"_SC.json")
pathBLASTjson = os.path.join(dicoInit['pathTmpDir'],titleBam+"_BLAST.json")
pathCumuljson = os.path.join(dicoInit['pathTmpDir'],titleBam+"_cumul.json")
#***** LOAD JSON files *****#
dicoBam = load_json(pathSCjson)
dicoDel = load_json(pathBLASTjson)
#**** SHIFT Deletions *****#
# Block one position and apply shift
dico_shift_del = {}
for delName in dicoDel.keys():
start = int(split(delName,"-")[0])
end = int(split(delName,"-")[1])
dico_index = len(dico_shift_del)
dico_shift_del[dico_index] = [delName]
for delName2 in dicoDel.keys():
if delName!=delName2:
start2 = int(split(delName2,"-")[0])
end2 = int(split(delName2,"-")[1])
# Same deletion start => shift end
if (start==start2 and (abs(end-end2)<=dicoInit["delShift"])) or (end==end2 and (abs(start-start2)<=dicoInit["delShift"])):
dico_shift_del[dico_index].append(delName2)
# Merge set
for key in dico_shift_del.keys():
for key2 in dico_shift_del.keys():
if key!=key2:
if len(set(dico_shift_del[key]).intersection(set(dico_shift_del[key2])))>0:
dico_shift_del[key] = set(dico_shift_del[key]).union(set(dico_shift_del[key2]))
dico_shift_del[key2] = set()
# Merge to one maximal blast position
for key in dico_shift_del.keys():
if len(dico_shift_del[key])>0:
nbBlast_F = 0 ; nbBlast_R = 0
lst_start_sc_fasta = [] ; lst_end_sc_fasta = []
maxBlast_F = 0 ; maxBlast_R = 0
maxBlast_start = "" ; maxBlast_end = ""
limit_max_start = -1 ; limit_max_end = -1
initial_SCposRead_F = [] ; initial_SCposRead_R = []
# Browse deletion
for delName in dico_shift_del[key]:
start = split(delName,"-")[0]
end = split(delName,"-")[1]
try: delnbblastF = dicoDel[delName]['scrF']['nbBlast']
except: delnbblastF = 0
try: delnbblastR = dicoDel[delName]['scrR']['nbBlast']
except: delnbblastR = 0
# Merge number of Blast
nbBlast_F+=delnbblastF
nbBlast_R+=delnbblastR
# Merge initial SC positions
try: initial_SCposRead_F.extend(dicoDel[delName]['scrF']['initial_SCposRead'])
except: pass
try: initial_SCposRead_R.extend(dicoDel[delName]['scrR']['initial_SCposRead'])
except: pass
# Find max blast positions for start and end
if delnbblastF>maxBlast_F:
maxBlast_F = delnbblastF
maxBlast_start = start
limit_max_start = dicoDel[delName]['scrF']['limit']
if delnbblastR>maxBlast_R:
maxBlast_R = delnbblastR
maxBlast_end = end
limit_max_end = dicoDel[delName]['scrR']['limit']
# Del entry
try : del(dicoDel[delName])
except: pass
if maxBlast_start=="": maxBlast_start = split(delName,"-")[0]
if maxBlast_end=="": maxBlast_end = split(delName,"-")[1]
dicoDel[maxBlast_start+"-"+maxBlast_end] = { 'scrF':{'nbBlast':nbBlast_F,'limit':limit_max_start,'initial_SCposRead':initial_SCposRead_F},\
'scrR':{'nbBlast':nbBlast_R,'limit':limit_max_end,'initial_SCposRead':initial_SCposRead_R},\
'freqF':0.0, 'freqR':0.0 }
#***** COMPUTE Frequencies *****#
for delName in dicoDel.keys():
# FILTERs
if dicoInit["bilateral"]==True and (dicoDel[delName]['scrF']['nbBlast']<dicoInit["minblast"] or dicoDel[delName]['scrR']['nbBlast']<dicoInit["minblast"]): del(dicoDel[delName])
elif dicoInit["bilateral"]==False and dicoDel[delName]['scrF']['nbBlast']<dicoInit["minblast"] and dicoDel[delName]['scrR']['nbBlast']<dicoInit["minblast"]: del(dicoDel[delName])
# Compute frequency
else:
start = int(split(delName,"-")[0])
end = int(split(delName,"-")[1])
nb_blast_F = float(dicoDel[delName]['scrF']['nbBlast'])
nb_blast_R = float(dicoDel[delName]['scrR']['nbBlast'])
nb_sc_fasta_F = 0 ; set_sc_fasta_pos_F = set()
nb_sc_fasta_R = 0 ; set_sc_fasta_pos_R = set()
if len(dicoDel[delName]['scrF']['initial_SCposRead'])==0: dicoDel[delName]['scrF']['initial_SCposRead'] = [start]
if len(dicoDel[delName]['scrR']['initial_SCposRead'])==0: dicoDel[delName]['scrR']['initial_SCposRead'] = [end]
# Check max total and soft-clipped reads in +-shifted pos
lst_nb_sc_reads_F = []
for init_pos in set(dicoDel[delName]['scrF']['initial_SCposRead']):
for i in range(max(1,init_pos-dicoInit["delShift"]),min(init_pos+dicoInit["delShift"],dicoInit["dicoGbk"]['refLength'])+1,1):
lst_nb_sc_reads_F.append(dicoBam[str(i)]['nb_sc_reads_F'])
if not i in set_sc_fasta_pos_F : nb_sc_fasta_F+=dicoBam[str(i)]['nb_sc_fasta_F'] ; set_sc_fasta_pos_F.add(i)
lst_nb_sc_reads_R = []
for init_pos in set(dicoDel[delName]['scrR']['initial_SCposRead']):
for i in range(max(1,init_pos-dicoInit["delShift"]),min(init_pos+dicoInit["delShift"],dicoInit["dicoGbk"]['refLength'])+1,1):
lst_nb_sc_reads_R.append(dicoBam[str(i)]['nb_sc_reads_R'])
if not i in set_sc_fasta_pos_R : nb_sc_fasta_R+=dicoBam[str(i)]['nb_sc_fasta_R'] ; set_sc_fasta_pos_R.add(i)
# Max occurence start/end
max_occ_start = max(dicoDel[delName]['scrF']['initial_SCposRead'],key=dicoDel[delName]['scrF']['initial_SCposRead'].count)
max_occ_end = max(dicoDel[delName]['scrR']['initial_SCposRead'],key=dicoDel[delName]['scrR']['initial_SCposRead'].count)
nb_reads_F = float(dicoBam[str(max_occ_start)]['nb_reads_F'])
nb_reads_R = float(dicoBam[str(max_occ_end)]['nb_reads_R'])
# Max number of sc reads
if len(lst_nb_sc_reads_F)==0: nb_sc_reads_F = 0
else: nb_sc_reads_F = float(max(lst_nb_sc_reads_F))
if len(lst_nb_sc_reads_R)==0: nb_sc_reads_R = 0
else: nb_sc_reads_R = float(max(lst_nb_sc_reads_R))
# Frequencies
if nb_sc_fasta_F==0: dicoDel[delName]['freqF'] = 0.0
else: dicoDel[delName]['freqF'] = (nb_sc_reads_F/nb_reads_F) * (nb_blast_F/nb_sc_fasta_F) *100.0
if nb_sc_fasta_R==0: dicoDel[delName]['freqR'] = 0.0
else: dicoDel[delName]['freqR'] = (nb_sc_reads_R/nb_reads_R) * (nb_blast_R/nb_sc_fasta_R) *100.0
dicoDel[delName]['depthF'] = nb_reads_F
dicoDel[delName]['depthR'] = nb_reads_R
#***** CUMULATIVE Frequency *****#
dicoCumulFreq = {}
for delPos in dicoDel.keys():
start = int(split(delPos,"-")[0])
end = int(split(delPos,"-")[1])
if start<end: posRange = range(start,end+1,1)
else: posRange = range(start,dicoInit["dicoGbk"]['refLength']+1,1) ; posRange.extend(range(1,end+1,1))
for pos in posRange:
if pos in dicoCumulFreq: dicoCumulFreq[pos]+=(dicoDel[delPos]['freqF']+dicoDel[delPos]['freqR'])/200.0
else: dicoCumulFreq[pos] = (dicoDel[delPos]['freqF']+dicoDel[delPos]['freqR'])/200.0
#***** WRITE .json results files *****#
JSON = open(pathBLASTjson,'wb')
JSON.write(json.JSONEncoder().encode(dicoDel))
JSON.close()
JSON = open(pathCumuljson,'wb')
JSON.write(json.JSONEncoder().encode(dicoCumulFreq))
JSON.close()
except:
exc_type, exc_value, exc_traceback = sys.exc_info()
lstError.append("DelPredThread \""+titleBam+"\": "+str(exc_value)+" (line "+str(exc_traceback.tb_lineno)+")")
#=================================================================================================================================================#
#=================================================================================================================================================#
#=================================================================================================================================================#
def create_results_table(dicoInit,lstError):
# Open output files
pathOutDel = os.path.join(dicoInit['pathOutputDir'],"eKLIPse_deletions.csv")
pathOutGenes = os.path.join(dicoInit['pathOutputDir'],"eKLIPse_genes.csv")
OUTDEL = open(pathOutDel,'w')
OUTGENES = open(pathOutGenes,'w')
# Write headers
headerDel = "\"Title\";\"5' breakpoint\";\"3' breakpoint\";\"Freq\";\"Freq For\";\"Freq Rev\";\"5' Blast\";\"3' Blast\";\"5' Depth\";\"3' Depth\";\"Repetition\"\n"
OUTDEL.write(headerDel)
headerGenes = "\"Gene\";\"Start\";\"End\";\"Type\""
for titleBam in dicoInit["dicoBam"].keys(): headerGenes = headerGenes+",\""+titleBam+"\""
OUTGENES.write(headerGenes+"\n")
#***** BROWSE input alignments *****#
dico_max_gene = {}
for titleBam in dicoInit["dicoBam"].keys():
# Load json results files
dicoDel = load_json(os.path.join(dicoInit['pathTmpDir'],titleBam+"_BLAST.json"))
dicoCumulFreq = load_json(os.path.join(dicoInit['pathTmpDir'],titleBam+"_cumul.json"))
#***** DELETIONS Results File *****#
# Sort deletion by deletion start and stop
dicoSortDel = {}
for deletion in dicoDel.keys():
start = int(split(deletion,"-")[0])
end = int(split(deletion,"-")[1])
if start in dicoSortDel: dicoSortDel[start][end] = deletion
else: dicoSortDel[start] = { end:deletion }
lst_start = dicoSortDel.keys() ; lst_start.sort()
# Browse sorted deletions
for start in lst_start:
lst_end = dicoSortDel[start].keys() ; lst_end.sort()
for end in lst_end:
# Search repetition (right)
repetition_right = "" ; cpt_right = 0
while dicoInit["dicoGbk"]['refSeq'][start+cpt_right]==dicoInit["dicoGbk"]['refSeq'][end-1+cpt_right]:
repetition_right = repetition_right+dicoInit["dicoGbk"]['refSeq'][start+cpt_right]
cpt_right+=1
# Search repetition (right)
repetition_left = "" ; cpt_left = 0
while dicoInit["dicoGbk"]['refSeq'][start-2-cpt_left]==dicoInit["dicoGbk"]['refSeq'][end-1-cpt_left]:
repetition_left = repetition_left+dicoInit["dicoGbk"]['refSeq'][start-1-cpt_left]
cpt_left+=1
repetition_left = repetition_left[::-1]
# Keep best one
if cpt_right==cpt_left==0: repetition = "none"
elif cpt_right>=cpt_left:
repetition = str(start+1)+"-"+repetition_right+"-"+str(start+cpt_right)+" <> "+str(end)+"-"+repetition_right+"-"+str(end-1+cpt_right)
else:
repetition = str(start-cpt_left)+"-"+repetition_left+"-"+str(start-1)+" <> "+str(end+1-cpt_left)+"-"+repetition_left+"-"+str(end)
# Write deletion line
deletion = dicoSortDel[start][end]
delPercent = ((dicoDel[deletion]['freqF']+dicoDel[deletion]['freqR'])/2.0)
ToWrite = "\""+titleBam+"\";\""+str(start)+"\";\""+str(end)+"\";\""+str(delPercent).replace(".",",")+\
"\";\""+str(dicoDel[deletion]['freqF']).replace(".",",")+"\";\""+str(dicoDel[deletion]['freqR']).replace(".",",")+\
"\";\""+str(dicoDel[deletion]['scrF']['nbBlast'])+"\";\""+str(dicoDel[deletion]['scrR']['nbBlast'])+"\";\""+str(dicoDel[deletion]['depthF'])+"\";\""+str(dicoDel[deletion]['depthR'])+"\";\""+repetition+"\"\n"
OUTDEL.write(ToWrite)
#***** GENES max per genes hashtable *****#
for features in dicoInit["dicoGbk"]['lstGene']:
try: dico_max_gene[features[0]][titleBam] = 0.0
except: dico_max_gene[features[0]] = { titleBam:0.0 }
for pos in dicoCumulFreq.keys():
if int(pos)>=features[1] and int(pos)<=features[2]: dico_max_gene[features[0]][titleBam] = max(dico_max_gene[features[0]][titleBam],dicoCumulFreq[pos])
#***** GENES Results File *****#
for features in dicoInit["dicoGbk"]['lstGene']:
ToWrite = "\""+features[0]+"\";\""+str(features[1])+"\";\""+str(features[2])+"\";\""+features[3]+"\""
for titleBam in dicoInit["dicoBam"].keys():
ToWrite = ToWrite+";\""+str(dico_max_gene[features[0]][titleBam]*100.0).replace(".",",")+"\""
OUTGENES.write(ToWrite+"\n")
# Close Files
OUTDEL.close()
OUTGENES.close()