def makemut(args, hc, avoid, alignopts):
mutid_list = []
for site in hc:
mutid_list.append(site['chrom'] + '_' + str(site['start']) + '_' +
str(site['end']) + '_' + str(site['vaf']) + '_' +
str(site['altbase']))
try:
if args.seed is not None:
random.seed(int(args.seed) + int(hc[0]['start']))
bamfile = pysam.Samfile(args.bamFileName, 'rb')
bammate = pysam.Samfile(
args.bamFileName, 'rb') # use for mates to avoid iterator problems
reffile = pysam.Fastafile(args.refFasta)
tmpbams = []
#snvfrac = float(args.snvfrac)
chrom = None
vaf = None
mutpos_list = []
altbase_list = []
for site in hc:
if chrom is None:
chrom = site['chrom']
else:
assert chrom == site[
'chrom'], "haplotype clusters cannot span multiple chromosomes!"
if vaf is None:
vaf = site['vaf']
elif vaf != site['vaf']:
logger.warning(
"multiple VAFs for single haplotype, using first encountered VAF: %f"
% vaf)
mutpos = int(random.uniform(site['start'], site['end'] +
1)) # position of mutation in genome
mutpos_list.append(mutpos) # FIXME
altbase_list.append(site['altbase'])
mutbase_list = []
refbase_list = []
mutstr_list = []
for n, mutpos in enumerate(mutpos_list):
refbase = reffile.fetch(chrom, mutpos - 1, mutpos)
altbase = altbase_list[n]
refbase_list.append(refbase)
if altbase == refbase.upper() and not args.ignoreref:
logger.warning(
"%s specified ALT base matches reference, skipping mutation"
% mutid_list[n])
return None
try:
mutbase = mut(refbase, altbase)
mutbase_list.append(mutbase)
except ValueError as e:
logger.warning(mutid_list[n] + " " + ' '.join(
("skipped site:", chrom, str(hc[n]['start']),
str(hc[n]['end']), "due to N base:", str(e), "\n")))
return None
mutstr_list.append(refbase + "-->" + str(mutbase))
# optional CNV file
cnv = None
if (args.cnvfile):
cnv = pysam.Tabixfile(args.cnvfile, 'r')
hapstr = "_".join(
('haplo', chrom, str(min(mutpos_list)), str(max(mutpos_list))))
log = open(
'addsnv_logs_' + os.path.basename(args.outBamFile) + '/' +
os.path.basename(args.outBamFile) + "." + hapstr + ".log", 'w')
tmpoutbamname = args.tmpdir + "/" + hapstr + ".tmpbam." + str(
uuid4()) + ".bam"
logger.info("%s creating tmp bam: %s" % (hapstr, tmpoutbamname))
outbam_muts = pysam.Samfile(tmpoutbamname, 'wb', template=bamfile)
mutfail, hasSNP, maxfrac, outreads, mutreads, mutmates = mutation.mutate(
args,
log,
bamfile,
bammate,
chrom,
min(mutpos_list),
max(mutpos_list) + 1,
mutpos_list,
avoid=avoid,
mutid_list=mutid_list,
is_snv=True,
mutbase_list=mutbase_list,
reffile=reffile)
if mutfail:
outbam_muts.close()
os.remove(tmpoutbamname)
return None
# pick reads to change
readlist = []
for extqname, read in outreads.iteritems():
if read.seq != mutreads[extqname]:
readlist.append(extqname)
logger.info("%s len(readlist): %s" % (hapstr, str(len(readlist))))
readlist.sort()
random.shuffle(readlist)
if len(readlist) < int(args.mindepth):
logger.warning("%s too few reads in region (%s) skipping..." %
(hapstr, str(len(readlist))))
outbam_muts.close()
os.remove(tmpoutbamname)
return None
if vaf is None:
vaf = float(
args.mutfrac
) # default minor allele freq if not otherwise specified
if cnv: # cnv file is present
if chrom in cnv.contigs:
for cnregion in cnv.fetch(chrom, min(mutpos_list),
max(mutpos_list) + 1):
cn = float(cnregion.strip().split()
[3]) # expect chrom,start,end,CN
logger.info(hapstr + "\t" +
' '.join(("copy number in snp region:", chrom,
str(min(mutpos_list)),
str(max(mutpos_list)), "=",
str(cn))))
if float(cn) > 0.0:
vaf = 1.0 / float(cn)
else:
vaf = 0.0
logger.info("%s adjusted VAF: %f" % (hapstr, vaf))
else:
logger.info("%s selected VAF: %f" % (hapstr, vaf))
lastread = int(len(readlist) * vaf)
# pick at least args.minmutreads if possible
if lastread < int(args.minmutreads):
if len(readlist) > int(args.minmutreads):
lastread = int(args.minmutreads)
logger.warning("%s forced %d reads." % (hapstr, lastread))
else:
logger.warning(
"%s dropped site with fewer reads than --minmutreads" %
hapstr)
os.remove(tmpoutbamname)
return None
readtrack = dd(list)
for readname in readlist:
orig_name, readpos, pairend = readname.split(',')
readtrack[orig_name].append('%s,%s' % (readpos, pairend))
usedreads = 0
newreadlist = []
for orig_name in readtrack:
for read_instance in readtrack[orig_name]:
newreadlist.append(orig_name + ',' + read_instance)
usedreads += 1
if usedreads >= lastread:
break
readlist = newreadlist
logger.info("%s picked: %d" % (hapstr, len(readlist)))
wrote = 0
nmut = 0
mut_out = {}
# change reads from .bam to mutated sequences
for extqname, read in outreads.iteritems():
if read.seq != mutreads[extqname]:
if not args.nomut and extqname in readlist:
qual = read.qual # changing seq resets qual (see pysam API docs)
read.seq = mutreads[extqname] # make mutation
read.qual = qual
nmut += 1
if not hasSNP or args.force:
wrote += 1
mut_out[extqname] = read
muts_written = {}
for extqname in mut_out:
if extqname not in muts_written:
outbam_muts.write(mut_out[extqname])
muts_written[extqname] = True
if mutmates[extqname] is not None:
# is mate also in mutated list?
mate_read = mutmates[extqname]
pairname = 'F' # read is first in pair
if mate_read.is_read2:
pairname = 'S' # read is second in pair
if not mate_read.is_paired:
pairname = 'U' # read is unpaired
mateqname = ','.join(
(mate_read.qname, str(mate_read.pos), pairname))
if mateqname in mut_out:
# yes: output mutated mate
outbam_muts.write(mut_out[mateqname])
muts_written[mateqname] = True
else:
# no: output original mate
outbam_muts.write(mate_read)
logger.info("%s wrote: %d, mutated: %d" % (hapstr, wrote, nmut))
if not hasSNP or args.force:
outbam_muts.close()
aligners.remap_bam(args.aligner,
tmpoutbamname,
args.refFasta,
alignopts,
mutid=hapstr,
paired=(not args.single),
picardjar=args.picardjar,
insane=args.insane)
outbam_muts = pysam.Samfile(tmpoutbamname, 'rb')
coverwindow = 1
incover = countReadCoverage(bamfile, chrom,
min(mutpos_list) - coverwindow,
max(mutpos_list) + coverwindow)
outcover = countReadCoverage(outbam_muts, chrom,
min(mutpos_list) - coverwindow,
max(mutpos_list) + coverwindow)
avgincover = float(sum(incover)) / float(len(incover))
avgoutcover = float(sum(outcover)) / float(len(outcover))
logger.info("%s avgincover: %f, avgoutcover: %f" %
(hapstr, avgincover, avgoutcover))
spikein_snvfrac = 0.0
if wrote > 0:
spikein_snvfrac = float(nmut) / float(wrote)
# qc cutoff for final snv depth
if (avgoutcover > 0 and avgincover > 0 and avgoutcover / avgincover
>= float(args.coverdiff)) or args.force:
tmpbams.append(tmpoutbamname)
for n, site in enumerate(hc):
snvstr = chrom + ":" + str(site['start']) + "-" + str(
site['end']) + " (VAF=" + str(vaf) + ")"
log.write("\t".join(("snv", snvstr,
str(mutpos_list[n]), mutstr_list[n],
str(avgoutcover), str(avgoutcover),
str(spikein_snvfrac), str(maxfrac))) +
"\n")
else:
outbam_muts.close()
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
logger.warning("%s dropped for outcover/incover < %s" %
(hapstr, str(args.coverdiff)))
return None
outbam_muts.close()
bamfile.close()
bammate.close()
log.close()
return tmpbams
except Exception, e:
sys.stderr.write("*" * 60 + "\nERROR\t" + now() +
"\tencountered error in mutation spikein: " +
str(mutid_list) + "\n")
traceback.print_exc(file=sys.stdout)
sys.stderr.write("*" * 60 + "\n")
if os.path.exists(tmpoutbamname):
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
return None
def makemut(args, chrom, start, end, vaf, ins, avoid, alignopts):
''' is ins is a sequence, it will is inserted at start, otherwise delete from start to end'''
if args.seed is not None: random.seed(int(args.seed) + int(start))
mutid = chrom + '_' + str(start) + '_' + str(end) + '_' + str(vaf)
if ins is None:
mutid += ':DEL'
else:
mutid += ':INS:' + ins
try:
bamfile = pysam.Samfile(args.bamFileName, 'rb')
bammate = pysam.Samfile(args.bamFileName, 'rb') # use for mates to avoid iterator problems
reffile = pysam.Fastafile(args.refFasta)
tmpbams = []
is_insertion = ins is not None
is_deletion = ins is None
snvfrac = float(args.snvfrac)
mutstr = get_mutstr(chrom, start, end, ins, reffile)
del_ln = 0
if is_deletion:
del_ln = end-start
mutpos = start
mutpos_list = [start]
# optional CNV file
cnv = None
if (args.cnvfile):
cnv = pysam.Tabixfile(args.cnvfile, 'r')
log = open('addindel_logs_' + os.path.basename(args.outBamFile) + '/' + os.path.basename(args.outBamFile) + "." + "_".join((chrom,str(start),str(end))) + ".log",'w')
tmpoutbamname = args.tmpdir + "/" + mutid + ".tmpbam." + str(uuid4()) + ".bam"
print "INFO\t" + now() + "\t" + mutid + "\tcreating tmp bam: ",tmpoutbamname #DEBUG
outbam_muts = pysam.Samfile(tmpoutbamname, 'wb', template=bamfile)
mutfail, hasSNP, maxfrac, outreads, mutreads, mutmates = mutation.mutate(args, log, bamfile, bammate, chrom, mutpos, mutpos+del_ln+1, mutpos_list, avoid=avoid, mutid_list=[mutid], is_insertion=is_insertion, is_deletion=is_deletion, ins_seq=ins, reffile=reffile, indel_start=start, indel_end=end)
if mutfail:
outbam_muts.close()
os.remove(tmpoutbamname)
return None
# pick reads to change
readlist = []
for extqname,read in outreads.iteritems():
if read.seq != mutreads[extqname]:
readlist.append(extqname)
print "len(readlist):",str(len(readlist))
readlist.sort()
random.shuffle(readlist)
if len(readlist) < int(args.mindepth):
sys.stderr.write("WARN\t" + now() + "\t" + mutid + "\tskipped, too few reads in region: " + str(len(readlist)) + "\n")
outbam_muts.close()
os.remove(tmpoutbamname)
return None
if vaf is None:
vaf = float(args.mutfrac) # default minor allele freq if not otherwise specified
if cnv: # cnv file is present
if chrom in cnv.contigs:
for cnregion in cnv.fetch(chrom,start,end):
cn = float(cnregion.strip().split()[3]) # expect chrom,start,end,CN
sys.stdout.write("INFO\t" + now() + "\t" + mutid + "\t" + ' '.join(("copy number in snp region:",chrom,str(start),str(end),"=",str(cn))) + "\n")
if float(cn) > 0.0:
vaf = 1.0/float(cn)
else:
vaf = 0.0
sys.stdout.write("INFO\t" + now() + "\t" + mutid + "\tadjusted VAF: " + str(vaf) + "\n")
else:
sys.stdout.write("INFO\t" + now() + "\t" + mutid + "\tselected VAF: " + str(vaf) + "\n")
lastread = int(len(readlist)*vaf)
# pick at least args.minmutreads if possible
if lastread < int(args.minmutreads):
if len(readlist) > int(args.minmutreads):
lastread = int(args.minmutreads)
sys.stdout.write("WARN\t" + now() + "\t" + mutid + "\tforced " + str(lastread) + " reads.\n")
else:
print "WARN\t" + now() + "\t" + mutid + "\tdropped site with fewer reads than --minmutreads"
os.remove(tmpoutbamname)
return None
readtrack = dd(list)
for readname in readlist:
orig_name, readpos, pairend = readname.split(',')
readtrack[orig_name].append('%s,%s' % (readpos, pairend))
usedreads = 0
newreadlist = []
for orig_name in readtrack:
for read_instance in readtrack[orig_name]:
newreadlist.append(orig_name + ',' + read_instance)
usedreads += 1
if usedreads >= lastread:
break
readlist = newreadlist
print "INFO\t" + now() + "\t" + mutid + "\tpicked: " + str(len(readlist)) + " reads"
wrote = 0
nmut = 0
mut_out = {}
# change reads from .bam to mutated sequences
for extqname,read in outreads.iteritems():
if read.seq != mutreads[extqname]:
if not args.nomut and extqname in readlist:
qual = read.qual # changing seq resets qual (see pysam API docs)
read.seq = mutreads[extqname] # make mutation
read.qual = qual
nmut += 1
if not hasSNP or args.force:
wrote += 1
mut_out[extqname] = read
muts_written = {}
for extqname in mut_out:
if extqname not in muts_written:
outbam_muts.write(mut_out[extqname])
muts_written[extqname] = True
if mutmates[extqname] is not None:
# is mate also in mutated list?
mate_read = mutmates[extqname]
pairname = 'F' # read is first in pair
if mate_read.is_read2:
pairname = 'S' # read is second in pair
if not mate_read.is_paired:
pairname = 'U' # read is unpaired
mateqname = ','.join((mate_read.qname,str(mate_read.pos),pairname))
if mateqname in mut_out:
# yes: output mutated mate
outbam_muts.write(mut_out[mateqname])
muts_written[mateqname] = True
else:
# no: output original mate
outbam_muts.write(mate_read)
print "INFO\t" + now() + "\t" + mutid + "\twrote: " + str(wrote) + " reads, mutated: " + str(nmut) + " reads"
if not hasSNP or args.force:
outbam_muts.close()
aligners.remap_bam(args.aligner, tmpoutbamname, args.refFasta, alignopts, mutid=mutid, paired=(not args.single), picardjar=args.picardjar)
outbam_muts = pysam.Samfile(tmpoutbamname,'rb')
coverwindow = 1
incover = countReadCoverage(bamfile,chrom,mutpos-coverwindow,mutpos+del_ln+coverwindow)
outcover = countReadCoverage(outbam_muts,chrom,mutpos-coverwindow,mutpos+del_ln+coverwindow)
avgincover = float(sum(incover))/float(len(incover))
avgoutcover = float(sum(outcover))/float(len(outcover))
spikein_frac = 0.0
if wrote > 0:
spikein_frac = float(nmut)/float(wrote)
# qc cutoff for final snv depth
if (avgoutcover > 0 and avgincover > 0 and avgoutcover/avgincover >= float(args.coverdiff)) or args.force:
tmpbams.append(tmpoutbamname)
indelstr = ''
if is_insertion:
indelstr = ':'.join(('INS', chrom, str(start), ins))
else:
indelstr = ':'.join(('DEL', chrom, str(start), str(end)))
snvstr = chrom + ":" + str(start) + "-" + str(end) + " (VAF=" + str(vaf) + ")"
log.write("\t".join(("indel",indelstr,str(mutpos),mutstr,str(avgincover),str(avgoutcover),str(spikein_frac),str(maxfrac)))+"\n")
else:
outbam_muts.close()
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
print "WARN\t" + now() + "\t" + mutid + "\tdropped for outcover/incover < " + str(args.coverdiff)
return None
outbam_muts.close()
bamfile.close()
bammate.close()
log.close()
return sorted(tmpbams)
except Exception, e:
sys.stderr.write("*"*60 + "\nencountered error in mutation spikein: " + mutid + "\n")
traceback.print_exc(file=sys.stdout)
sys.stderr.write("*"*60 + "\n")
if os.path.exists(tmpoutbamname):
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
return None
def makemut(args, chrom, start, end, vaf, ins, avoid, alignopts):
''' is ins is a sequence, it will is inserted at start, otherwise delete from start to end'''
if args.seed is not None: random.seed(int(args.seed) + int(start))
mutid = chrom + '_' + str(start) + '_' + str(end) + '_' + str(vaf)
if ins is None:
mutid += ':DEL'
else:
mutid += ':INS:' + ins
try:
bamfile = pysam.Samfile(args.bamFileName, 'rb')
bammate = pysam.Samfile(
args.bamFileName, 'rb') # use for mates to avoid iterator problems
reffile = pysam.Fastafile(args.refFasta)
tmpbams = []
is_insertion = ins is not None
is_deletion = ins is None
snvfrac = float(args.snvfrac)
mutstr = get_mutstr(chrom, start, end, ins, reffile)
del_ln = 0
if is_deletion:
del_ln = end - start
mutpos = start
mutpos_list = [start]
# optional CNV file
cnv = None
if (args.cnvfile):
cnv = pysam.Tabixfile(args.cnvfile, 'r')
log = open(
'addindel_logs_' + os.path.basename(args.outBamFile) + '/' +
os.path.basename(args.outBamFile) + "." + "_".join(
(chrom, str(start), str(end))) + ".log", 'w')
tmpoutbamname = args.tmpdir + "/" + mutid + ".tmpbam." + str(
uuid4()) + ".bam"
print "INFO\t" + now(
) + "\t" + mutid + "\tcreating tmp bam: ", tmpoutbamname #DEBUG
outbam_muts = pysam.Samfile(tmpoutbamname, 'wb', template=bamfile)
mutfail, hasSNP, maxfrac, outreads, mutreads, mutmates = mutation.mutate(
args,
log,
bamfile,
bammate,
chrom,
mutpos,
mutpos + del_ln + 1,
mutpos_list,
avoid=avoid,
mutid_list=[mutid],
is_insertion=is_insertion,
is_deletion=is_deletion,
ins_seq=ins,
reffile=reffile,
indel_start=start,
indel_end=end)
if mutfail:
outbam_muts.close()
os.remove(tmpoutbamname)
return None
# pick reads to change
readlist = []
for extqname, read in outreads.iteritems():
if read.seq != mutreads[extqname]:
readlist.append(extqname)
print "len(readlist):", str(len(readlist))
readlist.sort()
random.shuffle(readlist)
if len(readlist) < int(args.mindepth):
sys.stderr.write("WARN\t" + now() + "\t" + mutid +
"\tskipped, too few reads in region: " +
str(len(readlist)) + "\n")
outbam_muts.close()
os.remove(tmpoutbamname)
return None
if vaf is None:
vaf = float(
args.mutfrac
) # default minor allele freq if not otherwise specified
if cnv: # cnv file is present
if chrom in cnv.contigs:
for cnregion in cnv.fetch(chrom, start, end):
cn = float(cnregion.strip().split()
[3]) # expect chrom,start,end,CN
sys.stdout.write("INFO\t" + now() + "\t" + mutid + "\t" +
' '.join(("copy number in snp region:",
chrom, str(start), str(end),
"=", str(cn))) + "\n")
if float(cn) > 0.0:
vaf = 1.0 / float(cn)
else:
vaf = 0.0
sys.stdout.write("INFO\t" + now() + "\t" + mutid +
"\tadjusted VAF: " + str(vaf) + "\n")
else:
sys.stdout.write("INFO\t" + now() + "\t" + mutid +
"\tselected VAF: " + str(vaf) + "\n")
lastread = int(len(readlist) * vaf)
# pick at least args.minmutreads if possible
if lastread < int(args.minmutreads):
if len(readlist) > int(args.minmutreads):
lastread = int(args.minmutreads)
sys.stdout.write("WARN\t" + now() + "\t" + mutid +
"\tforced " + str(lastread) + " reads.\n")
else:
print "WARN\t" + now(
) + "\t" + mutid + "\tdropped site with fewer reads than --minmutreads"
os.remove(tmpoutbamname)
return None
readtrack = dd(list)
for readname in readlist:
orig_name, readpos, pairend = readname.split(',')
readtrack[orig_name].append('%s,%s' % (readpos, pairend))
usedreads = 0
newreadlist = []
for orig_name in readtrack:
for read_instance in readtrack[orig_name]:
newreadlist.append(orig_name + ',' + read_instance)
usedreads += 1
if usedreads >= lastread:
break
readlist = newreadlist
print "INFO\t" + now() + "\t" + mutid + "\tpicked: " + str(
len(readlist)) + " reads"
wrote = 0
nmut = 0
mut_out = {}
# change reads from .bam to mutated sequences
for extqname, read in outreads.iteritems():
if read.seq != mutreads[extqname]:
if not args.nomut and extqname in readlist:
qual = read.qual # changing seq resets qual (see pysam API docs)
read.seq = mutreads[extqname] # make mutation
read.qual = qual
nmut += 1
if not hasSNP or args.force:
wrote += 1
mut_out[extqname] = read
muts_written = {}
for extqname in mut_out:
if extqname not in muts_written:
outbam_muts.write(mut_out[extqname])
muts_written[extqname] = True
if mutmates[extqname] is not None:
# is mate also in mutated list?
mate_read = mutmates[extqname]
pairname = 'F' # read is first in pair
if mate_read.is_read2:
pairname = 'S' # read is second in pair
if not mate_read.is_paired:
pairname = 'U' # read is unpaired
mateqname = ','.join(
(mate_read.qname, str(mate_read.pos), pairname))
if mateqname in mut_out:
# yes: output mutated mate
outbam_muts.write(mut_out[mateqname])
muts_written[mateqname] = True
else:
# no: output original mate
outbam_muts.write(mate_read)
print "INFO\t" + now() + "\t" + mutid + "\twrote: " + str(
wrote) + " reads, mutated: " + str(nmut) + " reads"
if not hasSNP or args.force:
outbam_muts.close()
aligners.remap_bam(args.aligner,
tmpoutbamname,
args.refFasta,
alignopts,
mutid=mutid,
paired=(not args.single),
picardjar=args.picardjar,
insane=args.insane)
outbam_muts = pysam.Samfile(tmpoutbamname, 'rb')
coverwindow = 1
incover = countReadCoverage(bamfile, chrom, mutpos - coverwindow,
mutpos + del_ln + coverwindow)
outcover = countReadCoverage(outbam_muts, chrom,
mutpos - coverwindow,
mutpos + del_ln + coverwindow)
avgincover = float(sum(incover)) / float(len(incover))
avgoutcover = float(sum(outcover)) / float(len(outcover))
spikein_frac = 0.0
if wrote > 0:
spikein_frac = float(nmut) / float(wrote)
# qc cutoff for final snv depth
if (avgoutcover > 0 and avgincover > 0 and avgoutcover / avgincover
>= float(args.coverdiff)) or args.force:
tmpbams.append(tmpoutbamname)
indelstr = ''
if is_insertion:
indelstr = ':'.join(('INS', chrom, str(start), ins))
else:
indelstr = ':'.join(('DEL', chrom, str(start), str(end)))
snvstr = chrom + ":" + str(start) + "-" + str(
end) + " (VAF=" + str(vaf) + ")"
log.write("\t".join(("indel", indelstr, str(mutpos), mutstr,
str(avgincover), str(avgoutcover),
str(spikein_frac), str(maxfrac))) + "\n")
else:
outbam_muts.close()
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
print "WARN\t" + now(
) + "\t" + mutid + "\tdropped for outcover/incover < " + str(
args.coverdiff)
return None
outbam_muts.close()
bamfile.close()
bammate.close()
log.close()
return sorted(tmpbams)
except Exception, e:
sys.stderr.write("*" * 60 +
"\nencountered error in mutation spikein: " + mutid +
"\n")
traceback.print_exc(file=sys.stdout)
sys.stderr.write("*" * 60 + "\n")
if os.path.exists(tmpoutbamname):
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
return None
def makemut(args, hc, avoid, alignopts):
mutid_list = []
for site in hc:
mutid_list.append(site['chrom'] + '_' + str(site['start']) + '_' + str(site['end']) + '_' + str(site['vaf']) + '_' + str(site['altbase']))
try:
if args.seed is not None: random.seed(int(args.seed) + int(hc[0]['start']))
bamfile = pysam.Samfile(args.bamFileName, 'rb')
bammate = pysam.Samfile(args.bamFileName, 'rb') # use for mates to avoid iterator problems
reffile = pysam.Fastafile(args.refFasta)
tmpbams = []
#snvfrac = float(args.snvfrac)
chrom = None
vaf = None
mutpos_list = []
altbase_list = []
for site in hc:
if chrom is None:
chrom = site['chrom']
else:
assert chrom == site['chrom'], "haplotype clusters cannot span multiple chromosomes!"
if vaf is None:
vaf = site['vaf']
elif vaf != site['vaf']:
sys.stderr.write("WARN\t" + now() + "\tmultiple VAFs for single haplotype, using first encountered VAF: " + str(vaf) + "\n")
mutpos = int(random.uniform(site['start'],site['end']+1)) # position of mutation in genome
mutpos_list.append(mutpos) # FIXME
altbase_list.append(site['altbase'])
mutbase_list = []
refbase_list = []
mutstr_list = []
for n, mutpos in enumerate(mutpos_list):
refbase = reffile.fetch(chrom,mutpos-1,mutpos)
altbase = altbase_list[n]
refbase_list.append(refbase)
if altbase == refbase.upper() and not args.ignoreref:
sys.stderr.write("WARN\t" + now() + "\t" + mutid_list[n] + "\tspecified ALT base matches reference, skipping mutation\n")
return None
try:
mutbase = mut(refbase, altbase)
mutbase_list.append(mutbase)
except ValueError as e:
sys.stderr.write("WARN\t" + now() + "\t" + mutid_list[n] + "\t" + ' '.join(("skipped site:",chrom,str(hc[n]['start']),str(hc[n]['end']),"due to N base:",str(e),"\n")))
return None
mutstr_list.append(refbase + "-->" + str(mutbase))
# optional CNV file
cnv = None
if (args.cnvfile):
cnv = pysam.Tabixfile(args.cnvfile, 'r')
hapstr = "_".join(('haplo',chrom,str(min(mutpos_list)),str(max(mutpos_list))))
log = open('addsnv_logs_' + os.path.basename(args.outBamFile) + '/' + os.path.basename(args.outBamFile) + "." + hapstr + ".log",'w')
tmpoutbamname = args.tmpdir + "/" + hapstr + ".tmpbam." + str(uuid4()) + ".bam"
print "INFO\t" + now() + "\t" + hapstr + "\tcreating tmp bam: ",tmpoutbamname
outbam_muts = pysam.Samfile(tmpoutbamname, 'wb', template=bamfile)
mutfail, hasSNP, maxfrac, outreads, mutreads, mutmates = mutation.mutate(args, log, bamfile, bammate, chrom, min(mutpos_list), max(mutpos_list)+1, mutpos_list, avoid=avoid, mutid_list=mutid_list, is_snv=True, mutbase_list=mutbase_list, reffile=reffile)
if mutfail:
outbam_muts.close()
os.remove(tmpoutbamname)
return None
# pick reads to change
readlist = []
for extqname,read in outreads.iteritems():
if read.seq != mutreads[extqname]:
readlist.append(extqname)
print "INFO\t" + now() + "\t" + hapstr + "\tlen(readlist): " + str(len(readlist))
readlist.sort()
random.shuffle(readlist)
if len(readlist) < int(args.mindepth):
print "WARN\t" + now() + "\t" + hapstr + "\ttoo few reads in region (" + str(len(readlist)) + ") skipping..."
outbam_muts.close()
os.remove(tmpoutbamname)
return None
if vaf is None:
vaf = float(args.mutfrac) # default minor allele freq if not otherwise specified
if cnv: # cnv file is present
if chrom in cnv.contigs:
for cnregion in cnv.fetch(chrom,min(mutpos_list),max(mutpos_list)+1):
cn = float(cnregion.strip().split()[3]) # expect chrom,start,end,CN
print "INFO\t" + now() + "\t" + hapstr + "\t" + ' '.join(("copy number in snp region:",chrom,str(min(mutpos_list)),str(max(mutpos_list)),"=",str(cn))) + "\n"
if float(cn) > 0.0:
vaf = 1.0/float(cn)
else:
vaf = 0.0
print "adjusted VAF: " + str(vaf) + "\n"
else:
print "INFO\t" + now() + "\t" + hapstr + "\tselected VAF: " + str(vaf) + "\n"
lastread = int(len(readlist)*vaf)
# pick at least args.minmutreads if possible
if lastread < int(args.minmutreads):
if len(readlist) > int(args.minmutreads):
lastread = int(args.minmutreads)
sys.stdout.write("WARN\t" + now() + "\t" + hapstr + "\tforced " + str(lastread) + " reads.\n")
else:
print "WARN\t" + now() + "\t" + hapstr + "\tdropped site with fewer reads than --minmutreads"
os.remove(tmpoutbamname)
return None
readtrack = dd(list)
for readname in readlist:
orig_name, readpos, pairend = readname.split(',')
readtrack[orig_name].append('%s,%s' % (readpos, pairend))
usedreads = 0
newreadlist = []
for orig_name in readtrack:
for read_instance in readtrack[orig_name]:
newreadlist.append(orig_name + ',' + read_instance)
usedreads += 1
if usedreads >= lastread:
break
readlist = newreadlist
print "INFO\t" + now() + "\t" + hapstr + "\tpicked:",str(len(readlist))
wrote = 0
nmut = 0
mut_out = {}
# change reads from .bam to mutated sequences
for extqname,read in outreads.iteritems():
if read.seq != mutreads[extqname]:
if not args.nomut and extqname in readlist:
qual = read.qual # changing seq resets qual (see pysam API docs)
read.seq = mutreads[extqname] # make mutation
read.qual = qual
nmut += 1
if not hasSNP or args.force:
wrote += 1
mut_out[extqname] = read
muts_written = {}
for extqname in mut_out:
if extqname not in muts_written:
outbam_muts.write(mut_out[extqname])
muts_written[extqname] = True
if mutmates[extqname] is not None:
# is mate also in mutated list?
mate_read = mutmates[extqname]
pairname = 'F' # read is first in pair
if mate_read.is_read2:
pairname = 'S' # read is second in pair
if not mate_read.is_paired:
pairname = 'U' # read is unpaired
mateqname = ','.join((mate_read.qname,str(mate_read.pos),pairname))
if mateqname in mut_out:
# yes: output mutated mate
outbam_muts.write(mut_out[mateqname])
muts_written[mateqname] = True
else:
# no: output original mate
outbam_muts.write(mate_read)
print "INFO\t" + now() + "\t" + hapstr + "\twrote: ",wrote,"mutated:",nmut
if not hasSNP or args.force:
outbam_muts.close()
aligners.remap_bam(args.aligner, tmpoutbamname, args.refFasta, alignopts, mutid=hapstr, paired=(not args.single), picardjar=args.picardjar)
outbam_muts = pysam.Samfile(tmpoutbamname,'rb')
coverwindow = 1
incover = countReadCoverage(bamfile,chrom,min(mutpos_list)-coverwindow,max(mutpos_list)+coverwindow)
outcover = countReadCoverage(outbam_muts,chrom,min(mutpos_list)-coverwindow,max(mutpos_list)+coverwindow)
avgincover = float(sum(incover))/float(len(incover))
avgoutcover = float(sum(outcover))/float(len(outcover))
print "INFO\t" + now() + "\t" + hapstr + "\tavgincover: " + str(avgincover) + " avgoutcover: " + str(avgoutcover)
spikein_snvfrac = 0.0
if wrote > 0:
spikein_snvfrac = float(nmut)/float(wrote)
# qc cutoff for final snv depth
if (avgoutcover > 0 and avgincover > 0 and avgoutcover/avgincover >= float(args.coverdiff)) or args.force:
tmpbams.append(tmpoutbamname)
for n,site in enumerate(hc):
snvstr = chrom + ":" + str(site['start']) + "-" + str(site['end']) + " (VAF=" + str(vaf) + ")"
log.write("\t".join(("snv",snvstr,str(mutpos_list[n]),mutstr_list[n],str(avgoutcover),str(avgoutcover),str(spikein_snvfrac),str(maxfrac)))+"\n")
else:
outbam_muts.close()
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
print "WARN\t" + now() + "\t" + hapstr + "\tdropped for outcover/incover < " + str(args.coverdiff)
return None
outbam_muts.close()
bamfile.close()
bammate.close()
log.close()
return tmpbams
except Exception, e:
sys.stderr.write("*"*60 + "\nERROR\t" + now() + "\tencountered error in mutation spikein: " + str(mutid_list) + "\n")
traceback.print_exc(file=sys.stdout)
sys.stderr.write("*"*60 + "\n")
if os.path.exists(tmpoutbamname):
os.remove(tmpoutbamname)
if os.path.exists(tmpoutbamname + '.bai'):
os.remove(tmpoutbamname + '.bai')
return None