def main():
"""The main function
"""
parser = cmdline_parser()
args = parser.parse_args()
if args.verbose:
LOG.setLevel(logging.INFO)
if args.debug:
LOG.setLevel(logging.DEBUG)
assert os.path.exists(args.bam), ("BAM file %s does not exist" % args.bam)
samfh = pysam.Samfile(args.bam)
# setup vcf_reader
#
if args.vcfin == '-':
vcf_reader = vcf.VCFReader(sys.stdin)
else:
vcf_reader = vcf.VCFReader(filename=args.vcfin)
variants = [r for r in vcf_reader]
LOG.info("Loaded %d variants" % len(variants))
if args.mtc.lower() != 'None':
LOG.info("Will use %s for MTC on %s with alpha %f" %
(args.mtc, args.mtc_tag, args.mtc_alpha))
else:
LOG.info("No multiple testing correction will be done")
# setup vcf_writer
#
if args.vcfout == '-':
fh_out = sys.stdout
else:
if os.path.exists(args.vcfout):
LOG.fatal(
"Cowardly refusing to overwrite already existing file %s" %
(args.vcfout))
sys.exit(1)
if args.vcfout[-3:] == '.gz':
fh_out = gzip.open(args.vcfout, 'w')
else:
fh_out = open(args.vcfout, 'w')
# pyvcf needs template as arg to VCFWriter, whereas LoFreq's vcf clone didn't
vcf_writer = vcf.VCFWriter(fh_out, vcf_reader, lineterminator=os.linesep)
#vcf_writer = vcf.VCFWriter(fh_out)
#vcf_writer.meta_from_reader(vcf_reader)
pvalues = []
for (var_no, var) in enumerate(variants):
if var_no % 500 == 1:
LOG.info("Computing bias for var %d of %d" %
(var_no, len(variants)))
if var.INFO.has_key('INDEL'):
LOG.warn("Skipping unsupported indel variant %s:%d" %
(var.CHROM, var.POS))
continue
reads = list(
samfh.fetch(reference=var.CHROM, start=var.POS - 1, end=var.POS))
LOG.debug("%s %d: %d (unfiltered) reads covering position" %
(var.CHROM, var.POS, len(reads)))
ref_mquals = []
alt_mquals = []
ref_bquals = []
alt_bquals = []
# only for PE
#ref_isize = []
#alt_isize = []
# following two meant to test
#alt_vpos = []
#rlens = []
for r in reads:
if skip_read(r):
continue
orphan = (r.flag & 0x1) and not (r.flag & 0x2)
if orphan and not args.use_orphan:
continue
if r.mapq < args.min_mq:
continue
vpos_on_read = [
vpos_on_read for (vpos_on_read, vpos_on_ref) in r.aligned_pairs
if vpos_on_ref == var.POS - 1
]
assert len(vpos_on_read) == 1
vpos_on_read = vpos_on_read[0]
if vpos_on_read == None: # skip deletions
continue
#alt_vpos.append(vpos_on_read)
#rlens.append(r.rlen)
b = r.query[vpos_on_read]
bq = ord(r.qqual[vpos_on_read]) - 33
mq = r.mapq
if bq < args.min_bq:
continue
assert len(var.REF) == 1 and len(var.ALT) == 1
if b.upper() == var.REF[0].upper():
ref_mquals.append(mq)
ref_bquals.append(bq)
#if not args.use_orphan:
# ref_isize.append(abs(r.tlen))
elif b.upper() == str(var.ALT[0]).upper():
alt_mquals.append(mq)
alt_bquals.append(bq)
#if not args.use_orphan:
# alt_isize.append(abs(r.tlen))
else:
LOG.debug("Skipping non-ref-alt base %s at %s:%d" %
(b, var.CHROM, var.POS))
continue
LOG.debug("After filtering at %s:%d: %d ref mquals and %d alt mquals" %
(var.CHROM, var.POS, len(ref_mquals), len(alt_mquals)))
# mannwhitneyu fails if all values the same
if len(set(ref_mquals).union(alt_mquals)) == 1:
m_pv = 1.0
elif len(ref_mquals) == 0 or len(alt_mquals) == 0:
m_pv = 1.0
else:
# compute only if alternate quals are smaller on average
if mean(alt_mquals) < mean(ref_mquals):
ustat = mannwhitneyu(ref_mquals, alt_mquals)
m_pv = ustat[1]
else:
m_pv = 1.0
# same for bqs
if len(set(ref_bquals).union(alt_bquals)) == 1:
b_pv = 1.0
elif len(ref_bquals) == 0 or len(alt_bquals) == 0:
b_pv = 1.0
else:
if mean(alt_bquals) < mean(ref_bquals):
ustat = mannwhitneyu(ref_bquals, alt_bquals)
b_pv = ustat[1]
else:
b_pv = 1.0
# same for isize-qs
#if len(ref_isize) and len(alt_isize):
# if len(set(ref_isize).union(alt_isize))==1:
# i_pv = 1
# else:
# ustat = mannwhitneyu(ref_isize, alt_isize)
# i_pv = ustat[1]
#else:
# i_pv = 1
c_pv = fisher_comb(m_pv, b_pv)
#import pdb; pdb.set_trace()
LOG.debug("%s %d: mb %f bb %f cb %f" %
(var.CHROM, var.POS, m_pv, b_pv, c_pv))
var.INFO['MB'] = prob_to_phredqual(m_pv)
var.INFO['BB'] = prob_to_phredqual(b_pv)
#var.INFO['IB'] = prob_to_phredqual(i_pv)
var.INFO['CB'] = prob_to_phredqual(c_pv)
if args.mtc.lower() != 'none':
pvalues.append(phredqual_to_prob(int(var.INFO[args.mtc_tag])))
if args.mtc.lower() != 'none':
ftag = "%s<%f" % (args.mtc, args.mtc_alpha)
rej_idxs = []
if args.mtc == 'bonf':
rej_idxs = [
i for (i, p) in enumerate(
multiple_testing.Bonferroni(pvalues).corrected_pvals)
if p < args.mtc_alpha
]
elif args.mtc == 'holmbonf':
rej_idxs = [
i for (i, p) in enumerate(
multiple_testing.Bonferroni(pvalues).corrected_pvals)
if p < args.mtc_alpha
]
elif args.mtc == 'fdr':
rej_idxs = fdr.fdr(pvalues, a=args.mtc_alpha)
else:
raise ValueError("unknown MTC method %s" % args.mtc)
for i in rej_idxs:
# pyvcf filter is empty if not set. lofreq's vcf clone was . or PASS
#if not variants[i].FILTER or variants[i].FILTER in [".", "PASS"]:
# new_f = [ftag]
#else:
# new_f = "%s;%s" % (variants[i].FILTER, ftag)
#variants[i] = variants[i]._replace(FILTER=new_f)
variants[i].FILTER.append(ftag)
LOG.info("%d of %d variants didn't pass filter" %
(len(rej_idxs), len(variants)))
# pyvcf doesn't need write_metainfo or write_header
#vcf_writer.write_metainfo()
#vcf_writer.write_header()
for var in variants:
filtered = len(var.FILTER) > 0 and var.FILTER not in [".", "PASS"]
if args.pass_only and filtered:
continue
# LoFreq's vcf clone called this write_rec()
vcf_writer.write_record(var)
if fh_out != sys.stdout:
fh_out.close()
def main():
"""main function
"""
tmp_vcf_markup = []
parser = cmdline_parser()
# WARNING: undocumented arg to remove all defaults (and the reason
# why we have to use OptParse)
if '--no-defaults' in sys.argv:
for (k, v) in parser.defaults.items():
parser.defaults[k] = None
sys.argv = [x for x in sys.argv if x != "--no-defaults"]
(opts, args) = parser.parse_args()
if len(args):
parser.error("Unrecognized arguments found: %s." % (
' '.join(args)))
sys.exit(1)
if opts.verbose:
LOG.setLevel(logging.INFO)
if opts.debug:
LOG.setLevel(logging.DEBUG)
for (in_file, descr) in [(opts.vcf_in, "VCF")]:
if not in_file:
parser.error("%s input file argument missing." % descr)
sys.exit(1)
if not os.path.exists(in_file) and in_file != "-":
sys.stderr.write(
"file '%s' does not exist.\n" % in_file)
sys.exit(1)
for (out_file, descr) in [(opts.vcf_out, "VCF output file")]:
if not out_file:
parser.error("%s output file argument missing." % descr)
sys.exit(1)
if os.path.exists(out_file) and out_file!="-":
sys.stderr.write("Cowardly refusing to overwrite existing"
" output file '%s'.\n" % out_file)
sys.exit(1)
if opts.vcf_in == '-':
vcf_reader = vcf.VCFReader(sys.stdin)
else:
if opts.vcf_in[-3:] == '.gz':
vcf_reader = vcf.VCFReader(gzip.open(opts.vcf_in,'r'))
else:
vcf_reader = vcf.VCFReader(open(opts.vcf_in,'r'))
snvs = [r for r in vcf_reader]
LOG.info("Parsed %d SNVs from %s" % (len(snvs), opts.vcf_in))
# list of tuples: first element is a filter func, which takes a
# snv and a filter-id as input. second is the filter id. variant
# will be marked as filtered if func returns True
filters = []
if opts.min_af != None:
vcf_filter = vcf._Filter(
id=("minaf%f" % opts.min_af).rstrip('0'),
desc="Minimum allele frequency")
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
filters.append((
lambda s, f_id: f_id if s.INFO['AF'] < opts.min_af else None,
vcf_filter.id
))
if opts.max_cov != None:
if not all([s.INFO.has_key('DP') for s in snvs]):
LOG.error("At least one SNV was not annotated with depth info (DP)"
" (was this file produced with LoFreq?).")
sys.exit(1)
vcf_filter = vcf._Filter(
id="maxcov%d" % opts.max_cov,
desc="Maximum coverage")
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
filters.append((
lambda s, f_id: f_id if s.INFO['DP'] > opts.max_cov else None,
vcf_filter.id
))
if opts.min_cov != None:
if not all([s.INFO.has_key('DP') for s in snvs]):
LOG.error("At least one SNV was not annotated with depth info (DP)"
" (was this file produced with LoFreq?).")
sys.exit(1)
vcf_filter = vcf._Filter(
id="mincov%d" % opts.min_cov,
desc="Minimum coverage")
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
filters.append((
lambda s, f_id: f_id if s.INFO['DP'] < opts.min_cov else None,
vcf_filter.id
))
# structured as opts.snv_qual filtering, but keeps corrected
# values.
if opts.strandbias != None:
if opts.strandbias in ['bonf', 'holm-bonf']:
if not opts.strandbias_alpha:
LOG.fatal("Need alpha/significance threshold for strandbias"
" multiple testing correction")
sys.exit(1)
vcf_filter = vcf._Filter(
id="strandbias%s" % opts.strandbias.replace("-", ""),
desc="Strand-bias filter (%s corrected < %g)" % (
opts.strandbias, opts.strandbias_alpha))
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
if opts.strandbias == 'bonf':
vcf_info_id = "SBBC"
elif opts.strandbias == 'holm-bonf':
vcf_info_id = "SBHBC"
else:
raise ValueError
vcf_info = vcf._Info(
id=vcf_info_id, num=1, type='Integer',
desc="Strand-bias %s corrected" % opts.strandbias)
vcf_reader.infos[vcf_info.id] = vcf_info
try:
pvals = (phredqual_to_prob(s.INFO['SB']) for s in snvs)
except (KeyError, AssertionError) as e:
LOG.error("At least one SNV was not annotated properly with"
" strandbias info (SB)"
" (was this file produced with LoFreq?)"
" You will need to switch strandbias filtering off")
sys.exit(1)
if opts.strandbias == 'bonf':
corr_pvals = multiple_testing.Bonferroni(
pvals).corrected_pvals
elif opts.strandbias == 'holm-bonf':
corr_pvals = multiple_testing.HolmBonferroni(
pvals).corrected_pvals
else:
raise ValueError
for (cp, s) in zip(corr_pvals, snvs):
s.INFO[vcf_info.id] = prob_to_phredqual(cp)
if s.INFO[vcf_info.id] > MAX_INT:
s.INFO[vcf_info.id] = MAX_INT
filters.append((
lambda s, f_id: f_id if s.INFO[vcf_info.id] > prob_to_phredqual(opts.strandbias_alpha) else None,
vcf_filter.id
))
# int
elif opts.strandbias != 'off':
try:
max_strandbias_phred = int(opts.strandbias)
assert max_strandbias_phred >= 0
except (ValueError, AssertionError) as e:
LOG.fatal("Invalid strandbias argument: %s" % (opts.strandbias))
sys.exit(1)
vcf_filter = vcf._Filter(
max_strandbias_phred = int(
id="sbp%d" % opts.max_strandbias_phred,
desc="Phred-based strand-bias filter (max)"))
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
filters.append((
lambda s, f_id: f_id if float(s.INFO['SB']) > opts.max_strandbias_phred else None,
vcf_filter.id
))
# structured as opts.strandbias filtering, but doesn't keep
# corrected values.
if opts.snv_qual != None:
if opts.snv_qual in ['bonf', 'holm-bonf', 'fdr']:
if not opts.snv_qual_alpha:
LOG.fatal("Need alpha/significance threshold for snv quality"
" multiple testing correction")
sys.exit(1)
vcf_filter = vcf._Filter(
id="snvqual%s" % opts.snv_qual.replace("-", ""),
desc="SNV quality filter (%s corrected < %g)" % (
opts.snv_qual, opts.snv_qual_alpha))
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
vcf_info_id = "SNVQUALPASS" # tmp markup
tmp_vcf_markup.append(vcf_info_id)
pvals = []
pidx = []
for (i, s) in enumerate(snvs):
# if qual is not NA, convert to pvalue, else don't
# use filter (set filter to NA)
if s.QUAL != '.':
pvals.append(phredqual_to_prob(s.QUAL))
pidx.append(i)
s.INFO[vcf_info_id] = 0
else:
s.INFO[vcf_info_id] = '.'
if opts.snv_qual == 'bonf':
for (i, p) in enumerate(
multiple_testing.Bonferroni(
pvals, n=opts.snv_qual_numtests).corrected_pvals):
if p <= opts.snv_qual_alpha:
snvs[pidx[i]].INFO[vcf_info_id] = 1
elif opts.snv_qual == 'holm-bonf':
for (i, p) in enumerate(
multiple_testing.HolmBonferroni(
pvals, n=opts.snv_qual_numtests).corrected_pvals):
if p <= opts.snv_qual_alpha:
snvs[pidx[i]].INFO[vcf_info_id] = 1
elif opts.snv_qual == 'fdr':
for i in fdr.fdr(pvals, a=opts.snv_qual_alpha,
n=opts.snv_qual_numtests):
snvs[pidx[i]].INFO[vcf_info_id] = 1
else:
raise ValueError
filters.append((
lambda s, f_id: f_id if s.INFO[vcf_info_id] != '.' and s.INFO[vcf_info_id] == 0 else None,
vcf_filter.id
))
elif opts.snv_qual != 'off':
try:
min_qual = int(opts.snv_qual)
assert min_qual >= 0
except (ValueError, AssertionError) as e:
LOG.fatal("Invalid snv quality argument: %s" % (opts.snv_qual))
sys.exit(1)
vcf_filter = vcf._Filter(
id="minqual%d" % min_qual,
desc="Minimum SNV quality")
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
filters.append((
lambda s, f_id: f_id if s.QUAL != '.' and s.QUAL < min_qual else None,
vcf_filter.id
))
if opts.window_size != None:
vcf_filter = vcf._Filter(
id="snvwin%d" % opts.window_size,
desc="SNV window filter (SNVs within %d bp distance)" % (
opts.window_size))
vcf_reader.filters[vcf_filter.id] = vcf_filter# reader serves as template for writer
vcf_info_id = "SNVWINPASS" # tmp markup
tmp_vcf_markup.append(vcf_info_id)
snvs_on_cur_chrom = []
last_chrom = None
seen_chroms = []
for (i, cur_snv) in enumerate(snvs): # assumes snvs are sorted by chrom
if i == 0:
last_chrom = cur_snv.CHROM
if cur_snv.CHROM != last_chrom:
assert cur_snv.CHROM not in seen_chroms, (
"SNV input not ordered by chromosome."
" Sure this file was procuced by LoFreq?")
win_filter(snvs_on_cur_chrom, opts.window_size, vcf_info_id)
seen_chroms.append(last_chrom)
last_chrom = cur_snv.CHROM
snvs_on_cur_chrom = [cur_snv]
else:
snvs_on_cur_chrom.append(cur_snv)
# don't forget last chrom
win_filter(snvs_on_cur_chrom, opts.window_size, vcf_info_id)
filters.append((
lambda s, f_id: f_id if s.INFO[vcf_info_id] != '.' and s.INFO[vcf_info_id] == 0 else None,
vcf_filter.id
))
# The actual filtering: if filter function returns 1 the
# corresponding snv has to be filtered
#
# FIXME can't this be done easier with map()?
#
if len(filters) == 0:
LOG.error("No filters activated.")
sys.exit(1)
#import pdb; pdb.set_trace()
for (filter_func, filter_id) in filters:
for (i, s) in enumerate(snvs):
f = filter_func(s, filter_id)
if f:
# just s = s.__replace() can't work
if s.FILTER == '.' or s.FILTER == 'PASS':
snvs[i] = s._replace(FILTER=f)
else:
snvs[i] = s._replace(FILTER="%s;%s" % (s.FILTER, f))
# should all also work if we get already PASSed input
n_passed = 0
for (i, s) in enumerate(snvs):
if s.FILTER == '.':
snvs[i] = s._replace(FILTER="PASS")
n_passed += 1
LOG.info("%d SNVs passed all filters." % n_passed)
# remove temporary markup
for tmpkey in tmp_vcf_markup:
for s in snvs:
if s.INFO.has_key(tmpkey):
del s.INFO[tmpkey]
if opts.pass_only:
snvs = (s for s in snvs if s.FILTER == 'PASS')
if opts.vcf_out == '-':
fh_out = sys.stdout
else:
if opts.vcf_out[-3:] == '.gz':
fh_out = gzip.open(opts.vcf_out, 'w')
else:
fh_out = open(opts.vcf_out, 'w')
vcf_writer = vcf.VCFWriter(fh_out)
vcf_writer.meta_from_reader(vcf_reader)
vcf_writer.write(snvs)
if fh_out != sys.stdout:
fh_out.close()
def main():
"""The main function
"""
parser = cmdline_parser()
args = parser.parse_args()
if args.verbose:
LOG.setLevel(logging.INFO)
if args.debug:
LOG.setLevel(logging.DEBUG)
assert os.path.exists(args.bam), (
"BAM file %s does not exist" % args.bam)
samfh = pysam.Samfile(args.bam)
# setup vcf_reader
#
if args.vcfin == '-':
vcf_reader = vcf.VCFReader(sys.stdin)
else:
vcf_reader = vcf.VCFReader(filename=args.vcfin)
variants = [r for r in vcf_reader]
LOG.info("Loaded %d variants" % len(variants))
if args.mtc.lower() != 'None':
LOG.info("Will use %s for MTC on %s with alpha %f" % (
args.mtc, args.mtc_tag, args.mtc_alpha))
else:
LOG.info("No multiple testing correction will be done")
# setup vcf_writer
#
if args.vcfout == '-':
fh_out = sys.stdout
else:
if os.path.exists(args.vcfout):
LOG.fatal("Cowardly refusing to overwrite already existing file %s" % (args.vcfout))
sys.exit(1)
if args.vcfout[-3:] == '.gz':
fh_out = gzip.open(args.vcfout, 'w')
else:
fh_out = open(args.vcfout, 'w')
# pyvcf needs template as arg to VCFWriter, whereas LoFreq's vcf clone didn't
vcf_writer = vcf.VCFWriter(fh_out, vcf_reader, lineterminator=os.linesep)
#vcf_writer = vcf.VCFWriter(fh_out)
#vcf_writer.meta_from_reader(vcf_reader)
pvalues = []
for (var_no, var) in enumerate(variants):
if var_no%500==1:
LOG.info("Computing bias for var %d of %d" % (var_no, len(variants)))
if var.INFO.has_key('INDEL'):
LOG.warn("Skipping unsupported indel variant %s:%d" % (var.CHROM, var.POS))
continue
reads = list(samfh.fetch(reference=var.CHROM,
start=var.POS-1, end=var.POS))
LOG.debug("%s %d: %d (unfiltered) reads covering position" % (
var.CHROM, var.POS, len(reads)))
ref_mquals = []
alt_mquals = []
ref_bquals = []
alt_bquals = []
# only for PE
#ref_isize = []
#alt_isize = []
# following two meant to test
#alt_vpos = []
#rlens = []
for r in reads:
if skip_read(r):
continue
orphan = (r.flag & 0x1) and not (r.flag & 0x2)
if orphan and not args.use_orphan:
continue
if r.mapq < args.min_mq:
continue
vpos_on_read = [vpos_on_read
for (vpos_on_read, vpos_on_ref) in r.aligned_pairs
if vpos_on_ref==var.POS-1]
assert len(vpos_on_read)==1
vpos_on_read = vpos_on_read[0]
if vpos_on_read == None:# skip deletions
continue
#alt_vpos.append(vpos_on_read)
#rlens.append(r.rlen)
b = r.query[vpos_on_read]
bq = ord(r.qqual[vpos_on_read])-33
mq = r.mapq
if bq < args.min_bq:
continue
assert len(var.REF)==1 and len(var.ALT)==1
if b.upper() == var.REF[0].upper():
ref_mquals.append(mq)
ref_bquals.append(bq)
#if not args.use_orphan:
# ref_isize.append(abs(r.tlen))
elif b.upper() == str(var.ALT[0]).upper():
alt_mquals.append(mq)
alt_bquals.append(bq)
#if not args.use_orphan:
# alt_isize.append(abs(r.tlen))
else:
LOG.debug("Skipping non-ref-alt base %s at %s:%d" % (b, var.CHROM, var.POS))
continue
LOG.debug("After filtering at %s:%d: %d ref mquals and %d alt mquals" % (
var.CHROM, var.POS, len(ref_mquals), len(alt_mquals)))
# mannwhitneyu fails if all values the same
if len(set(ref_mquals).union(alt_mquals))==1:
m_pv = 1.0
elif len(ref_mquals)==0 or len(alt_mquals)==0:
m_pv = 1.0
else:
# compute only if alternate quals are smaller on average
if mean(alt_mquals) < mean(ref_mquals):
ustat = mannwhitneyu(ref_mquals, alt_mquals)
m_pv = ustat[1]
else:
m_pv = 1.0
# same for bqs
if len(set(ref_bquals).union(alt_bquals))==1:
b_pv = 1.0
elif len(ref_bquals)==0 or len(alt_bquals)==0:
b_pv = 1.0
else:
if mean(alt_bquals) < mean(ref_bquals):
ustat = mannwhitneyu(ref_bquals, alt_bquals)
b_pv = ustat[1]
else:
b_pv = 1.0
# same for isize-qs
#if len(ref_isize) and len(alt_isize):
# if len(set(ref_isize).union(alt_isize))==1:
# i_pv = 1
# else:
# ustat = mannwhitneyu(ref_isize, alt_isize)
# i_pv = ustat[1]
#else:
# i_pv = 1
c_pv = fisher_comb(m_pv, b_pv)
#import pdb; pdb.set_trace()
LOG.debug("%s %d: mb %f bb %f cb %f" % (var.CHROM, var.POS, m_pv, b_pv, c_pv))
var.INFO['MB'] = prob_to_phredqual(m_pv)
var.INFO['BB'] = prob_to_phredqual(b_pv)
#var.INFO['IB'] = prob_to_phredqual(i_pv)
var.INFO['CB'] = prob_to_phredqual(c_pv)
if args.mtc.lower() != 'none':
pvalues.append(phredqual_to_prob(int(var.INFO[args.mtc_tag])))
if args.mtc.lower() != 'none':
ftag = "%s<%f" % (args.mtc, args.mtc_alpha)
rej_idxs = []
if args.mtc == 'bonf':
rej_idxs = [i for (i, p) in
enumerate(multiple_testing.Bonferroni(pvalues).corrected_pvals)
if p<args.mtc_alpha]
elif args.mtc == 'holmbonf':
rej_idxs = [i for (i, p) in
enumerate(multiple_testing.Bonferroni(pvalues).corrected_pvals)
if p<args.mtc_alpha]
elif args.mtc == 'fdr':
rej_idxs = fdr.fdr(pvalues, a=args.mtc_alpha)
else:
raise ValueError("unknown MTC method %s" % args.mtc)
for i in rej_idxs:
# pyvcf filter is empty if not set. lofreq's vcf clone was . or PASS
#if not variants[i].FILTER or variants[i].FILTER in [".", "PASS"]:
# new_f = [ftag]
#else:
# new_f = "%s;%s" % (variants[i].FILTER, ftag)
#variants[i] = variants[i]._replace(FILTER=new_f)
variants[i].FILTER.append(ftag)
LOG.info("%d of %d variants didn't pass filter" % (
len(rej_idxs), len(variants)))
# pyvcf doesn't need write_metainfo or write_header
#vcf_writer.write_metainfo()
#vcf_writer.write_header()
for var in variants:
filtered = len(var.FILTER)>0 and var.FILTER not in [".", "PASS"]
if args.pass_only and filtered:
continue
# LoFreq's vcf clone called this write_rec()
vcf_writer.write_record(var)
if fh_out != sys.stdout:
fh_out.close()
def main():
"""main function
"""
tmp_vcf_markup = []
parser = cmdline_parser()
# WARNING: undocumented arg to remove all defaults (and the reason
# why we have to use OptParse)
if '--no-defaults' in sys.argv:
for (k, v) in parser.defaults.items():
parser.defaults[k] = None
sys.argv = [x for x in sys.argv if x != "--no-defaults"]
(opts, args) = parser.parse_args()
if len(args):
parser.error("Unrecognized arguments found: %s." % (' '.join(args)))
sys.exit(1)
if opts.verbose:
LOG.setLevel(logging.INFO)
if opts.debug:
LOG.setLevel(logging.DEBUG)
for (in_file, descr) in [(opts.vcf_in, "VCF")]:
if not in_file:
parser.error("%s input file argument missing." % descr)
sys.exit(1)
if not os.path.exists(in_file) and in_file != "-":
sys.stderr.write("file '%s' does not exist.\n" % in_file)
sys.exit(1)
for (out_file, descr) in [(opts.vcf_out, "VCF output file")]:
if not out_file:
parser.error("%s output file argument missing." % descr)
sys.exit(1)
if os.path.exists(out_file) and out_file != "-":
sys.stderr.write("Cowardly refusing to overwrite existing"
" output file '%s'.\n" % out_file)
sys.exit(1)
if opts.vcf_in == '-':
vcf_reader = vcf.VCFReader(sys.stdin)
else:
if opts.vcf_in[-3:] == '.gz':
vcf_reader = vcf.VCFReader(gzip.open(opts.vcf_in, 'r'))
else:
vcf_reader = vcf.VCFReader(open(opts.vcf_in, 'r'))
snvs = [r for r in vcf_reader]
LOG.info("Parsed %d SNVs from %s" % (len(snvs), opts.vcf_in))
# list of tuples: first element is a filter func, which takes a
# snv and a filter-id as input. second is the filter id. variant
# will be marked as filtered if func returns True
filters = []
if opts.min_af != None:
vcf_filter = vcf._Filter(id=("minaf%f" % opts.min_af).rstrip('0'),
desc="Minimum allele frequency")
vcf_reader.filters[
vcf_filter.id] = vcf_filter # reader serves as template for writer
filters.append(
(lambda s, f_id: f_id
if s.INFO['AF'] < opts.min_af else None, vcf_filter.id))
if opts.max_cov != None:
if not all([s.INFO.has_key('DP') for s in snvs]):
LOG.error("At least one SNV was not annotated with depth info (DP)"
" (was this file produced with LoFreq?).")
sys.exit(1)
vcf_filter = vcf._Filter(id="maxcov%d" % opts.max_cov,
desc="Maximum coverage")
vcf_reader.filters[
vcf_filter.id] = vcf_filter # reader serves as template for writer
filters.append(
(lambda s, f_id: f_id
if s.INFO['DP'] > opts.max_cov else None, vcf_filter.id))
if opts.min_cov != None:
if not all([s.INFO.has_key('DP') for s in snvs]):
LOG.error("At least one SNV was not annotated with depth info (DP)"
" (was this file produced with LoFreq?).")
sys.exit(1)
vcf_filter = vcf._Filter(id="mincov%d" % opts.min_cov,
desc="Minimum coverage")
vcf_reader.filters[
vcf_filter.id] = vcf_filter # reader serves as template for writer
filters.append(
(lambda s, f_id: f_id
if s.INFO['DP'] < opts.min_cov else None, vcf_filter.id))
# structured as opts.snv_qual filtering, but keeps corrected
# values.
if opts.strandbias != None:
if opts.strandbias in ['bonf', 'holm-bonf']:
if not opts.strandbias_alpha:
LOG.fatal("Need alpha/significance threshold for strandbias"
" multiple testing correction")
sys.exit(1)
vcf_filter = vcf._Filter(
id="strandbias%s" % opts.strandbias.replace("-", ""),
desc="Strand-bias filter (%s corrected < %g)" %
(opts.strandbias, opts.strandbias_alpha))
vcf_reader.filters[
vcf_filter.
id] = vcf_filter # reader serves as template for writer
if opts.strandbias == 'bonf':
vcf_info_id = "SBBC"
elif opts.strandbias == 'holm-bonf':
vcf_info_id = "SBHBC"
else:
raise ValueError
vcf_info = vcf._Info(id=vcf_info_id,
num=1,
type='Integer',
desc="Strand-bias %s corrected" %
opts.strandbias)
vcf_reader.infos[vcf_info.id] = vcf_info
try:
pvals = (phredqual_to_prob(s.INFO['SB']) for s in snvs)
except (KeyError, AssertionError) as e:
LOG.error("At least one SNV was not annotated properly with"
" strandbias info (SB)"
" (was this file produced with LoFreq?)"
" You will need to switch strandbias filtering off")
sys.exit(1)
if opts.strandbias == 'bonf':
corr_pvals = multiple_testing.Bonferroni(pvals).corrected_pvals
elif opts.strandbias == 'holm-bonf':
corr_pvals = multiple_testing.HolmBonferroni(
pvals).corrected_pvals
else:
raise ValueError
for (cp, s) in zip(corr_pvals, snvs):
s.INFO[vcf_info.id] = prob_to_phredqual(cp)
if s.INFO[vcf_info.id] > MAX_INT:
s.INFO[vcf_info.id] = MAX_INT
filters.append(
(lambda s, f_id: f_id
if s.INFO[vcf_info.id] > prob_to_phredqual(
opts.strandbias_alpha) else None, vcf_filter.id))
# int
elif opts.strandbias != 'off':
try:
max_strandbias_phred = int(opts.strandbias)
assert max_strandbias_phred >= 0
except (ValueError, AssertionError) as e:
LOG.fatal("Invalid strandbias argument: %s" %
(opts.strandbias))
sys.exit(1)
vcf_filter = vcf._Filter(max_strandbias_phred=int(
id="sbp%d" % opts.max_strandbias_phred,
desc="Phred-based strand-bias filter (max)"))
vcf_reader.filters[
vcf_filter.
id] = vcf_filter # reader serves as template for writer
filters.append(
(lambda s, f_id: f_id
if float(s.INFO['SB']) > opts.max_strandbias_phred else None,
vcf_filter.id))
# structured as opts.strandbias filtering, but doesn't keep
# corrected values.
if opts.snv_qual != None:
if opts.snv_qual in ['bonf', 'holm-bonf', 'fdr']:
if not opts.snv_qual_alpha:
LOG.fatal("Need alpha/significance threshold for snv quality"
" multiple testing correction")
sys.exit(1)
vcf_filter = vcf._Filter(
id="snvqual%s" % opts.snv_qual.replace("-", ""),
desc="SNV quality filter (%s corrected < %g)" %
(opts.snv_qual, opts.snv_qual_alpha))
vcf_reader.filters[
vcf_filter.
id] = vcf_filter # reader serves as template for writer
vcf_info_id = "SNVQUALPASS" # tmp markup
tmp_vcf_markup.append(vcf_info_id)
pvals = []
pidx = []
for (i, s) in enumerate(snvs):
# if qual is not NA, convert to pvalue, else don't
# use filter (set filter to NA)
if s.QUAL != '.':
pvals.append(phredqual_to_prob(s.QUAL))
pidx.append(i)
s.INFO[vcf_info_id] = 0
else:
s.INFO[vcf_info_id] = '.'
if opts.snv_qual == 'bonf':
for (i, p) in enumerate(
multiple_testing.Bonferroni(
pvals, n=opts.snv_qual_numtests).corrected_pvals):
if p <= opts.snv_qual_alpha:
snvs[pidx[i]].INFO[vcf_info_id] = 1
elif opts.snv_qual == 'holm-bonf':
for (i, p) in enumerate(
multiple_testing.HolmBonferroni(
pvals, n=opts.snv_qual_numtests).corrected_pvals):
if p <= opts.snv_qual_alpha:
snvs[pidx[i]].INFO[vcf_info_id] = 1
elif opts.snv_qual == 'fdr':
for i in fdr.fdr(pvals,
a=opts.snv_qual_alpha,
n=opts.snv_qual_numtests):
snvs[pidx[i]].INFO[vcf_info_id] = 1
else:
raise ValueError
filters.append((lambda s, f_id: f_id
if s.INFO[vcf_info_id] != '.' and s.INFO[
vcf_info_id] == 0 else None, vcf_filter.id))
elif opts.snv_qual != 'off':
try:
min_qual = int(opts.snv_qual)
assert min_qual >= 0
except (ValueError, AssertionError) as e:
LOG.fatal("Invalid snv quality argument: %s" % (opts.snv_qual))
sys.exit(1)
vcf_filter = vcf._Filter(id="minqual%d" % min_qual,
desc="Minimum SNV quality")
vcf_reader.filters[
vcf_filter.
id] = vcf_filter # reader serves as template for writer
filters.append((lambda s, f_id: f_id
if s.QUAL != '.' and s.QUAL < min_qual else None,
vcf_filter.id))
if opts.window_size != None:
vcf_filter = vcf._Filter(
id="snvwin%d" % opts.window_size,
desc="SNV window filter (SNVs within %d bp distance)" %
(opts.window_size))
vcf_reader.filters[
vcf_filter.id] = vcf_filter # reader serves as template for writer
vcf_info_id = "SNVWINPASS" # tmp markup
tmp_vcf_markup.append(vcf_info_id)
snvs_on_cur_chrom = []
last_chrom = None
seen_chroms = []
for (i,
cur_snv) in enumerate(snvs): # assumes snvs are sorted by chrom
if i == 0:
last_chrom = cur_snv.CHROM
if cur_snv.CHROM != last_chrom:
assert cur_snv.CHROM not in seen_chroms, (
"SNV input not ordered by chromosome."
" Sure this file was procuced by LoFreq?")
win_filter(snvs_on_cur_chrom, opts.window_size, vcf_info_id)
seen_chroms.append(last_chrom)
last_chrom = cur_snv.CHROM
snvs_on_cur_chrom = [cur_snv]
else:
snvs_on_cur_chrom.append(cur_snv)
# don't forget last chrom
win_filter(snvs_on_cur_chrom, opts.window_size, vcf_info_id)
filters.append((lambda s, f_id: f_id if s.INFO[vcf_info_id] != '.' and
s.INFO[vcf_info_id] == 0 else None, vcf_filter.id))
# The actual filtering: if filter function returns 1 the
# corresponding snv has to be filtered
#
# FIXME can't this be done easier with map()?
#
if len(filters) == 0:
LOG.error("No filters activated.")
sys.exit(1)
#import pdb; pdb.set_trace()
for (filter_func, filter_id) in filters:
for (i, s) in enumerate(snvs):
f = filter_func(s, filter_id)
if f:
# just s = s.__replace() can't work
if s.FILTER == '.' or s.FILTER == 'PASS':
snvs[i] = s._replace(FILTER=f)
else:
snvs[i] = s._replace(FILTER="%s;%s" % (s.FILTER, f))
# should all also work if we get already PASSed input
n_passed = 0
for (i, s) in enumerate(snvs):
if s.FILTER == '.':
snvs[i] = s._replace(FILTER="PASS")
n_passed += 1
LOG.info("%d SNVs passed all filters." % n_passed)
# remove temporary markup
for tmpkey in tmp_vcf_markup:
for s in snvs:
if s.INFO.has_key(tmpkey):
del s.INFO[tmpkey]
if opts.pass_only:
snvs = (s for s in snvs if s.FILTER == 'PASS')
if opts.vcf_out == '-':
fh_out = sys.stdout
else:
if opts.vcf_out[-3:] == '.gz':
fh_out = gzip.open(opts.vcf_out, 'w')
else:
fh_out = open(opts.vcf_out, 'w')
vcf_writer = vcf.VCFWriter(fh_out)
vcf_writer.meta_from_reader(vcf_reader)
vcf_writer.write(snvs)
if fh_out != sys.stdout:
fh_out.close()
