def main(argv=None):
"""script main.
"""
if not argv:
argv = sys.argv
# setup command line parser
parser = E.OptionParser(version="%prog version: $Id$",
usage=globals()["__doc__"])
parser.add_option("-o",
"--output-format",
dest="output_format",
type="choice",
choices=("bedgraph", "wiggle", "bigbed", "bigwig",
"bed"),
help="output format [default=%default]")
parser.add_option("-s",
"--shift-size",
dest="shift",
type="int",
help="shift reads by a certain amount (ChIP-Seq) "
"[%default]")
parser.add_option("-e",
"--extend",
dest="extend",
type="int",
help="extend reads by a certain amount "
"(ChIP-Seq) [%default]")
parser.add_option("-p",
"--wiggle-span",
dest="span",
type="int",
help="span of a window in wiggle tracks "
"[%default]")
parser.add_option("-m",
"--merge-pairs",
dest="merge_pairs",
action="store_true",
help="merge paired-ended reads into a single "
"bed interval [default=%default].")
parser.add_option("--scale-base",
dest="scale_base",
type="float",
help="number of reads/pairs to scale bigwig file to. "
"The default is to scale to 1M reads "
"[default=%default]")
parser.add_option("--scale-method",
dest="scale_method",
type="choice",
choices=(
"none",
"reads",
),
help="scale bigwig output. 'reads' will normalize by "
"the total number reads in the bam file that are used "
"to construct the bigwig file. If --merge-pairs is used "
"the number of pairs output will be used for "
"normalization. 'none' will not scale the bigwig file"
"[default=%default]")
parser.add_option("--max-insert-size",
dest="max_insert_size",
type="int",
help="only merge if insert size less that "
"# bases. 0 turns of this filter "
"[default=%default].")
parser.add_option("--min-insert-size",
dest="min_insert_size",
type="int",
help="only merge paired-end reads if they are "
"at least # bases apart. "
"0 turns of this filter. [default=%default]")
parser.set_defaults(
samfile=None,
output_format="wiggle",
shift=0,
extend=0,
span=1,
merge_pairs=None,
min_insert_size=0,
max_insert_size=0,
scale_method='none',
scale_base=1000000,
)
# add common options (-h/--help, ...) and parse command line
(options, args) = E.start(parser, argv=argv, add_output_options=True)
if len(args) >= 1:
options.samfile = args[0]
if len(args) == 2:
options.output_filename_pattern = args[1]
if not options.samfile:
raise ValueError("please provide a bam file")
# Read BAM file using Pysam
samfile = pysam.AlignmentFile(options.samfile, "rb")
# Create temporary files / folders
tmpdir = tempfile.mkdtemp()
E.debug("temporary files are in %s" % tmpdir)
tmpfile_wig = os.path.join(tmpdir, "wig")
tmpfile_sizes = os.path.join(tmpdir, "sizes")
# Create dictionary of contig sizes
contig_sizes = dict(list(zip(samfile.references, samfile.lengths)))
# write contig sizes
outfile_size = IOTools.open_file(tmpfile_sizes, "w")
for contig, size in sorted(contig_sizes.items()):
outfile_size.write("%s\t%s\n" % (contig, size))
outfile_size.close()
# Shift and extend only available for bigwig format
if options.shift or options.extend:
if options.output_format != "bigwig":
raise ValueError(
"shift and extend only available for bigwig output")
# Output filename required for bigwig / bigbed computation
if options.output_format == "bigwig":
if not options.output_filename_pattern:
raise ValueError(
"please specify an output file for bigwig computation.")
# Define executable to use for binary conversion
if options.output_format == "bigwig":
executable_name = "wigToBigWig"
else:
raise ValueError("unknown output format `%s`" %
options.output_format)
# check required executable file is in the path
executable = IOTools.which(executable_name)
if not executable:
raise OSError("could not find %s in path." % executable_name)
# Open outout file
outfile = IOTools.open_file(tmpfile_wig, "w")
E.info("starting output to %s" % tmpfile_wig)
else:
outfile = IOTools.open_file(tmpfile_wig, "w")
E.info("starting output to stdout")
# Set up output write functions
if options.output_format in ("wiggle", "bigwig"):
# wiggle is one-based, so add 1, also step-size is 1, so need
# to output all bases
if options.span == 1:
outf = lambda outfile, contig, start, end, val: \
outfile.write(
"".join(["%i\t%i\n" % (x, val)
for x in range(start + 1, end + 1)]))
else:
outf = SpanWriter(options.span)
elif options.output_format == "bedgraph":
# bed is 0-based, open-closed
outf = lambda outfile, contig, start, end, val: \
outfile.write("%s\t%i\t%i\t%i\n" % (contig, start, end, val))
# initialise counters
ninput, nskipped, ncontigs = 0, 0, 0
# set output file name
output_filename_pattern = options.output_filename_pattern
if output_filename_pattern:
output_filename = os.path.abspath(output_filename_pattern)
# shift and extend or merge pairs. Output temporay bed file
if options.shift > 0 or options.extend > 0 or options.merge_pairs:
# Workflow 1: convert to bed intervals and use bedtools
# genomecov to build a coverage file.
# Convert to bigwig with UCSC tools bedGraph2BigWig
if options.merge_pairs:
# merge pairs using bam2bed
E.info("merging pairs to temporary file")
counter = _bam2bed.merge_pairs(
samfile,
outfile,
min_insert_size=options.min_insert_size,
max_insert_size=options.max_insert_size,
bed_format=3)
E.info("merging results: {}".format(counter))
if counter.output == 0:
raise ValueError("no pairs output after merging")
else:
# create bed file with shifted/extended tags
shift, extend = options.shift, options.extend
shift_extend = shift + extend
counter = E.Counter()
for contig in samfile.references:
E.debug("output for %s" % contig)
lcontig = contig_sizes[contig]
for read in samfile.fetch(contig):
pos = read.pos
if read.is_reverse:
start = max(0, read.pos + read.alen - shift_extend)
else:
start = max(0, read.pos + shift)
# intervals extending beyond contig are removed
if start >= lcontig:
continue
end = min(lcontig, start + extend)
outfile.write("%s\t%i\t%i\n" % (contig, start, end))
counter.output += 1
outfile.close()
if options.scale_method == "reads":
scale_factor = float(options.scale_base) / counter.output
E.info("scaling: method=%s scale_quantity=%i scale_factor=%f" %
(options.scale_method, counter.output, scale_factor))
scale = "-scale %f" % scale_factor
else:
scale = ""
# Convert bed file to coverage file (bedgraph)
tmpfile_bed = os.path.join(tmpdir, "bed")
E.info("computing coverage")
# calculate coverage - format is bedgraph
statement = """bedtools genomecov -bg -i %(tmpfile_wig)s %(scale)s
-g %(tmpfile_sizes)s > %(tmpfile_bed)s""" % locals()
E.run(statement)
# Convert bedgraph to bigwig
E.info("converting to bigwig")
tmpfile_sorted = os.path.join(tmpdir, "sorted")
statement = ("sort -k 1,1 -k2,2n %(tmpfile_bed)s > %(tmpfile_sorted)s;"
"bedGraphToBigWig %(tmpfile_sorted)s %(tmpfile_sizes)s "
"%(output_filename_pattern)s" % locals())
E.run(statement)
else:
# Workflow 2: use pysam column iterator to build a
# wig file. Then convert to bigwig of bedgraph file
# with UCSC tools.
def column_iter(iterator):
start = None
end = 0
n = None
for t in iterator:
if t.pos - end > 1 or n != t.n:
if start is not None:
yield start, end, n
start = t.pos
end = t.pos
n = t.n
end = t.pos
yield start, end, n
if options.scale_method != "none":
raise NotImplementedError(
"scaling not implemented for pileup method")
# Bedgraph track definition
if options.output_format == "bedgraph":
outfile.write("track type=bedGraph\n")
for contig in samfile.references:
# if contig != "chrX": continue
E.debug("output for %s" % contig)
lcontig = contig_sizes[contig]
# Write wiggle header
if options.output_format in ("wiggle", "bigwig"):
outfile.write("variableStep chrom=%s span=%i\n" %
(contig, options.span))
# Generate pileup per contig using pysam and iterate over columns
for start, end, val in column_iter(samfile.pileup(contig)):
# patch: there was a problem with bam files and reads
# overextending at the end. These are usually Ns, but
# need to check as otherwise wigToBigWig fails.
if lcontig <= end:
E.warn("read extending beyond contig: %s: %i > %i" %
(contig, end, lcontig))
end = lcontig
if start >= end:
continue
if val > 0:
outf(outfile, contig, start, end, val)
ncontigs += 1
# Close output file
if type(outf) == type(SpanWriter):
outf.flush(outfile)
else:
outfile.flush()
E.info("finished output")
# Report counters
E.info("ninput=%i, ncontigs=%i, nskipped=%i" %
(ninput, ncontigs, nskipped))
# Convert to binary formats
if options.output_format == "bigwig":
outfile.close()
E.info("starting %s conversion" % executable)
try:
retcode = subprocess.call(" ".join(
(executable, tmpfile_wig, tmpfile_sizes,
output_filename_pattern)),
shell=True)
if retcode != 0:
E.warn("%s terminated with signal: %i" %
(executable, -retcode))
return -retcode
except OSError as msg:
E.warn("Error while executing bigwig: %s" % msg)
return 1
E.info("finished bigwig conversion")
else:
with open(tmpfile_wig) as inf:
sys.stdout.write(inf.read())
# Cleanup temp files
shutil.rmtree(tmpdir)
E.stop()
def main(argv=None):
"""script main.
parses command line options in sys.argv, unless *argv* is given.
"""
if not argv:
argv = sys.argv
# setup command line parser
parser = E.OptionParser(version="%prog version: $Id$",
usage=globals()["__doc__"])
parser.add_option("-m",
"--merge-pairs",
dest="merge_pairs",
action="store_true",
help="merge paired-ended reads and output interval "
"for entire fragment [default=%default]. ")
parser.add_option("--max-insert-size",
dest="max_insert_size",
type="int",
help="only merge paired-end reads if they are less than "
"# bases apart. "
" 0 turns off this filter [default=%default]. ")
parser.add_option("--min-insert-size",
dest="min_insert_size",
type="int",
help="only merge paired-end reads if they are at "
"least # bases apart. "
" 0 turns off this filter [default=%default]. ")
parser.add_option("--bed-format",
dest="bed_format",
type="choice",
choices=('3', '4', '5', '6'),
help="bed format to output. "
" [default=%default]")
parser.set_defaults(
region=None,
call_peaks=None,
merge_pairs=None,
min_insert_size=0,
max_insert_size=0,
bed_format='6',
)
(options, args) = E.start(parser, argv=argv)
if len(args) == 0:
args.append("-")
samfile = pysam.AlignmentFile(args[0], "rb")
options.bed_format = int(options.bed_format)
if options.merge_pairs is not None:
counter = _bam2bed.merge_pairs(samfile,
options.stdout,
min_insert_size=options.min_insert_size,
max_insert_size=options.max_insert_size,
bed_format=options.bed_format)
E.info("category\tcounts\n%s\n" % counter.asTable())
else:
# use until_eof. Files from stdin have no index
it = samfile.fetch(until_eof=True)
# more comfortable cigar parsing will
# come with the next pysam release
BAM_CMATCH = 0
BAM_CDEL = 2
BAM_CREF_SKIP = 3
take = (BAM_CMATCH, BAM_CDEL, BAM_CREF_SKIP)
outfile = options.stdout
for read in it:
if read.is_unmapped:
continue
t = 0
for op, l in read.cigar:
if op in take:
t += l
if read.is_reverse:
strand = "-"
else:
strand = "+"
# IMS: converted rname to reference name
outfile.write("%s\t%d\t%d\t%s\t%d\t%c\n" %
(samfile.getrname(read.rname), read.pos,
read.pos + t, read.qname, read.mapq, strand))
E.stop()
def main(argv=None):
"""script main.
parses command line options in sys.argv, unless *argv* is given.
"""
if not argv:
argv = sys.argv
# setup command line parser
parser = E.OptionParser(
version="%prog version: $Id$", usage=globals()["__doc__"])
parser.add_option("-m", "--merge-pairs", dest="merge_pairs",
action="store_true",
help="merge paired-ended reads and output interval "
"for entire fragment [default=%default]. ")
parser.add_option("--max-insert-size", dest="max_insert_size", type="int",
help="only merge paired-end reads if they are less than "
"# bases apart. "
" 0 turns off this filter [default=%default]. ")
parser.add_option("--min-insert-size", dest="min_insert_size", type="int",
help="only merge paired-end reads if they are at "
"least # bases apart. "
" 0 turns off this filter [default=%default]. ")
parser.add_option("--bed-format", dest="bed_format", type="choice",
choices=('3', '4', '5', '6'),
help="bed format to output. "
" [default=%default]")
parser.set_defaults(
region=None,
call_peaks=None,
merge_pairs=None,
min_insert_size=0,
max_insert_size=0,
bed_format='6',
)
(options, args) = E.Start(parser, argv=argv)
if len(args) == 0:
args.append("-")
samfile = pysam.Samfile(args[0], "rb")
options.bed_format = int(options.bed_format)
if options.merge_pairs is not None:
counter = _bam2bed.merge_pairs(samfile,
options.stdout,
min_insert_size=options.min_insert_size,
max_insert_size=options.max_insert_size,
bed_format=options.bed_format)
E.info("category\tcounts\n%s\n" % counter.asTable())
else:
# use until_eof. Files from stdin have no index
it = samfile.fetch(until_eof=True)
# more comfortable cigar parsing will
# come with the next pysam release
BAM_CMATCH = 0
BAM_CDEL = 2
BAM_CREF_SKIP = 3
take = (BAM_CMATCH, BAM_CDEL, BAM_CREF_SKIP)
outfile = options.stdout
for read in it:
if read.is_unmapped:
continue
t = 0
for op, l in read.cigar:
if op in take:
t += l
if read.is_reverse:
strand = "-"
else:
strand = "+"
# IMS: converted rname to reference name
outfile.write("%s\t%d\t%d\t%s\t%d\t%c\n" %
(samfile.getrname(read.rname),
read.pos,
read.pos + t,
read.qname,
read.mapq,
strand))
E.Stop()
def main(argv=None):
"""script main.
"""
if not argv:
argv = sys.argv
# setup command line parser
parser = E.OptionParser(
version="%prog version: $Id$",
usage=globals()["__doc__"])
parser.add_option("-o", "--output-format", dest="output_format",
type="choice",
choices=(
"bedgraph", "wiggle", "bigbed",
"bigwig", "bed"),
help="output format [default=%default]")
parser.add_option("-s", "--shift-size", dest="shift", type="int",
help="shift reads by a certain amount (ChIP-Seq) "
"[%default]")
parser.add_option("-e", "--extend", dest="extend", type="int",
help="extend reads by a certain amount "
"(ChIP-Seq) [%default]")
parser.add_option("-p", "--wiggle-span", dest="span", type="int",
help="span of a window in wiggle tracks "
"[%default]")
parser.add_option("-m", "--merge-pairs", dest="merge_pairs",
action="store_true",
help="merge paired-ended reads into a single "
"bed interval [default=%default].")
parser.add_option("--scale-base", dest="scale_base", type="float",
help="number of reads/pairs to scale bigwig file to. "
"The default is to scale to 1M reads "
"[default=%default]")
parser.add_option("--scale-method", dest="scale_method", type="choice",
choices=("none", "reads",),
help="scale bigwig output. 'reads' will normalize by "
"the total number reads in the bam file that are used "
"to construct the bigwig file. If --merge-pairs is used "
"the number of pairs output will be used for "
"normalization. 'none' will not scale the bigwig file"
"[default=%default]")
parser.add_option("--max-insert-size", dest="max_insert_size",
type="int",
help="only merge if insert size less that "
"# bases. 0 turns of this filter "
"[default=%default].")
parser.add_option("--min-insert-size", dest="min_insert_size",
type="int",
help="only merge paired-end reads if they are "
"at least # bases apart. "
"0 turns of this filter. [default=%default]")
parser.set_defaults(
samfile=None,
output_format="wiggle",
shift=0,
extend=0,
span=1,
merge_pairs=None,
min_insert_size=0,
max_insert_size=0,
scale_method='none',
scale_base=1000000,
)
# add common options (-h/--help, ...) and parse command line
(options, args) = E.Start(parser, argv=argv, add_output_options=True)
if len(args) >= 1:
options.samfile = args[0]
if len(args) == 2:
options.output_filename_pattern = args[1]
if not options.samfile:
raise ValueError("please provide a bam file")
# Read BAM file using Pysam
samfile = pysam.Samfile(options.samfile, "rb")
# Create temporary files / folders
tmpdir = tempfile.mkdtemp()
E.debug("temporary files are in %s" % tmpdir)
tmpfile_wig = os.path.join(tmpdir, "wig")
tmpfile_sizes = os.path.join(tmpdir, "sizes")
# Create dictionary of contig sizes
contig_sizes = dict(list(zip(samfile.references, samfile.lengths)))
# write contig sizes
outfile_size = IOTools.openFile(tmpfile_sizes, "w")
for contig, size in sorted(contig_sizes.items()):
outfile_size.write("%s\t%s\n" % (contig, size))
outfile_size.close()
# Shift and extend only available for bigwig format
if options.shift or options.extend:
if options.output_format != "bigwig":
raise ValueError(
"shift and extend only available for bigwig output")
# Output filename required for bigwig / bigbed computation
if options.output_format == "bigwig":
if not options.output_filename_pattern:
raise ValueError(
"please specify an output file for bigwig computation.")
# Define executable to use for binary conversion
if options.output_format == "bigwig":
executable_name = "wigToBigWig"
else:
raise ValueError("unknown output format `%s`" %
options.output_format)
# check required executable file is in the path
executable = IOTools.which(executable_name)
if not executable:
raise OSError("could not find %s in path." % executable_name)
# Open outout file
outfile = IOTools.openFile(tmpfile_wig, "w")
E.info("starting output to %s" % tmpfile_wig)
else:
outfile = IOTools.openFile(tmpfile_wig, "w")
E.info("starting output to stdout")
# Set up output write functions
if options.output_format in ("wiggle", "bigwig"):
# wiggle is one-based, so add 1, also step-size is 1, so need
# to output all bases
if options.span == 1:
outf = lambda outfile, contig, start, end, val: \
outfile.write(
"".join(["%i\t%i\n" % (x, val)
for x in range(start + 1, end + 1)]))
else:
outf = SpanWriter(options.span)
elif options.output_format == "bedgraph":
# bed is 0-based, open-closed
outf = lambda outfile, contig, start, end, val: \
outfile.write("%s\t%i\t%i\t%i\n" % (contig, start, end, val))
# initialise counters
ninput, nskipped, ncontigs = 0, 0, 0
# set output file name
output_filename_pattern = options.output_filename_pattern
if output_filename_pattern:
output_filename = os.path.abspath(output_filename_pattern)
# shift and extend or merge pairs. Output temporay bed file
if options.shift > 0 or options.extend > 0 or options.merge_pairs:
# Workflow 1: convert to bed intervals and use bedtools
# genomecov to build a coverage file.
# Convert to bigwig with UCSC tools bedGraph2BigWig
if options.merge_pairs:
# merge pairs using bam2bed
E.info("merging pairs to temporary file")
counter = _bam2bed.merge_pairs(
samfile,
outfile,
min_insert_size=options.min_insert_size,
max_insert_size=options.max_insert_size,
bed_format=3)
E.info("merging results: {}".format(counter))
if counter.output == 0:
raise ValueError("no pairs output after merging")
else:
# create bed file with shifted/extended tags
shift, extend = options.shift, options.extend
shift_extend = shift + extend
counter = E.Counter()
for contig in samfile.references:
E.debug("output for %s" % contig)
lcontig = contig_sizes[contig]
for read in samfile.fetch(contig):
pos = read.pos
if read.is_reverse:
start = max(0, read.pos + read.alen - shift_extend)
else:
start = max(0, read.pos + shift)
# intervals extending beyond contig are removed
if start >= lcontig:
continue
end = min(lcontig, start + extend)
outfile.write("%s\t%i\t%i\n" % (contig, start, end))
counter.output += 1
outfile.close()
if options.scale_method == "reads":
scale_factor = float(options.scale_base) / counter.output
E.info("scaling: method=%s scale_quantity=%i scale_factor=%f" %
(options.scale_method,
counter.output,
scale_factor))
scale = "-scale %f" % scale_factor
else:
scale = ""
# Convert bed file to coverage file (bedgraph)
tmpfile_bed = os.path.join(tmpdir, "bed")
E.info("computing coverage")
# calculate coverage - format is bedgraph
statement = """bedtools genomecov -bg -i %(tmpfile_wig)s %(scale)s
-g %(tmpfile_sizes)s > %(tmpfile_bed)s""" % locals()
E.run(statement)
# Convert bedgraph to bigwig
E.info("converting to bigwig")
tmpfile_sorted = os.path.join(tmpdir, "sorted")
statement = ("sort -k 1,1 -k2,2n %(tmpfile_bed)s > %(tmpfile_sorted)s;"
"bedGraphToBigWig %(tmpfile_sorted)s %(tmpfile_sizes)s "
"%(output_filename_pattern)s" % locals())
E.run(statement)
else:
# Workflow 2: use pysam column iterator to build a
# wig file. Then convert to bigwig of bedgraph file
# with UCSC tools.
def column_iter(iterator):
start = None
end = 0
n = None
for t in iterator:
if t.pos - end > 1 or n != t.n:
if start is not None:
yield start, end, n
start = t.pos
end = t.pos
n = t.n
end = t.pos
yield start, end, n
if options.scale_method != "none":
raise NotImplementedError(
"scaling not implemented for pileup method")
# Bedgraph track definition
if options.output_format == "bedgraph":
outfile.write("track type=bedGraph\n")
for contig in samfile.references:
# if contig != "chrX": continue
E.debug("output for %s" % contig)
lcontig = contig_sizes[contig]
# Write wiggle header
if options.output_format in ("wiggle", "bigwig"):
outfile.write("variableStep chrom=%s span=%i\n" %
(contig, options.span))
# Generate pileup per contig using pysam and iterate over columns
for start, end, val in column_iter(samfile.pileup(contig)):
# patch: there was a problem with bam files and reads
# overextending at the end. These are usually Ns, but
# need to check as otherwise wigToBigWig fails.
if lcontig <= end:
E.warn("read extending beyond contig: %s: %i > %i" %
(contig, end, lcontig))
end = lcontig
if start >= end:
continue
if val > 0:
outf(outfile, contig, start, end, val)
ncontigs += 1
# Close output file
if type(outf) == type(SpanWriter):
outf.flush(outfile)
else:
outfile.flush()
E.info("finished output")
# Report counters
E.info("ninput=%i, ncontigs=%i, nskipped=%i" %
(ninput, ncontigs, nskipped))
# Convert to binary formats
if options.output_format == "bigwig":
outfile.close()
E.info("starting %s conversion" % executable)
try:
retcode = subprocess.call(
" ".join((executable,
tmpfile_wig,
tmpfile_sizes,
output_filename_pattern)),
shell=True)
if retcode != 0:
E.warn("%s terminated with signal: %i" %
(executable, -retcode))
return -retcode
except OSError as msg:
E.warn("Error while executing bigwig: %s" % msg)
return 1
E.info("finished bigwig conversion")
else:
with open(tmpfile_wig) as inf:
sys.stdout.write(inf.read())
# Cleanup temp files
shutil.rmtree(tmpdir)
E.Stop()
