def convert_to_fastq(fp_obj, logger=None):
"""Convert a SAM or BAM file to FASTQ file(s) for alignment
"""
input_file = fp_obj.input_file
output_dir = fp_obj.output_dir
fastq_dir = join(output_dir, 'fastq_input')
fp_obj.check_output_dir(fastq_dir)
protoname = fp_obj.protoname
if fp_obj.paired_end:
fastq_filenames = (join(fastq_dir, '%s.1.txt.gz' % protoname),
join(fastq_dir, '%s.2.txt.gz' % protoname))
logger.info('Converting file %s to FASTQ files %s, %s',
input_file, fastq_filenames[0], fastq_filenames[1])
in_args = [sys.executable, '-m',
'seriesoftubes.converters.bamtofastq2',
'--gzip', input_file,
fastq_filenames[0], fastq_filenames[1]]
else:
fastq_filename = join(fastq_dir, '%s.txt.gz' % protoname)
logger.info('Converting file %s to FASTQ file %s',
input_file, fastq_filename)
in_args = [sys.executable, '-m',
'seriesoftubes.converters.bamtofastq2',
input_file, fastq_filename]
logger.debug('Launching %s', ' '.join(in_args))
polledpipe = PolledPipe(logger=logger, level=logging.ERROR)
job = Popen(in_args, stdout=polledpipe.w, stderr=STDOUT)
wait_for_job(job, [polledpipe], logger)
if fp_obj.paired_end:
logger.debug('Settings input_file to %s', fastq_filenames[0])
fp_obj.input_file = fastq_filenames[0]
logger.debug('Settings second_file to %s', fastq_filenames[1])
fp_obj.second_file = fastq_filenames[1]
else:
logger.debug('Settings input_file to %s', fastq_filename)
fp_obj.input_file = fastq_filenames[0]
logger.debug('Setting use_pysam to False')
fp_obj.use_pysam = False
logger.debug('Setting format to FASTQ')
fp_obj.format = 'FASTQ'
logger.debug('Ignoring open_func, it will not be used')
if not job.returncode == 0:
logger.critical('Conversion FAILED!')
else:
logger.info('Conversion successful')
return
def align_once(fp_obj, flags, ref, use_quality=False,
path_to_bowtie2=None, path_to_samtools=None, logger=None,
passthru_args=None,
**kwargs):
if use_quality:
if fp_obj.use_pysam:
flags.append('--phred33')
else:
flags.append('--phred64')
refname = os.path.basename(ref)
path_to_unsorted = fp_obj.tmp_filename(refname)
output_dir = os.path.split(path_to_unsorted)[0]
fp_obj.check_output_dir(output_dir)
filename1 = os.path.abspath(fp_obj.input_file)
second_file = fp_obj.second_file
if second_file is not None:
filename2 = os.path.abspath(second_file)
else:
filename2 = None
if fp_obj.paired_end:
file_args = ['-x', ref, '-1', filename1, '-2', filename2]
else:
file_args = ['-x', ref, '-U', filename1]
if passthru_args:
bowtie2_args = [path_to_bowtie2] + flags + passthru_args + file_args
else:
bowtie2_args = [path_to_bowtie2] + flags + file_args
# finish parsing input here
bowtie2_stderr = PolledPipe(logger=logger, level=logging.ERROR)
logger.info('Launching bowtie2 (output will be piped to samtools for BAM '
'encoding)')
logger.info(' '.join(bowtie2_args))
bowtie2_aligner = Popen(bowtie2_args, stdout=PIPE, stderr=bowtie2_stderr.w,
bufsize=-1)
samtools_args = [path_to_samtools, 'view', '-b', '-S', '-o',
path_to_unsorted, '-']
logger.info('Launching samtools to encode bowtie2 output as BAM')
logger.info(' '.join(samtools_args))
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
samtools_viewer = Popen(samtools_args, stdin=bowtie2_aligner.stdout,
stdout=samtools_stdout.w,
stderr=samtools_stderr.w, bufsize=-1)
logger.debug('Waiting for bowtie2 to finish')
pollables = [bowtie2_stderr, samtools_stdout, samtools_stderr]
wait_for_job(bowtie2_aligner, pollables, logger)
if not bowtie2_aligner.returncode == 0:
logger.critical("bowtie2 did not run properly [%d]",
bowtie2_aligner.returncode)
samtools_viewer.terminate()
samtools_viewer.poll()
logger.critical("samtools terminated")
return
logger.debug('Alignment successfully completed')
logger.debug('Waiting for samtools to finish')
wait_for_job(samtools_viewer, [samtools_stdout, samtools_stderr], logger)
if not samtools_viewer.returncode == 0:
logger.critical("samtools view did not run properly [%d]",
samtools_viewer.returncode)
return
logger.debug('Unsorted BAM file successfully written')
logger.info('Launching samtools again to sort BAM output')
output_dir, output_file = os.path.split(path_to_unsorted)
bam_file = os.path.splitext(output_file)[0]
sorter_args = [path_to_samtools, 'sort', output_file, bam_file]
logger.info(' '.join(sorter_args))
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
samtools_sorter = Popen(sorter_args, stdout=samtools_stdout.w,
stderr=samtools_stderr.w, cwd=output_dir)
wait_for_job(samtools_sorter, [samtools_stdout, samtools_stderr], logger)
if not samtools_sorter.returncode == 0:
logger.critical("samtools sort did not run properly [%d]",
samtools_sorter.returncode)
return
# don't destroy the files until we're sure we succeeded!
assert_path(os.path.join(output_dir, bam_file + '.bam'))
logger.debug('Removing unsorted file %s', path_to_unsorted)
os.remove(path_to_unsorted)
logger.debug('Launching samtools again to index sorted BAM output')
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
index_args = [path_to_samtools, 'index', bam_file + '.bam']
samtools_indexer = Popen(index_args, stdout=samtools_stdout.w,
stderr=samtools_stderr.w, cwd=output_dir)
wait_for_job(samtools_indexer, [samtools_stdout, samtools_stderr], logger)
if not samtools_indexer.returncode == 0:
logger.critical("samtools index did not run properly [%d]",
samtools_indexer.returncode)
return
# Make sure indexing succeeds
assert_path(os.path.join(output_dir, bam_file + '.bam.bai'))
return
def counteralign_once(fp_obj, flags, ref, use_quality=False,
path_to_bowtie2=None, path_to_samtools=None, logger=None,
passthru_args=None,
**kwargs):
"""Produce counter-alignements"""
if use_quality:
if fp_obj.use_pysam:
flags.append('--phred33')
else:
flags.append('--phred64')
refname = os.path.basename(ref)
output_dir, output_file = os.path.split(fp_obj.tmp_filename(refname))
fp_obj.check_output_dir(output_dir)
fp_obj.check_output_dir(join(output_dir, 'counteraligned'))
filename1 = os.path.abspath(fp_obj.input_file)
input_file = fp_obj.input_file
second_file = fp_obj.second_file
if second_file is not None:
filename2 = os.path.abspath(second_file)
else:
filename2 = None
if fp_obj.paired_end:
try:
paired_file = make_paired_name(input_file, second_file)
counteraligned = os.path.abspath(join(output_dir, 'counteraligned',
paired_file))
except ValueError:
counteraligned = os.path.abspath(join(output_dir, 'counteraligned',
input_file))
file_args = ['-x', ref, '-1', filename1, '-2', filename2,
'--al-conc-gz', counteraligned,
'--un-conc-gz', join(output_dir, paired_file)]
new_filenames = (join(output_dir, input_file),
join(output_dir, second_file))
else:
file_args = ['-x', ref, '-U', filename1,
'--al-gz', join(output_dir, 'counteraligned', input_file),
'--un-gz', join(output_dir, input_file)]
new_filenames = (join(output_dir, input_file), None)
if passthru_args is not None:
bowtie2_args = [path_to_bowtie2] + flags + passthru_args + file_args
else:
bowtie2_args = [path_to_bowtie2] + flags + file_args
# finish parsing input here
bowtie2_stderr = PolledPipe(logger=logger, level=logging.ERROR)
logger.info('Launching bowtie2 (output will be piped to samtools for '
'BAM encoding)')
logger.info(' '.join(bowtie2_args))
bowtie2_aligner = Popen(bowtie2_args, stdout=open(devnull, 'w'),
stderr=bowtie2_stderr.w,
bufsize=-1)
logger.info(' '.join(bowtie2_args))
logger.info('counteraligned reads will be saved as GZIPed FASTQ files '
'in counteraligned/')
logger.debug('Waiting for bowtie2 to finish')
pollables = [bowtie2_stderr]
wait_for_job(bowtie2_aligner, pollables, logger)
if not bowtie2_aligner.returncode == 0:
logger.critical("bowtie2 did not run properly [%d]",
bowtie2_aligner.returncode)
return
logger.debug('Alignment successfully completed')
return new_filenames
def align_once(fp_obj, flags, ref, match_type, use_quality=False,
quals_type='solexa1.3',
path_to_bowtie=None, path_to_samtools=None, logger=None,
**kwargs):
refname = os.path.basename(ref)
path_to_unsorted = fp_obj.tmp_filename(refname, match_type)
output_dir = os.path.split(path_to_unsorted)[0]
fp_obj.check_output_dir(output_dir)
filename1 = os.path.abspath(fp_obj.input_file)
second_file = fp_obj.second_file
if second_file is not None:
filename2 = os.path.abspath(second_file)
else:
filename2 = None
if use_quality:
if fp_obj.use_pysam:
flags.append('--phred33-quals')
else:
flags.append(''.join(['--', quals_type, '-quals']))
if fp_obj.paired_end:
file_args = [ref, '--12', '-']
logger.info('Automagically interpreting %s files', fp_obj.format)
else:
logger.info('Automagically interpreting %s file', fp_obj.format)
file_args = [ref, '-']
bowtie_args = [path_to_bowtie] + flags + file_args
# finish parsing input here
input_stderr = PolledPipe(logger=logger, level=logging.ERROR)
bowtie_stderr = PolledPipe(logger=logger, level=logging.ERROR)
if fp_obj.use_pysam:
if fp_obj.paired_end:
in_args = [sys.executable, '-m',
'seriesoftubes.converters.bamtotab',
filename1]
else:
in_args = [sys.executable, '-m',
'seriesoftubes.converters.bamtofastq',
'--no-gzip',
filename1]
elif fp_obj.paired_end and fp_obj.format == 'FASTQ':
in_args = [sys.executable, '-m', 'seriesoftubes.converters.fastqtotab',
filename1, filename2]
elif fp_obj.format == 'FASTQ':
in_args = [sys.executable, '-m', 'seriesoftubes.converters.cat',
filename1]
else:
logger.critical("Couldn't figure out what to do with file "
"%s of format %s",
fp_obj.input_file, fp_obj.format)
logger.info(' '.join(in_args))
input_reader = Popen(in_args, stdout=PIPE, stderr=input_stderr.w,
bufsize=-1)
logger.info('Launching bowtie (output will be piped to samtools)')
logger.info(' '.join(bowtie_args))
bowtie_aligner = Popen(bowtie_args, stdin=input_reader.stdout,
stdout=PIPE, stderr=bowtie_stderr.w,
bufsize=-1)
samtools_args = [path_to_samtools, 'view', '-b', '-S', '-o',
path_to_unsorted, '-']
logger.info('Launching samtools to encode bowtie output as BAM')
logger.info(' '.join(samtools_args))
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
samtools_viewer = Popen(samtools_args, stdin=bowtie_aligner.stdout,
stdout=samtools_stdout.w,
stderr=samtools_stderr.w, bufsize=-1)
logger.debug('Waiting for bowtie to finish')
pollables = [input_stderr, bowtie_stderr, samtools_stdout, samtools_stderr]
wait_for_job(bowtie_aligner, pollables, logger)
if not bowtie_aligner.returncode == 0:
logger.critical("bowtie did not run properly [%d]",
bowtie_aligner.returncode)
samtools_viewer.terminate()
samtools_viewer.poll()
logger.critical("samtools terminated")
return
logger.debug('Alignment successfully completed')
logger.debug('Waiting for samtools to finish')
wait_for_job(samtools_viewer, [samtools_stdout, samtools_stderr], logger)
if not samtools_viewer.returncode == 0:
logger.critical("samtools view did not run properly [%d]",
samtools_viewer.returncode)
return
logger.debug('Unsorted BAM file successfully written')
logger.info('Launching samtools again to sort BAM output')
output_dir, output_file = os.path.split(path_to_unsorted)
bam_file = os.path.splitext(output_file)[0]
sorter_args = [path_to_samtools, 'sort', output_file, bam_file]
logger.info(' '.join(sorter_args))
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
samtools_sorter = Popen(sorter_args, stdout=samtools_stdout.w,
stderr=samtools_stderr.w, cwd=output_dir)
wait_for_job(samtools_sorter, [samtools_stdout, samtools_stderr], logger)
if not samtools_sorter.returncode == 0:
logger.critical("samtools sort did not run properly [%d]",
samtools_sorter.returncode)
return
# don't destroy the files until we're sure we succeeded!
assert_path(os.path.join(output_dir, bam_file + '.bam'))
logger.debug('Removing unsorted file %s', path_to_unsorted)
os.remove(path_to_unsorted)
logger.debug('Launching samtools again to index sorted BAM output')
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
index_args = [path_to_samtools, 'index', bam_file + '.bam']
samtools_indexer = Popen(index_args, stdout=samtools_stdout.w,
stderr=samtools_stderr.w, cwd=output_dir)
wait_for_job(samtools_indexer, [samtools_stdout, samtools_stderr], logger)
if not samtools_indexer.returncode == 0:
logger.critical("samtools index did not run properly [%d]",
samtools_indexer.returncode)
return
# Make sure indexing succeeds
assert_path(os.path.join(output_dir, bam_file + '.bam.bai'))
return
def counteralign_once(fp_obj, flags, ref,
match_type, use_quality=False,
quals_type='solexa1.3',
path_to_bowtie=None, path_to_samtools=None, logger=None,
**kwargs):
"""Produce counter-alignements"""
refname = os.path.basename(ref)
output_dir, output_file = os.path.split(fp_obj.tmp_filename(refname))
fp_obj.check_output_dir(output_dir)
filename1 = os.path.abspath(fp_obj.input_file)
second_file = fp_obj.second_file
if second_file is not None:
filename2 = os.path.abspath(second_file)
else:
filename2 = None
if use_quality:
if fp_obj.use_pysam:
flags.append('--phred33-quals')
else:
flags.append(''.join(['--', quals_type, '-quals']))
if fp_obj.paired_end:
file_args = [ref, '--12', '-']
logger.info('Automagically interpreting %s files', fp_obj.format)
else:
logger.info('Automagically interpreting %s file', fp_obj.format)
file_args = [ref, '-']
bowtie_args = [path_to_bowtie] + flags + file_args
# finish parsing input here
input_stderr = PolledPipe(logger=logger, level=logging.ERROR)
bowtie_stderr = PolledPipe(logger=logger, level=logging.ERROR)
if fp_obj.use_pysam:
if fp_obj.paired_end:
in_args = [sys.executable, '-m',
'seriesoftubes.converters.bamtotab',
filename1]
else:
in_args = [sys.executable, '-m',
'seriesoftubes.converters.bamtofastq',
'--no-gzip',
filename1]
elif fp_obj.paired_end and fp_obj.format == 'FASTQ':
in_args = [sys.executable, '-m', 'seriesoftubes.converters.fastqtotab',
filename1, filename2]
elif fp_obj.format == 'FASTQ':
in_args = [sys.executable, '-m', 'seriesoftubes.converters.cat',
filename1]
else:
logger.critical("Couldn't figure out what to do with file "
"%s of format %s",
fp_obj.input_file, fp_obj.format)
logger.info(' '.join(in_args))
input_reader = Popen(in_args, stdout=PIPE, stderr=input_stderr.w,
bufsize=-1)
logger.info('Launching bowtie (output will be piped to samtools)')
logger.info(' '.join(bowtie_args))
bowtie_aligner = Popen(bowtie_args, stdin=input_reader.stdout,
stdout=PIPE, stderr=bowtie_stderr.w,
bufsize=-1)
logger.info('Only unaligned reads will be saved.')
samtools_args = [path_to_samtools, 'view', '-b', '-S', '-o',
'-f', '0x4', # ONLY SAVE UNALIGNED READS
join(output_dir, output_file), '-']
logger.info('Launching samtools to encode bowtie output as BAM')
logger.info(' '.join(samtools_args))
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
samtools_viewer = Popen(samtools_args, stdin=bowtie_aligner.stdout,
stdout=samtools_stdout.w,
stderr=samtools_stderr.w, bufsize=-1)
logger.debug('Waiting for bowtie to finish')
pollables = [input_stderr, bowtie_stderr, samtools_stdout, samtools_stderr]
wait_for_job(bowtie_aligner, pollables, logger)
if not bowtie_aligner.returncode == 0:
logger.critical("bowtie did not run properly [%d]",
bowtie_aligner.returncode)
samtools_viewer.terminate()
samtools_viewer.poll()
logger.critical("samtools terminated")
return
logger.debug('Alignment successfully completed')
logger.debug('Waiting for samtools to finish')
wait_for_job(samtools_viewer, [samtools_stdout, samtools_stderr], logger)
if not samtools_viewer.returncode == 0:
logger.critical("samtools view did not run properly [%d]",
samtools_viewer.returncode)
return
logger.debug('Unsorted BAM file successfully written')
return (join(output_dir, output_file), None)
def run_macs(f, subpeaks=True, path_to_macs=None, logging_level=10,
user_gsize=None, qvalue=0.01, passthru_args=None,
**kwargs):
"""Run MACS on a BAM file
"""
logger = get_logger(logging_level)
if path_to_macs is None:
path_to_macs = path_to_executable("macs2")
input_file = f.input_file
control_file = f.control_file
logger.debug('Processing %s', input_file)
if control_file is not None:
logger.debug('with control %s', control_file)
# determine genome name and size
if user_gsize:
genome_size = user_gsize
try:
genome_build = guess_bam_genome(input_file)
except NoMatchFoundError:
genome_build = None
else:
try:
genome_build = guess_bam_genome(input_file)
except NoMatchFoundError:
raise Usage('\
Could not determine genome / genome size for file %s' % input_file)
gname = ''.join([x for x in genome_build if x.isalpha()])
if gname == 'hg':
genome_size = 'hs'
elif gname in ['mm', 'ce', 'dm']:
genome_size = gname
else:
genome_size = '%.1e' % sum(genome(genome_build).itervalues())
fmt = decide_format(input_file, control_file, logger)
name = f.sample_name.replace(' ', '_')
if passthru_args is not None:
for i in range(len(passthru_args)):
passthru_args[i] = passthru_args[i].replace('+', '-')
logger.debug('Passing thru arguments %s', ' '.join(passthru_args))
macs_options = ['--trackline',
'-f', fmt, # correct file format BAM or BAMPE
'-B', '--SPMR', # bedgraphs, SPMR
'-g', genome_size,
'-q', qvalue,
'-n', name, # run name
'-t', join(getcwd(), input_file)] # treatment
if control_file is not None:
macs_options.extend(['-c', join(getcwd(), control_file)])
if subpeaks:
macs_options.append('--call-summits')
if passthru_args is not None:
macs_options.extend(passthru_args)
step = [path_to_macs, 'callpeak'] + macs_options
if platform.system() is 'Windows':
step.insert(sys.executable, 0)
macs_stdout = PolledPipe(logger=logger, level=WARN)
macs_stderr = PolledPipe(logger=logger, level=ERROR)
logger.debug('Launching %s', ' '.join(step))
job = Popen(step, stdout=macs_stdout.w, stderr=macs_stderr.w,
cwd=f.output_dir)
pollables = [macs_stdout, macs_stderr]
wait_for_job(job, pollables, logger)
return '%s\n\n' % ' '.join(step)
bwa_aligner = Popen(bwa_args, stdout=PIPE, stderr=bwa_stderr.w,
bufsize=-1)
samtools_args = [path_to_samtools, 'view', '-b', '-S', '-o',
path_to_unsorted, '-']
logger.info('Launching samtools to encode bwa output as BAM')
logger.info(' '.join(samtools_args))
samtools_stdout = PolledPipe(logger=logger, level=logging.WARN)
samtools_stderr = PolledPipe(logger=logger, level=logging.ERROR)
samtools_viewer = Popen(samtools_args, stdin=bwa_aligner.stdout,
stdout=samtools_stdout.w,
stderr=samtools_stderr.w, bufsize=-1)
logger.debug('Waiting for bwa to finish')
pollables = [bwa_stderr, samtools_stdout, samtools_stderr]
wait_for_job(bwa_aligner, pollables, logger)
if not bwa_aligner.returncode == 0:
logger.critical("bwa did not run properly [%d]",
bwa_aligner.returncode)
samtools_viewer.terminate()
samtools_viewer.poll()
logger.critical("samtools terminated")
return
logger.debug('Alignment successfully completed')
logger.debug('Waiting for samtools to finish')
wait_for_job(samtools_viewer, [samtools_stdout, samtools_stderr], logger)
if not samtools_viewer.returncode == 0:
logger.critical("samtools view did not run properly [%d]",
samtools_viewer.returncode)
