def align_dataset_parallel(
dataset,
blocks,
reference,
alignmentArgs,
ionstatsArgs,
BASECALLER_RESULTS,
basecaller_meta_information,
library_key,
graph_max_x,
ALIGNMENT_RESULTS,
do_realign,
do_ionstats,
do_mark_duplicates,
do_indexing,
align_threads,
barcodeInfo,
):
do_sorting = True
try:
# process block by block
memTotalGb = _get_total_memory_gb()
maxReads = 20000000
if memTotalGb > 140:
maxReads = 60000000
if reference and len(blocks) > 1 and int(dataset["read_count"]) > maxReads:
printtime(
"DEBUG: TRADITIONAL BLOCK PROCESSING ------ prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------"
% (dataset["file_prefix"], reference, dataset["read_count"])
)
# start alignment for each block and current barcode with reads
# TODO: in how many blocks are reads with this barcode
for block in blocks:
printtime("DEBUG: ALIGN ONLY ONE BLOCK: %s" % block)
align(
[block],
os.path.join(BASECALLER_RESULTS, dataset["basecaller_bam"]),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats=False,
do_sorting=do_sorting,
do_mark_duplicates=False,
do_indexing=False,
output_dir=os.path.join(block, ALIGNMENT_RESULTS),
output_basename=dataset["file_prefix"],
threads=align_threads,
barcode_info=barcodeInfo,
)
bamdir = "." # TODO , do we need this ?
bamBase = dataset["file_prefix"]
bamfile = dataset["file_prefix"] + ".bam"
block_bam_list = [
os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks
]
block_bam_list = [
block_bam_filename
for block_bam_filename in block_bam_list
if os.path.exists(block_bam_filename)
]
printtime("blocks with reads: %s" % len(block_bam_list))
bamFile = dataset["file_prefix"] + ".bam"
composite_bam_filepath = dataset["file_prefix"] + ".bam"
blockprocessing.extract_and_merge_bam_header(
block_bam_list, composite_bam_filepath
)
# Usage: samtools merge [-nr] [-h inh.sam] <out.bam> <in1.bam> <in2.bam> [...]
cmd = "samtools merge -l1 [email protected]"
if do_ionstats:
cmd += " - "
else:
cmd += " %s" % (composite_bam_filepath)
for bamfile in block_bam_list:
cmd += " %s" % bamfile
cmd += " -h %s.header.sam" % composite_bam_filepath
if do_ionstats:
bam_filenames = ["/dev/stdin"]
ionstats_alignment_filename = (
"%s.ionstats_alignment.json" % bamBase
) # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_alignment.json')
ionstats_alignment_h5_filename = (
"%s.ionstats_error_summary.h5" % bamBase
) # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_error_summary.h5')
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs,
bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information,
library_key,
graph_max_x,
)
cmd += " | tee >(%s)" % ionstats_cmd
if do_mark_duplicates:
json_name = (
"BamDuplicates.%s.json" % bamBase
if bamBase != "rawlib"
else "BamDuplicates.json"
)
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile, json_name)
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(["/bin/bash", "-c", cmd]).wait()
if ret != 0:
printtime("ERROR: merging failed, return code: %d" % ret)
raise RuntimeError("exit code: %d" % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd, shell=True)
else:
printtime(
"DEBUG: MERGED BLOCK PROCESSING ----------- prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------"
% (dataset["file_prefix"], reference, dataset["read_count"])
)
# TODO: try a python multiprocessing pool
align(
blocks,
os.path.join(BASECALLER_RESULTS, dataset["basecaller_bam"]),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir=ALIGNMENT_RESULTS if reference else BASECALLER_RESULTS,
output_basename=dataset["file_prefix"],
threads=align_threads,
barcode_info=barcodeInfo,
)
except Exception:
traceback.print_exc()
def align(
blocks,
basecaller_bam_filename, # e.g. 'basecaller_results/IonXpress001.basecaller.bam'
alignerArgs,
ionstatsArgs,
referenceName,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir,
output_basename,
threads=0):
try:
threads = threads or multiprocessing.cpu_count()
bamBase = os.path.normpath(output_dir + "/" + output_basename)
bamFile = bamBase + ".bam"
printtime("reference: %s" % referenceName)
printtime("input blocks: %s" % blocks)
printtime("input reads: %s" % basecaller_bam_filename)
printtime("output dir: %s" % output_dir)
printtime("output basename: %s" % output_basename)
printtime("full output base: %s" % bamBase)
printtime("full output file: %s" % bamFile) # TODO: not always used
if 'tmap' in alignerArgs:
aligner = 'tmap'
if '...' in alignerArgs:
alist = alignerArgs.split('...')
cmd = alist[0]
tmap_stage_options = alist[1]
else:
cmd = 'tmap mapall'
tmap_stage_options = 'stage1 map4'
elif 'bowtie2' in alignerArgs:
aligner = 'bowtie2'
cmd = alignerArgs
else:
printtime("ERROR: Aligner command not specified")
raise
if not referenceName:
# 1. create merged unmapped bam, 2. call ionstats
# TODO: long term: move ionstats basecaller into basecaller binary
cmd = ""
composite_bam_filepath = bamBase+'.basecaller.bam'
if blocks:
bamdir = '.' # TODO , do we need this ?
bamfile = basecaller_bam_filename
block_bam_list = [os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks]
block_bam_list = [block_bam_filename for block_bam_filename in block_bam_list if os.path.exists(block_bam_filename)]
if len(block_bam_list) >= 2:
blockprocessing.extract_and_merge_bam_header(block_bam_list, composite_bam_filepath)
cmd = 'samtools cat -h %s.header.sam -o /dev/stdout' % (composite_bam_filepath)
for blockbamfile in block_bam_list:
cmd = cmd + ' %s' % blockbamfile
elif len(block_bam_list) == 1:
# cmd = "samtools reheader %s.header.sam %s -" % (composite_bam_filepath,block_bam_list[0])
cmd = "cat %s" % (block_bam_list[0])
else:
return
'''
if block_bam_list:
composite_bai_filepath=""
mark_duplicates=False
method='samtools'
blockprocessing.merge_bam_files(block_bam_list, composite_bam_filepath, composite_bai_filepath, mark_duplicates, method)
'''
bam_filenames = ["/dev/stdin"]
else:
bam_filenames = [basecaller_bam_filename]
if do_ionstats:
ionstats_cmd = ionstats.generate_ionstats_basecaller_cmd(
bam_filenames,
bamBase+'.ionstats_basecaller.json',
library_key,
graph_max_x)
if blocks:
cmd += " | tee >(%s)" % ionstats_cmd
cmd += " > %s" % composite_bam_filepath
else:
cmd = ionstats_cmd
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: unmapped bam merging failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
return
if aligner == 'tmap':
referenceFastaFile = '/results/referenceLibrary/tmap-f3/' + referenceName + '/' + referenceName + '.fasta'
if blocks:
bamdir = '.' # TODO , do we need this ?
bamfile = basecaller_bam_filename
# printtime("DEBUG: BLOCKS for BAMFILE %s: %s" % (bamfile, blocks))
block_bam_list = [os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
block_bam_list = [block_bam_filename for block_bam_filename in block_bam_list if os.path.exists(block_bam_filename)]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
printtime("blocks with reads: %s" % len(block_bam_list))
if len(block_bam_list) >= 2:
blockprocessing.extract_and_merge_bam_header(block_bam_list, basecaller_bam_filename)
mergecmd = 'samtools cat -h %s.header.sam -o /dev/stdout' % basecaller_bam_filename
for blockbamfile in block_bam_list:
mergecmd = mergecmd + ' %s' % blockbamfile
'''
mergecmd = 'java -Xmx8g -jar /opt/picard/picard-tools-current/picard.jar MergeSamFiles'
for blockbamfile in block_bam_list:
mergecmd = mergecmd + ' I=%s' % blockbamfile
mergecmd = mergecmd + ' O=/dev/stdout'
mergecmd = mergecmd + ' VERBOSITY=WARNING' # suppress INFO on stderr
mergecmd = mergecmd + ' QUIET=true' # suppress job-summary on stderr
mergecmd = mergecmd + ' VALIDATION_STRINGENCY=SILENT'
'''
cmd = mergecmd + " | " + cmd
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -i bam"
elif len(block_bam_list) == 1:
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -r %s" % block_bam_list[0]
else:
printtime("ERROR: all blocks filtered")
return
else:
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -r %s" % basecaller_bam_filename
cmd += " -v"
cmd += " -Y"
cmd += " -u --prefix-exclude 5" # random seed based on read name after ignoring first 5 characters
if do_realign:
cmd += " --do-realign"
cmd += " -o 2" # -o 0: SAM, -o 2: uncompressed BAM
cmd += " %s" % tmap_stage_options
cmd += " 2>> " + bamBase + '.alignmentQC_out.txt' # logfile
elif aligner == 'bowtie2':
referenceFastaDir = '/results/referenceLibrary/bowtie2/' + referenceName + '/' + referenceName
cmd = "java -Xmx8g -jar /opt/picard/picard-tools-current/picard.jar SamToFastq I=%s F=/dev/stdout" % basecaller_bam_filename
cmd += " | /results/plugins/bowtielauncher/bowtie2 -p%d -x %s -U /dev/stdin" % (threads, referenceFastaDir)
cmd += " | samtools view -ubS -"
if do_ionstats:
bam_filenames = ["/dev/stdin"]
ionstats_alignment_filename = "%s.ionstats_alignment.json" % bamBase
ionstats_alignment_h5_filename = "%s.ionstats_error_summary.h5" % bamBase
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs,
bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information,
library_key,
graph_max_x)
cmd += " | tee >(%s)" % ionstats_cmd
if do_sorting:
if do_mark_duplicates:
# TODO: implement alternative, maybe with named pipes
cmd += " | samtools sort -m 1000M -l1 [email protected] -o - -"
json_name = 'BamDuplicates.%s.json' % bamBase if bamBase != 'rawlib' else 'BamDuplicates.json'
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile, json_name)
else:
# cmd += " | ( samtools sort -m 1000M -l1 [email protected] - %s <&0 & )" % bamBase
cmd += " | samtools sort -m 1000M -l1 [email protected] - %s" % bamBase
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: alignment failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd, shell=True)
'''
if do_indexing:
try:
composite_bam_filepath = os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.bam')
composite_bai_filepath = os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.bam.bai')
blockprocessing.create_index_file(composite_bam_filepath, composite_bai_filepath)
except:
traceback.print_exc()
'''
except:
raise
def align(
blocks,
basecaller_bam_filename, # e.g. 'basecaller_results/IonXpress001.basecaller.bam'
alignerArgs,
ionstatsArgs,
referenceName,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir,
output_basename,
threads=0,
barcode_info={},
):
try:
threads = threads or multiprocessing.cpu_count()
memGb = _get_total_memory_gb()
bamBase = os.path.normpath(output_dir + "/" + output_basename)
bamFile = bamBase + ".bam"
printtime("reference: %s" % referenceName)
printtime("input blocks: %s" % blocks)
printtime("input reads: %s" % basecaller_bam_filename)
printtime("output dir: %s" % output_dir)
printtime("output basename: %s" % output_basename)
printtime("full output base: %s" % bamBase)
printtime("full output file: %s" % bamFile) # TODO: not always used
# process args before the splits
alignerArgs = process_tmap_bed_file_args(
input_args=alignerArgs,
bam_filename=output_basename,
barcode_info=barcode_info,
)
if "tmap" in alignerArgs:
aligner = "tmap"
if "..." in alignerArgs:
alist = alignerArgs.split("...")
cmd = alist[0]
if memGb and memGb <= 40:
cmd = _add_read_queue_size(cmd, 50000)
tmap_stage_options = alist[1]
else:
cmd = "tmap mapall"
if memGb and memGb <= 40:
cmd = _add_read_queue_size(cmd, 50000)
tmap_stage_options = "stage1 map4"
elif "bowtie2" in alignerArgs:
aligner = "bowtie2"
cmd = alignerArgs
else:
printtime("ERROR: Aligner command not specified")
raise
if not referenceName:
# 1. create merged unmapped bam, 2. call ionstats
# TODO: long term: move ionstats basecaller into basecaller binary
cmd = ""
composite_bam_filepath = bamBase + ".basecaller.bam"
if blocks:
bamdir = "." # TODO , do we need this ?
bamfile = basecaller_bam_filename
block_bam_list = [
os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks
]
block_bam_list = [
block_bam_filename
for block_bam_filename in block_bam_list
if os.path.exists(block_bam_filename)
]
if len(block_bam_list) >= 2:
blockprocessing.extract_and_merge_bam_header(
block_bam_list, composite_bam_filepath
)
cmd = "samtools cat -h %s.header.sam -o /dev/stdout" % (
composite_bam_filepath
)
for blockbamfile in block_bam_list:
cmd = cmd + " %s" % blockbamfile
elif len(block_bam_list) == 1:
# cmd = "samtools reheader %s.header.sam %s -" % (composite_bam_filepath,block_bam_list[0])
cmd = "cat %s" % (block_bam_list[0])
else:
return
"""
if block_bam_list:
composite_bai_filepath=""
mark_duplicates=False
method='samtools'
blockprocessing.merge_bam_files(block_bam_list, composite_bam_filepath, composite_bai_filepath, mark_duplicates, method)
"""
bam_filenames = ["/dev/stdin"]
else:
bam_filenames = [basecaller_bam_filename]
if do_ionstats:
ionstats_cmd = ionstats.generate_ionstats_basecaller_cmd(
bam_filenames,
bamBase + ".ionstats_basecaller.json",
library_key,
graph_max_x,
)
if blocks:
cmd += " | tee >(%s)" % ionstats_cmd
cmd += " > %s" % composite_bam_filepath
else:
cmd = ionstats_cmd
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(["/bin/bash", "-c", cmd]).wait()
if ret != 0:
printtime("ERROR: unmapped bam merging failed, return code: %d" % ret)
raise RuntimeError("exit code: %d" % ret)
return
if aligner == "tmap":
referenceFastaFile = (
ion.referenceBasePath + referenceName + "/" + referenceName + ".fasta"
)
if blocks:
bamdir = "." # TODO , do we need this ?
bamfile = basecaller_bam_filename
# printtime("DEBUG: BLOCKS for BAMFILE %s: %s" % (bamfile, blocks))
block_bam_list = [
os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks
]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
block_bam_list = [
block_bam_filename
for block_bam_filename in block_bam_list
if os.path.exists(block_bam_filename)
]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
printtime("blocks with reads: %s" % len(block_bam_list))
if len(block_bam_list) >= 2:
blockprocessing.extract_and_merge_bam_header(
block_bam_list, basecaller_bam_filename
)
mergecmd = (
"samtools cat -h %s.header.sam -o /dev/stdout"
% basecaller_bam_filename
)
for blockbamfile in block_bam_list:
mergecmd = mergecmd + " %s" % blockbamfile
"""
mergecmd = 'java -Xmx8g -jar ' + ion.picardPath + ' MergeSamFiles'
for blockbamfile in block_bam_list:
mergecmd = mergecmd + ' I=%s' % blockbamfile
mergecmd = mergecmd + ' O=/dev/stdout'
mergecmd = mergecmd + ' VERBOSITY=WARNING' # suppress INFO on stderr
mergecmd = mergecmd + ' QUIET=true' # suppress job-summary on stderr
mergecmd = mergecmd + ' VALIDATION_STRINGENCY=SILENT'
"""
cmd = mergecmd + " | " + cmd
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -i bam"
elif len(block_bam_list) == 1:
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -r %s" % block_bam_list[0]
else:
printtime("ERROR: all blocks filtered")
return
else:
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -r %s" % basecaller_bam_filename
cmd += " -v"
cmd += " -Y"
cmd += (
" -u --prefix-exclude 5"
) # random seed based on read name after ignoring first 5 characters
if do_realign:
cmd += " --do-realign"
cmd += " -o 2" # -o 0: SAM, -o 2: uncompressed BAM
cmd += " %s" % tmap_stage_options
cmd += " 2>> " + bamBase + ".alignmentQC_out.txt" # logfile
elif aligner == "bowtie2":
referenceFastaDir = (
"/results/referenceLibrary/bowtie2/"
+ referenceName
+ "/"
+ referenceName
)
cmd = "java -Xmx8g -jar %s SamToFastq I=%s F=/dev/stdout" % (
ion.picardPath,
basecaller_bam_filename,
)
cmd += (
" | /results/plugins/bowtielauncher/bowtie2 -p%d -x %s -U /dev/stdin"
% (threads, referenceFastaDir)
)
cmd += " | samtools view -ubS -"
if do_ionstats:
bam_filenames = ["/dev/stdin"]
ionstats_alignment_filename = "%s.ionstats_alignment.json" % bamBase
ionstats_alignment_h5_filename = "%s.ionstats_error_summary.h5" % bamBase
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs,
bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information,
library_key,
graph_max_x,
)
cmd += " | tee >(%s)" % ionstats_cmd
# use number align_threads if smaller than 12
samtool_threads = min(threads, 12)
if do_sorting:
if do_mark_duplicates:
# use '-T' option to avoid temp file name collision among barcodes
cmd += " | samtools sort -m 1000M -l1 [email protected]%d -T tmp_%s -O bam -o - -" % (
samtool_threads,
bamBase,
)
json_name = (
"BamDuplicates.%s.json" % bamBase
if bamBase != "rawlib"
else "BamDuplicates.json"
)
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile, json_name)
else:
# use '-T' option to avoid temp file name collision among barcodes
cmd += " | samtools sort -m 1000M -l1 [email protected]{thread_num} -T tmp_{output_prefix} -O bam -o - - > {output_prefix}.bam".format(
thread_num=samtool_threads, output_prefix=bamBase
)
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(["/bin/bash", "-c", cmd]).wait()
if ret != 0:
printtime("ERROR: alignment failed, return code: %d" % ret)
raise RuntimeError("exit code: %d" % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd, shell=True)
"""
if do_indexing:
try:
composite_bam_filepath = os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.bam')
composite_bai_filepath = os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.bam.bai')
blockprocessing.create_index_file(composite_bam_filepath, composite_bai_filepath)
except:
traceback.print_exc()
"""
except Exception:
raise
def align_dataset_parallel(
dataset,
blocks,
reference,
alignmentArgs,
ionstatsArgs,
BASECALLER_RESULTS,
basecaller_meta_information,
library_key,
graph_max_x,
ALIGNMENT_RESULTS,
do_realign,
do_ionstats,
do_mark_duplicates,
do_indexing,
align_threads
):
do_sorting = True
try:
# process block by block
if reference and len(blocks) > 1 and int(dataset["read_count"]) > 20000000:
printtime("DEBUG: TRADITIONAL BLOCK PROCESSING ------ prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------" % (dataset['file_prefix'], reference, dataset["read_count"]))
# start alignment for each block and current barcode with reads
# TODO: in how many blocks are reads with this barcode
for block in blocks:
printtime("DEBUG: ALIGN ONLY ONE BLOCK: %s" % block)
align(
[block],
os.path.join(BASECALLER_RESULTS, dataset['basecaller_bam']),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats=False,
do_sorting=do_sorting,
do_mark_duplicates=False,
do_indexing=False,
output_dir=os.path.join(block, ALIGNMENT_RESULTS),
output_basename=dataset['file_prefix'],
threads=align_threads)
bamdir = '.' # TODO , do we need this ?
bamBase = dataset['file_prefix']
bamfile = dataset['file_prefix'] + ".bam"
block_bam_list = [os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks]
block_bam_list = [block_bam_filename for block_bam_filename in block_bam_list if os.path.exists(block_bam_filename)]
printtime("blocks with reads: %s" % len(block_bam_list))
bamFile = dataset['file_prefix'] + ".bam"
composite_bam_filepath = dataset['file_prefix'] + ".bam"
blockprocessing.extract_and_merge_bam_header(block_bam_list, composite_bam_filepath)
# Usage: samtools merge [-nr] [-h inh.sam] <out.bam> <in1.bam> <in2.bam> [...]
cmd = 'samtools merge -l1 [email protected]'
if do_ionstats:
cmd += ' - '
else:
cmd += ' %s' % (composite_bam_filepath)
for bamfile in block_bam_list:
cmd += ' %s' % bamfile
cmd += ' -h %s.header.sam' % composite_bam_filepath
if do_ionstats:
bam_filenames = ["/dev/stdin"]
ionstats_alignment_filename = "%s.ionstats_alignment.json" % bamBase # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_alignment.json')
ionstats_alignment_h5_filename = "%s.ionstats_error_summary.h5" % bamBase # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_error_summary.h5')
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs,
bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information,
library_key,
graph_max_x)
cmd += " | tee >(%s)" % ionstats_cmd
if do_mark_duplicates:
json_name = 'BamDuplicates.%s.json' % bamBase if bamBase != 'rawlib' else 'BamDuplicates.json'
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile, json_name)
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: merging failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd, shell=True)
else:
printtime("DEBUG: MERGED BLOCK PROCESSING ----------- prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------" % (dataset['file_prefix'], reference, dataset["read_count"]))
# TODO: try a python multiprocessing pool
align(
blocks,
os.path.join(BASECALLER_RESULTS, dataset['basecaller_bam']),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir=ALIGNMENT_RESULTS if reference else BASECALLER_RESULTS,
output_basename=dataset['file_prefix'],
threads=align_threads)
except:
traceback.print_exc()
def align(
blocks,
basecaller_bam_filename, # e.g. 'basecaller_results/IonXpress001.basecaller.bam'
alignerArgs,
ionstatsArgs,
referenceName,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir,
output_basename,
threads=0):
try:
threads = threads or multiprocessing.cpu_count()
bamBase = os.path.normpath(output_dir + "/" + output_basename)
bamFile = bamBase + ".bam"
printtime("reference: %s" % referenceName)
printtime("input blocks: %s" % blocks)
printtime("input reads: %s" % basecaller_bam_filename)
printtime("output dir: %s" % output_dir)
printtime("output basename: %s" % output_basename)
printtime("full output base: %s" % bamBase)
printtime("full output file: %s" %
bamFile) # TODO: not always used
if 'tmap' in alignerArgs:
aligner = 'tmap'
if '...' in alignerArgs:
alist = alignerArgs.split('...')
cmd = alist[0]
tmap_stage_options = alist[1]
else:
cmd = 'tmap mapall'
tmap_stage_options = 'stage1 map4'
elif 'bowtie2' in alignerArgs:
aligner = 'bowtie2'
cmd = alignerArgs
else:
printtime("ERROR: Aligner command not specified")
raise
if not referenceName:
# 1. create merged unmapped bam, 2. call ionstats
# TODO: long term: move ionstats basecaller into basecaller binary
cmd = ""
composite_bam_filepath = bamBase + '.basecaller.bam'
if blocks:
bamdir = '.' # TODO , do we need this ?
bamfile = basecaller_bam_filename
block_bam_list = [
os.path.join(blockdir, bamdir, bamfile)
for blockdir in blocks
]
block_bam_list = [
block_bam_filename for block_bam_filename in block_bam_list
if os.path.exists(block_bam_filename)
]
if len(block_bam_list) >= 2:
blockprocessing.extract_and_merge_bam_header(
block_bam_list, composite_bam_filepath)
cmd = 'samtools cat -h %s.header.sam -o /dev/stdout' % (
composite_bam_filepath)
for blockbamfile in block_bam_list:
cmd = cmd + ' %s' % blockbamfile
elif len(block_bam_list) == 1:
# cmd = "samtools reheader %s.header.sam %s -" % (composite_bam_filepath,block_bam_list[0])
cmd = "cat %s" % (block_bam_list[0])
else:
return
'''
if block_bam_list:
composite_bai_filepath=""
mark_duplicates=False
method='samtools'
blockprocessing.merge_bam_files(block_bam_list, composite_bam_filepath, composite_bai_filepath, mark_duplicates, method)
'''
bam_filenames = ["/dev/stdin"]
else:
bam_filenames = [basecaller_bam_filename]
if do_ionstats:
ionstats_cmd = ionstats.generate_ionstats_basecaller_cmd(
bam_filenames, bamBase + '.ionstats_basecaller.json',
library_key, graph_max_x)
if blocks:
cmd += " | tee >(%s)" % ionstats_cmd
cmd += " > %s" % composite_bam_filepath
else:
cmd = ionstats_cmd
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime(
"ERROR: unmapped bam merging failed, return code: %d" %
ret)
raise RuntimeError('exit code: %d' % ret)
return
if aligner == 'tmap':
referenceFastaFile = '/results/referenceLibrary/tmap-f3/' + referenceName + '/' + referenceName + '.fasta'
if blocks:
bamdir = '.' # TODO , do we need this ?
bamfile = basecaller_bam_filename
# printtime("DEBUG: BLOCKS for BAMFILE %s: %s" % (bamfile, blocks))
block_bam_list = [
os.path.join(blockdir, bamdir, bamfile)
for blockdir in blocks
]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
block_bam_list = [
block_bam_filename for block_bam_filename in block_bam_list
if os.path.exists(block_bam_filename)
]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
printtime("blocks with reads: %s" % len(block_bam_list))
if len(block_bam_list) >= 2:
blockprocessing.extract_and_merge_bam_header(
block_bam_list, basecaller_bam_filename)
mergecmd = 'samtools cat -h %s.header.sam -o /dev/stdout' % basecaller_bam_filename
for blockbamfile in block_bam_list:
mergecmd = mergecmd + ' %s' % blockbamfile
'''
mergecmd = 'java -Xmx8g -jar /opt/picard/picard-tools-current/picard.jar MergeSamFiles'
for blockbamfile in block_bam_list:
mergecmd = mergecmd + ' I=%s' % blockbamfile
mergecmd = mergecmd + ' O=/dev/stdout'
mergecmd = mergecmd + ' VERBOSITY=WARNING' # suppress INFO on stderr
mergecmd = mergecmd + ' QUIET=true' # suppress job-summary on stderr
mergecmd = mergecmd + ' VALIDATION_STRINGENCY=SILENT'
'''
cmd = mergecmd + " | " + cmd
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -i bam"
elif len(block_bam_list) == 1:
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -r %s" % block_bam_list[0]
else:
printtime("ERROR: all blocks filtered")
return
else:
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
cmd += " -r %s" % basecaller_bam_filename
cmd += " -v"
cmd += " -Y"
cmd += " -u --prefix-exclude 5" # random seed based on read name after ignoring first 5 characters
if do_realign:
cmd += " --do-realign"
cmd += " -o 2" # -o 0: SAM, -o 2: uncompressed BAM
cmd += " %s" % tmap_stage_options
cmd += " 2>> " + bamBase + '.alignmentQC_out.txt' # logfile
elif aligner == 'bowtie2':
referenceFastaDir = '/results/referenceLibrary/bowtie2/' + referenceName + '/' + referenceName
cmd = "java -Xmx8g -jar /opt/picard/picard-tools-current/picard.jar SamToFastq I=%s F=/dev/stdout" % basecaller_bam_filename
cmd += " | /results/plugins/bowtielauncher/bowtie2 -p%d -x %s -U /dev/stdin" % (
threads, referenceFastaDir)
cmd += " | samtools view -ubS -"
if do_ionstats:
bam_filenames = ["/dev/stdin"]
ionstats_alignment_filename = "%s.ionstats_alignment.json" % bamBase
ionstats_alignment_h5_filename = "%s.ionstats_error_summary.h5" % bamBase
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs, bam_filenames, ionstats_alignment_filename,
ionstats_alignment_h5_filename, basecaller_meta_information,
library_key, graph_max_x)
cmd += " | tee >(%s)" % ionstats_cmd
if do_sorting:
if do_mark_duplicates:
# TODO: implement alternative, maybe with named pipes
cmd += " | samtools sort -m 1000M -l1 [email protected] -o - -"
json_name = 'BamDuplicates.%s.json' % bamBase if bamBase != 'rawlib' else 'BamDuplicates.json'
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile,
json_name)
else:
# cmd += " | ( samtools sort -m 1000M -l1 [email protected] - %s <&0 & )" % bamBase
cmd += " | samtools sort -m 1000M -l1 [email protected] - %s" % bamBase
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: alignment failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd, shell=True)
'''
if do_indexing:
try:
composite_bam_filepath = os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.bam')
composite_bai_filepath = os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.bam.bai')
blockprocessing.create_index_file(composite_bam_filepath, composite_bai_filepath)
except:
traceback.print_exc()
'''
except:
raise
def align(
blocks,
alignerArgs,
ionstatsArgs,
referenceName,
basecaller_meta_information,
library_key,
graph_max_x,
readFile,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
logfile,
output_dir,
output_basename):
# Input : readFile
# Output : output_dir/output_basename.bam
try:
if 'tmap' in alignerArgs:
aligner = 'tmap'
if '...' in alignerArgs:
alist = alignerArgs.split('...')
cmd = alist[0]
tmap_stage_options = alist[1]
else:
cmd = 'tmap mapall'
tmap_stage_options = 'stage1 map4'
elif 'bowtie2' in alignerArgs:
aligner = 'bowtie2'
cmd = alignerArgs
else:
printtime("ERROR: Aligner command not specified")
raise
threads = multiprocessing.cpu_count()
bamBase = os.path.normpath(output_dir + "/" + output_basename)
bamFile = bamBase + ".bam"
blocks=[] # TODO
if aligner == 'tmap':
referenceFastaFile = '/results/referenceLibrary/tmap-f3/' + referenceName + '/' + referenceName + '.fasta'
if blocks:
mergecmd = 'java -Xmx8g -jar /opt/picard/picard-tools-current/picard.jar MergeSamFiles'
bamdir = '.'
bamfile = readFile
block_bam_list = [os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks]
block_bam_list = [block_bam_filename for block_bam_filename in block_bam_list if os.path.exists(block_bam_filename)]
for bamfile in block_bam_list:
mergecmd = mergecmd + ' I=%s' % bamfile
mergecmd = mergecmd + ' O=/dev/stdout'
mergecmd = mergecmd + ' VERBOSITY=WARNING' # suppress INFO on stderr
mergecmd = mergecmd + ' QUIET=true' # suppress job-summary on stderr
mergecmd = mergecmd + ' VALIDATION_STRINGENCY=SILENT'
cmd = mergecmd + " | " + cmd
cmd += " -n %d" % threads
cmd += " -f %s" % referenceFastaFile
if blocks:
cmd += " -i bam"
else:
cmd += " -r %s" % readFile
cmd += " -v"
cmd += " -Y"
cmd += " -u --prefix-exclude 5" # random seed based on read name after ignoring first 5 characters
if do_realign:
cmd += " --do-realign"
cmd += " -o 2" # -o 0: SAM, -o 2: uncompressed BAM
cmd += " %s" % tmap_stage_options
cmd += " 2>> " + logfile
elif aligner == 'bowtie2':
referenceFastaDir = '/results/referenceLibrary/bowtie2/' + referenceName + '/' + referenceName
cmd="java -Xmx8g -jar /opt/picard/picard-tools-current/picard.jar SamToFastq I=%s F=/dev/stdout" % readFile
cmd+=" | /results/plugins/bowtielauncher/bowtie2 -p%d -x %s -U /dev/stdin" % (threads, referenceFastaDir)
cmd+=" | samtools view -ubS -"
if do_ionstats:
bam_filenames=["/dev/stdin"]
ionstats_alignment_filename="%s.ionstats_alignment.json" % bamBase
ionstats_alignment_h5_filename="%s.ionstats_error_summary.h5" % bamBase
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs,
bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information,
library_key,
graph_max_x)
cmd += " | tee >(%s)" % ionstats_cmd
if do_sorting:
if do_mark_duplicates:
#TODO: implement alternative, maybe with named pipes
cmd += " | samtools sort -m 1000M -l1 [email protected] -o - -"
json_name = 'BamDuplicates.%s.json' % bamBase if bamBase!='rawlib' else 'BamDuplicates.json'
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile, json_name)
else:
# cmd += " | ( samtools sort -m 1000M -l1 [email protected] - %s <&0 & )" % bamBase
cmd += " | samtools sort -m 1000M -l1 [email protected] - %s" % bamBase
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: alignment failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
print cmd
subprocess.call(cmd,shell=True)
except:
raise
def process_datasets(blocks, alignmentArgs, ionstatsArgs, BASECALLER_RESULTS,
basecaller_meta_information, library_key, graph_max_x,
basecaller_datasets, ALIGNMENT_RESULTS, do_realign,
do_ionstats, do_mark_duplicates, do_indexing,
barcodeInfo):
printtime("Attempt to align")
printtime("DEBUG: PROCESS DATASETS blocks: '%s'" % blocks)
do_sorting = True
# TODO: compare with pipeline/python/ion/utils/ionstats.py
ionstats_basecaller_file_list = []
ionstats_alignment_file_list = []
ionstats_basecaller_filtered_file_list = []
ionstats_alignment_filtered_file_list = []
for dataset in basecaller_datasets["datasets"]:
read_group = dataset['read_groups'][0]
reference = basecaller_datasets['read_groups'][read_group]['reference']
#print "DEBUG: reference: %s' % reference
filtered = True
for rg_name in dataset["read_groups"]:
if not basecaller_datasets["read_groups"][rg_name].get(
'filtered', False):
filtered = False
# skip non-existing bam file
if int(dataset["read_count"]) == 0:
continue
try:
# process block by block
if reference and len(blocks) > 1 and int(
dataset["read_count"]) > 20000000:
printtime(
"DEBUG: TRADITIONAL BLOCK PROCESSING ------ prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------"
%
(dataset['file_prefix'], reference, dataset["read_count"]))
# start alignment for each block and current barcode with reads
# TODO: in how many blocks are reads with this barcode
for block in blocks:
printtime("DEBUG: ALIGN ONLY ONE BLOCK: %s" % block)
align([block],
os.path.join(BASECALLER_RESULTS,
dataset['basecaller_bam']),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats=False,
do_sorting=do_sorting,
do_mark_duplicates=False,
do_indexing=False,
output_dir=os.path.join(block, ALIGNMENT_RESULTS),
output_basename=dataset['file_prefix'])
bamdir = '.' # TODO , do we need this ?
bamBase = dataset['file_prefix']
bamfile = dataset['file_prefix'] + ".bam"
# printtime("DEBUG: BLOCKS for BAMFILE %s: %s" % (bamfile, blocks))
block_bam_list = [
os.path.join(blockdir, bamdir, bamfile)
for blockdir in blocks
]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
block_bam_list = [
block_bam_filename for block_bam_filename in block_bam_list
if os.path.exists(block_bam_filename)
]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
printtime("blocks with reads: %s" % len(block_bam_list))
bamFile = dataset['file_prefix'] + ".bam"
composite_bam_filepath = dataset['file_prefix'] + ".bam"
blockprocessing.extract_and_merge_bam_header(
block_bam_list, composite_bam_filepath)
# Usage: samtools merge [-nr] [-h inh.sam] <out.bam> <in1.bam> <in2.bam> [...]
cmd = 'samtools merge -l1 [email protected]'
if do_ionstats:
cmd += ' - '
else:
cmd += ' %s' % (composite_bam_filepath)
for bamfile in block_bam_list:
cmd += ' %s' % bamfile
cmd += ' -h %s.header.sam' % composite_bam_filepath
if do_ionstats:
bam_filenames = ["/dev/stdin"]
ionstats_alignment_filename = "%s.ionstats_alignment.json" % bamBase # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_alignment.json')
ionstats_alignment_h5_filename = "%s.ionstats_error_summary.h5" % bamBase # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_error_summary.h5')
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs, bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information, library_key, graph_max_x)
cmd += " | tee >(%s)" % ionstats_cmd
if do_mark_duplicates:
json_name = 'BamDuplicates.%s.json' % bamBase if bamBase != 'rawlib' else 'BamDuplicates.json'
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile,
json_name)
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: merging failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd, shell=True)
else:
printtime(
"DEBUG: MERGED BLOCK PROCESSING ----------- prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------"
%
(dataset['file_prefix'], reference, dataset["read_count"]))
# TODO: try a python multiprocessing pool
align(blocks,
os.path.join(BASECALLER_RESULTS,
dataset['basecaller_bam']),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir=ALIGNMENT_RESULTS
if reference else BASECALLER_RESULTS,
output_basename=dataset['file_prefix'])
except:
traceback.print_exc()
if reference:
if filtered:
ionstats_alignment_filtered_file_list.append(
os.path.join(
ALIGNMENT_RESULTS,
dataset['file_prefix'] + '.ionstats_alignment.json'))
else:
ionstats_alignment_file_list.append(
os.path.join(
ALIGNMENT_RESULTS,
dataset['file_prefix'] + '.ionstats_alignment.json'))
else:
if filtered:
ionstats_basecaller_filtered_file_list.append(
os.path.join(
BASECALLER_RESULTS,
dataset['file_prefix'] + '.ionstats_basecaller.json'))
else:
ionstats_basecaller_file_list.append(
os.path.join(
BASECALLER_RESULTS,
dataset['file_prefix'] + '.ionstats_basecaller.json'))
if do_ionstats:
# Merge ionstats files from individual (barcoded) datasets
if len(ionstats_alignment_file_list) > 0:
ionstats.reduce_stats(
ionstats_alignment_file_list,
os.path.join(ALIGNMENT_RESULTS, 'ionstats_alignment.json'))
else: # barcode classification filtered all barcodes or no reads available
# TODO: ionstats needs to produce initial json file
try:
#cmd = "echo $'@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
cmd = "echo '@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.call(cmd, shell=True)
if ret != 0:
printtime(
"ERROR: empty bam file generation failed, return code: %d"
% ret)
raise RuntimeError('exit code: %d' % ret)
ionstats.generate_ionstats_alignment(
ionstatsArgs, ['empty_dummy.bam'],
os.path.join(ALIGNMENT_RESULTS, 'ionstats_alignment.json'),
os.path.join(ALIGNMENT_RESULTS,
'ionstats_error_summary.h5'),
basecaller_meta_information, library_key, graph_max_x)
except:
raise
if len(ionstats_basecaller_file_list) > 0:
ionstats.reduce_stats(
ionstats_basecaller_file_list,
os.path.join(BASECALLER_RESULTS,
'ionstats_tmp_basecaller.json'))
else: # barcode classification filtered all barcodes or no reads available
# TODO: ionstats needs to produce initial json file
try:
#cmd = "echo $'@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
cmd = "echo '@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.call(cmd, shell=True)
if ret != 0:
printtime(
"ERROR: empty bam file generation failed, return code: %d"
% ret)
raise RuntimeError('exit code: %d' % ret)
ionstats.generate_ionstats_basecaller(
['empty_dummy.bam'],
os.path.join(BASECALLER_RESULTS,
'ionstats_tmp_basecaller.json'), library_key,
graph_max_x)
except:
raise
ionstatslist = []
a = os.path.join(ALIGNMENT_RESULTS, 'ionstats_alignment.json')
b = os.path.join(BASECALLER_RESULTS, 'ionstats_tmp_basecaller.json')
if os.path.exists(a):
ionstatslist.append(a)
if os.path.exists(b):
ionstatslist.append(b)
if len(ionstatslist) > 0:
ionstats.reduce_stats(
ionstatslist,
os.path.join(BASECALLER_RESULTS,
'ionstats_basecaller_with_aligninfos.json'))
ionstats.reduce_stats(
reversed(ionstatslist),
os.path.join(BASECALLER_RESULTS, 'ionstats_basecaller.json'))
# if len(ionstats_alignment_h5_file_list) > 0:
# ionstats.reduce_stats_h5(ionstats_alignment_h5_file_list,os.path.join(ALIGNMENT_RESULTS,'ionstats_error_summary.h5'))
printtime("**** Alignment completed ****")
def process_datasets(
blocks,
alignmentArgs,
ionstatsArgs,
BASECALLER_RESULTS,
basecaller_meta_information,
library_key,
graph_max_x,
basecaller_datasets,
ALIGNMENT_RESULTS,
do_realign,
do_ionstats,
do_mark_duplicates,
do_indexing,
barcodeInfo):
printtime("Attempt to align")
printtime("DEBUG: PROCESS DATASETS blocks: '%s'" % blocks)
do_sorting = True
# TODO: compare with pipeline/python/ion/utils/ionstats.py
ionstats_basecaller_file_list = []
ionstats_alignment_file_list = []
ionstats_basecaller_filtered_file_list = []
ionstats_alignment_filtered_file_list = []
for dataset in basecaller_datasets["datasets"]:
read_group = dataset['read_groups'][0]
reference = basecaller_datasets['read_groups'][read_group]['reference']
#print "DEBUG: reference: %s' % reference
filtered = True
for rg_name in dataset["read_groups"]:
if not basecaller_datasets["read_groups"][rg_name].get('filtered',False):
filtered = False
# skip non-existing bam file
if int(dataset["read_count"]) == 0:
continue
try:
# process block by block
if reference and len(blocks) > 1 and int(dataset["read_count"]) > 20000000:
printtime("DEBUG: TRADITIONAL BLOCK PROCESSING ------ prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------" % (dataset['file_prefix'], reference, dataset["read_count"]))
# start alignment for each block and current barcode with reads
# TODO: in how many blocks are reads with this barcode
for block in blocks:
printtime("DEBUG: ALIGN ONLY ONE BLOCK: %s" % block)
align(
[block],
os.path.join(BASECALLER_RESULTS, dataset['basecaller_bam']),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats=False,
do_sorting=do_sorting,
do_mark_duplicates=False,
do_indexing=False,
output_dir=os.path.join(block,ALIGNMENT_RESULTS),
output_basename=dataset['file_prefix'])
bamdir = '.' # TODO , do we need this ?
bamBase = dataset['file_prefix']
bamfile = dataset['file_prefix'] + ".bam"
# printtime("DEBUG: BLOCKS for BAMFILE %s: %s" % (bamfile, blocks))
block_bam_list = [os.path.join(blockdir, bamdir, bamfile) for blockdir in blocks]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
block_bam_list = [block_bam_filename for block_bam_filename in block_bam_list if os.path.exists(block_bam_filename)]
# printtime("DEBUG: block_bam_list: %s" % block_bam_list)
printtime("blocks with reads: %s" % len(block_bam_list))
bamFile = dataset['file_prefix'] + ".bam"
composite_bam_filepath = dataset['file_prefix'] + ".bam"
blockprocessing.extract_and_merge_bam_header(block_bam_list,composite_bam_filepath)
# Usage: samtools merge [-nr] [-h inh.sam] <out.bam> <in1.bam> <in2.bam> [...]
cmd = 'samtools merge -l1 [email protected]'
if do_ionstats:
cmd += ' - '
else:
cmd += ' %s' % (composite_bam_filepath)
for bamfile in block_bam_list:
cmd += ' %s' % bamfile
cmd += ' -h %s.header.sam' % composite_bam_filepath
if do_ionstats:
bam_filenames=["/dev/stdin"]
ionstats_alignment_filename="%s.ionstats_alignment.json" % bamBase # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_alignment.json')
ionstats_alignment_h5_filename="%s.ionstats_error_summary.h5" % bamBase # os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_error_summary.h5')
ionstats_cmd = ionstats.generate_ionstats_alignment_cmd(
ionstatsArgs,
bam_filenames,
ionstats_alignment_filename,
ionstats_alignment_h5_filename,
basecaller_meta_information,
library_key,
graph_max_x)
cmd += " | tee >(%s)" % ionstats_cmd
if do_mark_duplicates:
json_name = 'BamDuplicates.%s.json' % bamBase if bamBase!='rawlib' else 'BamDuplicates.json'
cmd = "BamDuplicates -i <(%s) -o %s -j %s" % (cmd, bamFile, json_name)
else:
cmd += " > %s.bam" % bamBase
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.Popen(['/bin/bash', '-c', cmd]).wait()
if ret != 0:
printtime("ERROR: merging failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
# TODO: piping into samtools index or create index in sort process ?
if do_indexing and do_sorting:
cmd = "samtools index " + bamFile
printtime("DEBUG: Calling '%s':" % cmd)
subprocess.call(cmd,shell=True)
else:
printtime("DEBUG: MERGED BLOCK PROCESSING ----------- prefix: %20s ----------- reference: %20s ---------- reads: %10s ----------" % (dataset['file_prefix'], reference, dataset["read_count"]))
# TODO: try a python multiprocessing pool
align(
blocks,
os.path.join(BASECALLER_RESULTS, dataset['basecaller_bam']),
alignmentArgs,
ionstatsArgs,
reference,
basecaller_meta_information,
library_key,
graph_max_x,
do_realign,
do_ionstats,
do_sorting,
do_mark_duplicates,
do_indexing,
output_dir=ALIGNMENT_RESULTS if reference else BASECALLER_RESULTS,
output_basename=dataset['file_prefix'])
except:
traceback.print_exc()
if reference:
if filtered:
ionstats_alignment_filtered_file_list.append(os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_alignment.json'))
else:
ionstats_alignment_file_list.append(os.path.join(ALIGNMENT_RESULTS, dataset['file_prefix']+'.ionstats_alignment.json'))
else:
if filtered:
ionstats_basecaller_filtered_file_list.append(os.path.join(BASECALLER_RESULTS, dataset['file_prefix']+'.ionstats_basecaller.json'))
else:
ionstats_basecaller_file_list.append(os.path.join(BASECALLER_RESULTS, dataset['file_prefix']+'.ionstats_basecaller.json'))
if do_ionstats:
# Merge ionstats files from individual (barcoded) datasets
if len(ionstats_alignment_file_list) > 0:
ionstats.reduce_stats(ionstats_alignment_file_list,os.path.join(ALIGNMENT_RESULTS,'ionstats_alignment.json'))
else: # barcode classification filtered all barcodes or no reads available
# TODO: ionstats needs to produce initial json file
try:
#cmd = "echo $'@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
cmd = "echo '@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.call(cmd,shell=True)
if ret != 0:
printtime("ERROR: empty bam file generation failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
ionstats.generate_ionstats_alignment(
ionstatsArgs,
['empty_dummy.bam'],
os.path.join(ALIGNMENT_RESULTS, 'ionstats_alignment.json'),
os.path.join(ALIGNMENT_RESULTS, 'ionstats_error_summary.h5'),
basecaller_meta_information,
library_key,
graph_max_x)
except:
raise
if len(ionstats_basecaller_file_list) > 0:
ionstats.reduce_stats(ionstats_basecaller_file_list,os.path.join(BASECALLER_RESULTS,'ionstats_tmp_basecaller.json'))
else: # barcode classification filtered all barcodes or no reads available
# TODO: ionstats needs to produce initial json file
try:
#cmd = "echo $'@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
cmd = "echo '@HD\tVN:1.5\tSO:coordinate\[email protected]\tSN:ref\tLN:4\[email protected]\tID:filename\tSM:filename' | samtools view -F4 -S -b - > empty_dummy.bam"
printtime("DEBUG: Calling '%s':" % cmd)
ret = subprocess.call(cmd,shell=True)
if ret != 0:
printtime("ERROR: empty bam file generation failed, return code: %d" % ret)
raise RuntimeError('exit code: %d' % ret)
ionstats.generate_ionstats_basecaller(
['empty_dummy.bam'],
os.path.join(BASECALLER_RESULTS, 'ionstats_tmp_basecaller.json'),
library_key,
graph_max_x)
except:
raise
ionstatslist = []
a = os.path.join(ALIGNMENT_RESULTS,'ionstats_alignment.json')
b = os.path.join(BASECALLER_RESULTS,'ionstats_tmp_basecaller.json')
if os.path.exists(a):
ionstatslist.append(a)
if os.path.exists(b):
ionstatslist.append(b)
if len(ionstatslist) > 0:
ionstats.reduce_stats( ionstatslist, os.path.join(BASECALLER_RESULTS,'ionstats_basecaller_with_aligninfos.json'))
ionstats.reduce_stats( reversed(ionstatslist), os.path.join(BASECALLER_RESULTS,'ionstats_basecaller.json'))
# if len(ionstats_alignment_h5_file_list) > 0:
# ionstats.reduce_stats_h5(ionstats_alignment_h5_file_list,os.path.join(ALIGNMENT_RESULTS,'ionstats_error_summary.h5'))
printtime("**** Alignment completed ****")
