def align(fastq_file, pair_file, ref_file, names, align_dir, data):
assert data["analysis"].lower().startswith(
"wgbs-seq"), "No comparible alignment."
config = data["config"]
sample = dd.get_sample_name(data)
out_prefix = os.path.join(align_dir, dd.get_lane(data))
out_dir = os.path.join(align_dir, "%s_bismark" % dd.get_lane(data))
if not ref_file:
logger.error(
"bismark index not found. We don't provide the STAR indexes "
"by default because they are very large. You can install "
"the index for your genome with: bcbio_nextgen.py upgrade "
"--aligners bismark --genomes genome-build-name --data")
sys.exit(1)
final_out = os.path.join(align_dir, "{0}.bam".format(sample))
if file_exists(final_out):
data = dd.set_work_bam(data, final_out)
data["bam_report"] = glob.glob(os.path.join(out_dir, "*report.txt"))[0]
return data
bismark = config_utils.get_program("bismark", config)
# bismark uses 5 threads/sample and ~12GB RAM/sample (hg38)
resources = config_utils.get_resources("bismark", data["config"])
max_cores = resources.get("cores", 1)
max_mem = config_utils.convert_to_bytes(resources.get("memory", "1G"))
n = min(max(int(max_cores / 5), 1),
max(int(max_mem / config_utils.convert_to_bytes("12G")), 1))
kit = kits.KITS.get(dd.get_kit(data), None)
directional = "--non_directional" if kit and not kit.is_directional else ""
other_opts = resources.get("options", [])
other_opts = " ".join([str(x) for x in other_opts]).strip()
fastq_files = " ".join([fastq_file, pair_file
]) if pair_file else fastq_file
safe_makedir(align_dir)
cmd = "{bismark} {other_opts} {directional} --bowtie2 --temp_dir {tx_out_dir} --gzip --multicore {n} -o {tx_out_dir} --unmapped {ref_file} {fastq_file}"
if pair_file:
fastq_file = "-1 %s -2 %s" % (fastq_file, pair_file)
raw_bam = glob.glob(out_dir + "/*bismark*bt2*bam")
if not raw_bam:
with tx_tmpdir() as tx_out_dir:
run_message = "Running Bismark aligner on %s and %s" % (fastq_file,
ref_file)
do.run(cmd.format(**locals()), run_message, None)
shutil.move(tx_out_dir, out_dir)
raw_bam = glob.glob(out_dir + "/*bismark*bt2*bam")
process_bam = _process_bam(raw_bam[0], fastq_files, sample,
dd.get_sam_ref(data), config)
utils.symlink_plus(process_bam, final_out)
data = dd.set_work_bam(data, final_out)
data["bam_report"] = glob.glob(os.path.join(out_dir, "*report.txt"))[0]
return data
def _cram_to_fastq_region(cram_file, work_dir, base_name, region, data):
"""Convert CRAM to fastq in a specified region.
"""
ref_file = tz.get_in(["reference", "fasta", "base"], data)
resources = config_utils.get_resources("bamtofastq", data["config"])
cores = tz.get_in(["config", "algorithm", "num_cores"], data, 1)
max_mem = config_utils.convert_to_bytes(resources.get("memory",
"1G")) * cores
rext = "-%s" % region.replace(":", "_").replace("-",
"_") if region else "full"
out_s, out_p1, out_p2, out_o1, out_o2 = [
os.path.join(work_dir, "%s%s-%s.fq.gz" % (base_name, rext, fext))
for fext in ["s1", "p1", "p2", "o1", "o2"]
]
if not utils.file_exists(out_p1):
with file_transaction(data, out_s, out_p1, out_p2, out_o1, out_o2) as \
(tx_out_s, tx_out_p1, tx_out_p2, tx_out_o1, tx_out_o2):
cram_file = objectstore.cl_input(cram_file)
sortprefix = "%s-sort" % utils.splitext_plus(tx_out_s)[0]
cmd = (
"bamtofastq filename={cram_file} inputformat=cram T={sortprefix} "
"gz=1 collate=1 colsbs={max_mem} exclude=SECONDARY,SUPPLEMENTARY "
"F={tx_out_p1} F2={tx_out_p2} S={tx_out_s} O={tx_out_o1} O2={tx_out_o2} "
"reference={ref_file}")
if region:
cmd += " ranges='{region}'"
do.run(cmd.format(**locals()),
"CRAM to fastq %s" % region if region else "")
return [[out_p1, out_p2, out_s]]
def align(fastq_file, pair_file, ref_file, names, align_dir, data):
assert data["analysis"].lower().startswith("wgbs-seq"), "No comparible alignment."
config = data["config"]
sample = dd.get_sample_name(data)
out_prefix = os.path.join(align_dir, dd.get_lane(data))
out_dir = os.path.join(align_dir, "%s_bismark" % dd.get_lane(data))
if not ref_file:
logger.error("bismark index not found. You can install "
"the index for your genome with: bcbio_nextgen.py upgrade "
"--aligners bismark --genomes genome-build-name --data")
sys.exit(1)
final_out = os.path.join(align_dir, "{0}.bam".format(sample))
if file_exists(final_out):
data = dd.set_work_bam(data, final_out)
data["bam_report"] = glob.glob(os.path.join(out_dir, "*report.txt"))[0]
data = dd.update_summary_qc(data, "bismark", base=data["bam_report"])
return data
bismark = config_utils.get_program("bismark", config)
# bismark uses 5 threads/sample and ~12GB RAM/sample (hg38)
resources = config_utils.get_resources("bismark", data["config"])
max_cores = dd.get_num_cores(data)
max_mem = config_utils.convert_to_bytes(resources.get("memory", "1G")) / (1024.0 * 1024.0)
instances = calculate_bismark_instances(max_cores, max_mem * max_cores)
# override instances if specified in the config
if resources and resources.get("bismark_threads"):
instances = resources.get("bismark_threads")
logger.info(f"Using {instances} bismark instances - overriden by resources")
bowtie_threads = 1
if resources and resources.get("bowtie_threads"):
bowtie_threads = resources.get("bowtie_threads")
logger.info(f"Using {bowtie_threads} bowtie threads per bismark instance")
kit = kits.KITS.get(dd.get_kit(data), None)
directional = "--non_directional" if kit and not kit.is_directional else ""
other_opts = resources.get("options", [])
other_opts = " ".join([str(x) for x in other_opts]).strip()
fastq_files = " ".join([fastq_file, pair_file]) if pair_file else fastq_file
safe_makedir(align_dir)
cmd = "{bismark} {other_opts} {directional} --bowtie2 --temp_dir {tx_out_dir} --gzip --parallel {instances} -p {bowtie_threads} -o {tx_out_dir} --unmapped {ref_file} {fastq_file} "
if pair_file:
fastq_file = "-1 %s -2 %s" % (fastq_file, pair_file)
raw_bam = glob.glob(out_dir + "/*bismark*bt2*bam")
if not raw_bam:
with tx_tmpdir() as tx_out_dir:
run_message = "Running Bismark aligner on %s and %s" % (fastq_file, ref_file)
do.run(cmd.format(**locals()), run_message, None)
shutil.move(tx_out_dir, out_dir)
raw_bam = glob.glob(out_dir + "/*bismark*bt2*bam")
# don't process bam in the bismark pipeline!
utils.symlink_plus(raw_bam[0], final_out)
data = dd.set_work_bam(data, final_out)
data["bam_report"] = glob.glob(os.path.join(out_dir, "*report.txt"))[0]
data = dd.update_summary_qc(data, "bismark", base=data["bam_report"])
return data
def _bgzip_from_bam(bam_file, dirs, config, is_retry=False, output_infix=''):
"""Create bgzipped fastq files from an input BAM file.
"""
# tools
bamtofastq = config_utils.get_program("bamtofastq", config)
resources = config_utils.get_resources("bamtofastq", config)
cores = config["algorithm"].get("num_cores", 1)
max_mem = config_utils.convert_to_bytes(resources.get("memory",
"1G")) * cores
bgzip = tools.get_bgzip_cmd(config, is_retry)
# files
work_dir = utils.safe_makedir(os.path.join(dirs["work"], "align_prep"))
out_file_1 = os.path.join(
work_dir, "%s%s-1.fq.gz" %
(os.path.splitext(os.path.basename(bam_file))[0], output_infix))
out_file_2 = out_file_1.replace("-1.fq.gz", "-2.fq.gz")
needs_retry = False
if is_retry or not utils.file_exists(out_file_1):
if not bam.is_paired(bam_file):
out_file_2 = None
with file_transaction(config, out_file_1) as tx_out_file:
for f in [tx_out_file, out_file_1, out_file_2]:
if f and os.path.exists(f):
os.remove(f)
fq1_bgzip_cmd = "%s -c /dev/stdin > %s" % (bgzip, tx_out_file)
sortprefix = "%s-sort" % os.path.splitext(tx_out_file)[0]
if bam.is_paired(bam_file):
fq2_bgzip_cmd = "%s -c /dev/stdin > %s" % (bgzip, out_file_2)
out_str = (
"F=>({fq1_bgzip_cmd}) F2=>({fq2_bgzip_cmd}) S=/dev/null O=/dev/null "
"O2=/dev/null collate=1 colsbs={max_mem}")
else:
out_str = "S=>({fq1_bgzip_cmd})"
bam_file = objectstore.cl_input(bam_file)
cmd = "{bamtofastq} filename={bam_file} T={sortprefix} " + out_str
try:
do.run(cmd.format(**locals()),
"BAM to bgzipped fastq",
checks=[do.file_reasonable_size(tx_out_file, bam_file)],
log_error=False)
except subprocess.CalledProcessError as msg:
if not is_retry and "deflate failed" in str(msg):
logger.info(
"bamtofastq deflate IO failure preparing %s. Retrying with single core."
% (bam_file))
needs_retry = True
else:
logger.exception()
raise
if needs_retry:
return _bgzip_from_bam(bam_file, dirs, config, is_retry=True)
else:
return [
x for x in [out_file_1, out_file_2]
if x is not None and utils.file_exists(x)
]
def _bgzip_from_bam(bam_file, dirs, data, is_retry=False, output_infix=''):
"""Create bgzipped fastq files from an input BAM file.
"""
# tools
config = data["config"]
bamtofastq = config_utils.get_program("bamtofastq", config)
resources = config_utils.get_resources("bamtofastq", config)
cores = config["algorithm"].get("num_cores", 1)
max_mem = config_utils.convert_to_bytes(resources.get("memory", "1G")) * cores
bgzip = tools.get_bgzip_cmd(config, is_retry)
# files
work_dir = utils.safe_makedir(os.path.join(dirs["work"], "align_prep"))
out_file_1 = os.path.join(work_dir, "%s%s-1.fq.gz" % (os.path.splitext(os.path.basename(bam_file))[0], output_infix))
out_file_2 = out_file_1.replace("-1.fq.gz", "-2.fq.gz")
needs_retry = False
if is_retry or not utils.file_exists(out_file_1):
if not bam.is_paired(bam_file):
out_file_2 = None
with file_transaction(config, out_file_1) as tx_out_file:
for f in [tx_out_file, out_file_1, out_file_2]:
if f and os.path.exists(f):
os.remove(f)
fq1_bgzip_cmd = "%s -c /dev/stdin > %s" % (bgzip, tx_out_file)
prep_cmd = _seqtk_fastq_prep_cl(data, read_num=0)
if prep_cmd:
fq1_bgzip_cmd = prep_cmd + " | " + fq1_bgzip_cmd
sortprefix = "%s-sort" % os.path.splitext(tx_out_file)[0]
if bam.is_paired(bam_file):
prep_cmd = _seqtk_fastq_prep_cl(data, read_num=1)
fq2_bgzip_cmd = "%s -c /dev/stdin > %s" % (bgzip, out_file_2)
if prep_cmd:
fq2_bgzip_cmd = prep_cmd + " | " + fq2_bgzip_cmd
out_str = ("F=>({fq1_bgzip_cmd}) F2=>({fq2_bgzip_cmd}) S=/dev/null O=/dev/null "
"O2=/dev/null collate=1 colsbs={max_mem}")
else:
out_str = "S=>({fq1_bgzip_cmd})"
bam_file = objectstore.cl_input(bam_file)
extra_opts = " ".join([str(x) for x in resources.get("options", [])])
cmd = "{bamtofastq} filename={bam_file} T={sortprefix} {extra_opts} " + out_str
try:
do.run(cmd.format(**locals()), "BAM to bgzipped fastq",
checks=[do.file_reasonable_size(tx_out_file, bam_file)],
log_error=False)
except subprocess.CalledProcessError as msg:
if not is_retry and "deflate failed" in str(msg):
logger.info("bamtofastq deflate IO failure preparing %s. Retrying with single core."
% (bam_file))
needs_retry = True
else:
logger.exception()
raise
if needs_retry:
return _bgzip_from_bam(bam_file, dirs, data, is_retry=True)
else:
return [x for x in [out_file_1, out_file_2] if x is not None and utils.file_exists(x)]
def _cram_to_fastq_region(cram_file, work_dir, base_name, region, data):
"""Convert CRAM to fastq in a specified region.
"""
ref_file = tz.get_in(["reference", "fasta", "base"], data)
resources = config_utils.get_resources("bamtofastq", data["config"])
cores = tz.get_in(["config", "algorithm", "num_cores"], data, 1)
max_mem = config_utils.convert_to_bytes(resources.get("memory", "1G")) * cores
rext = "-%s" % region.replace(":", "_").replace("-", "_") if region else "full"
out_s, out_p1, out_p2, out_o1, out_o2 = [os.path.join(work_dir, "%s%s-%s.fq.gz" %
(base_name, rext, fext))
for fext in ["s1", "p1", "p2", "o1", "o2"]]
if not utils.file_exists(out_p1):
with file_transaction(data, out_s, out_p1, out_p2, out_o1, out_o2) as \
(tx_out_s, tx_out_p1, tx_out_p2, tx_out_o1, tx_out_o2):
cram_file = objectstore.cl_input(cram_file)
sortprefix = "%s-sort" % utils.splitext_plus(tx_out_s)[0]
cmd = ("bamtofastq filename={cram_file} inputformat=cram T={sortprefix} "
"gz=1 collate=1 colsbs={max_mem} exclude=SECONDARY,SUPPLEMENTARY "
"F={tx_out_p1} F2={tx_out_p2} S={tx_out_s} O={tx_out_o1} O2={tx_out_o2} "
"reference={ref_file}")
if region:
cmd += " ranges='{region}'"
do.run(cmd.format(**locals()), "CRAM to fastq %s" % region if region else "")
return [[out_p1, out_p2, out_s]]
