def sortIndexBAMs(self, path_to_exe=False, force=False, max_cpus=-1):
if not path_to_exe:
path_to_exe = _get_exe_path('samtools')
processes = set()
max_processes = _decide_max_processes(max_cpus)
paths_to_BAMs_dd_si = []
for SAM in self.paths_to_BAMs_dd:
BAM_out = SAM[:-4] + '_si.bam'
if not _os.path.exists(BAM_out) or force:
cmd = '{0} sort {1} {2}_si; {0} index {2}_si.bam'.format(
path_to_exe, SAM, SAM[:-4])
print('Called: %s' % cmd)
processes.add(_subprocess.Popen(cmd, shell=True))
if len(processes) >= max_processes:
(pid, exit_status) = _os.wait()
processes.difference_update(
[p for p in processes if p.poll() is not None])
else:
print('Found:')
print(BAM_out)
print('use "force = True" to overwrite')
paths_to_BAMs_dd_si += [BAM_out]
# Check if all the child processes were closed
for p in processes:
if p.poll() is None:
p.wait()
self.paths_to_BAMs_dd_si = paths_to_BAMs_dd_si
def removeDuplicates(self,
path_to_jar=False,
force=False,
mem_num_gigs=2,
max_cpus=-1):
if not path_to_jar:
path_to_jar = _get_jar_path('picard')
processes = set()
max_processes = _decide_max_processes(max_cpus)
print('Print: will use %s cpus for picard' % max_processes)
paths_to_BAMs_dd = []
for BAM in self.paths_to_BAMs:
BAM_out = BAM[:-4] + '_dd.bam'
if not _os.path.exists(BAM_out) or force:
# an alternative parallel code with polling not waiting
# while len(processes) >= max_processes:
# _sleep(1)
# print('processes: %s' % (len(processes)))
# processes.difference_update(
# [p for p in processes if p.poll() is not None]
# )
picard_command = [
'MarkDuplicates', 'I=', BAM, 'O=', BAM_out, 'M=',
BAM[:-4] + '_dd.log'
] #, 'VALIDATION_STRINGENCY=','LENIENT']
cmd = ['java',
'-Xmx%sg' % mem_num_gigs, '-jar', path_to_jar
] + picard_command
processes.add(_subprocess.Popen(cmd, shell=False))
print('Called: %s' % (' '.join(map(str, cmd))))
#_subprocess.call(cmd, shell=False)
print('processes: %s, max_processes: %s' %
(len(processes), max_processes))
if len(processes) >= max_processes:
(pid, exit_status) = _os.wait()
processes.difference_update(
[p for p in processes if p.poll() is not None])
else:
print('Found:')
print(BAM_out)
print('use "force = True" to overwrite')
paths_to_BAMs_dd += [BAM_out]
# Check if all the child processes were closed
for p in processes:
if p.poll() is None:
p.wait()
self.paths_to_BAMs_dd = paths_to_BAMs_dd
def run_SPAdes(cmd):
proc = _subprocess.Popen(cmd,
stdout=_subprocess.PIPE,
stderr=_subprocess.PIPE)
# allow for failed SPAdes runs (possibly caused by small fastq files) <== but also check they were actually built properly
try:
stdout_value, stderr_value = proc.communicate()
checkthese = []
getline = False
for line in stdout_value.split('\n'):
if 'Warnings saved to' in line:
getline = False
if getline:
l = line.rstrip()
if len(l):
checkthese += [l]
if 'SPAdes pipeline finished WITH WARNINGS!' in line:
getline = True
if len(checkthese):
print('SPAdes completed with warnings:\n{}\n'.format(
'\n'.join(checkthese)))
else:
print('SPAdes completed without warnings')
# with open('___SPAdes_{}_good_{}.log'.format(cnum, thetime), 'w') as fout:
# fout.write(stdout_value)
path2contigs = _os.path.sep.join(
[this_output_path, 'contigs.fasta'])
except _subprocess.CalledProcessError as e:
print('SPAdes probably did not complete: error returned ({})'.
format(proc.returncode))
print('Error: {}'.format(e))
print(
'Writing some info relevent to SPAdes crash to ___SPAdes_{}_bad_{}.log'
.format(cnum, thetime))
with open('___SPAdes_{}_bad_{}.log'.format(cnum, thetime),
'w') as fout:
fout.write(dir(proc))
fout.write('\n' + str(e.returncode) + '\n')
fout.write(
_os.path.sep.join([this_output_path, 'contigs.fasta']))
path2contigs = None
return (path2contigs)
def check_no_error(path='',
system=False,
java_commands=False,
extra=False,
extra_pre=False,
success_returncode=1):
'''
check a binary executable can be called.
A failure is considered as OSError, not a non-zero exit status.
Can check in default baga location or a specified path or the system path
or java.
Additional commands and be appended with 'extra' list or pre-pended with
'extra-pre' list e.g., specifying 'python2' for when python3 defaults but
python >3.3 is installed but not supported.
'''
if java_commands:
p = _subprocess.Popen(['java'] + java_commands,
stdout=_subprocess.PIPE,
stderr=_subprocess.STDOUT)
output = p.stdout.read()
if 'Error:' in output:
print(output)
return (False)
else:
print(output)
return (True)
else:
if system:
cmd = path[-1:]
elif path[0][0] == _os.path.sep or path[0] == '':
# absolute path to elsewhere provided
cmd = [_os.path.sep.join(path)]
else:
cmd = [_os.path.sep.join(['external_programs'] + path)]
if extra:
cmd += extra
if extra_pre:
cmd = extra_pre + cmd
try:
o = _subprocess.check_output(cmd)
o = '\n'.join(['external> ' + l for l in o.split('\n')]) + '\n'
print(o) # eventually put in the debugging log <== too much noise
# some programs output a help screen if commands not given
# . . . they are installed and available
return (True)
except _subprocess.CalledProcessError as e:
if e.returncode == success_returncode:
# some programs that exit return non-zero code if commands not given
# usually this is 1, but occasionally it is 2 (e.g. cutadapt)
# but a fail on 2 is e.g. python calling a non-existent python program
# should be set in the dependencies dictionary if not 1 (default)
# . . . but they are installed and available
return (True)
else:
return (False)
except OSError as e:
print('{}: {}'.format(cmd[0], e))
return (False)
def trim(self, path_to_exe=False, force=False, max_cpus=-1):
if not path_to_exe:
exe_sickle = _get_exe_path('sickle')
else:
exe_sickle = _os.path.sep.join(path_to_exe)
e1 = 'Could not find "adaptorcut_read_files" attribute. \
Before quality score trimming, reads must be cleaned of \
library preparation sequences. Please run cutAdaptors() \
method on this Reads instance.'
assert hasattr(self, 'adaptorcut_read_files'), e1
e2 = 'Could not find %s. Either run cutAdaptors() again \
or ensure file exists'
for pairname, files in self.adaptorcut_read_files.items():
assert _os.path.exists(files[1]), e2 % files[1]
assert _os.path.exists(files[1]), e2 % files[1]
trimmed_read_files = {}
print(sorted(self.adaptorcut_read_files))
cmds = []
processed_paths_to_do = []
for pairname, files in self.adaptorcut_read_files.items():
processed_path_1 = insert_suffix(files[1], '_qual')
processed_path_2 = insert_suffix(files[2], '_qual')
processed_path_s = insert_suffix(files[2], '_singletons_qual')
# Illumina quality using CASAVA >= 1.8 is Sanger encoded
QSscore_scale = 'sanger'
cmd = [
exe_sickle,
'pe',
'-f',
files[1],
'-r',
files[2],
'-t',
QSscore_scale,
'-o',
processed_path_1,
'-p',
processed_path_2,
'-s',
processed_path_s,
# quality 25, length 50 (of 150)
'-q',
'25',
'-l',
'50'
]
if not all([_os.path.exists(processed_path_1),
_os.path.exists(processed_path_2),
_os.path.exists(processed_path_s)]) \
or force:
# collect expected outputs
processed_paths_to_do += [(processed_path_1, processed_path_2,
processed_path_s)]
# collect all the commands to be issued
cmds += [(pairname, cmd)]
else:
print('Found:')
print(processed_path_1)
print(processed_path_2)
print(processed_path_s)
print('use "force = True" to overwrite')
trimmed_read_files[pairname] = {}
trimmed_read_files[pairname][1] = processed_path_1
trimmed_read_files[pairname][2] = processed_path_2
if len(cmds):
max_processes = _decide_max_processes(max_cpus)
processes = {}
### how to combine this which hangs on _os.wait()
for pairname, cmd in cmds:
print('Called: "%s"' % ' '.join(cmd))
# process is key, open file being piped to is value
# baga CollectReads currently includes path in pairname
this_stdout_file = open(pairname + '_sickle.log', "w")
thisprocess = _subprocess.Popen(cmd,
shell=False,
stdout=this_stdout_file)
processes[thisprocess] = this_stdout_file
if len(processes) >= max_processes:
_os.wait()
finished = dict([(p, f) for p, f in processes.items()
if p.poll() is not None])
# close files for finished processes
for process, stdout_file in finished.items():
stdout_file.close()
# update active processes
del processes[process]
# Check if all the child processes were closed
for p in processes:
if p.poll() is None:
p.wait()
fails = []
for (pairname, cmd), (processed_path_1, processed_path_2,
processed_path_s) in zip(cmds,
processed_paths_to_do):
if _os.path.exists(processed_path_1) and _os.path.exists(
processed_path_2):
print('Found:')
print(processed_path_1)
print(processed_path_2)
trimmed_read_files[pairname] = {}
trimmed_read_files[pairname][1] = processed_path_1
trimmed_read_files[pairname][2] = processed_path_2
else:
print('Processing of the following pair seems to have failed')
print(processed_path_1)
print(processed_path_2)
fails += [(processed_path_1, processed_path_2)]
assert len(
fails
) == 0, 'There was a problem finding all of the output from sickle. Try repeating this or an earlier step with the --force option to overwite previous, possibly incomplete, files'
self.trimmed_read_files = trimmed_read_files
def cutAdaptors(self, path_to_exe=False, force=False, max_cpus=-1):
if not path_to_exe:
path_to_exe = _get_exe_path('cutadapt')
adaptorcut_read_files = {}
adaptor_seqs = [
'AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC',
'AGATCGGAAGAGCACACGTCT',
'AGATCGGAAGAGC',
'GATCGGAAGAGCGGTTCAGCAGGAATGCCGAG',
'ACACTCTTTCCCTACACGACGCTCTTCCGATCT',
]
cmds = []
processed_paths_to_do = []
for cnum, (pairname, files) in enumerate(self.read_files.items()):
processed_path_1 = insert_suffix(files[1], '_adpt')
processed_path_2 = insert_suffix(files[2], '_adpt')
# print(files[1], processed_path_1)
# print(files[2], processed_path_2)
# single end
cmd = [path_to_exe] + \
[a for b in [('-a', a) for a in adaptor_seqs] for a in b] + \
['-o', processed_path_1, files[1]]
# paired end
cmd = [path_to_exe] + \
[a for b in [('-a', a) for a in adaptor_seqs] for a in b] + \
[a for b in [('-A', a) for a in adaptor_seqs] for a in b] + \
['-o', processed_path_1, '-p', processed_path_2] + \
[files[1], files[2]]
if not all([_os.path.exists(processed_path_1),
_os.path.exists(processed_path_2)]) \
or force:
# collect expected outputs
processed_paths_to_do += [(processed_path_1, processed_path_2)]
# collect all the commands to be issued
cmds += [(pairname, cmd)]
else:
print('Found:')
print(processed_path_1)
print(processed_path_2)
print('use "force = True" to overwrite')
adaptorcut_read_files[pairname] = {}
adaptorcut_read_files[pairname][1] = processed_path_1
adaptorcut_read_files[pairname][2] = processed_path_2
if len(cmds):
max_processes = _decide_max_processes(max_cpus)
processes = {}
### how to combine this which hangs on _os.wait()
for pairname, cmd in cmds:
print('Called: "%s"' % ' '.join(cmd))
# process is key, open file being piped to is value
# baga CollectReads currently includes path in pairname
this_stdout_file = open(pairname + '_cutadapt.log', "w")
thisprocess = _subprocess.Popen(cmd,
shell=False,
stdout=this_stdout_file)
processes[thisprocess] = this_stdout_file
if len(processes) >= max_processes:
_os.wait()
finished = dict([(p, f) for p, f in processes.items()
if p.poll() is not None])
# close files for finished processes
for process, stdout_file in finished.items():
stdout_file.close()
# update active processes
del processes[process]
# Check if all the child processes were closed
for p in processes:
if p.poll() is None:
p.wait()
fails = []
for (pairname, cmd), (processed_path_1,
processed_path_2) in zip(cmds,
processed_paths_to_do):
if _os.path.exists(processed_path_1) and _os.path.exists(
processed_path_2):
print('Found:')
print(processed_path_1)
print(processed_path_2)
adaptorcut_read_files[pairname] = {}
adaptorcut_read_files[pairname][1] = processed_path_1
adaptorcut_read_files[pairname][2] = processed_path_2
else:
print('Processing of the following pair seems to have failed')
print(processed_path_1)
print(processed_path_2)
fails += [(processed_path_1, processed_path_2)]
assert len(
fails
) == 0, 'There was a problem finding all of the output from cutadapt. Try repeating this or an eralier step with the --force option to overwite previous, possibly incomplete, files'
self.adaptorcut_read_files = adaptorcut_read_files
def IndelRealignGATK(self,
jar=[
'external_programs', 'GenomeAnalysisTK',
'GenomeAnalysisTK.jar'
],
picard_jar=False,
samtools_exe=False,
use_java='java',
force=False,
mem_num_gigs=2,
max_cpus=-1):
# GATK is manually downloaded by user and placed in folder of their choice
jar = _os.path.sep.join(jar)
if not picard_jar:
picard_jar = _get_jar_path('picard')
if not samtools_exe:
samtools_exe = _get_exe_path('samtools')
genome_fna = 'genome_sequences/{}.fna'.format(self.genome_id)
e1 = 'Could not find "paths_to_BAMs_dd_si" attribute. Before starting GATK analysis, read alignments must have duplicates removed. Please run: .toBAMS(), .removeDuplicates(), .sortIndexBAMs() methods on this SAMs instance, or --deduplicate if using baga_cli.py.'
assert hasattr(self, 'paths_to_BAMs_dd_si'), e1
e2 = 'Could not find %s. Please ensure file exists'
for BAM in self.paths_to_BAMs_dd_si:
assert _os.path.exists(BAM), e2 % BAM
# always (re)generate dict in case of upstream changes in data
print('Creating sequence dictionary for %s' % genome_fna)
_subprocess.call([
use_java, '-jar', picard_jar, 'CreateSequenceDictionary', 'R=',
genome_fna, 'O=', genome_fna[:-4] + '.dict'
])
# always (re)index in case of upstream changes in data
print('Writing index files for %s' % genome_fna)
_subprocess.call([samtools_exe, 'faidx', genome_fna])
processes = set()
max_processes = _decide_max_processes(max_cpus)
for BAM in self.paths_to_BAMs_dd_si:
intervals = BAM[:-4] + '.intervals'
if not _os.path.exists(intervals) or force:
cmd = [
use_java,
'-Xmx%sg' % mem_num_gigs, '-jar', jar, '-T',
'RealignerTargetCreator', '-R', genome_fna, '-I', BAM,
'-o', intervals
] #, '--validation_strictness', 'LENIENT']
print(' '.join(map(str, cmd)))
processes.add(_subprocess.Popen(cmd, shell=False))
if len(processes) >= max_processes:
(pid, exit_status) = _os.wait()
processes.difference_update(
[p for p in processes if p.poll() is not None])
else:
print('Found:')
print(intervals)
print('use "force = True" to overwrite')
# Check if all the child processes were closed
for p in processes:
if p.poll() is None:
p.wait()
paths_to_BAMs_dd_si_ra = []
for BAM in self.paths_to_BAMs_dd_si:
intervals = BAM[:-4] + '.intervals'
bam_out = BAM[:-4] + '_realn.bam'
if not _os.path.exists(bam_out) or force:
cmd = [
use_java, '-Xmx4g', '-jar', jar, '-T', 'IndelRealigner',
'-R', genome_fna, '-I', BAM, '-targetIntervals', intervals,
'-o', bam_out, '--filter_bases_not_stored'
]
print(' '.join(map(str, cmd)))
processes.add(_subprocess.Popen(cmd, shell=False))
if len(processes) >= max_processes:
_os.wait()
processes.difference_update(
[p for p in processes if p.poll() is not None])
else:
print('Found:')
print(bam_out)
print('use "force = True" to overwrite')
paths_to_BAMs_dd_si_ra += [bam_out]
for p in processes:
if p.poll() is None:
p.wait()
# the last list of BAMs in ready_BAMs is input for CallgVCFsGATK
# both IndelRealignGATK and recalibBaseScoresGATK put here
self.ready_BAMs = [paths_to_BAMs_dd_si_ra]
def generateReads(self,
path_to_exe=False,
paths_to_genomes=False,
readcov=60,
readlen=100,
fraglen=350,
sterrfraglen=20,
model=4,
max_cpus=-1):
'''
Call GemSIM to generate reads
Need to have written genome sequences to generate from, possibly with
generated SNPs, small indels and large deletions.
'''
#max_cpus etc
if paths_to_genomes:
use_genomes = sorted(paths_to_genomes)
elif hasattr(self, 'written_genomes'):
use_genomes = sorted(self.written_genomes)
else:
raise ValueError(
'provide either paths_to_genomes or generate some then .writeSequences()'
)
if not path_to_exe:
path_to_exe = _get_exe_path('gemsim')
comment2 = '''
to generate reads put GemSIM v1.6 into subfolder GemSIM_v1.6 and issue these commands:
GemSIM_v1.6/GemReads.py -r LESB58_for_GemSim_01.fasta -n 1980527 -l d -u 350 -s 20 -m GemSIM_v1.6/models/ill100v4_p.gzip -c -q 33 -p -o GemSimLESB58_01
'''
num_pairs = len(self.genome.sequence) * readcov / (readlen * 2)
if model == 4:
path_to_model = _os.path.sep.join(
path_to_exe.split(_os.path.sep)[:-1] +
['models', 'ill100v4_p.gzip'])
elif model == 5:
path_to_model = _os.path.sep.join(
path_to_exe.split(_os.path.sep)[:-1] +
['models', 'ill100v5_p.gzip'])
print('Using error model: {}'.format(path_to_model))
print(
'Generating {:,} {}bp read pairs for {}x coverage depth of a {}bp genome ({})'
.format(num_pairs, readlen, readcov, len(self.genome.sequence),
self.genome.id))
processes = set()
max_processes = _decide_max_processes(max_cpus)
import time
start = time.time()
out_raw = []
for i, genome_in in enumerate(use_genomes):
# could use per genome length . . less consistent than using reference
# genome_len = len(_SeqIO.read(genome_in,'fasta').seq)
# num_pairs = genome_len * readcov / (readlen*2)
outprefix = 'GemSim_{}_{:02d}'.format(self.genome.id, i + 1)
cmd = [
path_to_exe, '-r', genome_in, '-n', num_pairs, '-l', 'd', '-u',
fraglen, '-s', sterrfraglen, '-m', path_to_model, '-c', '-q',
33, '-p', '-o', outprefix
]
out_raw += [outprefix + '_fir.fastq', outprefix + '_sec.fastq']
# this would be better to rename and compress all in one
# maybe as a shell script? Then resuming (--force) would be easier.
if _os.path.exists(outprefix+'_fir.fastq') and \
_os.path.exists(outprefix+'_sec.fastq'):
print('Found output for {}_fir.fastq (and sec), not regenerating, '\
'delete these to start from scratch'.format(outprefix))
else:
cmd = map(str, cmd)
print(' '.join(cmd))
processes.add(_subprocess.Popen(cmd, shell=False))
if len(processes) >= max_processes:
(pid, exit_status) = _os.wait()
processes.difference_update(
[p for p in processes if p.poll() is not None])
# Check if all the child processes were closed
for p in processes:
if p.poll() is None:
p.wait()
missing = []
for o in out_raw:
if not _os.path.exists(o):
missing += [o]
assert len(missing) == 0, 'Could not find:\n{}'.format(
'\n'.join(missing))
print('all finished after {} minutes'.format(
int(round((time.time() - start) / 60.0))))
outdir = _os.path.sep.join(['simulated_reads', self.genome.id])
try:
_os.makedirs(outdir)
except OSError:
pass
for o in out_raw:
new = _os.path.sep.join(
[outdir, o.replace('fir', 'R1').replace('sec', 'R2')])
print('{} ==> {}'.format(o, new))
_os.rename(o, new)
cmd = ['gzip', new]
print(' '.join(cmd))
_subprocess.call(cmd)
