def trimmomatic(jar_path_trimmomatic, sampleName, trimmomatic_folder, threads, adaptersFasta, script_path, doNotSearchAdapters, fastq_files, maxReadsLength, doNotTrimCrops, crop, headCrop, leading, trailing, slidingWindow, minLength, nts2clip_based_ntsContent, jarMaxMemory, fastq_encoding):
trimmomatic_out_files = []
for fastq in fastq_files:
trimmomatic_out_files.append(os.path.join(trimmomatic_folder, str(os.path.splitext(os.path.splitext(os.path.basename(fastq))[0])[0] + 'P.fastq.gz')))
trimmomatic_out_files.append(os.path.join(trimmomatic_folder, str(os.path.splitext(os.path.splitext(os.path.basename(fastq))[0])[0] + 'U.fastq.gz')))
# Run Trimmomatic
command = ['java', '', '-jar', jar_path_trimmomatic, 'PE', '-threads', str(threads), '', ' '.join(fastq_files), ' '.join(trimmomatic_out_files), '', '', '', str('SLIDINGWINDOW:' + slidingWindow), str('LEADING:' + str(leading)), str('TRAILING:' + str(trailing)), str('MINLEN:' + str(minLength)), 'TOPHRED33']
if str(jarMaxMemory) != 'off':
command[1] = '-Xmx' + str(int(round(jarMaxMemory * 1024, 0))) + 'M'
if not doNotTrimCrops:
if maxReadsLength is not None:
if crop is not None:
crop = maxReadsLength - crop[0]
command[10] = str('CROP:' + str(crop))
else:
if nts2clip_based_ntsContent is not None:
crop = nts2clip_based_ntsContent[1]
print str(crop) + ' nucleotides will be clipped at the end of reads'
crop = maxReadsLength - crop
command[10] = str('CROP:' + str(crop))
else:
print 'Because FastQC did not run successfully, --trimCrop option will not be considered'
if headCrop is not None:
command[11] = str('HEADCROP:' + str(headCrop[0]))
else:
if nts2clip_based_ntsContent is not None:
headCrop = nts2clip_based_ntsContent[0]
print str(headCrop) + ' nucleotides will be clipped at the beginning of reads'
command[11] = str('HEADCROP:' + str(headCrop))
if not doNotSearchAdapters:
if adaptersFasta is not None:
print 'Removing adapters contamination using ' + adaptersFasta
command[12] = 'ILLUMINACLIP:' + adaptersFasta + ':3:30:10:6:true'
else:
trimmomatic_adapters_folder = os.path.join(os.path.dirname(script_path), 'src', 'Trimmomatic-0.36', 'adapters')
adapters_files = [os.path.join(trimmomatic_adapters_folder, 'Nextera_XT_INNUca.fasta'), os.path.join(trimmomatic_adapters_folder, 'NexteraPE-PE.fa'), os.path.join(trimmomatic_adapters_folder, 'TruSeq2-PE.fa'), os.path.join(trimmomatic_adapters_folder, 'TruSeq3-PE-2.fa')]
print 'Removing adapters contamination using ' + str(adapters_files)
adaptersFasta = concatenateFastaFiles(adapters_files, trimmomatic_folder, 'concatenated_adaptersFile.fasta')
command[12] = 'ILLUMINACLIP:' + adaptersFasta + ':3:30:10:6:true'
if fastq_encoding is not None:
if fastq_encoding == 33:
command[7] = '-phred33'
elif fastq_encoding == 64:
command[7] = '-phred64'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
else:
print 'Trimmomatic fail! Trying run with Phred+33 enconding defined...'
command[7] = '-phred33'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
print 'Trimmomatic fail again! Trying run with Phred+64 enconding defined...'
command[7] = '-phred64'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
return run_successfully
def indexSequenceBowtie2(referenceFile, threads):
if os.path.isfile(str(referenceFile + '.1.bt2')):
run_successfully = True
else:
command = ['bowtie2-build', '--threads', str(threads), referenceFile, referenceFile]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
return run_successfully
def compress_decompress(compressed_file, decompressed_file, compressed_True):
run_successfully = False
malformated_fastq = False
length_sequence = None
compression_type = None
if not compressed_True:
compression_type = utils.compressionType(compressed_file)
if compression_type is not None or compressed_True:
command = ['', '', '--stdout', '--keep', '', '>', '']
if not compressed_True:
command[0] = compression_type[0]
command[1] = '--decompress'
command[4] = compressed_file
command[6] = decompressed_file
else:
command[0] = 'gzip'
command[4] = decompressed_file
command[6] = compressed_file
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, True)
if run_successfully and not compressed_True:
malformated_fastq, length_sequence = check_uncompression_fastq(decompressed_file)
elif compression_type is None and not compressed_True:
run_successfully = True
malformated_fastq, length_sequence = check_uncompression_fastq(compressed_file)
decompressed_file = compressed_file
if malformated_fastq:
run_successfully = False
utils.saveVariableToPickle([run_successfully, compressed_file if compressed_True else decompressed_file, length_sequence], os.path.dirname(decompressed_file), os.path.splitext(os.path.basename(decompressed_file))[0])
def pilon(jar_path_pilon, assembly, bam_file, outdir, jarMaxMemory):
assembly_polished = os.path.splitext(assembly)[0] + '.polished.fasta'
command = ['java', '', '-jar', jar_path_pilon, '--genome', assembly, '--frags', bam_file, '--outdir', outdir, '--output', os.path.basename(os.path.splitext(assembly_polished)[0]), '--changes', '--vcf']
if str(jarMaxMemory) != 'off':
command[1] = '-Xmx' + str(int(round(jarMaxMemory * 1024, 0))) + 'M'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
assembly_polished = None
return run_successfully, assembly_polished
def sortAlignment(alignment_file, output_file, sortByName_True, threads):
outFormat_string = os.path.splitext(output_file)[1][1:].lower()
command = ['samtools', 'sort', '-o', output_file, '-O', outFormat_string, '', '-@', str(threads), alignment_file]
if sortByName_True:
command[6] = '-n'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
output_file = None
return run_successfully, output_file
def runMlst(contigs, scheme, outdir, species_genus, mlst_scheme_genus):
pass_qc = False
failing = {}
failing['sample'] = False
warnings = {}
novel_alleles = os.path.join(outdir, 'mlst_novel_alleles.fasta')
command = ['mlst', '--novel', novel_alleles, contigs]
run_successfully, stdout, _ = utils.runCommandPopenCommunicate(command, False, None, True)
if run_successfully:
scheme_mlst = stdout.splitlines()[0].split('\t')[1].split('_')[0]
st = stdout.splitlines()[0].split('\t')[2]
profile = stdout.splitlines()[0].split('\t')[3:]
if st == '-':
clean_novel_alleles(novel_alleles=novel_alleles, scheme_mlst=scheme_mlst, profile=profile)
else:
if os.path.isfile(novel_alleles):
os.remove(novel_alleles)
report = 'MLST found ST ' + str(st) + ' from scheme ' + scheme_mlst
print(report)
with open(os.path.join(outdir, 'mlst_report.txt'), 'wt') as writer:
writer.write('#scheme' + '\n' + scheme_mlst + '\n' + '#ST' + '\n' + st + '\n')
writer.write('#profile' + '\n' + ' '.join(profile) + '\n')
writer.flush()
if scheme_mlst.split('_', 1)[0] == scheme.split('_', 1)[0]:
pass_qc = True
else:
if scheme == 'unknown' and scheme_mlst != '-':
pass_qc = True
warnings['sample'] = 'Found {scheme_mlst} scheme for a species with unknown' \
' scheme'.format(scheme_mlst=scheme_mlst)
elif scheme == 'unknown' and scheme_mlst == '-':
pass_qc = True
elif species_genus == 'yersinia' and mlst_scheme_genus == 'yersinia':
pass_qc = True
warnings['sample'] = 'Found a Yersinia scheme ({scheme_mlst}), but it is different from what it was' \
' expected ({scheme})'.format(scheme_mlst=scheme_mlst, scheme=scheme)
else:
if mlst_scheme_genus is not None and scheme_mlst == scheme == mlst_scheme_genus:
pass_qc = True
else:
failing['sample'] = 'MLST scheme found ({scheme_mlst}) and provided ({scheme}) are not the' \
' same'.format(scheme_mlst=scheme_mlst, scheme=scheme)
print(failing['sample'])
else:
failing['sample'] = 'Did not run'
if len(warnings) > 0:
print(warnings['sample'])
return run_successfully, pass_qc, failing, warnings
def get_bam_subset(alignment_file, sequences_2_keep, threads):
bam_subset = os.path.splitext(alignment_file)[0] + '.subset.bam'
command = ['samtools', 'view', '-buh', '-F', '4', '-o', bam_subset, '-@', str(threads), alignment_file, ' '.join(sequences_2_keep)]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, False)
if not run_successfully:
bam_subset = None
return run_successfully, bam_subset
def getScheme(species):
command = ['which', 'mlst']
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, False)
mlst_folder = os.path.abspath(os.path.realpath(stdout.splitlines()[0]))
mlst_db_path, species_scheme_map_new = get_species_scheme_map_version(mlst_folder)
scheme, genus_mlst_scheme = parse_species_scheme_map(species.lower().split(' '), mlst_db_path, species_scheme_map_new)
print '\n' + 'MLST scheme found for {species}: {scheme}'.format(species=species, scheme=scheme)
return scheme, species.lower().split(' ')[0], genus_mlst_scheme
def gzip_files(file_2_compress, pickle_prefix, outdir):
if file_2_compress.endswith('.temp'):
out_file = os.path.splitext(file_2_compress)[0]
else:
out_file = file_2_compress
command = ['gzip', '--stdout', '--best', file_2_compress, '>', str(out_file + '.gz')]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, True)
if run_successfully:
os.remove(file_2_compress)
utils.saveVariableToPickle(run_successfully, outdir, str(pickle_prefix + '.' + os.path.basename(file_2_compress)))
def index_fasta_samtools(fasta, region_None, region_outfile_none,
print_comand_True):
command = ['samtools', 'faidx', fasta, '', '', '']
shell_true = False
if region_None is not None:
command[3] = region_None
if region_outfile_none is not None:
command[4] = '>'
command[5] = region_outfile_none
shell_true = True
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, shell_true, None, print_comand_True)
return run_successfully, stdout
def get_bam_subset(alignment_file, sequences_2_keep, threads):
bam_subset = os.path.splitext(alignment_file)[0] + '.subset.bam'
command = [
'samtools', 'view', '-buh', '-F', '4', '-o', bam_subset, '-@',
str(threads), alignment_file, ' '.join(sequences_2_keep)
]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, False)
if not run_successfully:
bam_subset = None
return run_successfully, bam_subset
def runMlst(contigs, scheme, outdir, species_genus, mlst_scheme_genus):
pass_qc = False
failing = {}
failing['sample'] = False
warnings = {}
command = ['mlst', contigs]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
if run_successfully:
scheme_mlst = stdout.splitlines()[0].split('\t')[1].split('_')[0]
st = stdout.splitlines()[0].split('\t')[2]
profile = stdout.splitlines()[0].split('\t')[3:]
report = 'MLST found ST ' + str(st) + ' from scheme ' + scheme_mlst
print report
with open(os.path.join(outdir, 'mlst_report.txt'), 'wt') as writer:
writer.write('#scheme' + '\n' + scheme_mlst + '\n' + '#ST' + '\n' +
st + '\n')
writer.write('#profile' + '\n' + ' '.join(profile) + '\n')
writer.flush()
if scheme_mlst.split('_', 1)[0] == scheme.split('_', 1)[0]:
pass_qc = True
else:
if scheme == 'unknown' and scheme_mlst != '-':
pass_qc = True
warnings[
'sample'] = 'Found {scheme_mlst} scheme for a species with unknown scheme'.format(
scheme_mlst=scheme_mlst)
elif scheme == 'unknown' and scheme_mlst == '-':
pass_qc = True
elif species_genus == 'yersinia' and mlst_scheme_genus == 'yersinia':
pass_qc = True
warnings[
'sample'] = 'Found a Yersinia scheme ({scheme_mlst}), but it is different from what it was expected({scheme})'.format(
scheme_mlst=scheme_mlst, scheme=scheme)
else:
failing[
'sample'] = 'MLST scheme found (' + scheme_mlst + ') and provided (' + scheme + ') are not the same'
print failing['sample']
else:
warnings['sample'] = 'Did not run;'
pass_qc = True
if len(warnings) > 0:
print warnings['sample']
return run_successfully, pass_qc, failing, warnings
def download_with_aspera(aspera_file_path, aspera_key, outdir, pickle_prefix, sra, ena_id):
command = ['ascp', '-QT', '-l', '300m', '', '-i', aspera_key, '', outdir]
if not sra:
command[4] = '-P33001'
command[7] = str('era-fasp@' + aspera_file_path)
pickle = pickle_prefix + '.' + aspera_file_path.rsplit('/', 1)[1]
else:
command[7] = '[email protected]:/sra/sra-instant/reads/ByRun/sra/{a}/{b}/{c}/{c}.sra'.format(
a=ena_id[:3], b=ena_id[:6], c=ena_id)
pickle = pickle_prefix + '.' + ena_id
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, 3600, True)
utils.saveVariableToPickle(run_successfully, outdir, pickle)
def countSequencedBases(fastq_file, outdir):
run_successfully = False
bases = None
# Determine compression type
compression_type = utils.compressionType(fastq_file)
if compression_type is not None:
command = [compression_type[1], '--keep', '--stdout', fastq_file, '|', 'grep', '--after-context=1', '"@"', '|', 'grep', '--invert-match', '"^--$"', '|', 'grep', '--invert-match', '"@"', '|', 'wc', '']
# Number of characters
command[18] = '--chars'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
if run_successfully:
bases = int(stdout.splitlines()[0])
# Number of lines
command[18] = '--lines'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
if run_successfully:
lines = int(stdout.splitlines()[0])
bases = bases - lines
utils.saveVariableToPickle([run_successfully, bases], outdir, str('estimate_coverage.' + os.path.basename(fastq_file)))
def sortAlignment(alignment_file, output_file, sortByName_True, threads):
outFormat_string = os.path.splitext(output_file)[1][1:].lower()
command = [
'samtools', 'sort', '-o', output_file, '-O', outFormat_string, '',
'-@',
str(threads), alignment_file
]
if sortByName_True:
command[6] = '-n'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
if not run_successfully:
output_file = None
return run_successfully, output_file
def download_with_wget(ftp_file_path, outdir, pickle_prefix, sra, ena_id):
command = ['wget', '--tries=1', '', '-O', '']
if not sra:
command[2] = ftp_file_path
file_download = ftp_file_path.rsplit('/', 1)[1]
command[4] = os.path.join(outdir, file_download)
pickle = pickle_prefix + '.' + file_download
else:
command[2] = 'ftp://ftp-trace.ncbi.nih.gov/sra/sra-instant/reads/ByRun/sra/{a}/{b}/{c}/{c}.sra'.format(
a=ena_id[:3], b=ena_id[:6], c=ena_id)
command[4] = os.path.join(outdir, ena_id + '.sra')
pickle = pickle_prefix + '.' + ena_id
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, 3600, True)
utils.saveVariableToPickle(run_successfully, outdir, pickle)
def countSequencedBases(fastq_file, outdir):
run_successfully = False
bases = None
# Determine compression type
compression_type = utils.compressionType(fastq_file)
if compression_type is not None:
command = [compression_type[1], '--keep', '--stdout', fastq_file, '|', 'grep', '--after-context=1', '"@"', '|',
'grep', '--invert-match', '"^--$"', '|', 'grep', '--invert-match', '"@"', '|', 'wc', '']
# Number of characters
command[18] = '--chars'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
if run_successfully:
bases = int(stdout.splitlines()[0])
# Number of lines
command[18] = '--lines'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
if run_successfully:
lines = int(stdout.splitlines()[0])
bases = bases - lines
utils.saveVariableToPickle([run_successfully, bases], outdir, str('estimate_coverage.' + os.path.basename(fastq_file)))
def pilon(jar_path_pilon, assembly, bam_file, outdir, jarMaxMemory):
assembly_polished = os.path.splitext(assembly)[0] + '.polished.fasta'
command = [
'java', '', '-jar', jar_path_pilon, '--genome', assembly, '--frags',
bam_file, '--outdir', outdir, '--output',
os.path.basename(os.path.splitext(assembly_polished)[0]), '--changes',
'--vcf'
]
if str(jarMaxMemory) != 'off':
command[1] = '-Xmx' + str(int(round(jarMaxMemory * 1024, 0))) + 'M'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
if not run_successfully:
assembly_polished = None
return run_successfully, assembly_polished
def sra_2_fastq(download_dir, ena_id):
command = ['fastq-dump', '-I', '-O', download_dir, '--split-files', '{download_dir}{ena_id}.sra'.format(
download_dir=download_dir, ena_id=ena_id)]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, 3600, True)
if run_successfully:
files = [os.path.join(download_dir, f) for f in os.listdir(download_dir)
if not f.startswith('.') and os.path.isfile(os.path.join(download_dir, f)) and f.endswith('.fastq')]
pool = multiprocessing.Pool(processes=2)
results = []
p = pool.map_async(rename_header_sra, files, callback=results.extend)
p.wait()
run_successfully = all(results)
return run_successfully
def create_vcf(bam_file, sequence_to_analyse, outdir, counter, reference_file):
gene_vcf = os.path.join(
outdir, 'samtools_mpileup.sequence_' + str(counter) + '.vcf')
command = [
'samtools', 'mpileup', '--count-orphans', '--no-BAQ', '--min-BQ', '0',
'--min-MQ', '0', '--fasta-ref', reference_file, '--region',
sequence_to_analyse, '--output', gene_vcf, '--VCF', '--uncompressed',
'--output-tags', 'INFO/AD,AD,DP', bam_file
]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, False)
if not run_successfully:
gene_vcf = None
return run_successfully, gene_vcf
def getScheme(species):
command = ['which', 'mlst']
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, False)
mlst_folder = os.path.abspath(os.path.realpath(stdout.splitlines()[0]))
mlst_db_path, species_scheme_map_new = get_species_scheme_map_version(
mlst_folder)
scheme, genus_mlst_scheme = parse_species_scheme_map(
species.lower().split(' '), mlst_db_path, species_scheme_map_new)
print('\n' + 'MLST scheme found for {species}: {scheme}'.format(
species=species, scheme=scheme))
return scheme, species.lower().split(' ')[0], genus_mlst_scheme
def mapping_bowtie2(fastq_files, reference_file, outdir, keep_bam=False, threads=1):
"""
Map reads against a reference fasta file
Parameters
----------
fastq_files : list
List of fastq files
reference_file : str
Path to the reference file (the assembly)
outdir : str
Path to the output directory
keep_bam : bool, default False
True if want to keep the BAM file produced (with mapped and unmapped reads)
threads : int, default 1
Number of threads to be used
Returns
-------
run_successfully : bool
Boolean stating if INNUca Assembly_Mapping module ran successfully or not
sam_file : str or None
If everything went fine, it returns the path to the sam file, otherwise it returns None
"""
sam_file = os.path.join(outdir, str('alignment.sam'))
# Index reference file
run_successfully = indexSequenceBowtie2(reference_file, threads)
if run_successfully:
command = ['bowtie2', '-q', '--very-sensitive-local', '--threads', str(threads), '-x', reference_file, '',
'', '-S', sam_file]
if len(fastq_files) == 1:
command[7] = '-U ' + fastq_files[0]
else:
command[7] = '-1 ' + fastq_files[0] + ' -2 ' + fastq_files[1]
if not keep_bam:
command[8] = '--no-unal'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
sam_file = None
return run_successfully, sam_file
def mapping_bowtie2(fastq_files, reference_file, outdir, keep_bam=False, threads=1):
"""
Map reads against a reference fasta file
Parameters
----------
fastq_files : list
List of fastq files
reference_file : str
Path to the reference file (the assembly)
outdir : str
Path to the output directory
keep_bam : bool, default False
True if want to keep the BAM file produced (with mapped and unmapped reads)
threads : int, default 1
Number of threads to be used
Returns
-------
run_successfully : bool
Boolean stating if INNUca Assembly_Mapping module ran successfully or not
sam_file : str or None
If everything went fine, it returns the path to the sam file, otherwise it returns None
"""
sam_file = os.path.join(outdir, str('alignment.sam'))
# Index reference file
run_successfully = indexSequenceBowtie2(reference_file, threads)
if run_successfully:
command = ['bowtie2', '-q', '--very-sensitive-local', '--threads', str(threads), '-x', reference_file, '',
'', '--fr', '-I', '0', '-X', '2000', '-S', sam_file]
if len(fastq_files) == 1:
command[7] = '-U ' + fastq_files[0]
else:
command[7] = '-1 ' + fastq_files[0] + ' -2 ' + fastq_files[1]
if not keep_bam:
command[8] = '--no-unal'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
sam_file = None
return run_successfully, sam_file
def controlForZeroReads(fastq_files):
not_empty_fastq = False
fastq = fastq_files[0]
compression_type = utils.compressionType(fastq)
if compression_type is not None:
command = [compression_type[1], '--stdout', '--keep', fastq, '|', 'head', '-n', '4']
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
if run_successfully:
stdout = stdout.splitlines()
if len(stdout) == 4:
not_empty_fastq = True
return not_empty_fastq
def spades(spades_folder, threads, fastq_files, notUseCareful, maxMemory, minCoverageAssembly, kmers, assembled_se_reads):
contigs = os.path.join(spades_folder, 'contigs.fasta')
command = ['spades.py', '', '--only-assembler', '--threads', str(threads), '--memory', str(maxMemory), '--cov-cutoff', str(minCoverageAssembly), '', '-1', fastq_files[0], '-2', fastq_files[1], '', '-o', spades_folder]
if not notUseCareful:
command[1] = '--careful'
if len(kmers) > 0:
kmers = ','.join(map(str, kmers))
command[9] = str('-k ' + kmers)
if assembled_se_reads is not None:
command[14] = str('-s ' + assembled_se_reads)
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
return run_successfully, contigs
def getting_mapping_statistics(alignment_file):
command = ['samtools', 'flagstat', alignment_file]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, True)
dict_mapping_statistics = {}
if run_successfully:
stdout = stdout.splitlines()
for line in stdout:
line = line.splitlines()[0]
if len(line) > 0:
line = line.split(' ', 3)
field = line[3].split('(', 1)
if len(field) == 0:
field = field[0].replace(' ', '_')
else:
field = field[0].rsplit(' ', 1)[0].replace(' ', '_')
dict_mapping_statistics[field] = {'qc_passed': int(line[0]), 'qc_failed': int(line[2])}
return run_successfully, dict_mapping_statistics
def mappingBowtie2(fastq_files, referenceFile, threads, outdir):
sam_file = os.path.join(outdir, str('alignment.sam'))
# Index reference file
run_successfully = indexSequenceBowtie2(referenceFile, threads)
if run_successfully:
command = ['bowtie2', '-q', '--very-sensitive-local', '--threads', str(threads), '-x', referenceFile, '', '--no-unal', '-S', sam_file]
if len(fastq_files) == 1:
command[8] = '-U ' + fastq_files[0]
else:
command[8] = '-1 ' + fastq_files[0] + ' -2 ' + fastq_files[1]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
sam_file = None
return run_successfully, sam_file
def runMlst(contigs, scheme, outdir, species_genus, mlst_scheme_genus):
pass_qc = False
failing = {}
failing['sample'] = False
warnings = {}
command = ['mlst', contigs]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if run_successfully:
scheme_mlst = stdout.splitlines()[0].split('\t')[1].split('_')[0]
st = stdout.splitlines()[0].split('\t')[2]
profile = stdout.splitlines()[0].split('\t')[3:]
report = 'MLST found ST ' + str(st) + ' from scheme ' + scheme_mlst
print report
with open(os.path.join(outdir, 'mlst_report.txt'), 'wt') as writer:
writer.write('#scheme' + '\n' + scheme_mlst + '\n' + '#ST' + '\n' + st + '\n')
writer.write('#profile' + '\n' + ' '.join(profile) + '\n')
writer.flush()
if scheme_mlst.split('_', 1)[0] == scheme.split('_', 1)[0]:
pass_qc = True
else:
if scheme == 'unknown' and scheme_mlst != '-':
pass_qc = True
warnings['sample'] = 'Found {scheme_mlst} scheme for a species with unknown scheme'.format(scheme_mlst=scheme_mlst)
elif scheme == 'unknown' and scheme_mlst == '-':
pass_qc = True
elif species_genus == 'yersinia' and mlst_scheme_genus == 'yersinia':
pass_qc = True
warnings['sample'] = 'Found a Yersinia scheme ({scheme_mlst}), but it is different from what it was expected ({scheme})'.format(scheme_mlst=scheme_mlst, scheme=scheme)
else:
failing['sample'] = 'MLST scheme found (' + scheme_mlst + ') and provided (' + scheme + ') are not the same'
print failing['sample']
else:
warnings['sample'] = 'Did not run;'
pass_qc = True
if len(warnings) > 0:
print warnings['sample']
return run_successfully, pass_qc, failing, warnings
def fastQC(fastqc_folder, threads, adaptersFasta, fastq_files):
# Create temporary FastQC foldes
os.mkdir(os.path.join(fastqc_folder, 'temp.fastqc_temporary_dir', ''))
# Run FastQC
command = ['fastqc', '-o', fastqc_folder, '--extract', '--nogroup', '--format', 'fastq', '--threads', str(threads), '', '--dir', os.path.join(fastqc_folder, 'temp.fastqc_temporary_dir', '')]
command = command + fastq_files
if adaptersFasta is not None:
adaptersTEMP = adapters2fastQC(fastqc_folder, adaptersFasta)
print 'Scanning for adapters contamination using ' + adaptersFasta
command[9] = '--adapters ' + adaptersTEMP
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
# Remove temporary files
os.rmdir(os.path.join(fastqc_folder, 'temp.fastqc_temporary_dir', ''))
if adaptersFasta is not None:
os.remove(adaptersTEMP)
return run_successfully
def compute_consensus_sequence(reference_file, sequence_to_analyse,
compressed_vcf_file, outdir, sufix):
sequence_dict = None
gene_fasta = os.path.join(outdir, str(sequence_to_analyse + '.fasta'))
run_successfully, stdout = index_fasta_samtools(reference_file,
sequence_to_analyse,
gene_fasta, False)
if run_successfully:
command = [
'bcftools', 'consensus', '-f', gene_fasta, compressed_vcf_file
]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, False)
if run_successfully:
sequence_dict = parse_fasta_inMemory(stdout)
return run_successfully, sequence_dict
def fastQintegrity(fastq, outdir):
run_successfully = False
temporary_output_file = os.path.join(outdir, os.path.splitext(os.path.basename(fastq))[0])
compression_type = utils.compressionType(fastq)
encoding, min_reads_length, max_reads_length = None, None, None
if compression_type is not None:
command = [compression_type[1], '--stdout', '--keep', fastq, '>', temporary_output_file]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
if run_successfully:
encoding, min_reads_length, max_reads_length = run_guess_encoding_single_thread(temporary_output_file, None, outdir)
if os.path.isfile(temporary_output_file):
os.remove(temporary_output_file)
utils.saveVariableToPickle([run_successfully, encoding, min_reads_length, max_reads_length], outdir, os.path.basename(fastq))
def controlForZeroReads(fastq_files):
not_empty_fastq = False
fastq = fastq_files[0]
compression_type = utils.compressionType(fastq)
if compression_type is not None:
command = [
compression_type[1], '--stdout', '--keep', fastq, '|', 'head',
'-n', '4'
]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, True, None, False)
if run_successfully:
stdout = stdout.splitlines()
if len(stdout) == 4:
not_empty_fastq = True
return not_empty_fastq
def getting_mapping_statistics(alignment_file):
command = ['samtools', 'flagstat', alignment_file]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, True, None, True)
dict_mapping_statistics = {}
if run_successfully:
stdout = stdout.splitlines()
for line in stdout:
line = line.splitlines()[0]
if len(line) > 0:
line = line.split(' ', 3)
field = line[3].split('(', 1)
if len(field) == 0:
field = field[0].replace(' ', '_')
else:
field = field[0].rsplit(' ', 1)[0].replace(' ', '_')
dict_mapping_statistics[field] = {
'qc_passed': int(line[0]),
'qc_failed': int(line[2])
}
return run_successfully, dict_mapping_statistics
def mappingBowtie2(fastq_files, referenceFile, threads, outdir):
sam_file = os.path.join(outdir, str('alignment.sam'))
# Index reference file
run_successfully = indexSequenceBowtie2(referenceFile, threads)
if run_successfully:
command = [
'bowtie2', '-q', '--very-sensitive-local', '--threads',
str(threads), '-x', referenceFile, '', '-S', sam_file
]
if len(fastq_files) == 1:
command[8] = '-U ' + fastq_files
else:
command[8] = '-1 ' + fastq_files[0] + ' -2 ' + fastq_files[1]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
if not run_successfully:
sam_file = None
return run_successfully, sam_file
def controlForZeroReads(fastq_files): not_empty_fastq = False fastq = fastq_files[0] command = ['', '--stdout', '--keep', fastq, '|', 'head', '-n', '4'] filetype = utils.compressionType(fastq) if filetype == 'gz': command[0] = 'gunzip' elif filetype == 'bz2': command[0] = 'bunzip2' if command[0] != '': run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False) if run_successfully: stdout = stdout.splitlines() if len(stdout) == 4: not_empty_fastq = True return not_empty_fastq
def spades(spades_folder, threads, fastq_files, notUseCareful, maxMemory,
minCoverageAssembly, kmers):
contigs = os.path.join(spades_folder, 'contigs.fasta')
command = [
'spades.py', '', '--only-assembler', '--threads',
str(threads), '--memory',
str(maxMemory), '--cov-cutoff',
str(minCoverageAssembly), '', '-1', fastq_files[0], '-2',
fastq_files[1], '-o', spades_folder
]
if not notUseCareful:
command[1] = '--careful'
if len(kmers) > 0:
kmers = ','.join(map(str, kmers))
command[9] = str('-k ' + kmers)
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
return run_successfully, contigs
def fastQintegrity(fastq, outdir):
run_successfully = False
temporary_output_file = os.path.join(
outdir,
os.path.splitext(os.path.basename(fastq))[0])
command = ['', '--stdout', '--keep', fastq, '>', temporary_output_file]
filetype = utils.compressionType(fastq)
if filetype == 'gz':
command[0] = 'gunzip'
elif filetype == 'bz2':
command[0] = 'bunzip2'
if command[0] != '':
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, True, None, False)
if os.path.isfile(temporary_output_file):
os.remove(temporary_output_file)
utils.saveVariableToPickle(run_successfully, outdir,
os.path.basename(fastq))
def alignmentToFastq(alignment_file, outdir, threads, pair_end_type): fastq_basename = os.path.splitext(alignment_file)[0] outfiles = None bamFile = fastq_basename + '.temp.bam' # sort cram run_successfully, bamFile = sortAlignment(alignment_file, bamFile, True, threads) if run_successfully: command = ['samtools', 'fastq', '', bamFile] if pair_end_type.lower() == 'paired': command[2] = '-1 ' + str(fastq_basename + '_1.fq') + ' -2 ' + str(fastq_basename + '_2.fq') elif pair_end_type == 'single': command[2] = '-0 ' + str(fastq_basename + '.fq') run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True) if run_successfully: if pair_end_type.lower() == 'paired': outfiles = [str(fastq_basename + '_1.fq'), str(fastq_basename + '_2.fq')] elif pair_end_type.lower() == 'single': outfiles = [str(fastq_basename + '.fq')] if os.path.isfile(bamFile): os.remove(bamFile) return run_successfully, outfiles
def indexAlignment(alignment_file):
command = ['samtools', 'index', alignment_file]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
return run_successfully
def getBlastPath():
print '\n' + 'The following blastn will be used'
command = ['which', 'blastn']
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
print stdout
def compute_genome_coverage_data(alignment_file, sequence_to_analyse, outdir, counter):
genome_coverage_data_file = os.path.join(outdir, 'samtools_depth.sequence_' + str(counter) + '.tab')
command = ['samtools', 'depth', '-a', '-r', sequence_to_analyse, alignment_file, '>', genome_coverage_data_file]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, False)
return run_successfully, genome_coverage_data_file
def curl_installed():
command = ['which', 'curl']
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, False)
return run_successfully
def runMlst(contigs, scheme, outdir, species_genus, mlst_scheme_genus):
pass_qc = False
failing = {}
failing['sample'] = False
warnings = {}
novel_alleles = os.path.join(outdir, 'mlst_novel_alleles.fasta')
command = ['mlst', '--novel', novel_alleles, contigs]
run_successfully, stdout, _ = utils.runCommandPopenCommunicate(
command, False, None, True)
if run_successfully:
scheme_mlst = stdout.splitlines()[0].split('\t')[1].split('_')[0]
st = stdout.splitlines()[0].split('\t')[2]
profile = stdout.splitlines()[0].split('\t')[3:]
if st == '-' and os.path.isfile(novel_alleles):
clean_novel_alleles(novel_alleles=novel_alleles,
scheme_mlst=scheme_mlst,
profile=profile)
else:
if os.path.isfile(novel_alleles):
os.remove(novel_alleles)
report = 'MLST found ST ' + str(st) + ' from scheme ' + scheme_mlst
print(report)
with open(os.path.join(outdir, 'mlst_report.txt'), 'wt') as writer:
writer.write('#scheme' + '\n' + scheme_mlst + '\n' + '#ST' + '\n' +
st + '\n')
writer.write('#profile' + '\n' + ' '.join(profile) + '\n')
writer.flush()
if scheme_mlst.split('_', 1)[0] == scheme.split('_', 1)[0]:
pass_qc = True
else:
if scheme == 'unknown' and scheme_mlst != '-':
pass_qc = True
warnings['sample'] = 'Found {scheme_mlst} scheme for a species with unknown' \
' scheme'.format(scheme_mlst=scheme_mlst)
elif scheme == 'unknown' and scheme_mlst == '-':
pass_qc = True
elif scheme != 'unknown' and scheme_mlst == '-':
pass_qc = True
warnings[
'sample'] = 'Could not find a scheme for a species with known scheme ({})'.format(
scheme)
elif species_genus == 'yersinia' and mlst_scheme_genus == 'yersinia':
pass_qc = True
warnings['sample'] = 'Found a Yersinia scheme ({scheme_mlst}), but it is different from what it was' \
' expected ({scheme})'.format(scheme_mlst=scheme_mlst, scheme=scheme)
else:
if mlst_scheme_genus is not None and scheme_mlst == scheme == mlst_scheme_genus:
pass_qc = True
else:
failing['sample'] = 'MLST scheme found ({scheme_mlst}) and provided ({scheme}) are not the' \
' same'.format(scheme_mlst=scheme_mlst, scheme=scheme)
print(failing['sample'])
else:
failing['sample'] = 'Did not run'
if len(warnings) > 0:
print(warnings['sample'])
return run_successfully, pass_qc, failing, warnings
def downloadWithFtp(ftp_file_path, outdir, pickle_prefix):
file_download = ftp_file_path.rsplit('/', 1)[1]
command = ['wget', ftp_file_path, '-O', os.path.join(outdir, file_download)]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, 3600, True)
utils.saveVariableToPickle(run_successfully, outdir, str(pickle_prefix + '.' + file_download))
def downloadWithAspera(aspera_file_path, asperaKey, outdir, pickle_prefix):
command = ['ascp', '-QT', '-l', '300m', '-i', asperaKey, str('era-fasp@' + aspera_file_path), outdir]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, 3600, True)
utils.saveVariableToPickle(run_successfully, outdir, str(pickle_prefix + '.' + aspera_file_path.rsplit('/', 1)[1]))
def getBlastPath():
print('\n' + 'The following blastn will be used')
command = ['which', 'blastn']
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
print(stdout)
def run_pear(decompressed_reads_list, sample_name, threads, outdir, fastq_encoding, trimmomatic_run_successfully, minimum_overlap_reads):
pass_qc = False
failing = {}
command = ['pear', '--forward-fastq', decompressed_reads_list[0], '--reverse-fastq', decompressed_reads_list[1], '--output', os.path.join(outdir, sample_name), '--p-value', str(1.0), '--min-assembly-length', str(minimum_overlap_reads), '--phred-base', '', '--cap', str(0), '--threads', str(threads), '--memory', str(str(threads) + 'G'), '--keep-original']
if trimmomatic_run_successfully:
command[12] = '33'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, True, None, True)
else:
if fastq_encoding is not None:
command[12] = str(fastq_encoding[1])
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
else:
print 'Pear fail! Trying run with Phred+33 enconding defined...'
command[12] = '33'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
if not run_successfully:
print 'Pear fail again! Trying run with Phred+64 enconding defined...'
command[12] = '64'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(command, False, None, True)
with open(os.path.join(outdir, str(sample_name + '.pear_out.txt')), 'wt') as writer:
for line in stdout:
writer.write(line)
unassembled_pe_reads, assembled_se_reads = None, None
assembled_reads, unassembled_reads, discarded_reads = None, None, None
if run_successfully:
assembled_reads, unassembled_reads, discarded_reads = parse_pearOutput_getAssembled(stdout)
unassembled_pe_reads_uncompressed, assembled_se_reads_uncompressed = get_pear_output(outdir, sample_name)
if assembled_reads == 0:
assembled_se_reads = None
else:
compress_decompress(str(assembled_se_reads_uncompressed + '.gz'), assembled_se_reads_uncompressed, True)
run_successfully, assembled_se_reads = get_compressed_decompressed_reads(outdir)
assembled_se_reads = assembled_se_reads[0] if run_successfully else assembled_se_reads
if unassembled_reads == 0:
unassembled_pe_reads = None
else:
if run_successfully:
pool = multiprocessing.Pool(processes=threads)
for fastq in unassembled_pe_reads_uncompressed:
pool.apply_async(compress_decompress, args=(str(fastq + '.gz'), fastq, True,))
pool.close()
pool.join()
run_successfully, unassembled_pe_reads = get_compressed_decompressed_reads(outdir)
os.remove(assembled_se_reads_uncompressed)
for fastq in unassembled_pe_reads_uncompressed:
os.remove(fastq)
if float(assembled_reads) / float(assembled_reads + unassembled_reads) < 0.75:
pass_qc = True
failing['sample'] = False
else:
failing['sample'] = 'Number of overlapping reads is >= 75% of total reads'
print failing
return run_successfully, pass_qc, failing, assembled_se_reads, unassembled_pe_reads, assembled_reads, unassembled_reads, discarded_reads
def indexAlignment(alignment_file):
command = ['samtools', 'index', alignment_file]
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
return run_successfully
def trimmomatic(jar_path_trimmomatic, sampleName, trimmomatic_folder, threads,
adaptersFasta, script_path, doNotSearchAdapters, fastq_files,
maxReadsLength, doNotTrimCrops, crop, headCrop, leading,
trailing, slidingWindow, minLength, nts2clip_based_ntsContent,
jarMaxMemory, fastq_encoding):
trimmomatic_out_files = []
for fastq in fastq_files:
trimmomatic_out_files.append(
os.path.join(
trimmomatic_folder,
str(
os.path.splitext(
os.path.splitext(os.path.basename(fastq))[0])[0] +
'P.fastq.gz')))
trimmomatic_out_files.append(
os.path.join(
trimmomatic_folder,
str(
os.path.splitext(
os.path.splitext(os.path.basename(fastq))[0])[0] +
'U.fastq.gz')))
# Run Trimmomatic
command = [
'java', '', '-jar', jar_path_trimmomatic, 'PE', '-threads',
str(threads), '', ' '.join(fastq_files),
' '.join(trimmomatic_out_files), '', '', '',
str('SLIDINGWINDOW:' + slidingWindow),
str('LEADING:' + str(leading)),
str('TRAILING:' + str(trailing)),
str('MINLEN:' + str(minLength)), 'TOPHRED33'
]
if str(jarMaxMemory) != 'off':
command[1] = '-Xmx' + str(int(round(jarMaxMemory * 1024, 0))) + 'M'
if not doNotTrimCrops:
if maxReadsLength is not None:
if crop is not None:
crop = maxReadsLength - crop[0]
command[10] = str('CROP:' + str(crop))
else:
if nts2clip_based_ntsContent is not None:
crop = nts2clip_based_ntsContent[1]
print str(
crop
) + ' nucleotides will be clipped at the end of reads'
crop = maxReadsLength - crop
command[10] = str('CROP:' + str(crop))
else:
print 'Because FastQC did not run successfully, --trimCrop option will not be considered'
if headCrop is not None:
command[11] = str('HEADCROP:' + str(headCrop[0]))
else:
if nts2clip_based_ntsContent is not None:
headCrop = nts2clip_based_ntsContent[0]
print str(
headCrop
) + ' nucleotides will be clipped at the beginning of reads'
command[11] = str('HEADCROP:' + str(headCrop))
if not doNotSearchAdapters:
if adaptersFasta is not None:
print 'Removing adapters contamination using ' + adaptersFasta
command[12] = 'ILLUMINACLIP:' + adaptersFasta + ':3:30:10:6:true'
else:
trimmomatic_adapters_folder = os.path.join(
os.path.dirname(script_path), 'src', 'Trimmomatic-0.36',
'adapters')
adapters_files = [
os.path.join(trimmomatic_adapters_folder,
'Nextera_XT_INNUca.fasta'),
# os.path.join(trimmomatic_adapters_folder, 'NexteraPE-PE.fa'),
os.path.join(trimmomatic_adapters_folder, 'TruSeq2-PE.fa'),
os.path.join(trimmomatic_adapters_folder, 'TruSeq3-PE-2.fa')
]
print 'Removing adapters contamination using ' + str(
adapters_files)
adaptersFasta = concatenateFastaFiles(
adapters_files, trimmomatic_folder,
'concatenated_adaptersFile.fasta')
command[12] = 'ILLUMINACLIP:' + adaptersFasta + ':3:30:10:6:true'
run_successfully = False
if fastq_encoding is not None:
if fastq_encoding == 33:
command[7] = '-phred33'
elif fastq_encoding == 64:
command[7] = '-phred64'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
if not run_successfully:
print 'Trying to run Trimmomatic with Phred+33 enconding defined...'
command[7] = '-phred33'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
if not run_successfully:
print 'Trimmomatic fail again! Trying to run with Phred+64 enconding defined...'
command[7] = '-phred64'
run_successfully, stdout, stderr = utils.runCommandPopenCommunicate(
command, False, None, True)
return run_successfully
