def get_xml(dirname, analysis_id, logger):
print "Downloading XML"
print "Analysis ID = %s" % analysis_id
xml_file = "%s.xml" %os.path.join(dirname, analysis_id)
cmd = ['cgquery', '-o' , xml_file, 'analysis_id=%s' %analysis_id]
pipelineUtil.log_function_time('cgquery', analysis_id, cmd, logger)
return xml_file
def decompress(filename, workdir):
""" Unpack fastq files """
if filename.endswith(".tar"):
cmd = ['tar', '-xvf', filename, '-C', workdir]
elif filename.endswith(".gz"):
cmd = ['tar', '-xzvf', filename, '-C', workdir]
else:
raise Exception('Unknown input file extension for file %s' % filename)
pipelineUtil.log_function_time("tar", filename, cmd)
def run_pipeline(args, workdir, analysis_id, logger):
""" align datasets using STAR and compute expression using cufflinks """
for filename in os.listdir(workdir):
if filename.endswith(".tar") or filename.endswith(".tar.gz"):
tar_file_in = os.path.join(workdir, filename)
break
star_output_dir = os.path.join(workdir, 'star_2_pass')
if os.path.isdir(star_output_dir):
pipelineUtil.remove_dir(star_output_dir)
os.mkdir(star_output_dir)
bam = "%s_star.bam" %os.path.join(star_output_dir, analysis_id)
if not os.path.isfile(bam):
star_cmd = ['time', '/usr/bin/time', 'python', args.star_pipeline,
'--genomeDir', args.genome_dir,
'--runThreadN', args.p,
'--tarFileIn', tar_file_in,
'--workDir', workdir,
'--out', bam,
'--genomeFastaFile', args.genome_fasta_file,
'--sjdbGTFfile', args.gtf
]
if args.quantMode != "":
star_cmd.append('--quantMode')
star_cmd.append(args.quantMode)
pipelineUtil.log_function_time("STAR", analysis_id, star_cmd, logger)
remote_bam_path = "%s_star.bam" % os.path.join(args.bucket, analysis_id, analysis_id)
pipelineUtil.upload_to_cleversafe(logger, remote_bam_path, bam)
cufflinks_cmd = ['time', '/usr/bin/time', 'python', args.cufflinks_pipeline,
'--bam', bam,
'--gtf', args.gtf,
'--analysis_id', analysis_id,
'--out', star_output_dir,
'--p', args.p,
'--multi_read_correct', 'True'
]
pipelineUtil.log_function_time("CUFFLINKS", analysis_id, cufflinks_cmd, logger)
cuffout_genes_local = os.path.join(star_output_dir, "genes.fpkm_tracking")
cuffout_genes_remote = os.path.join(args.bucket, "cufflinks", "star_gene", "%s.genes.fpkm_tracking" %analysis_id)
pipelineUtil.upload_to_cleversafe(logger, cuffout_genes_remote, cuffout_genes_local)
cuffout_isoforms_local = os.path.join(star_output_dir, "isoforms.fpkm_tracking")
cuffout_isoforms_remote = os.path.join(args.bucket, "cufflinks", "star_iso", "%s.isoforms.fpkm_tracking" %analysis_id)
pipelineUtil.upload_to_cleversafe(logger, cuffout_isoforms_remote, cuffout_isoforms_local)
pipelineUtil.remove_dir(star_output_dir)
def cufflinks_compute(args, logger=None):
""" compute rna-seq expression using cufflinks """
cmd = ['cufflinks']
if args.multi_read_correct == 'True':
cmd.append('--multi-read-correct')
if args.frag_bias_correct == 'True':
cmd.append('--frag-bias-correct')
cmd += [
'--GTF', args.gtf,
'--output-dir', args.out,
'--num-threads', str(args.p),
args.bam
]
print cmd
pipelineUtil.log_function_time('cufflinks', args.analysis_id, cmd, logger)
def rna_seq_qc(rna_seq_qc_path, bam_file, uuid, outdir, ref_genome, gtf, logger=None):
""" Perform RNA-seqQC on post alignment BAM file """
if os.path.isfile(bam_file) and os.path.isfile(rna_seq_qc_path) and os.path.isfile(gtf):
cmd = ['java', '-jar', rna_seq_qc_path, '-o', outdir, '-r', ref_genome, '-s',
'%s|%s|%s' %(uuid, bam_file, uuid), '-t', gtf]
exit_code = pipelineUtil.log_function_time('RNAseq_qc', uuid, cmd, logger)
else:
raise Exception("Cannot find one of rnaseq-qc %s, bam %s or gtf %s" %(rna_seq_qc_path, bam_file, gtf))
if not exit_code == 0:
if not logger == None:
logger.error("Broad's RNA-Seq-QC returned non-zero exit code %s" %exit_code)
return exit_code
def bam_index(bam_file, uuid, logger=None):
""" Index the resultant post alignment BAM file """
if os.path.isfile(bam_file):
cmd = ['samtools', 'index', '-b', bam_file]
exit_code = pipelineUtil.log_function_time("BamIndex", uuid, cmd, logger)
if exit_code == 0:
assert(os.path.isfile('%s.bai' %bam_file))
else:
raise Exception("Cannot file bam file %s" %bam_file)
if not exit_code == 0:
if not logger == None:
logger.error("Samtools Index returned non-zero exit code %s" %exit_code)
return exit_code
def fastqc(fastqc_path, reads_1, reads_2, rg_id_dir, analysis_id, logger=None):
""" perform pre-alignment qc checks using fastqc """
if not os.path.isdir(rg_id_dir):
raise Exception("Invalid directory: %s")
fastqc_results = "%s" %(os.path.join(rg_id_dir, "fastqc_results"))
if not os.path.isdir(fastqc_results):
os.mkdir(fastqc_results)
if not reads_2 == "":
cmd = [fastqc_path, reads_1, reads_2, '--outdir', fastqc_results, '--extract']
else:
cmd = [fastqc_path, reads_1, '--outdir', fastqc_results, '--extract']
exit_code = pipelineUtil.log_function_time("FastQC", analysis_id, cmd, logger)
if not exit_code == 0:
if not logger == None:
logger.error('FastQC returned a non-zero exit code: %s' %exit_code)
def bam_to_fastq(fastq_dir, bam_file, analysis_id, logger=None):
""" Convert input BAM to Fastq files """
tmp_fastq = os.path.join(fastq_dir, 'tmp')
cmd = ['bamtofastq', 'filename=%s' %bam_file, 'outputdir=%s' %fastq_dir,
'tryoq=1', 'collate=1', 'outputperreadgroup=1', 'T=%s' %tmp_fastq]
exit_code = pipelineUtil.log_function_time('Biobambam', analysis_id, cmd, logger)
if exit_code == 0:
for filename in os.listdir(fastq_dir):
if filename.endswith(".fq"):
new_filename = filename.replace(".fq", ".fastq")
os.rename(os.path.join(fastq_dir, filename), os.path.join(fastq_dir, new_filename))
else:
logger.error("Biobambam BamToFastq conversion of %s returned a non-zero exit code %s"
%(analysis_id, exit_code))
def reorder_bam(picard_path, bam_file, uuid, outdir, ref_genome, logger=None):
""" Reorder the BAM file according to the reference genome """
if os.path.isfile(bam_file) and os.path.isfile(picard_path) and os.path.isfile(ref_genome):
outbam = os.path.join(outdir, '%s.reorder.bam' %uuid)
tmp_dir = os.path.join(outdir, 'tmp')
if not os.path.isdir(tmp_dir):
os.mkdir(tmp_dir)
cmd = ['java', '-jar', picard_path, 'ReorderSam', 'I=%s' %bam_file, 'O=%s' %outbam, 'R=%s' %ref_genome,
'VALIDATION_STRINGENCY=LENIENT', 'TMP_DIR=%s' %tmp_dir]
exit_code = pipelineUtil.log_function_time("picard_reorder_sam", uuid, cmd, logger)
if exit_code == 0:
assert(os.path.isfile(outbam))
else:
raise Exception("Cannot find one of bam %s, picard path %s or reference genome %s" %(bam_file, picard_path, ref_genome))
if not exit_code == 0:
if not logger == None:
logger.error("Picard reorderBAM returned non-zero exit code %s" %exit_code)
return outbam
def collect_rna_seq_metrics(picard_path, bam_file, uuid, outdir, ref_flat, logger=None):
""" Collect RNA-seq metrics using Picard """
if os.path.isfile(picard_path) and os.path.isfile(bam_file):
tmp_dir = os.path.join(outdir, 'tmp')
outfile = os.path.join(outdir, "%s.rna_seq_metrics.txt" %uuid)
if not os.path.isdir(tmp_dir):
os.mkdir(tmp_dir)
cmd = ['java', '-jar', picard_path, "CollectRnaSeqMetrics", "METRIC_ACCUMULATION_LEVEL=READ_GROUP",
"I=%s" %bam_file, "O=%s" %outfile, "STRAND=NONE",
"REF_FLAT=%s" %ref_flat, "VALIDATION_STRINGENCY=LENIENT", "TMP_DIR=%s" %tmp_dir]
exit_code = pipelineUtil.log_function_time("RNAseq_metrics", uuid, cmd, logger)
if exit_code == 0:
assert(os.path.isfile(outfile))
else:
raise Exception("Invalid path to picard or bam")
if not exit_code == 0:
if not logger == None:
logger.error("Picard CollectRnaSeqMetrics returned non-zero exit code %s" %exit_code)
return exit_code
def validate_bam_file(picard_path, bam_file, uuid, outdir, logger=None):
""" Validate resulting post-alignment BAM file """
if os.path.isfile(picard_path) and os.path.isfile(bam_file):
outfile = os.path.join(outdir, "%s.validate" %uuid)
tmp_dir = os.path.join(outdir, 'tmp')
if not os.path.isdir(tmp_dir):
os.mkdir(tmp_dir)
cmd = ['java', '-jar', picard_path, "ValidateSamFile", "I=%s" %bam_file,
"O=%s" %outfile, "VALIDATION_STRINGENCY=LENIENT",
"TMP_DIR=%s" %tmp_dir]
exit_code = pipelineUtil.log_function_time("ValidateSAM", uuid, cmd, logger)
if exit_code == 0:
assert(os.path.isfile(outfile))
else:
raise Exception("Invalid path to picard or BAM")
if not exit_code == 0:
if not logger == None:
logger.error("Picard ValidateSamFile returned non-zero exit code %s" %exit_code)
return exit_code
def add_or_replace_read_group(picard_path, bam_file, outdir, uuid, rg_id, rg_lb="Unknown", rg_pl="Unknown", rg_pu="Unknown",rg_sm="Unknown", logger=None):
""" Replace the @RG tag in the reads and header """
outbam = '%s.addRG.bam' %os.path.join(outdir, uuid)
if os.path.isfile(bam_file) and os.path.isfile(picard_path):
tmp_dir = os.path.join(outdir, 'tmp')
if not os.path.isdir(tmp_dir):
os.mkdir(tmp_dir)
cmd = ['java', '-jar', picard_path, 'AddOrReplaceReadGroups', 'I=%s' %bam_file, 'O=%s' %outbam,
'RGID=%s'%rg_id, 'RGLB=%s' %rg_lb, 'RGPL=%s' %rg_pl, 'RGPU=%s' %rg_pu, 'RGSM=%s' %rg_sm,
'VALIDATION_STRINGENCY=LENIENT','TMP_DIR=%s' %tmp_dir]
exit_code = pipelineUtil.log_function_time('AddOrReplaceReadGroups', uuid, cmd, logger)
else:
raise Exception("Cannot find bam file %s or path to picard %s" %(bam_file, picard_path))
if not exit_code == 0:
if not logger == None:
logger.error("Picard AddOrReplaceReadGroups returned non-zero exit code %s" %exit_code)
if os.path.isfile(outbam):
print "returning file now %s" %outbam
return outbam
else:
raise Exception('Could not add or replace read groups. Check log file for errors')
log_file = os.path.join(sub_dir, log_file)
logp = open(log_file, "r")
for line in logp:
if "CUFFLINKS_TIME" in line:
line = line.split()
f.write("%s\t%s\t%s\n" %(line[4], line[5], metadata["downloadable_file_size"]))
f.close()
if __name__ == "__main__":
parser = argparse.ArgumentParser(prog='label_dataset.py')
parser.add_argument('--dirname', default='/home/ubuntu/SCRATCH/lung_results')
parser.add_argument('--disease', help='disease to be labeled')
args = parser.parse_args()
#collect_metrics(args.dirname)
for filename in os.listdir(args.dirname):
if filename.endswith('fpkm_tracking'):
analysis_id = filename.split(".")[0]
#metadata = extract_metadata(args.dirname, analysis_id, None)
#print 'disease= %s' %metadata['disease']
cmd = ['mv', '%s' % os.path.join(args.dirname, filename), '%s' %os.path.join(args.dirname, args.disease, '%s_%s' %(args.disease, analysis_id))]
"""
print metadata
if metadata['disease'] != "":
if not os.path.isdir(os.path.join(args.dirname, metadata['disease'])):
os.mkdir(os.path.join(args.dirname, metadata['disease']))
cmd = ['mv', '%s' % os.path.join(args.dirname, filename), '%s' %os.path.join(args.dirname, metadata["disease"], '%s_%s' %(metadata['disease'], analysis_id))]
"""
pipelineUtil.log_function_time('mv', analysis_id, cmd, None)
def run_pipeline(args, workdir, analysis_id, fastq_dir, logger):
""" align datasets using STAR and compute expression using cufflinks """
tar_file_in = args.input_file
qc_dir = os.path.join(workdir, 'qc')
if not os.path.isdir(qc_dir):
os.mkdir(qc_dir)
decompress(tar_file_in, fastq_dir)
for fname in os.listdir(fastq_dir):
if fname.endswith("_1.fastq.gz") or fname.endswith("_1.fastq"):
reads_1 = os.path.join(fastq_dir, fname)
if fname.endswith("_2.fastq.gz") or fname.endswith("_2.fastq"):
reads_2 = os.path.join(fastq_dir, fname)
qc.fastqc(args.fastqc_path, reads_1, reads_2, qc_dir, analysis_id, logger)
star_output_dir = os.path.join(workdir, 'star_2_pass')
if os.path.isdir(star_output_dir):
pipelineUtil.remove_dir(star_output_dir)
os.mkdir(star_output_dir)
bam = "%s_star.bam" %os.path.join(star_output_dir, analysis_id)
if not os.path.isfile(bam):
star_cmd = ['time', '/usr/bin/time', 'python', args.star_pipeline,
'--genomeDir', args.genome_dir,
'--runThreadN', args.p,
'--tarFileIn', tar_file_in,
'--workDir', workdir,
'--out', bam,
'--genomeFastaFile', args.genome_fasta_file,
'--sjdbGTFfile', args.gtf
]
if args.quantMode != "":
star_cmd.append('--quantMode')
star_cmd.append(args.quantMode)
pipelineUtil.log_function_time("STAR", analysis_id, star_cmd, logger)
exit_code = 1
#Fix mate information for BAM
exit_code, fix_mate_out = post_alignment_qc.fix_mate_information(args.picard, bam,
analysis_id, workdir, logger)
if exit_code == 0:
os.remove(bam)
assert(not os.path.isfile(bam))
os.rename(fix_mate_out, bam)
assert(os.path.isfile(bam))
#validate the post alignment BAM file
post_alignment_qc.validate_bam_file(args.picard, bam, analysis_id, qc_dir, logger)
#collect RNA-seq metrics
post_alignment_qc.collect_rna_seq_metrics(args.picard, bam, analysis_id,
qc_dir, args.ref_flat, logger)
#quantify using cufflinks
cufflinks_cmd = ['time', '/usr/bin/time', 'python', args.cufflinks_pipeline,
'--bam', bam,
'--gtf', args.gtf,
'--analysis_id', analysis_id,
'--out', star_output_dir,
'--p', args.p,
'--multi_read_correct', 'True'
]
pipelineUtil.log_function_time("CUFFLINKS", analysis_id, cufflinks_cmd, logger)
"--outSAMstrandField", str(args.outSAMstrandField),
"--outSAMunmapped", str(args.outSAMunmapped)
]
if args.keepJunctions:
cmd = cmd.append("--keepJunctions")
cmd = cmd.append(str(args.keepJunctions))
if not args.metaDataTab == None:
cmd = cmd.append("--metaDataTab")
cmd = cmd.append(str(args.metaDataTab))
if not args.outSAMattrRGline == None:
cmd = cmd.append("--outSAMattrRGline")
cmd = cmd.append(str(args.outSAMattrRGline))
if not args.outSAMattrRGfile == None:
cmd = cmd.append("--outSAMattrRGfile")
cmd = cmd.append(str(args.outSAMattrRGfile))
logger.info('Starting Alignment with STAR')
exit_code = pipelineUtil.log_function_time("STAR_ALIGN", args.id, cmd, logger)
if exit_code == 0:
logger.info('Starting post alignment QC')
post_aln_qc(args, args.out, logger)
else:
logger.error('STAR returned a non-zero exit code %s' %exit_code)
