def map_reads_with_gmap(reference_fpath, reads_fpath, out_bam_fpath,
parameters):
'It maps the reads with gmap'
threads = parameters['threads']
tmp_dir = parameters['tmp_dir'] if 'tmp_dir' in parameters else None
reference_dir, reference_file_name = os.path.split(reference_fpath)
reference_name = reference_file_name.split('.')[0]
if not reference_dir:
reference_dir = '.'
if not os.path.exists(os.path.join(reference_dir, reference_name,
reference_name + '.chromosome')):
create_gmap_reference(reference_dir, reference_fpath, reference_name,
parameters)
cmd = ['gmap', '-d', reference_name, '-D', reference_dir, '-f', 'samse']
# this gmap options doesn' detect deletions close to introns
cmd.append('--canonical-mode=0')
if threads:
cmd.extend(['-t', str(threads)])
cmd.append(reads_fpath)
out_sam_fhand = NamedTemporaryFile(suffix='.sam', dir=tmp_dir)
call(cmd, stdout=out_sam_fhand, raise_on_error=True)
if 'unmapped_fhand' in parameters and parameters['unmapped_fhand'] is not None:
out_sam_fhand2 = NamedTemporaryFile(dir=tmp_dir, suffix='.sam')
get_out_unmapped(out_sam_fhand, parameters['unmapped_fhand'],
out_sam_fhand2)
out_sam_fhand = out_sam_fhand2
sam2bam(out_sam_fhand.name, out_bam_fpath)
out_sam_fhand.close()
def main():
'The script itself'
#set parameters
work_dir, output, reference = set_parameters()
# make a working tempfir
temp_dir = NamedTemporaryDir()
# add readgroup tag to each alignment in bam
add_header_and_tags_bams(work_dir, temp_dir.name)
# Prepare files to merge
sams = get_opened_sams_from_dir(temp_dir.name)
temp_sam = NamedTemporaryFile()
# merge all the sam in one
merge_sam(sams, temp_sam, reference)
# Convert sam into a bam,(Temporary)
temp_bam = NamedTemporaryFile(suffix='.bam')
sam2bam(temp_sam.name, temp_bam.name)
# finally we need to order the bam
sort_bam_sam(temp_bam.name, output)
# and make and index of the bam
call(['samtools', 'index', output], raise_on_error=True)
temp_dir.close()
def test_general_mapping_stats():
'General mapping statistics'
sam = NamedTemporaryFile(suffix='.sam')
sam.write(SAM)
sam.flush()
bam_fhand = NamedTemporaryFile()
sam2bam(sam.name, bam_fhand.name)
out_fhand = StringIO()
bam_general_stats(bam_fhand, out_fhand)
result = out_fhand.getvalue()
assert 'illumina\t3\t100.0' in result
assert 'Secondary alignments: 1' in result
assert 'Reads with one X0 best alignment: 1' in result
assert 'Total number of reads: 7' in result
out_fhand = StringIO()
unmapped_fhand = NamedTemporaryFile()
unmapped_fhand.write('1\n2\n3\n')
unmapped_fhand.flush()
bam_general_stats(bam_fhand, out_fhand, unmapped_fhand)
result = out_fhand.getvalue()
assert 'illumina\t3\t100.0' in result
assert 'Secondary alignments: 1' in result
assert 'Reads with one X0 best alignment: 1' in result
assert 'Total number of reads: 10' in result
def sam_creator(fhand, out_bam_path, out_ref_path, read_repeats=None):
"""it creates a sam using an alignment file. The format of the alignment
file is:
ref aggttttataaaacAAAAaattaagtctacagagcaacta
sample aggttttataaaacAAA-aattaagtctacagagcaacta
read1 aggttttataaaacAA-Aaattaagtctacagagcaacta
read2 aggttttataaaacA-AAaattaagtctacagagcaacta
read3 aggttttataaaac-AAAaattaagtctacagagcaacta
"""
mapq = "250"
out_sam = NamedTemporaryFile(suffix=".sam")
header_done = False
ref_name = "ref"
if read_repeats is None:
read_repeats = 1
count = 0
for ref, read in _reads_in_alignment(fhand):
ref_seq = ref.replace("-", "").replace("*", "").strip()
if not header_done:
out_sam.write("@SQ\tSN:%s\tLN:%d\n" % (ref_name, len(ref_seq)))
header_done = True
cigar = _get_cigar(ref, read)
pos = _get_alignment_start(read)
for i in range(read_repeats):
count += 1
read_name = "read%d" % count
flag = "0"
rnext = "*"
pnext = "0"
tlen = "0"
seq = read.replace("-", "").replace("*", "").strip()
qual = "=" * len(seq)
sam_line = "%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\n" % (
read_name,
flag,
ref_name,
pos,
mapq,
cigar,
rnext,
pnext,
tlen,
seq,
qual,
)
out_sam.write(sam_line)
out_sam.flush()
sam2bam(out_sam.name, out_bam_path)
ref_fhand = open(out_ref_path, "w")
ref_fhand.write(">ref\n%s" % ref_seq)
ref_fhand.flush()
def testsam2bam():
'It test sam2bam function'
bampath = os.path.join(TEST_DATA_DIR, 'seq.bam')
sampath = NamedTemporaryFile(suffix='.sam').name
bam2sam(bampath, sampath, header=True)
assert 'SN:SGN-U572743' in open(sampath).readline()
newbam = NamedTemporaryFile(suffix='.bam')
sam2bam(sampath, newbam.name)
newsam = NamedTemporaryFile(suffix='.sam')
bam2sam(newbam.name, newsam.name, header=True)
newsam_content = open(newsam.name).read()
oldsam_content = open(sampath).read()
assert newsam_content == oldsam_content
def test_sample_bam():
'it tests sample bam function'
sam = NamedTemporaryFile(suffix='.sam')
sam.write(SAM)
sam.flush()
bam_fhand = NamedTemporaryFile()
sam2bam(sam.name, bam_fhand.name)
bam_fhand.flush()
out_bam = NamedTemporaryFile(suffix='.bam')
sample_bam(bam_fhand, out_bam, 2)
out_sam = NamedTemporaryFile(suffix='.sam')
bam2sam(out_bam.name, out_sam.name, header=True)
sam = open(out_sam.name).read().splitlines()
assert len(sam) == 6
def map_reads_with_bwa(reference_fpath, reads_fpath, bam_fpath, parameters):
'It maps the reads to the reference using bwa and returns a bam file'
colorspace = parameters['colorspace']
reads_length = parameters['reads_length']
threads = parameters['threads']
java_conf = parameters['java_conf']
tmp_dir = parameters['tmp_dir'] if 'tmp_dir' in parameters else None
threads = get_num_threads(threads)
#the reference should have an index
bwt_fpath = reference_fpath + '.bwt'
if not os.path.exists(bwt_fpath):
create_bwa_reference(reference_fpath, color=colorspace)
output_ali = 'output.ali'
bam_file_bam = 'bam_file.bam'
output_sai = 'output.sai'
if reads_length == 'short':
cmd = ['bwa', 'aln', reference_fpath, reads_fpath,
'-t', str(threads)]
if colorspace:
cmd.append('-c')
sai_fhand = NamedTemporaryFile(dir=tmp_dir, suffix=output_sai, mode='wb')
call(cmd, stdout=sai_fhand, raise_on_error=True)
cmd = ['bwa', 'samse', reference_fpath, sai_fhand.name, reads_fpath]
ali_fhand = NamedTemporaryFile(dir=tmp_dir, suffix=output_ali, mode='w')
call(cmd, stdout=ali_fhand, raise_on_error=True)
elif reads_length == 'long':
cmd = ['bwa', 'dbwtsw', reference_fpath, reads_fpath,
'-t', str(threads)]
ali_fhand = NamedTemporaryFile(dir=tmp_dir, suffix=output_ali)
call(cmd, stdout=ali_fhand, raise_on_error=True)
else:
raise ValueError('Reads length: short or long')
if 'unmapped_fhand' in parameters and parameters['unmapped_fhand'] is not None:
out_ali_fhand = NamedTemporaryFile(dir=tmp_dir, suffix=output_ali)
get_out_unmapped(ali_fhand, parameters['unmapped_fhand'], out_ali_fhand)
ali_fhand = out_ali_fhand
# From sam to Bam
# unsorted_bam = os.path.join(temp_dir.name, bam_file_bam)
unsorted_bam = NamedTemporaryFile(dir=tmp_dir, suffix=bam_file_bam)
sam2bam(ali_fhand.name, unsorted_bam.name)
# sort bam file
sort_bam_sam(unsorted_bam.name, bam_fpath, sort_method='coordinate',
java_conf=java_conf, strict_validation=False, tmp_dir=tmp_dir)
def test_get_read_group_info():
'Tests get_read_group_info'
sam_sample = '''@SQ\tSN:SGN-U576692\tLN:1714
@SQ\tSN:SGN-U572743\tLN:833
@RG\tID:g1\tLB:g1\tSM:g1\tPL:sanger
@RG\tID:g3\tLB:g3\tSM:g3\tPL:sanger
SGN-E200000\t0\tSGN-U572743\t317\t226\t14M\t*\t0\t0\tGGATGATKTTAGAG\t*\tAS:i:250\tXS:i:0\tXF:i:0\tXE:i:7\tXN:i:0\tRG:Z:g1
SGN-E40000\t0\tSGN-U576692\t1416\t207\t10M\t*\t0\t0\tAGCCTGATAA\t,,09377777\tAS:i:160\tXS:i:0\tXF:i:3\tXE:i:4\tXN:i:0\tRG:Z:g3
SGN-E40000\t20\tSGN-U576692\t1416\t207\t10M\t*\t0\t0\tAGCCTGATAA\t,,09377777\tAS:i:160\tXS:i:0\tXF:i:3\tXE:i:4\tXN:i:0\tRG:Z:g3
'''
sam_fhand = NamedTemporaryFile(suffix='.sam')
sam_fhand.write(sam_sample)
sam_fhand.flush()
bam_fhand = NamedTemporaryFile(suffix='.bam')
sam2bam(sam_fhand.name, bam_fhand.name)
bam_fhand.flush()
bam = pysam.Samfile(bam_fhand.name, 'rb')
read_gro_i = get_read_group_info(bam)
assert read_gro_i == {'g3': {'LB': 'g3', 'SM': 'g3', 'PL': 'sanger'},
'g1': {'LB': 'g1', 'SM': 'g1', 'PL': 'sanger'}}
def test_remove_unmapped_reads():
'Tests remove_unmapped_reads'
sam = NamedTemporaryFile(suffix='.sam')
sam.write(SAM)
sam.flush()
bam_fhand = NamedTemporaryFile()
sam2bam(sam.name, bam_fhand.name)
out_bam_fhand = NamedTemporaryFile()
out_removed_reads_fhand = NamedTemporaryFile()
remove_unmapped_reads(bam_fhand, out_bam_fhand, out_removed_reads_fhand)
reads = open(out_removed_reads_fhand.name).read()
assert '@SGN-E221406' in reads
assert 'FFMMMJJ@@755225889>0.' in reads
out_sam = NamedTemporaryFile(suffix='.sam')
bam2sam(out_bam_fhand.name, out_sam.name, header=True)
sam_out = open(out_sam.name).read()
assert 'SGN-U572743' in sam_out
assert 'SGN-E221403' in sam_out
def test_bam_distribs():
'test bam coverage distrib'
sam = NamedTemporaryFile(suffix='.sam')
sam.write(SAM)
sam.flush()
bam_fhand = NamedTemporaryFile()
sam2bam(sam.name, bam_fhand.name)
summary_fhand = StringIO()
distribs = bam_distribs(bam_fhand, 'coverage',
summary_fhand=summary_fhand)
expected = [2547]
assert distribs[('platform', '454')]['distrib'] == expected
assert 'average: 0.13' in summary_fhand.getvalue()
distribs = bam_distribs(bam_fhand, 'mapq')
assert distribs[('platform', '454')]['distrib'][0] == 1
distribs = bam_distribs(bam_fhand, 'mapq', sample_size=100)
assert distribs[('platform', '454')]['distrib'][0] == 1
distribs = bam_distribs(bam_fhand, 'edit_distance')
assert distribs[('platform', '454')]['distrib'][0] == 1
def run(self):
'''It runs the analysis.'''
self._log({'analysis_started':True})
settings = self._project_settings
project_path = settings['General_settings']['project_path']
tmp_dir = settings['General_settings']['tmpdir']
inputs = self._get_input_fpaths()
bam_paths = inputs['bams']
reference_path = inputs['reference']
output_dir = self._create_output_dirs()['result']
merged_bam_path = VersionedPath(os.path.join(output_dir,
BACKBONE_BASENAMES['merged_bam']))
merged_bam_fpath = merged_bam_path.next_version
#Do we have to add the default qualities to the sam file?
#do we have characters different from ACTGN?
add_qualities = settings['Sam_processing']['add_default_qualities']
#memory for the java programs
java_mem = settings['Other_settings']['java_memory']
picard_path = settings['Other_settings']['picard_path']
if add_qualities:
default_sanger_quality = settings['Other_settings']['default_sanger_quality']
default_sanger_quality = int(default_sanger_quality)
else:
default_sanger_quality = None
temp_dir = NamedTemporaryDir()
for bam_path in bam_paths:
bam_basename = bam_path.basename
temp_sam = NamedTemporaryFile(prefix='%s.' % bam_basename,
suffix='.sam')
sam_fpath = os.path.join(temp_dir.name, bam_basename + '.sam')
bam2sam(bam_path.last_version, temp_sam.name)
sam_fhand = open(sam_fpath, 'w')
# First we need to create the sam with added tags and headers
add_header_and_tags_to_sam(temp_sam, sam_fhand)
temp_sam.close()
sam_fhand.close()
#the standardization
temp_sam2 = NamedTemporaryFile(prefix='%s.' % bam_basename,
suffix='.sam', delete=False)
standardize_sam(open(sam_fhand.name), temp_sam2,
default_sanger_quality,
add_def_qual=add_qualities,
only_std_char=True)
temp_sam2.flush()
shutil.move(temp_sam2.name, sam_fhand.name)
temp_sam2.close()
get_sam_fpaths = lambda dir_: [os.path.join(dir_, fname) for fname in os.listdir(dir_) if fname.endswith('.sam')]
# Once the headers are ready we are going to merge
sams = get_sam_fpaths(temp_dir.name)
sams = [open(sam) for sam in sams]
temp_sam = NamedTemporaryFile(suffix='.sam')
reference_fhand = open(reference_path.last_version)
try:
merge_sam(sams, temp_sam, reference_fhand)
except Exception:
if os.path.exists(merged_bam_fpath):
os.remove(merged_bam_fpath)
raise
reference_fhand.close()
# close files
for sam in sams:
sam.close()
# Convert sam into a bam,(Temporary)
temp_bam = NamedTemporaryFile(suffix='.bam')
sam2bam(temp_sam.name, temp_bam.name)
# finally we need to order the bam
#print 'unsorted.bam', temp_bam.name
#raw_input()
sort_bam_sam(temp_bam.name, merged_bam_fpath,
java_conf={'java_memory':java_mem,
'picard_path':picard_path}, tmp_dir=tmp_dir )
temp_bam.close()
temp_sam.close()
create_bam_index(merged_bam_fpath)
self._log({'analysis_finished':True})
