def __init__(self, in_filenames, extractor, reads_interleaved, r1_length, r2_length):
""" Args:
in_filenames (dict of str -> str): Map of paths to fastq files
feature_ref (FeatureExtractor): for extracting feature barcodes
"""
self.in_fastqs = None
self.in_iter = iter([])
# Relevant read types
read_types = extractor.get_read_types()
if in_filenames:
in_filenames = get_fastqs_from_feature_ref(in_filenames,
reads_interleaved,
read_types)
if in_filenames != (None, None):
if reads_interleaved:
filename = in_filenames[0] if in_filenames[0] else in_filenames[1]
self.in_fastqs = (cr_io.open_maybe_gzip(filename, 'r') if filename[0] else None,
None)
else:
self.in_fastqs = (cr_io.open_maybe_gzip(in_filenames[0], 'r') if in_filenames[0] else None,
cr_io.open_maybe_gzip(in_filenames[1], 'r') if in_filenames[1] else None)
self.in_iter = get_feature_generator_fastq(files=self.in_fastqs,
extractor=extractor,
interleaved=reads_interleaved,
read_types=read_types,
r1_length=r1_length,
r2_length=r2_length)
def main(args, outs):
outs.coerce_strings()
paired_end = cr_chem.is_paired_end(args.chemistry_def)
outs.read1s = martian.make_path('reads_1.fastq' + h5_constants.LZ4_SUFFIX)
r1_fq_out = cr_io.open_maybe_gzip(outs.read1s, 'w')
if paired_end:
outs.read2s = martian.make_path('reads_2.fastq' +
h5_constants.LZ4_SUFFIX)
r2_fq_out = cr_io.open_maybe_gzip(outs.read2s, 'w')
else:
outs.read2s = None
r2_fq_out = None
barcodes_out = cr_io.open_maybe_gzip(outs.chunk_barcodes, 'w')
merge_by_barcode(args.fastqs, r1_fq_out, r2_fq_out, barcodes_out,
paired_end)
r1_fq_out.close()
if r2_fq_out is not None:
r2_fq_out.close()
barcodes_out.close()
def create_unaligned_bam(args, outs):
star_ref_path = cr_utils.get_reference_star_path(args.reference_path)
header_buf = cStringIO.StringIO()
header_buf.write('@HD\tVN:1.4\n')
# SQ header lines
with open(os.path.join(star_ref_path, 'chrNameLength.txt')) as f:
for line in f:
chr_name, chr_len = line.strip().split('\t')
header_buf.write('@SQ\tSN:{}\tLN:{}\n'.format(chr_name, chr_len))
# RG header lines
for packed_rg in args.read_groups:
header_buf.write(
re.sub('\\\\t', '\t', tk_bam.make_rg_header(packed_rg)) + '\n')
# Get read group ID for this chunk of reads
read_group = args.read_group
# pysam doesn't support reading SAM from a StringIO object
with open('tmphdr', 'w') as f:
f.write(header_buf.getvalue())
samfile = pysam.AlignmentFile('tmphdr', 'r', check_sq=False)
outbam = pysam.AlignmentFile(outs.genome_output, 'wb', template=samfile)
fastq_file1 = cr_io.open_maybe_gzip(args.read_chunk)
fastq_file2 = cr_io.open_maybe_gzip(
args.read2_chunk) if args.read2_chunk else None
read1s = tk_fasta.read_generator_fastq(fastq_file1)
read2s = tk_fasta.read_generator_fastq(fastq_file2) if fastq_file2 else []
record = pysam.AlignedSegment()
record.flag = 4
for read1, read2 in itertools.izip_longest(read1s, read2s):
name, seq, qual = read1
record.query_name, record.query_sequence = name.split(' ')[0], seq
record.query_qualities = tk_fasta.get_qvs(qual)
record.set_tag('RG', read_group, 'Z')
outbam.write(record)
if read2:
name, seq, qual = read2
record.query_name, record.query_sequence = name.split(' ')[0], seq
record.query_qualities = tk_fasta.get_qvs(qual)
record.set_tag('RG', read_group, 'Z')
outbam.write(record)
samfile.close()
fastq_file1.close()
if fastq_file2 is not None:
fastq_file2.close()
outbam.close()
def load_barcode_translate_map(bc_whitelist):
"""
Guide BC to Cell BC translate.
If the barcode whitelist needs to translate, return the mapping dictionary,
else, return None.
"""
if bc_whitelist is None:
return None
file_path = None
for extension in ['.txt', '.txt.gz']:
file_ext = os.path.join(cr_constants.BARCODE_WHITELIST_TRANSLATE_PATH,
bc_whitelist + extension)
if os.path.exists(file_ext):
file_path = file_ext
break
if file_path is None:
return None
else:
translate_map = {}
for line in cr_io.open_maybe_gzip(file_path, 'r'):
if line.startswith('#'):
continue
bcs = line.strip().split()
translate_map[bcs[0]] = bcs[1]
return translate_map
def _compute_r1_length(fastqs, reads_interleaved):
""" Infer the length of R1 """
num_reads = 0
r1_max_len = 0
def get_r1_noninterleaved(read_iter):
for _, seq, _ in read_iter:
yield seq
def get_r1_interleaved(read_iter):
for _, seq, _, _, _, _ in read_iter:
yield seq
get_r1 = get_r1_interleaved if reads_interleaved else get_r1_noninterleaved
for fastq in fastqs:
with cr_io.open_maybe_gzip(fastq, 'r') as fq_file:
reads = tk_fasta.read_generator_fastq(fq_file, reads_interleaved)
for r1 in get_r1(reads):
if num_reads == cr_constants.DETECT_CHEMISTRY_INITIAL_READS:
break
r1_max_len = max(len(r1), r1_max_len)
num_reads += 1
if num_reads == cr_constants.DETECT_CHEMISTRY_INITIAL_READS:
break
return r1_max_len
def load_barcode_tsv(filename, as_set=False):
barcodes = [
x.strip() for x in cr_io.open_maybe_gzip(filename, 'r')
if not ('#' in x)
]
barcode_set = set(barcodes)
if len(barcodes) != len(barcode_set):
raise Exception('Duplicates found in barcode whitelist: %s' % filename)
return barcode_set if as_set else barcodes
def save_features_tsv(feature_ref, base_dir, compress):
"""Save a FeatureReference to a tsv file"""
out_features_fn = os.path.join(base_dir, 'features.tsv')
if compress:
out_features_fn += '.gz'
with cr_io.open_maybe_gzip(out_features_fn, 'w') as f:
for feature_def in feature_ref.feature_defs:
f.write('\t'.join((feature_def.id, feature_def.name,
feature_def.feature_type)) + '\n')
def __init__(self, in_filenames, read_def, reads_interleaved, r1_length, r2_length):
""" Args:
in_filenames - Map of paths to fastq files
read_def - ReadDef
"""
self.in_fastq = None
self.in_iter = iter([])
self.read_def = read_def
if in_filenames:
in_filename = get_fastq_from_read_type(in_filenames,
read_def,
reads_interleaved)
if in_filename:
self.in_fastq = cr_io.open_maybe_gzip(in_filename, 'r')
self.in_iter = get_read_generator_fastq(self.in_fastq,
read_def=read_def,
reads_interleaved=reads_interleaved,
r1_length=r1_length,
r2_length=r2_length)
def save_mex(self,
base_dir,
save_features_func,
metadata=None,
compress=True):
"""Save in Matrix Market Exchange format.
Args:
base_dir (str): Path to directory to write files in.
save_features_func (func): Func that takes (FeatureReference, base_dir, compress) and writes
a file describing the features.
metadata (dict): Optional metadata to encode into the comments as JSON.
"""
self.tocoo()
cr_io.makedirs(base_dir, allow_existing=True)
out_matrix_fn = os.path.join(base_dir, 'matrix.mtx')
out_barcodes_fn = os.path.join(base_dir, 'barcodes.tsv')
if compress:
out_matrix_fn += '.gz'
out_barcodes_fn += '.gz'
# This method only supports an integer matrix.
assert self.m.dtype in ['uint32', 'int32', 'uint64', 'int64']
assert type(self.m) == sp_sparse.coo.coo_matrix
rows, cols = self.m.shape
# Header fields in the file
rep = 'coordinate'
field = 'integer'
symmetry = 'general'
metadata = metadata or {}
metadata.update({
'format_version': MATRIX_H5_VERSION,
})
metadata_str = json.dumps(metadata)
comment = 'metadata_json: %s' % metadata_str
with cr_io.open_maybe_gzip(out_matrix_fn, 'w') as stream:
# write initial header line
stream.write(
np.compat.asbytes('%%MatrixMarket matrix {0} {1} {2}\n'.format(
rep, field, symmetry)))
# write comments
for line in comment.split('\n'):
stream.write(np.compat.asbytes('%%%s\n' % (line)))
# write shape spec
stream.write(
np.compat.asbytes('%i %i %i\n' % (rows, cols, self.m.nnz)))
# write row, col, val in 1-based indexing
for r, c, d in itertools.izip(self.m.row + 1, self.m.col + 1,
self.m.data):
stream.write(np.compat.asbytes(("%i %i %i\n" % (r, c, d))))
# both GEX and ATAC provide an implementation of this in respective feature_ref.py
save_features_func(self.feature_ref, base_dir, compress=compress)
with cr_io.open_maybe_gzip(out_barcodes_fn, 'w') as f:
for bc in self.bcs:
f.write(bc + '\n')
def open_file(self, filename):
return cr_io.open_maybe_gzip(filename, 'w')
def main(args, outs):
# Martian coerces dict keys to string
# Coerce keys back to int
args.chunks_per_gem_group = {int(k): v for k, v in args.chunks_per_gem_group.iteritems()}
paired_end = args.read2s_chunk is not None
# Lazy load R1
r1_file = cr_io.open_maybe_gzip(args.read1s_chunk)
read1s = tk_fasta.read_generator_fastq(r1_file)
# Lazy load R2
if paired_end:
r2_file = cr_io.open_maybe_gzip(args.read2s_chunk)
read2s = tk_fasta.read_generator_fastq(r2_file)
else:
read2s = []
# Lazy load corrected BCs
bc_file = cr_io.open_maybe_gzip(args.bcs)
bcs = (line.strip() for line in bc_file)
buckets = {}
bucket_filenames = {}
for gem_group, bucket_name in enumerate_bucket_names(args.chunks_per_gem_group):
filename = martian.make_path("%s.fastq" % bucket_name)
bucket_filenames[bucket_name] = filename
buckets[bucket_name] = []
for read1, read2, barcode in itertools.izip_longest(read1s, read2s, bcs):
# Exclude unbarcoded reads
if barcode == '':
continue
# Exclude short reads
if len(read1[1]) < MIN_READ_LENGTH or (read2 is not None and len(read2[1]) < MIN_READ_LENGTH):
continue
# Attach processed barcode to reads
r1_hdr = cr_fastq.AugmentedFastqHeader(read1[0])
r1_hdr.set_tag(cr_constants.PROCESSED_BARCODE_TAG, barcode)
r1_new_qname = r1_hdr.to_string()
if paired_end:
r2_hdr = cr_fastq.AugmentedFastqHeader(read2[0])
r2_hdr.set_tag(cr_constants.PROCESSED_BARCODE_TAG, barcode)
r2_new_qname = r2_hdr.to_string()
barcode_seq, gem_group = cr_utils.split_barcode_seq(barcode)
bucket_name = get_bucket_name(gem_group, barcode_seq, args.chunks_per_gem_group[gem_group])
buckets[bucket_name].append((r1_new_qname, read1[1], read1[2]))
if paired_end:
buckets[bucket_name].append((r2_new_qname, read2[1], read2[2]))
outs.buckets = {}
# Sort and write each bucket
for bucket_name, bucket in buckets.iteritems():
bucket.sort(key=vdj_utils.fastq_barcode_sort_key)
# Don't create empty bucket files.
# This is common when the reads are ordered by gem group
# And a chunk sees only a single gem group.
if len(bucket) == 0:
continue
filename = bucket_filenames[bucket_name]
with cr_io.open_maybe_gzip(filename, 'w') as f:
for read in bucket:
tk_fasta.write_read_fastq(f, *read)
outs.buckets[bucket_name] = bucket_filenames[bucket_name]
def main(args, outs):
# Load barcode whitelist
if args.barcode_whitelist is not None:
barcode_whitelist = cr_utils.load_barcode_whitelist(
args.barcode_whitelist)
reporter = vdj_report.VdjReporter()
# Load barcode count distribution
barcode_dist = cr_utils.load_barcode_dist(args.barcode_counts,
barcode_whitelist,
args.gem_group,
args.library_type)
if args.barcode_whitelist is not None:
barcode_whitelist_set = set(barcode_whitelist)
else:
barcode_whitelist_set = None
in_read1_fastq = cr_io.open_maybe_gzip(args.read1_chunk)
in_read2_fastq = cr_io.open_maybe_gzip(
args.read2_chunk) if args.read2_chunk else []
outs.corrected_bcs += h5_constants.LZ4_SUFFIX
out_file = cr_io.open_maybe_gzip(outs.corrected_bcs, 'w')
bc_counter = cr_fastq.BarcodeCounter(args.barcode_whitelist,
outs.corrected_barcode_counts)
# Correct barcodes, add processed bc tag to fastq
read_pair_iter = itertools.izip_longest(tk_fasta.read_generator_fastq(in_read1_fastq), \
tk_fasta.read_generator_fastq(in_read2_fastq))
for read1, read2 in itertools.islice(read_pair_iter, args.initial_reads):
read1_header = cr_fastq.AugmentedFastqHeader(read1[0])
raw_bc = read1_header.get_tag(cr_constants.RAW_BARCODE_TAG)
bc_qual = read1_header.get_tag(cr_constants.RAW_BARCODE_QUAL_TAG)
processed_bc = None
if raw_bc:
if barcode_whitelist_set is not None and raw_bc not in barcode_whitelist_set:
processed_bc = cr_stats.correct_bc_error(
args.barcode_confidence_threshold, raw_bc, bc_qual,
barcode_dist)
else:
# Disallow Ns in no-whitelist case
if 'N' in raw_bc:
processed_bc = None
else:
processed_bc = raw_bc
if processed_bc:
bc_counter.count(None, processed_bc, None)
# Add gem group to barcode sequence
processed_bc = cr_utils.format_barcode_seq(
processed_bc, gem_group=args.gem_group)
reporter.vdj_barcode_cb(raw_bc, processed_bc)
out_file.write('%s\n' %
(processed_bc if processed_bc is not None else ''))
in_read1_fastq.close()
if in_read2_fastq:
in_read2_fastq.close()
out_file.close()
bc_counter.close()
reporter.save(outs.chunked_reporter)