def begin_output(self):
from nesoni import io
if self.output is not None:
return io.open_possibly_compressed_writer(self.output)
else:
return sys.stdout
def begin_output(self):
from nesoni import io
if self.output is not None:
return io.open_possibly_compressed_writer(self.output)
else:
return sys.stdout
def write_gff3(filename, items):
# IGV likes to index large GFFs, and needs them to be sorted for this
items = sorted(items, key=lambda item: (item.seqid, item.start))
with io.open_possibly_compressed_writer(filename) as f:
write_gff3_header(f)
for item in items:
print >> f, item.as_gff()
def write_gff3(filename, items, sort=True):
# IGV likes to index large GFFs, and needs them to be sorted for this
if sort:
items = sorted(items, key=lambda item: (item.seqid, item.start))
with io.open_possibly_compressed_writer(filename) as f:
write_gff3_header(f)
for item in items:
print >> f, item.as_gff()
def run(self):
min_quality = chr(33+self.quality)
with io.open_possibly_compressed_writer(self.prefix+'.csfastq.gz') as out_file:
n = 0
n_discarded = 0
n_clipped = 0
total_before = 0
total_clipped = 0
for filename in self.filenames:
for name, seq, qual in io.read_sequences(filename, qualities='required'):
score = 0
start = 0
for i in xrange(len(seq)-1):
if qual[i] >= min_quality:
if seq[i+1] == '0':
score += 1
else:
score = max(0, score-4)
if not score: start = i+2
n += 1
total_before += len(seq)
if start > self.length+1:
if start < len(seq):
n_clipped += 1
total_clipped += len(seq)-start
print >> out_file, '@'+name
print >> out_file, seq[:start]
print >> out_file, '+'
print >> out_file, qual[:start-1]
else:
n_discarded += 1
self.log.datum(self.sample,'reads',n)
if n:
self.log.datum(self.sample,'mean length before poly-A clipping',float(total_before)/n)
self.log.datum(self.sample,'reads discarded as too short after poly-A clipping',n_discarded)
self.log.datum(self.sample,'reads poly-A clipped and kept',n_clipped)
if n_clipped:
self.log.datum(self.sample,'mean length clipped',float(total_clipped)/n_clipped)
def run(self):
min_quality = chr(33+self.quality)
with io.open_possibly_compressed_writer(self.prefix+'.csfastq.gz') as out_file:
n = 0
n_discarded = 0
n_clipped = 0
total_before = 0
total_clipped = 0
for filename in self.filenames:
for name, seq, qual in io.read_sequences(filename, qualities='required'):
score = 0
start = 0
for i in xrange(len(seq)-1):
if qual[i] >= min_quality:
if seq[i+1] == '0':
score += 1
else:
score = max(0, score-4)
if not score: start = i+2
n += 1
total_before += len(seq)
if start > self.length+1:
if start < len(seq):
n_clipped += 1
total_clipped += len(seq)-start
print >> out_file, '@'+name
print >> out_file, seq[:start]
print >> out_file, '+'
print >> out_file, qual[:start-1]
else:
n_discarded += 1
self.log.datum(self.sample,'reads',n)
if n:
self.log.datum(self.sample,'mean length before poly-A clipping',float(total_before)/n)
self.log.datum(self.sample,'reads discarded as too short after poly-A clipping',n_discarded)
self.log.datum(self.sample,'reads poly-A clipped and kept',n_clipped)
if n_clipped:
self.log.datum(self.sample,'mean length clipped',float(total_clipped)/n_clipped)
def write_gff3(filename, items, header):
with io.open_possibly_compressed_writer(filename) as f:
f.write(header)
for item in items:
print >> f, item.as_gff()
def run(self):
assert self.extension is not None, '--extension must be specified'
#workspace = self.get_workspace()
workspace = working_directory.Working(self.working_dir, must_exist=True)
if self.annotations == None:
reference = workspace.get_reference()
annotations_filename = reference.annotations_filename()
else:
annotations_filename = self.annotations
types = [ item.lower() for item in self.types.split(',') ]
parts = self.parts or self.types
parts = [ item.lower() for item in parts.split(',') ]
all_annotations = list(annotation.read_annotations(annotations_filename))
annotation.link_up_annotations(all_annotations)
for item in all_annotations:
item.primary = None
annotations = [
item
for item in all_annotations
if item.type.lower() in types
]
part_annotations = [ ]
seen = set()
queue = [ (item,item) for item in annotations ]
while queue:
primary, item = queue.pop()
if item.type.lower() in parts:
assert item.primary is None, "Feature with multiple parents"
item.primary = primary
key = (id(primary),item.start,item.end,item.seqid,item.strand)
# Ignore duplicate exons (many isoforms will have the same exons)
if key not in seen:
seen.add(key)
part_annotations.append(item)
queue.extend( (primary, item2) for item2 in item.children )
del seen
del all_annotations
self.log.log('%d annotations\n' % len(annotations))
self.log.log('%d part annotations\n' % len(part_annotations))
#assert annotations, 'No annotations of specified types in file'
for item in part_annotations:
this_extension = self.extension
if "max_extension" in item.attr:
this_extension = min(this_extension,int(item.attr["max_extension"]))
if item.strand >= 0:
item.tail_pos = item.end
item.end += this_extension
else:
item.tail_pos = item.start
item.start -= this_extension
for item in annotations:
item.hits = [] # [ (tail_length, adaptor_bases) ]
index = span_index.index_annotations(part_annotations)
for alignment in sam.Bam_reader(workspace/'alignments_filtered_sorted.bam'):
if alignment.is_unmapped or alignment.is_secondary or alignment.is_supplementary:
continue
start = alignment.reference_start
end = alignment.reference_end
alignment_length = end-start
strand = -1 if alignment.flag&sam.FLAG_REVERSE else 1
fragment_feature = annotation.Annotation(
seqid=alignment.reference_name,
start=start,
end=end,
strand=strand
)
if strand >= 0:
tail_pos = end
else:
tail_pos = start
tail_length = 0
adaptor_bases = 0
for item in alignment.extra:
if item.startswith('AN:i:'):
tail_length = int(item[5:])
elif item.startswith('AD:i:'):
adaptor_bases = int(item[5:])
hits = index.get(fragment_feature, same_strand=True)
if hits:
gene = min(hits, key=lambda gene:
(abs(tail_pos - gene.tail_pos), gene.primary.get_id()))
# Nearest by tail_pos
# failing that, by id to ensure a deterministic choice
gene.primary.hits.append( (tail_length,adaptor_bases) )
for item in annotations:
del item.parents
del item.children
del item.primary
f = io.open_possibly_compressed_writer(self.prefix + '.pickle.gz')
pickle.dump((workspace.name, workspace.get_tags(), annotations), f, pickle.HIGHEST_PROTOCOL)
f.close()
def run(self):
"""
<sequence> <poly-A> <adaptor> <anything>
"""
clip_quality = chr(33+self.clip_quality)
ignore_quality = chr(33+self.ignore_quality)
with io.open_possibly_compressed_writer(self.prefix+'.fastq.gz') as out_file, \
io.open_possibly_compressed_writer(self.prefix+'.clips.gz') as out_clips_file:
print >> out_clips_file, '#Read\tread length\tpoly-A start\tpoly-A end\tpoly-A start, ignoring adaptor\tpoly-A end, ignoring adaptor\tadaptor bases matched'
n = 0
n_discarded = 0
n_clipped = 0
total_before = 0
total_clipped = 0
for filename in self.filenames:
for name, seq, qual in io.read_sequences(filename, qualities='required'):
# "Good quality" sequence ends at the first low quality base
#good_quality_end = 0
#while good_quality_end < len(seq) and qual[good_quality_end] >= clip_quality:
# good_quality_end += 1
goodness_score = 0
best_goodness_score = 0
good_quality_end = 0
i = 0
while True:
if goodness_score > best_goodness_score:
best_goodness_score = goodness_score
good_quality_end = i
if i >= len(seq):
break
if qual[i] >= clip_quality:
goodness_score += 1
else:
goodness_score -= 9
i += 1
best_score = 0
best_a_start = good_quality_end
best_a_end = good_quality_end
best_adaptor_bases = 0
best_aonly_score = 0
best_aonly_start = good_quality_end
best_aonly_end = good_quality_end
# Consider each possible start position for the poly(A)
for a_start in xrange(good_quality_end):
if a_start and seq[a_start-1] == 'A': continue
# Consider each possible end position for the poly(A)
a_end = a_start
aonly_score = 0
while True:
if aonly_score > best_aonly_score:
best_aonly_score = aonly_score
best_aonly_start = a_start
best_aonly_end = a_end
# The poly(A) should be followed by adaptor,
# at least until the end of good quality sequence.
# However if there is evidence of the adaptor beyond
# the end of good quality, we still want to know that,
# and count it towards the number of adaptor bases present.
score = aonly_score
adaptor_bases = 0
i = a_end
while True:
if (score > best_score and
(i >= good_quality_end or i >= a_end+len(self.adaptor))):
best_score = score
best_a_start = a_start
best_a_end = a_end
best_adaptor_bases = adaptor_bases
if i >= a_end+len(self.adaptor) or i >= len(seq):
break
if qual[i] >= ignore_quality:
if seq[i] == self.adaptor[i-a_end]:
score += 1
adaptor_bases += 1
else:
score -= 4
i += 1
#if a_end >= len(seq): break
# poly(A) tail only within good quality region.
if a_end >= good_quality_end: break
if qual[a_end] >= ignore_quality:
if seq[a_end] == 'A':
aonly_score += 1
else:
aonly_score -= 4
if aonly_score <= 0: break
a_end += 1
a_start = best_a_start
a_end = best_a_end
adaptor_bases = best_adaptor_bases
aonly_start = best_aonly_start
aonly_end = best_aonly_end
if self.debug: # and a_end == a_start and a_end < len(seq)-10:
print name
print ''.join(
'I' if item<ignore_quality
else ('C' if item<clip_quality else ' ')
for item in qual )
print '-' * good_quality_end
print seq
print ' '*a_start + 'A'*(a_end-a_start) + self.adaptor + ".%d %d"%(adaptor_bases,best_score)
#print ' '*aonly_start + 'A'*(aonly_end-aonly_start) + "."
print
sys.stdout.flush()
n += 1
total_before += len(seq)
# 0 - sequence name
# 1 - sequence length
# 2 - poly(A) start
# 3 - poly(A) end
# (4 - best run of As start, for debugging the need to detect adaptor seq)
# (5 - best run of As end)
# 6 - number of adaptor bases matched
print >> out_clips_file, '%s\t%d\t%d\t%d\t%d\t%d\t%d' % (name, len(seq) , a_start, a_end, aonly_start, aonly_end, adaptor_bases)
if a_start > self.length:
if a_start < len(seq):
n_clipped += 1
total_clipped += a_start
print >> out_file, '@'+name
print >> out_file, seq[:a_start]
print >> out_file, '+'
print >> out_file, qual[:a_start]
else:
n_discarded += 1
if n%10000 == 0:
grace.status('Clip-runs ' + self.sample + ' ' + grace.pretty_number(n)) # + ' (' + grace.pretty_number(len(dstates)) + ' dstates)')
grace.status('')
self.log.datum(self.sample,'reads',n)
if n:
self.log.datum(self.sample,'mean length before poly-A/adaptor clipping',float(total_before)/n)
self.log.datum(self.sample,'reads discarded as too short after poly-A/adaptor clipping',n_discarded)
self.log.datum(self.sample,'reads poly-A/adaptor clipped and kept',n_clipped)
if n_clipped:
self.log.datum(self.sample,'mean length clipped',float(total_clipped)/n_clipped)
def write_gff3(filename, items):
with io.open_possibly_compressed_writer(filename) as f:
write_gff3_header(f)
for item in items:
print >> f, item.as_gff()
def run(self):
log = self.log
#quality_cutoff, args = grace.get_option_value(args, '--quality', int, 10)
#qoffset, args = grace.get_option_value(args, '--qoffset', int, None)
#clip_ambiguous, args = grace.get_option_value(args, '--clip-ambiguous', grace.as_bool, True)
#length_cutoff, args = grace.get_option_value(args, '--length', int, 24)
#adaptor_cutoff, args = grace.get_option_value(args, '--match', int, 10)
#max_error, args = grace.get_option_value(args, '--max-errors', int, 1)
#adaptor_set, args = grace.get_option_value(args, '--adaptors', str, 'truseq-adapter,truseq-srna,genomic,multiplexing,pe,srna')
#disallow_homopolymers, args = grace.get_option_value(args, '--homopolymers', grace.as_bool, False)
#reverse_complement, args = grace.get_option_value(args, '--revcom', grace.as_bool, False)
#trim_start, args = grace.get_option_value(args, '--trim-start', int, 0)
#trim_end, args = grace.get_option_value(args, '--trim-end', int, 0)
#output_fasta, args = grace.get_option_value(args, '--fasta', grace.as_bool, False)
#use_gzip, args = grace.get_option_value(args, '--gzip', grace.as_bool, True)
#output_rejects, args = grace.get_option_value(args, '--rejects', grace.as_bool, False)
#grace.expect_no_further_options(args)
prefix = self.prefix
log_name = os.path.split(prefix)[1]
quality_cutoff = self.quality
qoffset = self.qoffset
clip_ambiguous = self.clip_ambiguous
length_cutoff = self.length
adaptor_cutoff = self.match
max_error = self.max_errors
adaptor_set = self.adaptors
disallow_homopolymers = self.homopolymers
reverse_complement = self.revcom
trim_start = self.trim_start
trim_end = self.trim_end
output_fasta = self.fasta
use_gzip = self.gzip
output_rejects = self.rejects
iterators = []
filenames = []
any_paired = False
for filename in self.reads:
filenames.append(filename)
iterators.append(
itertools.izip(io.read_sequences(filename, qualities=True)))
for pair_filenames in self.pairs:
assert len(pair_filenames
) == 2, 'Expected a pair of files for "pairs" section.'
filenames.extend(pair_filenames)
any_paired = True
iterators.append(
itertools.izip(
io.read_sequences(pair_filenames[0], qualities=True),
io.read_sequences(pair_filenames[1], qualities=True)))
for filename in self.interleaved:
filenames.extend(filename)
any_paired = True
iterators.append(
deinterleave(io.read_sequences(filename, qualities=True)))
fragment_reads = (2 if any_paired else 1)
read_in_fragment_names = ['read-1', 'read-2'
] if any_paired else ['read']
assert iterators, 'Nothing to clip'
if qoffset is None:
guesses = [
io.guess_quality_offset(filename) for filename in filenames
]
assert len(
set(guesses)
) == 1, 'Conflicting quality offset guesses, please specify manually.'
qoffset = guesses[0]
log.log('FASTQ offset seems to be %d\n' % qoffset)
quality_cutoff_char = chr(qoffset + quality_cutoff)
#log.log('Minimum quality: %d (%s)\n' % (quality_cutoff, quality_cutoff_char))
#log.log('Clip ambiguous bases: %s\n' % (grace.describe_bool(clip_ambiguous)))
#log.log('Minimum adaptor match: %d bases, %d errors\n' % (adaptor_cutoff, max_error))
#log.log('Minimum length: %d bases\n' % length_cutoff)
adaptor_seqs = []
adaptor_names = []
if adaptor_set and adaptor_set.lower() != 'none':
for item in adaptor_set.split(','):
item = item.strip().lower() + ' '
any = False
for line in ADAPTORS.strip().split('\n'):
if line.startswith('#'): continue
if not line.lower().startswith(item): continue
any = True
name, seq = line.rsplit(None, 1)
seq = seq.replace('U', 'T')
#if seq in adaptor_seqs: print 'Dup', name
adaptor_seqs.append(seq)
adaptor_names.append(name)
adaptor_seqs.append(bio.reverse_complement(seq))
adaptor_names.append(name)
if not any:
raise grace.Error('Unknown adaptor set: ' + item)
matcher = Matcher(adaptor_seqs, adaptor_names, max_error)
start_clips = [
collections.defaultdict(list) for i in xrange(fragment_reads)
]
end_clips = [
collections.defaultdict(list) for i in xrange(fragment_reads)
]
if output_fasta:
write_sequence = io.write_fasta_single_line
else:
write_sequence = io.write_fastq
f_single = io.open_possibly_compressed_writer(
self.reads_output_filenames()[0])
if fragment_reads == 2:
f_paired = io.open_possibly_compressed_writer(
self.interleaved_output_filenames()[0])
if output_rejects:
f_reject = io.open_possibly_compressed_writer(
self.rejects_output_filenames()[0])
n_single = 0
n_paired = 0
n_in_single = 0
n_in_paired = 0
total_in_length = [0] * fragment_reads
n_out = [0] * fragment_reads
n_q_clipped = [0] * fragment_reads
n_a_clipped = [0] * fragment_reads
n_homopolymers = [0] * fragment_reads
total_out_length = [0] * fragment_reads
#log.attach(open(prefix + '_log.txt', 'wb'))
for iterator in iterators:
for fragment in iterator:
if (n_in_single + n_in_paired) % 10000 == 0:
grace.status(
'Clipping fragment %s' %
grace.pretty_number(n_in_single + n_in_paired))
if len(fragment) == 1:
n_in_single += 1
else:
n_in_paired += 1
graduates = []
rejects = []
for i, (name, seq, qual) in enumerate(fragment):
name = name.split()[0]
seq = seq.upper()
total_in_length[i] += len(seq)
start = trim_start
best_start = 0
best_len = 0
for j in xrange(len(seq) - trim_end):
if qual[j] < quality_cutoff_char or \
(clip_ambiguous and seq[j] not in 'ACGT'):
if best_len < j - start:
best_start = start
best_len = j - start
start = j + 1
j = len(seq) - trim_end
if best_len < j - start:
best_start = start
best_len = j - start
clipped_seq = seq[best_start:best_start + best_len]
clipped_qual = qual[best_start:best_start + best_len]
if len(clipped_seq) < length_cutoff:
n_q_clipped[i] += 1
rejects.append((name, seq, qual, 'quality'))
continue
match = matcher.match(clipped_seq)
if match and match[0] >= adaptor_cutoff:
clipped_seq = clipped_seq[match[0]:]
clipped_qual = clipped_qual[match[0]:]
start_clips[i][match[0]].append(match[1][0])
if len(clipped_seq) < length_cutoff:
n_a_clipped[i] += 1
rejects.append((name, seq, qual, 'adaptor'))
continue
match = matcher.match(bio.reverse_complement(clipped_seq))
if match and match[0] >= adaptor_cutoff:
clipped_seq = clipped_seq[:len(clipped_seq) - match[0]]
clipped_qual = clipped_qual[:len(clipped_qual) -
match[0]]
end_clips[i][match[0]].append(match[1][0])
if len(clipped_seq) < length_cutoff:
n_a_clipped[i] += 1
rejects.append((name, seq, qual, 'adaptor'))
continue
if disallow_homopolymers and len(set(clipped_seq)) <= 1:
n_homopolymers[i] += 1
rejects.append((name, seq, qual, 'homopolymer'))
continue
graduates.append((name, clipped_seq, clipped_qual))
n_out[i] += 1
total_out_length[i] += len(clipped_seq)
if output_rejects:
for name, seq, qual, reason in rejects:
write_sequence(f_reject, name + ' ' + reason, seq,
qual)
if graduates:
if reverse_complement:
graduates = [(name, bio.reverse_complement(seq),
qual[::-1])
for name, seq, qual in graduates]
if len(graduates) == 1:
this_f = f_single
n_single += 1
else:
assert len(graduates) == 2
this_f = f_paired
n_paired += 1
for name, seq, qual in graduates:
write_sequence(this_f, name, seq, qual)
grace.status('')
if output_rejects:
f_reject.close()
if fragment_reads == 2:
f_paired.close()
f_single.close()
def summarize_clips(name, location, clips):
total = 0
for i in clips:
total += len(clips[i])
log.datum(log_name, name + ' adaptors clipped at ' + location,
total)
if not clips:
return
for i in xrange(min(clips), max(clips) + 1):
item = clips[i]
log.quietly_log('%3d bases: %10d ' % (i, len(item)))
if item:
avg_errors = float(sum(item2[0]
for item2 in item)) / len(item)
log.quietly_log(' avg errors: %5.2f ' % avg_errors)
counts = collections.defaultdict(int)
for item2 in item:
counts[item2[1]] += 1
#print counts
for no in sorted(counts,
key=lambda item2: counts[item2],
reverse=True)[:2]:
log.quietly_log('%dx%s ' %
(counts[no], matcher.names[no]))
if len(counts) > 2: log.quietly_log('...')
log.quietly_log('\n')
log.quietly_log('\n')
if n_in_paired:
log.datum(log_name, 'read-pairs', n_in_paired)
if n_in_single:
log.datum(log_name, 'single reads', n_in_single)
for i in xrange(fragment_reads):
if start_clips:
summarize_clips(read_in_fragment_names[i], 'start',
start_clips[i])
if end_clips:
summarize_clips(read_in_fragment_names[i], 'end', end_clips[i])
prefix = read_in_fragment_names[i]
log.datum(log_name, prefix + ' too short after quality clip',
n_q_clipped[i])
log.datum(log_name, prefix + ' too short after adaptor clip',
n_a_clipped[i])
if disallow_homopolymers:
log.datum(log_name, prefix + ' homopolymers',
n_homopolymers[i])
if fragment_reads > 1:
log.datum(log_name, prefix + ' kept', n_out[i])
log.datum(log_name, prefix + ' average input length',
float(total_in_length[i]) / (n_in_single + n_in_paired))
if n_out[i]:
log.datum(log_name, prefix + ' average output length',
float(total_out_length[i]) / n_out[i])
if fragment_reads == 2:
log.datum(log_name, 'pairs kept after clipping', n_paired)
log.datum(log_name, 'reads kept after clipping', n_single)
def run(self):
"""
<sequence> <poly-A> <adaptor> <anything>
"""
clip_quality = chr(33+self.clip_quality)
#ignore_quality = chr(33+self.ignore_quality)
with io.open_possibly_compressed_writer(self.prefix+'.fastq.gz') as out_file, \
io.open_possibly_compressed_writer(self.prefix+'.clips.gz') as out_clips_file:
print >> out_clips_file, '#Read\tread length\tpoly-A start\tpoly-A end\tpoly-A start, ignoring adaptor\tpoly-A end, ignoring adaptor\tadaptor bases matched'
n = 0
n_discarded = 0
n_clipped = 0
total_before = 0
total_clipped = 0
for filename in self.filenames:
for name, seq, qual in io.read_sequences(filename, qualities='required'):
# "Good quality" sequence ends at the first low quality base
#good_quality_end = 0
#while good_quality_end < len(seq) and qual[good_quality_end] >= clip_quality:
# good_quality_end += 1
goodness_score = 0
best_goodness_score = 0
good_quality_end = 0
i = 0
while True:
if goodness_score > best_goodness_score:
best_goodness_score = goodness_score
good_quality_end = i
if i >= len(seq):
break
if qual[i] >= clip_quality:
goodness_score += 1
else:
goodness_score -= 9
i += 1
best_score = self.min_score-1
best_a_start = good_quality_end
best_a_end = good_quality_end
best_adaptor_bases = 0
best_aonly_score = 0
best_aonly_start = good_quality_end
best_aonly_end = good_quality_end
# Consider each possible start position for the poly(A)
for a_start in xrange(len(seq)):
if a_start and seq[a_start-1] == 'A': continue
# Consider each possible end position for the poly(A)
a_end = a_start
aonly_score = 0
while True:
if aonly_score > best_aonly_score:
best_aonly_score = aonly_score
best_aonly_start = a_start
best_aonly_end = a_end
# The poly(A) should be followed by adaptor,
## at least until the end of good quality sequence.
# However if there is evidence of the adaptor beyond
# the end of good quality, we still want to know that,
# and count it towards the number of adaptor bases present.
score = aonly_score
adaptor_bases = 0
i = a_end
abort_score = best_score-len(self.adaptor)
abort_i = min(len(seq), a_end+len(self.adaptor))
while score >= abort_score:
#if (score > best_score and
# (i >= good_quality_end or i >= a_end+len(self.adaptor))):
if score > best_score:
best_score = score
best_a_start = a_start
best_a_end = a_end
best_adaptor_bases = adaptor_bases
if i >= abort_i:
break
if seq[i] == self.adaptor[i-a_end]:
score += 1
adaptor_bases += 1
else:
score -= 4
i += 1
#if a_end >= len(seq): break
# Modified 2018-03-21
# poly(A) tail only within good quality region.
#if a_end >= good_quality_end: break
#if qual[a_end] >= ignore_quality:
# if seq[a_end] == 'A':
# aonly_score += 1
# else:
# aonly_score -= 4
# if aonly_score <= 0: break
if a_end >= len(seq): break
if seq[a_end] == 'A':
aonly_score += 1
else: #if qual[a_end] >= ignore_quality:
aonly_score -= 4
#else:
# aonly_score -= 1
a_end += 1
# 2018-03-21
# Look for tail starting after good quality,
# however don't call a tail if starts after good quality
if best_a_start > good_quality_end:
best_a_start = good_quality_end
best_a_end = good_quality_end
best_adaptor_bases = 0
best_score = 0
a_start = best_a_start
a_end = best_a_end
adaptor_bases = best_adaptor_bases
aonly_start = best_aonly_start
aonly_end = best_aonly_end
if self.debug: # and a_end == a_start and a_end < len(seq)-10:
print name
print ''.join(
('C' if item<clip_quality else ' ')
for item in qual )
print '-' * good_quality_end
print seq
print ' '*a_start + 'A'*(a_end-a_start) + self.adaptor + ".%d %d"%(adaptor_bases,best_score)
#print ' '*aonly_start + 'A'*(aonly_end-aonly_start) + "."
print
sys.stdout.flush()
n += 1
total_before += len(seq)
# 0 - sequence name
# 1 - sequence length
# 2 - poly(A) start
# 3 - poly(A) end
# (4 - best run of As start, for debugging the need to detect adaptor seq)
# (5 - best run of As end)
# 6 - number of adaptor bases matched
print >> out_clips_file, '%s\t%d\t%d\t%d\t%d\t%d\t%d' % (name, len(seq) , a_start, a_end, aonly_start, aonly_end, adaptor_bases)
if a_start >= self.length:
if a_start < len(seq):
n_clipped += 1
total_clipped += a_start
print >> out_file, '@'+name
print >> out_file, seq[:a_start]
print >> out_file, '+'
print >> out_file, qual[:a_start]
else:
n_discarded += 1
if n%10000 == 0:
grace.status('Clip-runs ' + self.sample + ' ' + grace.pretty_number(n)) # + ' (' + grace.pretty_number(len(dstates)) + ' dstates)')
# Option to do a quick subsample
if self.only and self.only <= n:
break
grace.status('')
self.log.datum(self.sample,'reads',n)
if n:
self.log.datum(self.sample,'mean length before poly-A/adaptor clipping',float(total_before)/n)
self.log.datum(self.sample,'reads discarded as too short after poly-A/adaptor clipping',n_discarded)
self.log.datum(self.sample,'reads poly-A/adaptor clipped and kept',n_clipped)
if n_clipped:
self.log.datum(self.sample,'mean length clipped',float(total_clipped)/n_clipped)
def run(self):
log = self.log
#quality_cutoff, args = grace.get_option_value(args, '--quality', int, 10)
#qoffset, args = grace.get_option_value(args, '--qoffset', int, None)
#clip_ambiguous, args = grace.get_option_value(args, '--clip-ambiguous', grace.as_bool, True)
#length_cutoff, args = grace.get_option_value(args, '--length', int, 24)
#adaptor_cutoff, args = grace.get_option_value(args, '--match', int, 10)
#max_error, args = grace.get_option_value(args, '--max-errors', int, 1)
#adaptor_set, args = grace.get_option_value(args, '--adaptors', str, 'truseq-adapter,truseq-srna,genomic,multiplexing,pe,srna')
#disallow_homopolymers, args = grace.get_option_value(args, '--homopolymers', grace.as_bool, False)
#reverse_complement, args = grace.get_option_value(args, '--revcom', grace.as_bool, False)
#trim_start, args = grace.get_option_value(args, '--trim-start', int, 0)
#trim_end, args = grace.get_option_value(args, '--trim-end', int, 0)
#output_fasta, args = grace.get_option_value(args, '--fasta', grace.as_bool, False)
#use_gzip, args = grace.get_option_value(args, '--gzip', grace.as_bool, True)
#output_rejects, args = grace.get_option_value(args, '--rejects', grace.as_bool, False)
#grace.expect_no_further_options(args)
prefix = self.prefix
log_name = os.path.split(prefix)[1]
quality_cutoff = self.quality
qoffset = self.qoffset
clip_ambiguous = self.clip_ambiguous
length_cutoff = self.length
adaptor_cutoff = self.match
max_error = self.max_errors
disallow_homopolymers = self.homopolymers
reverse_complement = self.revcom
trim_start = self.trim_start
trim_end = self.trim_end
output_fasta = self.fasta
use_gzip = self.gzip
output_rejects = self.rejects
iterators = [ ]
filenames = [ ]
any_paired = False
for filename in self.reads:
filenames.append(filename)
iterators.append(itertools.izip(
io.read_sequences(filename, qualities=True)
))
for pair_filenames in self.pairs:
assert len(pair_filenames) == 2, 'Expected a pair of files for "pairs" section.'
filenames.extend(pair_filenames)
any_paired = True
iterators.append(itertools.izip(
io.read_sequences(pair_filenames[0], qualities=True),
io.read_sequences(pair_filenames[1], qualities=True)
))
for filename in self.interleaved:
filenames.append(filename)
any_paired = True
iterators.append(deinterleave(
io.read_sequences(filename, qualities=True)
))
fragment_reads = (2 if any_paired else 1)
read_in_fragment_names = [ 'read-1', 'read-2' ] if any_paired else [ 'read' ]
assert iterators, 'Nothing to clip'
io.check_name_uniqueness(self.reads, self.pairs, self.interleaved)
if qoffset is None:
guesses = [ io.guess_quality_offset(filename) for filename in filenames ]
assert len(set(guesses)) == 1, 'Conflicting quality offset guesses, please specify manually.'
qoffset = guesses[0]
log.log('FASTQ offset seems to be %d\n' % qoffset)
quality_cutoff_char = chr(qoffset + quality_cutoff)
#log.log('Minimum quality: %d (%s)\n' % (quality_cutoff, quality_cutoff_char))
#log.log('Clip ambiguous bases: %s\n' % (grace.describe_bool(clip_ambiguous)))
#log.log('Minimum adaptor match: %d bases, %d errors\n' % (adaptor_cutoff, max_error))
#log.log('Minimum length: %d bases\n' % length_cutoff)
adaptor_seqs = [ ]
adaptor_names = [ ]
if self.adaptor_clip:
if self.adaptor_file:
adaptor_iter = io.read_sequences(self.adaptor_file)
else:
adaptor_iter = ADAPTORS
for name, seq in adaptor_iter:
seq = seq.upper().replace('U','T')
adaptor_seqs.append(seq)
adaptor_names.append(name)
adaptor_seqs.append(bio.reverse_complement(seq))
adaptor_names.append(name)
matcher = Matcher(adaptor_seqs, adaptor_names, max_error)
start_clips = [ collections.defaultdict(list) for i in xrange(fragment_reads) ]
end_clips = [ collections.defaultdict(list) for i in xrange(fragment_reads) ]
if output_fasta:
write_sequence = io.write_fasta_single_line
else:
write_sequence = io.write_fastq
f_single = io.open_possibly_compressed_writer(self.reads_output_filenames()[0])
if fragment_reads == 2:
names = self.pairs_output_filenames()[0] if self.out_separate else self.interleaved_output_filenames()
f_paired = map(io.open_possibly_compressed_writer, names)
if output_rejects:
f_reject = io.open_possibly_compressed_writer(self.rejects_output_filenames()[0])
n_single = 0
n_paired = 0
n_in_single = 0
n_in_paired = 0
total_in_length = [ 0 ] * fragment_reads
n_out = [ 0 ] * fragment_reads
n_q_clipped = [ 0 ] * fragment_reads
n_a_clipped = [ 0 ] * fragment_reads
n_homopolymers = [ 0 ] * fragment_reads
total_out_length = [ 0 ] * fragment_reads
#log.attach(open(prefix + '_log.txt', 'wb'))
for iterator in iterators:
for fragment in iterator:
if (n_in_single+n_in_paired) % 10000 == 0:
grace.status('Clipping fragment %s' % grace.pretty_number(n_in_single+n_in_paired))
if len(fragment) == 1:
n_in_single += 1
else:
n_in_paired += 1
graduates = [ ]
rejects = [ ]
for i, (name, seq, qual) in enumerate(fragment):
seq = seq.upper()
total_in_length[i] += len(seq)
if self.trim_to:
seq = seq[:self.trim_to]
qual = qual[:self.trim_to]
start = trim_start
best_start = 0
best_len = 0
for j in xrange(len(seq)-trim_end):
if qual[j] < quality_cutoff_char or \
(clip_ambiguous and seq[j] not in 'ACGT'):
if best_len < j-start:
best_start = start
best_len = j-start
start = j + 1
j = len(seq)-trim_end
if best_len < j-start:
best_start = start
best_len = j-start
clipped_seq = seq[best_start:best_start+best_len]
clipped_qual = qual[best_start:best_start+best_len]
if len(clipped_seq) < length_cutoff:
n_q_clipped[i] += 1
rejects.append( (name,seq,qual,'quality') )
continue
match = matcher.match(clipped_seq)
if match and match[0] >= adaptor_cutoff:
clipped_seq = clipped_seq[match[0]:]
clipped_qual = clipped_qual[match[0]:]
start_clips[i][match[0]].append( match[1][0] )
if len(clipped_seq) < length_cutoff:
n_a_clipped[i] += 1
rejects.append( (name,seq,qual,'adaptor') )
continue
match = matcher.match(bio.reverse_complement(clipped_seq))
if match and match[0] >= adaptor_cutoff:
clipped_seq = clipped_seq[: len(clipped_seq)-match[0] ]
clipped_qual = clipped_qual[: len(clipped_qual)-match[0] ]
end_clips[i][match[0]].append( match[1][0] )
if len(clipped_seq) < length_cutoff:
n_a_clipped[i] += 1
rejects.append( (name,seq,qual,'adaptor') )
continue
if disallow_homopolymers and len(set(clipped_seq)) <= 1:
n_homopolymers[i] += 1
rejects.append( (name,seq,qual,'homopolymer') )
continue
graduates.append( (name, clipped_seq, clipped_qual) )
n_out[i] += 1
total_out_length[i] += len(clipped_seq)
if output_rejects:
for name,seq,qual,reason in rejects:
write_sequence(f_reject, name + ' ' + reason, seq, qual)
if graduates:
if reverse_complement:
graduates = [
(name, bio.reverse_complement(seq), qual[::-1])
for name, seq, qual in graduates
]
if len(graduates) == 1:
n_single += 1
(name, seq, qual) = graduates[0]
write_sequence(f_single, name, seq, qual)
else:
assert len(graduates) == 2
n_paired += 1
# Write the pair to an interleaved file or separate l/r files
for (lr,(name, seq, qual)) in enumerate(graduates):
write_sequence(f_paired[lr%len(f_paired)], name, seq, qual)
grace.status('')
if output_rejects:
f_reject.close()
if fragment_reads == 2:
map(lambda f: f.close(), f_paired)
f_single.close()
def summarize_clips(name, location, clips):
total = 0
for i in clips:
total += len(clips[i])
log.datum(log_name, name + ' adaptors clipped at ' + location, total)
if not clips:
return
for i in xrange(min(clips), max(clips)+1):
item = clips[i]
log.quietly_log('%3d bases: %10d ' % (i, len(item)))
if item:
avg_errors = float(sum( item2[0] for item2 in item )) / len(item)
log.quietly_log(' avg errors: %5.2f ' % avg_errors)
counts = collections.defaultdict(int)
for item2 in item: counts[item2[1]] += 1
#print counts
for no in sorted(counts,key=lambda item2:counts[item2],reverse=True)[:2]:
log.quietly_log('%dx%s ' % (counts[no], matcher.names[no]))
if len(counts) > 2: log.quietly_log('...')
log.quietly_log('\n')
log.quietly_log('\n')
if n_in_paired:
log.datum(log_name,'read-pairs', n_in_paired)
if n_in_single:
log.datum(log_name,'single reads', n_in_single)
for i in xrange(fragment_reads):
if start_clips:
summarize_clips(read_in_fragment_names[i], 'start', start_clips[i])
if end_clips:
summarize_clips(read_in_fragment_names[i], 'end', end_clips[i])
prefix = read_in_fragment_names[i]
log.datum(log_name, prefix + ' too short after quality clip', n_q_clipped[i])
log.datum(log_name, prefix + ' too short after adaptor clip', n_a_clipped[i])
if disallow_homopolymers:
log.datum(log_name, prefix + ' homopolymers', n_homopolymers[i])
if fragment_reads > 1:
log.datum(log_name, prefix + ' kept', n_out[i])
log.datum(log_name, prefix + ' average input length', float(total_in_length[i]) / (n_in_single+n_in_paired))
if n_out[i]:
log.datum(log_name, prefix + ' average output length', float(total_out_length[i]) / n_out[i])
if fragment_reads == 2:
log.datum(log_name,'pairs kept after clipping', n_paired)
log.datum(log_name, 'reads kept after clipping', n_single)
def run(self):
"""
<sequence> <poly-A> <adaptor> <anything>
"""
min_quality = chr(33+self.quality)
with io.open_possibly_compressed_writer(self.prefix+'.fastq.gz') as out_file, \
io.open_possibly_compressed_writer(self.prefix+'.clips.gz') as out_clips_file:
print >> out_clips_file, '#Read\tread length\tpoly-A start\tpoly-A end\tpoly-A start, ignoring adaptor\tpoly-A end, ignoring adaptor\tadaptor bases matched'
n = 0
n_discarded = 0
n_clipped = 0
total_before = 0
total_clipped = 0
for filename in self.filenames:
for name, seq, qual in io.read_sequences(filename, qualities='required'):
best_score = 0
best_a_start = len(seq)
best_a_end = len(seq)
best_adaptor_bases = 0
best_aonly_score = 0
best_aonly_start = len(seq)
best_aonly_end = len(seq)
for a_start in xrange(len(seq)):
if a_start and seq[a_start-1] == 'A': continue
a_end = a_start
aonly_score = 0
while True:
if aonly_score > best_aonly_score:
best_aonly_score = aonly_score
best_aonly_start = a_start
best_aonly_end = a_end
score = aonly_score
adaptor_bases = 0
for i in xrange(a_end,min(a_end+len(self.adaptor),len(seq))):
if qual[i] >= min_quality:
if seq[i] == self.adaptor[i-a_end]:
score += 1
adaptor_bases += 1
else:
score -= 4
if score > best_score:
best_score = score
best_a_start = a_start
best_a_end = a_end
best_adaptor_bases = adaptor_bases
if a_end >= len(seq): break
if qual[a_end] >= min_quality:
if seq[a_end] == 'A':
aonly_score += 1
else:
aonly_score -= 4
if aonly_score <= 0: break
a_end += 1
a_start = best_a_start
a_end = best_a_end
adaptor_bases = best_adaptor_bases
aonly_start = best_aonly_start
aonly_end = best_aonly_end
if self.debug: # and a_end == a_start and a_end < len(seq)-10:
print name
print ''.join( 'X' if item<min_quality else ' ' for item in qual )
print seq
print ' '*a_start + 'A'*(a_end-a_start) + self.adaptor
print ' '*aonly_start + 'A'*(aonly_end-aonly_start)
print
sys.stdout.flush()
n += 1
total_before += len(seq)
# 0 - sequence name
# 1 - sequence length
# 2 - poly(A) start
# 3 - poly(A) end
# (4 - best run of As start, for debugging the need to detect adaptor seq)
# (5 - best run of As end)
# 6 - number of adaptor bases matched
print >> out_clips_file, '%s\t%d\t%d\t%d\t%d\t%d\t%d' % (name, len(seq) , a_start, a_end, aonly_start, aonly_end, adaptor_bases)
if a_start > self.length:
if a_start < len(seq):
n_clipped += 1
total_clipped += a_start
print >> out_file, '@'+name
print >> out_file, seq[:a_start]
print >> out_file, '+'
print >> out_file, qual[:a_start]
else:
n_discarded += 1
if n%10000 == 0:
grace.status('Clip-runs ' + self.sample + ' ' + grace.pretty_number(n)) # + ' (' + grace.pretty_number(len(dstates)) + ' dstates)')
grace.status('')
self.log.datum(self.sample,'reads',n)
if n:
self.log.datum(self.sample,'mean length before poly-A/adaptor clipping',float(total_before)/n)
self.log.datum(self.sample,'reads discarded as too short after poly-A/adaptor clipping',n_discarded)
self.log.datum(self.sample,'reads poly-A/adaptor clipped and kept',n_clipped)
if n_clipped:
self.log.datum(self.sample,'mean length clipped',float(total_clipped)/n_clipped)
def run(self):
assert self.extension is not None, '--extension must be specified'
#workspace = self.get_workspace()
workspace = working_directory.Working(self.working_dir,
must_exist=True)
if self.annotations == None:
reference = workspace.get_reference()
annotations_filename = reference.annotations_filename()
else:
annotations_filename = self.annotations
types = [item.lower() for item in self.types.split(',')]
parts = self.parts or self.types
parts = [item.lower() for item in parts.split(',')]
all_annotations = list(
annotation.read_annotations(annotations_filename))
annotation.link_up_annotations(all_annotations)
for item in all_annotations:
item.primary = None
annotations = [
item for item in all_annotations if item.type.lower() in types
]
part_annotations = []
seen = set()
queue = [(item, item) for item in annotations]
while queue:
primary, item = queue.pop()
if item.type.lower() in parts:
assert item.primary is None, "Feature with multiple parents"
item.primary = primary
key = (id(primary), item.start, item.end, item.seqid,
item.strand)
# Ignore duplicate exons (many isoforms will have the same exons)
if key not in seen:
seen.add(key)
part_annotations.append(item)
queue.extend((primary, item2) for item2 in item.children)
del seen
del all_annotations
self.log.log('%d annotations\n' % len(annotations))
self.log.log('%d part annotations\n' % len(part_annotations))
#assert annotations, 'No annotations of specified types in file'
for item in part_annotations:
this_extension = self.extension
if "max_extension" in item.attr:
this_extension = min(this_extension,
int(item.attr["max_extension"]))
if item.strand >= 0:
item.tail_pos = item.end
item.end += this_extension
else:
item.tail_pos = item.start
item.start -= this_extension
for item in annotations:
item.hits = [] # [ (tail_length, adaptor_bases) ]
index = span_index.index_annotations(part_annotations)
for alignment in sam.Bam_reader(workspace /
'alignments_filtered_sorted.bam'):
if alignment.is_unmapped or alignment.is_secondary or alignment.is_supplementary:
continue
start = alignment.reference_start
end = alignment.reference_end
alignment_length = end - start
strand = -1 if alignment.flag & sam.FLAG_REVERSE else 1
fragment_feature = annotation.Annotation(
seqid=alignment.reference_name,
start=start,
end=end,
strand=strand)
if strand >= 0:
tail_pos = end
else:
tail_pos = start
tail_length = 0
adaptor_bases = 0
for item in alignment.extra:
if item.startswith('AN:i:'):
tail_length = int(item[5:])
elif item.startswith('AD:i:'):
adaptor_bases = int(item[5:])
hits = index.get(fragment_feature, same_strand=True)
if hits:
gene = min(
hits,
key=lambda gene:
(abs(tail_pos - gene.tail_pos), gene.primary.get_id()))
# Nearest by tail_pos
# failing that, by id to ensure a deterministic choice
gene.primary.hits.append((tail_length, adaptor_bases))
for item in annotations:
del item.parents
del item.children
del item.primary
f = io.open_possibly_compressed_writer(self.prefix + '.pickle.gz')
pickle.dump((workspace.name, workspace.get_tags(), annotations), f,
pickle.HIGHEST_PROTOCOL)
f.close()
def run(self):
assert self.extension is not None, '--extension must be specified'
#workspace = self.get_workspace()
workspace = working_directory.Working(self.working_dir, must_exist=True)
if self.annotations == None:
reference = workspace.get_reference()
annotations_filename = reference.annotations_filename()
else:
annotations_filename = self.annotations
types = [ item.lower() for item in self.types.split(',') ]
annotations = [
item
for item in annotation.read_annotations(annotations_filename)
if item.type.lower() in types
]
self.log.log('%d annotations\n' % len(annotations))
assert annotations, 'No annotations of specified types in file'
index = { }
for item in annotations:
if item.strand >= 0:
item.tail_pos = item.end
item.end += self.extension
else:
item.tail_pos = item.start
item.start -= self.extension
if item.seqid not in index:
index[item.seqid] = span_index.Span_index()
index[item.seqid].insert(item)
item.hits = [] # [ (rel_start, rel_end, tail_length) ]
for item in index.itervalues():
item.prepare()
for read_name, fragment_alignments, unmapped in sam.bam_iter_fragments(workspace/'alignments_filtered.bam'):
for fragment in fragment_alignments:
start = min(item.pos-1 for item in fragment)
end = max(item.pos+item.length-1 for item in fragment)
alignment_length = end-start
strand = -1 if fragment[0].flag&sam.FLAG_REVERSE else 1
if strand >= 0:
tail_pos = end
else:
tail_pos = start
tail_length = 0
adaptor_bases = 0
for item in fragment[0].extra:
if item.startswith('AN:i:'):
tail_length = int(item[5:])
elif item.startswith('AD:i:'):
adaptor_bases = int(item[5:])
if fragment[0].rname in index:
hits = [
gene
for gene in index[fragment[0].rname].get(start,end)
if gene.strand == strand
]
if hits:
gene = min(hits, key=lambda gene: (abs(tail_pos - gene.tail_pos), gene.get_id()))
# Nearest by tail_pos
# failing that, by id to ensure a deterministic choice
if strand > 0:
rel_start = start - gene.start
rel_end = end - gene.start
else:
rel_start = gene.end - end
rel_end = gene.end - start
gene.hits.append( (rel_start,rel_end,tail_length,adaptor_bases) )
f = io.open_possibly_compressed_writer(self.prefix + '.pickle.gz')
pickle.dump((workspace.name, workspace.get_tags(), annotations), f, pickle.HIGHEST_PROTOCOL)
f.close()
