def _run(self, _config, temp):
def keyfunc(bed):
return (bed.contig, bed.name, bed.start)
fastafile = pysam.Fastafile(self._reference)
seqs = collections.defaultdict(list)
with open(self._intervals) as bedfile:
intervals = text.parse_lines_by_contig(bedfile, pysam.asBed()).items()
for (contig, beds) in sorted(intervals):
beds.sort(key = keyfunc)
for (gene, gene_beds) in itertools.groupby(beds, lambda x: x.name):
gene_beds = tuple(gene_beds)
for bed in gene_beds:
seqs[(contig, gene)].append(fastafile.fetch(contig, bed.start, bed.end))
seq = "".join(seqs[(contig, gene)])
if any((bed.strand == "-") for bed in gene_beds):
assert all((bed.strand == "-") for bed in gene_beds)
seq = sequences.reverse_complement(seq)
seqs[(contig, gene)] = seq
temp_file = os.path.join(temp, "sequences.fasta")
with open(temp_file, "w") as out_file:
for ((_, gene), sequence) in sorted(seqs.items()):
fasta.print_fasta(gene, sequence, out_file)
move_file(temp_file, self._outfile)
def _run(self, _config, temp):
def keyfunc(bed):
return (bed.contig, bed.name, bed.start)
fastafile = pysam.Fastafile(self._reference)
seqs = collections.defaultdict(list)
with open(self._intervals) as bedfile:
intervals = text.parse_lines_by_contig(bedfile,
pysam.asBed()).items()
for (contig, beds) in sorted(intervals):
beds.sort(key=keyfunc)
for (gene,
gene_beds) in itertools.groupby(beds, lambda x: x.name):
gene_beds = tuple(gene_beds)
for bed in gene_beds:
seqs[(contig, gene)].append(
fastafile.fetch(contig, bed.start, bed.end))
seq = "".join(seqs[(contig, gene)])
if any((bed.strand == "-") for bed in gene_beds):
assert all((bed.strand == "-") for bed in gene_beds)
seq = sequences.reverse_complement(seq)
seqs[(contig, gene)] = seq
temp_file = os.path.join(temp, "sequences.fasta")
with open(temp_file, "w") as out_file:
for ((_, gene), sequence) in sorted(seqs.items()):
fasta.print_fasta(gene, sequence, out_file)
move_file(temp_file, self._outfile)
def _generate_reads(cls, options, rng, sample, minimum, pcr1):
reads = []
while len(reads) < minimum:
name, sequence = sample.get_fragment()
cur_forward = sequence + pcr1
cur_reverse = reverse_complement(PCR2 + sequence)
# Number of PCR copies -- minimum 1
num_dupes = toint(_rexp(options.library_pcr_lambda, rng)) + 1
for dupe_id in xrange(num_dupes):
cur_name = "%s_%s" % (name, dupe_id)
reads.append((cur_name, cur_forward, cur_reverse))
random.shuffle(reads)
return reads
def _generate_reads(cls, options, rng, sample, minimum, pcr1):
reads = []
while len(reads) < minimum:
name, sequence = sample.get_fragment()
cur_forward = sequence + pcr1
cur_reverse = reverse_complement(sequence) + PCR2
# Number of PCR copies -- minimum 1
num_dupes = toint(_rexp(options.library_pcr_lambda, rng)) + 1
for dupe_id in xrange(num_dupes):
cur_name = "%s_%s" % (name, dupe_id)
reads.append((cur_name, cur_forward, cur_reverse))
random.shuffle(reads)
return reads
def write_records(self, records):
for record in records:
seq = record.seq
qual = record.qual
if record.is_reverse:
seq = reverse_complement(seq)
qual = qual[::-1]
assert len(qual) == len(seq), record.qname
self._handle.write("@%s\n" % (record.qname,))
self._handle.write("%s\n" % (seq,))
self._handle.write("+\n")
self._handle.write("%s\n" % (qual,))
def write_records(self, records):
for record in records:
seq = record.seq
qual = record.qual
if record.is_reverse:
seq = reverse_complement(seq)
qual = qual[::-1]
assert len(qual) == len(seq), record.qname
self._handle.write("@%s\n" % (record.qname, ))
self._handle.write("%s\n" % (seq, ))
self._handle.write("+\n")
self._handle.write("%s\n" % (qual, ))
def _get_endogenous_sequence(self):
length = self._get_frag_len()
max_position = len(self._specimen.sequence) - length
position = self._random.randint(0, max_position)
strand = self._random.choice(("fw", "rv"))
sequence = self._specimen.sequence[position:position + length]
real_pos = self._specimen.positions[position]
if strand == "rv":
sequence = reverse_complement("".join(sequence))
self._endog_id += 1
name = "Seq_%i_%i_%i_%s" % (self._endog_id, real_pos, length, strand)
return (True, name, sequence)
def _get_endogenous_sequence(self):
length = self._get_frag_len()
max_position = len(self._specimen.sequence) - length
position = self._random.randint(0, max_position)
strand = self._random.choice(("fw", "rv"))
sequence = self._specimen.sequence[position:position + length]
real_pos = self._specimen.positions[position]
if strand == "rv":
sequence = reverse_complement("".join(sequence))
self._endog_id += 1
name = "Seq_%i_%i_%i_%s" % (self._endog_id, real_pos, length, strand)
return (True, name, sequence)
def _collect_sequence(cls, fastafile, beds):
sequence = []
for bed in beds:
fragment = fastafile.fetch(bed.contig, bed.start, bed.end)
if len(fragment) != (bed.end - bed.start):
cls._report_failure(bed, fragment)
sequence.append(fragment)
sequence = "".join(sequence)
if any((bed.strand == "-") for bed in beds):
assert all((bed.strand == "-") for bed in beds)
sequence = sequtils.reverse_complement(sequence)
return sequence
def _collect_sequence(cls, fastafile, beds):
sequence = []
for bed in beds:
fragment = fastafile.fetch(bed.contig, bed.start, bed.end)
if len(fragment) != (bed.end - bed.start):
cls._report_failure(bed, fragment)
sequence.append(fragment)
sequence = "".join(sequence)
if any((bed.strand == "-") for bed in beds):
assert all((bed.strand == "-") for bed in beds)
sequence = sequtils.reverse_complement(sequence)
return sequence
def build_genes(options, genotype, regions):
def keyfunc(bed):
return (bed.contig, bed.name, bed.start)
regions.sort(key=keyfunc)
for (gene, beds) in itertools.groupby(regions, lambda x: x.name):
sequence, beds = [], tuple(beds)
for bed in beds:
sequence.extend(build_region(options, genotype, bed))
sequence = "".join(sequence)
if any((bed.strand == "-") for bed in beds):
assert all((bed.strand == "-") for bed in beds)
sequence = sequences.reverse_complement(sequence)
yield (gene, sequence)
def build_genes(options, genotype, regions):
def keyfunc(bed):
return (bed.contig, bed.name, bed.start)
regions.sort(key=keyfunc)
for (gene, beds) in itertools.groupby(regions, lambda x: x.name):
sequence, beds = [], tuple(beds)
for bed in beds:
sequence.extend(build_region(options, genotype, bed))
sequence = "".join(sequence)
if any((bed.strand == "-") for bed in beds):
assert all((bed.strand == "-") for bed in beds)
sequence = sequences.reverse_complement(sequence)
yield (gene, sequence)
def _validate_makefile_adapters(makefile):
"""Checks for the default adapter sequences specified in the wrong
orientation for AdapterRemoval, which is a typical mistake when using
the --pcr2 option.
"""
# The non-reverse complemented mate 2 adapter, as seen in raw FASTQ reads
adapter_2 = "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT"
tests = {
# --pcr2 expects the reverse complement of the mate 2 adapter seq.
"--pcr2": adapter_2,
# --adapter2 (AdapterRemoval v2) expects the regular sequence
"--adapter2": sequences.reverse_complement(adapter_2)
}
def check_options(options, results):
for key, value in tests.iteritems():
if options.get(key) == value:
results[key] = True
results = dict.fromkeys(tests, False)
for (_, _, _, _, record) in _iterate_over_records(makefile):
adapterrm_opt = record.get("Options", {}).get("AdapterRemoval", {})
check_options(adapterrm_opt, results)
adapterrm_opt = makefile.get("Options", {}).get("AdapterRemoval", {})
check_options(adapterrm_opt, results)
if any(results.itervalues()):
print_warn(
"WARNING: An adapter specified for AdapterRemoval "
"corresponds to the default sequence, but is reverse "
"complemented. Please make sure that this is intended! ",
end="")
if results["--pcr2"]:
print_warn("For --pcr2, the sequence given should be the "
"reverse complement of the sequence observed in the "
"mate 2 FASTQ file.\n")
if results["--adapter2"]:
print_warn("For --adapter2 (AdapterRemoval v2, only) the value "
"should be exactly as observed in the FASTQ reads.\n")
def _validate_makefile_adapters(makefile):
"""Checks for the default adapter sequences specified in the wrong
orientation for AdapterRemoval, which is a typical mistake when using
the --pcr2 option.
"""
# The non-reverse complemented mate 2 adapter, as seen in raw FASTQ reads
adapter_2 = "AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT"
tests = {
# --pcr2 expects the reverse complement of the mate 2 adapter seq.
"--pcr2": adapter_2,
# --adapter2 (AdapterRemoval v2) expects the regular sequence
"--adapter2": sequences.reverse_complement(adapter_2)
}
def check_options(options, results):
for key, value in tests.iteritems():
if options.get(key) == value:
results[key] = True
results = dict.fromkeys(tests, False)
for (_, _, _, _, record) in _iterate_over_records(makefile):
adapterrm_opt = record.get("Options", {}).get("AdapterRemoval", {})
check_options(adapterrm_opt, results)
adapterrm_opt = makefile.get("Options", {}).get("AdapterRemoval", {})
check_options(adapterrm_opt, results)
if any(results.itervalues()):
print_warn("WARNING: An adapter specified for AdapterRemoval "
"corresponds to the default sequence, but is reverse "
"complemented. Please make sure that this is intended! ",
end="")
if results["--pcr2"]:
print_warn("For --pcr2, the sequence given should be the "
"reverse complement of the sequence observed in the "
"mate 2 FASTQ file.\n")
if results["--adapter2"]:
print_warn("For --adapter2 (AdapterRemoval v2, only) the value "
"should be exactly as observed in the FASTQ reads.\n")
def build_regions(options, genotype, beds, reverse_compl):
for bed in beds:
sequence = build_region(options, genotype, bed)
if reverse_compl:
sequence = sequences.reverse_complement(sequence)
yield sequence
def test_reverse_complement():
assert_equal(reverse_complement(_REF_SRC), _REF_DST[::-1])
def test_reverse_complement():
assert_equal(reverse_complement(_REF_SRC), _REF_DST[::-1])
def build_regions(options, genotype, beds, reverse_compl):
for bed in beds:
sequence = build_region(options, genotype, bed)
if reverse_compl:
sequence = sequences.reverse_complement(sequence)
yield sequence
