def combine_mappings(self):
num_unmapped = 0
num_nonunique = 0
num_unique = 0
mappings = pysam.Samfile(self.file_names['accepted_hits'])
unmapped = pysam.Samfile(self.file_names['unmapped_bam'])
merged = sam.merge_by_name(mappings, unmapped)
grouped = utilities.group_by(merged, lambda m: m.qname)
alignment_sorter = sam.AlignmentSorter(mappings.references,
mappings.lengths,
self.file_names['bam'],
)
with alignment_sorter:
for qname, group in grouped:
unmapped = any(m.is_unmapped for m in group)
if unmapped:
num_unmapped += 1
continue
nonunique = len(group) > 1 or any(m.mapq < 40 for m in group)
if nonunique:
num_nonunique += 1
else:
num_unique += 1
for mapping in group:
alignment_sorter.write(mapping)
self.summary.extend(
[('Unmapped', num_unmapped),
('Nonunique', num_nonunique),
('Unique', num_unique),
],
)
def combine_mappings(self):
num_unmapped = 0
num_nonunique = 0
num_unique = 0
mappings = pysam.Samfile(self.file_names['accepted_hits'])
unmapped = pysam.Samfile(self.file_names['unmapped_bam'])
merged = sam.merge_by_name(mappings, unmapped)
grouped = utilities.group_by(merged, lambda m: m.qname)
alignment_sorter = sam.AlignmentSorter(
mappings.references,
mappings.lengths,
self.file_names['bam'],
)
with alignment_sorter:
for qname, group in grouped:
unmapped = any(m.is_unmapped for m in group)
if unmapped:
num_unmapped += 1
continue
nonunique = len(group) > 1 or any(m.mapq < 40 for m in group)
if nonunique:
num_nonunique += 1
else:
num_unique += 1
for mapping in group:
alignment_sorter.write(mapping)
self.summary.extend([
('Unmapped', num_unmapped),
('Nonunique', num_nonunique),
('Unique', num_unique),
], )
def combine_mappings(self):
num_unmapped = 0
num_five_unmapped = 0
num_three_unmapped = 0
num_nonunique = 0
num_discordant = 0
num_concordant = 0
five_prime_mappings = pysam.Samfile(self.file_names['five_prime_accepted_hits'])
five_prime_unmapped = pysam.Samfile(self.file_names['five_prime_unmapped'])
all_five_prime = sam.merge_by_name(five_prime_mappings, five_prime_unmapped)
five_prime_grouped = utilities.group_by(all_five_prime, lambda m: m.qname)
three_prime_mappings = pysam.Samfile(self.file_names['three_prime_accepted_hits'])
three_prime_unmapped = pysam.Samfile(self.file_names['three_prime_unmapped'])
all_three_prime = sam.merge_by_name(three_prime_mappings, three_prime_unmapped)
three_prime_grouped = utilities.group_by(all_three_prime, lambda m: m.qname)
group_pairs = izip(five_prime_grouped, three_prime_grouped)
alignment_sorter = sam.AlignmentSorter(five_prime_mappings.references,
five_prime_mappings.lengths,
self.file_names['combined_extended'],
)
region_fetcher = genomes.build_region_fetcher(self.file_names['genome'],
load_references=True,
sam_file=five_prime_mappings,
)
with alignment_sorter:
for (five_qname, five_group), (three_qname, three_group) in group_pairs:
five_annotation = trim.PayloadAnnotation.from_identifier(five_qname)
three_annotation = trim.PayloadAnnotation.from_identifier(three_qname)
if five_annotation['original_name'] != three_annotation['original_name']:
# Ensure that the iteration through pairs is in sync.
print five_qname, three_qname
raise ValueError
five_unmapped = any(m.is_unmapped for m in five_group)
three_unmapped = any(m.is_unmapped for m in three_group)
if five_unmapped:
num_five_unmapped += 1
if three_unmapped:
num_three_unmapped += 1
if five_unmapped or three_unmapped:
num_unmapped += 1
continue
five_nonunique = len(five_group) > 1 or any(m.mapq < 40 for m in five_group)
three_nonunique = len(three_group) > 1 or any(m.mapq < 40 for m in three_group)
if five_nonunique or three_nonunique:
num_nonunique += 1
continue
five_m = five_group.pop()
three_m = three_group.pop()
five_strand = '-' if five_m.is_reverse else '+'
three_strand = '-' if three_m.is_reverse else '+'
tlen = max(five_m.aend, three_m.aend) - min(five_m.pos, three_m.pos)
discordant = (five_m.tid != three_m.tid) or (five_strand) != (three_strand) or (tlen > 10000)
if discordant:
num_discordant += 1
continue
if five_strand == '+':
first_read = five_m
second_read = three_m
elif five_strand == '-':
first_read = three_m
second_read = five_m
gap = second_read.pos - first_read.aend
if gap < 0:
num_discordant += 1
continue
combined_read = pysam.AlignedRead()
# qname needs to come from three_m to include trimmed As
combined_read.qname = three_m.qname
combined_read.tid = five_m.tid
combined_read.seq = first_read.seq + second_read.seq
combined_read.qual = first_read.qual + second_read.qual
combined_read.cigar = first_read.cigar + [(3, gap)] + second_read.cigar
combined_read.pos = first_read.pos
combined_read.is_reverse = first_read.is_reverse
combined_read.mapq = min(first_read.mapq, second_read.mapq)
combined_read.rnext = -1
combined_read.pnext = -1
num_concordant += 1
extended_mapping = trim.extend_polyA_end(combined_read,
region_fetcher,
)
alignment_sorter.write(extended_mapping)
self.summary.extend(
[('Unmapped', num_unmapped),
('Five prime unmapped', num_five_unmapped),
('Three prime unmapped', num_three_unmapped),
('Nonunique', num_nonunique),
('Discordant', num_discordant),
('Concordant', num_concordant),
],
)
def combine_mappings(self):
num_unmapped = 0
num_R1_unmapped = 0
num_R2_unmapped = 0
num_nonunique = 0
num_discordant = 0
num_disoriented = 0
num_concordant = 0
tlens = Counter()
R1_mappings = pysam.Samfile(self.file_names['R1_accepted_hits'])
R1_unmapped = pysam.Samfile(self.file_names['R1_unmapped'])
all_R1 = sam.merge_by_name(R1_mappings, R1_unmapped)
R1_grouped = utilities.group_by(all_R1, lambda m: m.qname)
R2_mappings = pysam.Samfile(self.file_names['R2_accepted_hits'])
R2_unmapped = pysam.Samfile(self.file_names['R2_unmapped'])
all_R2 = sam.merge_by_name(R2_mappings, R2_unmapped)
R2_grouped = utilities.group_by(all_R2, lambda m: m.qname)
group_pairs = izip(R1_grouped, R2_grouped)
alignment_sorter = sam.AlignmentSorter(R1_mappings.references,
R1_mappings.lengths,
self.file_names['combined'],
)
with alignment_sorter:
for (R1_qname, R1_group), (R2_qname, R2_group) in group_pairs:
#print R1_qname, R2_qname
if fastq.get_pair_name(R1_qname) != fastq.get_pair_name(R2_qname):
# Ensure that the iteration through pairs is in sync.
print R1_qname, R2_qname
raise ValueError
R1_unmapped = any(m.is_unmapped for m in R1_group)
R2_unmapped = any(m.is_unmapped for m in R2_group)
if R1_unmapped:
num_R1_unmapped += 1
if R2_unmapped:
num_R2_unmapped += 1
if R1_unmapped or R2_unmapped:
num_unmapped += 1
continue
R1_nonunique = len(R1_group) > 1 or any(m.mapq < 40 for m in R1_group)
R2_nonunique = len(R2_group) > 1 or any(m.mapq < 40 for m in R2_group)
if R1_nonunique or R2_nonunique:
num_nonunique += 1
continue
R1_m = R1_group.pop()
R2_m = R2_group.pop()
R1_strand = sam.get_strand(R1_m)
R2_strand = sam.get_strand(R2_m)
tlen = max(R1_m.aend, R2_m.aend) - min(R1_m.pos, R2_m.pos)
discordant = (R1_m.tid != R2_m.tid) or (R1_strand) == (R2_strand) or (tlen > 10000)
if discordant:
num_discordant += 1
continue
# Reminder: the protocol produces anti-sense reads.
if R1_strand == '-':
if R1_m.pos < R2_m.pos:
num_disoriented += 1
continue
elif R1_strand == '+':
if R2_m.pos < R1_m.pos:
num_disoriented += 1
continue
combined_read = paired_end.combine_paired_mappings(R1_m, R2_m)
tlens[tlen] += 1
if combined_read:
# Flip combined_read back to the sense strand.
if combined_read.is_reverse:
combined_read.is_reverse = False
else:
combined_read.is_reverse = True
trim.set_nongenomic_length(combined_read, 0)
alignment_sorter.write(combined_read)
num_concordant += 1
self.summary.extend(
[('Unmapped', num_unmapped),
('R1 unmapped', num_R1_unmapped),
('R2 unmapped', num_R2_unmapped),
('Nonunique', num_nonunique),
('Discordant', num_discordant),
('Unexpected orientation', num_disoriented),
('Concordant', num_concordant),
],
)
tlens = utilities.counts_to_array(tlens)
self.write_file('tlens', tlens)
