def get_allele_counts_insertions_from_file_unfiltered(bamfilename,
length,
qual_min=30,
match_len_min=10,
maxreads=-1,
VERBOSE=0):
'''Get the allele counts and insertions
Parameters:
- maxreads: limit the counts to a random subset of the reads of this size
'''
# Prepare output structures
counts = np.zeros((len(read_types), len(alpha), length), int)
# Note: the data structure for inserts is a nested dict with:
# position --> string --> read type --> count
# (dict) (dict) (list) (int)
inserts = defaultdict(
lambda: defaultdict(lambda: np.zeros(len(read_types), int)))
# Open BAM file
# Note: the reads should already be filtered of unmapped stuff at this point
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if maxreads != -1:
from hivwholeseq.utils.mapping import extract_mapped_reads_subsample_open
read_iter = extract_mapped_reads_subsample_open(bamfile,
maxreads,
VERBOSE=VERBOSE,
pairs=False)
else:
read_iter = bamfile
# Iterate over single reads
for i, read in enumerate(read_iter):
# Max number of reads
if i == maxreads:
if VERBOSE >= 2:
print 'Max reads reached:', maxreads
break
# Print output
if (VERBOSE >= 3) and (not ((i + 1) % 10000)):
print(i + 1)
# NOTE: since we change the consensus all the time, mapping is never
# safe, and we have to filter the results thoroughly.
# If unmapped/unpaired, trash
if read.is_unmapped or (not read.is_proper_pair) or (read.isize
== 0):
if VERBOSE >= 3:
print 'Read ' + read.qname + ': unmapped/unpaired/no isize'
continue
# Get good CIGARs
(good_cigars, first_good_cigar, last_good_cigar) = \
get_ind_good_cigars(read.cigar, match_len_min=match_len_min,
full_output=True)
# If no good CIGARs, give up
if not good_cigars.any():
continue
# Divide by read 1/2 and forward/reverse
js = 2 * read.is_read2 + read.is_reverse
# Read CIGARs
seq = np.fromstring(read.seq, 'S1')
qual = np.fromstring(read.qual, np.int8) - 33
pos = read.pos
cigar = read.cigar
len_cig = len(cigar)
# Iterate over CIGARs
for ic, (block_type, block_len) in enumerate(cigar):
# Check for pos: it should never exceed the length of the fragment
if (block_type in [0, 1, 2]) and (pos > length):
raise ValueError('Pos exceeded the length of the fragment')
# Inline block
if block_type == 0:
# Keep only stuff from good CIGARs
if first_good_cigar <= ic <= last_good_cigar:
seqb = seq[:block_len]
qualb = qual[:block_len]
# Increment counts
for j, a in enumerate(alpha):
posa = ((seqb == a) &
(qualb >= qual_min)).nonzero()[0]
if len(posa):
counts[js, j, pos + posa] += 1
# Chop off this block
if ic != len_cig - 1:
seq = seq[block_len:]
qual = qual[block_len:]
pos += block_len
# Deletion
elif block_type == 2:
# Keep only stuff from good CIGARs
if first_good_cigar <= ic <= last_good_cigar:
# Increment gap counts
counts[js, 4, pos:pos + block_len] += 1
# Chop off pos, but not sequence
pos += block_len
# Insertion
# an insert @ pos 391 means that seq[:391] is BEFORE the insert,
# THEN the insert, FINALLY comes seq[391:]
elif block_type == 1:
# Keep only stuff from good CIGARs
if first_good_cigar <= ic <= last_good_cigar:
seqb = seq[:block_len]
qualb = qual[:block_len]
# Accept only high-quality inserts
if (qualb >= qual_min).all():
inserts[pos][seqb.tostring()][js] += 1
# Chop off seq, but not pos
if ic != len_cig - 1:
seq = seq[block_len:]
qual = qual[block_len:]
# Other types of cigar?
else:
raise ValueError('CIGAR type ' + str(block_type) +
' not recognized')
return (counts, inserts)
def get_allele_counts_insertions_from_file_unfiltered(bamfilename, length, qual_min=30,
match_len_min=10,
maxreads=-1, VERBOSE=0):
'''Get the allele counts and insertions
Parameters:
- maxreads: limit the counts to a random subset of the reads of this size
'''
# Prepare output structures
counts = np.zeros((len(read_types), len(alpha), length), int)
# Note: the data structure for inserts is a nested dict with:
# position --> string --> read type --> count
# (dict) (dict) (list) (int)
inserts = defaultdict(lambda: defaultdict(lambda: np.zeros(len(read_types), int)))
# Open BAM file
# Note: the reads should already be filtered of unmapped stuff at this point
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if maxreads != -1:
from hivwholeseq.utils.mapping import extract_mapped_reads_subsample_open
read_iter = extract_mapped_reads_subsample_open(bamfile, maxreads,
VERBOSE=VERBOSE,
pairs=False)
else:
read_iter = bamfile
# Iterate over single reads
for i, read in enumerate(read_iter):
# Max number of reads
if i == maxreads:
if VERBOSE >= 2:
print 'Max reads reached:', maxreads
break
# Print output
if (VERBOSE >= 3) and (not ((i +1) % 10000)):
print (i+1)
# NOTE: since we change the consensus all the time, mapping is never
# safe, and we have to filter the results thoroughly.
# If unmapped/unpaired, trash
if read.is_unmapped or (not read.is_proper_pair) or (read.isize == 0):
if VERBOSE >= 3:
print 'Read '+read.qname+': unmapped/unpaired/no isize'
continue
# Get good CIGARs
(good_cigars, first_good_cigar, last_good_cigar) = \
get_ind_good_cigars(read.cigar, match_len_min=match_len_min,
full_output=True)
# If no good CIGARs, give up
if not good_cigars.any():
continue
# Divide by read 1/2 and forward/reverse
js = 2 * read.is_read2 + read.is_reverse
# Read CIGARs
seq = np.fromstring(read.seq, 'S1')
qual = np.fromstring(read.qual, np.int8) - 33
pos = read.pos
cigar = read.cigar
len_cig = len(cigar)
# Iterate over CIGARs
for ic, (block_type, block_len) in enumerate(cigar):
# Check for pos: it should never exceed the length of the fragment
if (block_type in [0, 1, 2]) and (pos > length):
raise ValueError('Pos exceeded the length of the fragment')
# Inline block
if block_type == 0:
# Keep only stuff from good CIGARs
if first_good_cigar <= ic <= last_good_cigar:
seqb = seq[:block_len]
qualb = qual[:block_len]
# Increment counts
for j, a in enumerate(alpha):
posa = ((seqb == a) & (qualb >= qual_min)).nonzero()[0]
if len(posa):
counts[js, j, pos + posa] += 1
# Chop off this block
if ic != len_cig - 1:
seq = seq[block_len:]
qual = qual[block_len:]
pos += block_len
# Deletion
elif block_type == 2:
# Keep only stuff from good CIGARs
if first_good_cigar <= ic <= last_good_cigar:
# Increment gap counts
counts[js, 4, pos:pos + block_len] += 1
# Chop off pos, but not sequence
pos += block_len
# Insertion
# an insert @ pos 391 means that seq[:391] is BEFORE the insert,
# THEN the insert, FINALLY comes seq[391:]
elif block_type == 1:
# Keep only stuff from good CIGARs
if first_good_cigar <= ic <= last_good_cigar:
seqb = seq[:block_len]
qualb = qual[:block_len]
# Accept only high-quality inserts
if (qualb >= qual_min).all():
inserts[pos][seqb.tostring()][js] += 1
# Chop off seq, but not pos
if ic != len_cig - 1:
seq = seq[block_len:]
qual = qual[block_len:]
# Other types of cigar?
else:
raise ValueError('CIGAR type '+str(block_type)+' not recognized')
return (counts, inserts)
if sample[fragment] not in ['ok', 'low']:
if VERBOSE >= 1:
print 'not "ok". skipping'
continue
if VERBOSE >= 1:
print 'ok'
bamfilename = sample.get_mapped_filtered_filename(
fragment, decontaminated=True)
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if maxreads == -1:
reads = pair_generator(bamfile)
else:
reads = extract_mapped_reads_subsample_open(
bamfile, maxreads, VERBOSE=VERBOSE)
dists[sample.name] = get_distance_reads_sequence(
refseq,
reads,
VERBOSE=VERBOSE,
score_match=3,
score_mismatch=-3)
hs = {}
binmax = max(map(max, dists.itervalues()))
bins = np.arange(0, binmax, 6)
bincenters = 0.5 * (bins[1:] + bins[:-1])
for samplename, dist in dists.iteritems():
hs[samplename] = np.histogram(dist, bins=bins, density=True)[0]
def get_local_block(bamfilename,
start,
end,
VERBOSE=0,
maxreads=-1,
refroi=None):
'''Extract reads fully covering the region, discarding insertions'''
import sys
import pysam
from hivwholeseq.utils.mapping import pair_generator
from hivwholeseq.utils.mapping import extract_mapped_reads_subsample_open
with pysam.Samfile(bamfilename, 'rb') as bamfile:
block = []
if maxreads == -1:
reads_iter = pair_generator(bamfile)
else:
reads_iter = extract_mapped_reads_subsample_open(bamfile,
maxreads,
VERBOSE=VERBOSE,
pairs=True)
for irp, reads in enumerate(reads_iter):
if VERBOSE >= 2:
if not ((irp + 1) % 10000):
if irp + 1 != 10000:
sys.stdout.write("\x1b[1A\n")
sys.stdout.write(str(irp + 1))
sys.stdout.flush()
# Sort fwd read first
is_fwd = reads[0].is_reverse
reads = [reads[is_fwd], reads[not is_fwd]]
# Check for coverage of the region
start_fwd = reads[0].pos
end_fwd = start_fwd + sum(
bl for (bt, bl) in reads[0].cigar if bt in (0, 2))
start_rev = reads[1].pos
end_rev = start_rev + sum(
bl for (bt, bl) in reads[1].cigar if bt in (0, 2))
if start_fwd > start:
continue
if end_rev < end:
continue
if (end_fwd < end) and (start_rev > start) and (start_rev >
end_fwd):
continue
if VERBOSE >= 3:
print ' '.join(
map('{:>4d}'.format,
[start_fwd, end_fwd, start_rev, end_rev]))
# Gather info from both reads, merge by putting ambiguous nucleotides
seqs = []
st_ens = [[start_fwd, end_fwd], [start_rev, end_rev]]
for ir, read in enumerate(reads):
(start_read, end_read) = st_ens[ir]
if (end_read <= start) or (start_read >= end):
seqs.append(None)
continue
seq = []
pos_ref = start_read
pos_read = 0
start_block = max(start, start_read) - start
end_block = min(end, end_read) - start
for (bt, bl) in read.cigar:
if bt == 1:
pos_read += bl
elif bt == 2:
if pos_ref + bl > start:
st = max(0, start - pos_ref)
en = min(bl, end - pos_ref)
seq.append('-' * (en - st))
if pos_ref + bl >= end:
break
pos_ref += bl
elif bt == 0:
if pos_ref + bl > start:
st = max(0, start - pos_ref)
en = min(bl, end - pos_ref)
seq.append(read.seq[pos_read + st:pos_read + en])
if pos_ref + bl >= end:
break
pos_ref += bl
pos_read += bl
seq = ''.join(seq)
seqs.append((start_block, end_block, seq))
# Merge sequences if both fwd and rev cover part of it
if seqs[0] is None:
seq = seqs[1][2]
elif seqs[1] is None:
seq = seqs[0][2]
else:
# The fwd read starts before the rev, because of our insert sizes
end_block_fwd = seqs[0][1]
start_block_rev = seqs[1][0]
overlap = [
seqs[0][2][start_block_rev:],
seqs[1][2][:end_block_fwd - start_block_rev]
]
# The two reads in a pair should have the same length in the overlap
if len(overlap[0]) != len(overlap[1]):
if VERBOSE >= 3:
print 'WARNING:', reads[
0].qname, 'not same length in overlap!'
continue
ol_fwd = np.fromstring(overlap[0], 'S1')
ol_rev = np.fromstring(overlap[1], 'S1')
ol_fwd[ol_fwd != ol_rev] = 'N'
seq = seqs[0][2][:start_block_rev] + \
ol_fwd.tostring() + \
seqs[1][2][end_block_fwd - start_block_rev:]
block.append(seq)
if VERBOSE >= 2:
print ''
return block
print sample.name,
if sample[fragment] not in ['ok', 'low']:
if VERBOSE >= 1:
print 'not "ok". skipping'
continue
if VERBOSE >= 1:
print 'ok'
bamfilename = sample.get_mapped_filtered_filename(fragment, decontaminated=True)
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if maxreads == -1:
reads = pair_generator(bamfile)
else:
reads = extract_mapped_reads_subsample_open(bamfile, maxreads,
VERBOSE=VERBOSE)
dists[sample.name] = get_distance_reads_sequence(refseq, reads,
VERBOSE=VERBOSE,
score_match=3,
score_mismatch=-3)
hs = {}
binmax = max(map(max, dists.itervalues()))
bins = np.arange(0, binmax, 6)
bincenters = 0.5 * (bins[1:] + bins[:-1])
for samplename, dist in dists.iteritems():
hs[samplename] = np.histogram(dist, bins=bins, density=True)[0]
fig, ax = plt.subplots(figsize=(10, 5))
def get_local_haplotypes(bamfilename, start, end, VERBOSE=0, maxreads=-1,
label=''):
'''Extract reads fully covering the region, discarding insertions'''
import sys
import pysam
from hivwholeseq.utils.mapping import pair_generator
from hivwholeseq.utils.mapping import extract_mapped_reads_subsample_open
from collections import Counter
haplotypes = Counter()
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if maxreads == -1:
reads_iter = pair_generator(bamfile)
else:
reads_iter = extract_mapped_reads_subsample_open(bamfile, maxreads,
VERBOSE=VERBOSE,
pairs=True)
for irp, reads in enumerate(reads_iter):
if VERBOSE >= 2:
if not ((irp + 1) % 10000):
if irp + 1 != 10000:
sys.stdout.write("\x1b[1A\n")
if label:
sys.stdout.write(label+'\t')
sys.stdout.write(str(irp + 1))
sys.stdout.flush()
# Sort fwd read first: this is important because with our insert
# size we know the fwd read starts <= the rev read
is_fwd = reads[0].is_reverse
reads = [reads[is_fwd], reads[not is_fwd]]
# Check for coverage of the region
start_fwd = reads[0].pos
end_fwd = start_fwd + sum(bl for (bt, bl) in reads[0].cigar if bt in (0, 2))
start_rev = reads[1].pos
end_rev = start_rev + sum(bl for (bt, bl) in reads[1].cigar if bt in (0, 2))
overlap_len = max(0, end_fwd - start_rev)
# Various scenarios possible
if start_fwd > start:
continue
if end_rev < end:
continue
# No single read covers the whole region AND (the insert has a whole
# OR a very short overlap)
if (end_fwd < end) and (start_rev > start) and (overlap_len < 20):
continue
# Now the good cases
if (start_fwd <= start) and (end_fwd >= end):
seq = trim_read_roi(reads[0], start, end)
elif (start_rev <= start) and (end_rev >= end):
seq = trim_read_roi(reads[1], start, end)
else:
seqs = [trim_read_roi(read, start, end) for read in reads]
seq = merge_read_pair(*seqs)
haplotypes[seq] += 1
if VERBOSE >= 4:
import ipdb; ipdb.set_trace()
if VERBOSE >= 2:
if irp >= 10000:
sys.stdout.write('\n')
sys.stdout.flush()
return haplotypes
def get_local_haplotypes(bamfilename,
start,
end,
VERBOSE=0,
maxreads=-1,
label=''):
'''Extract reads fully covering the region, discarding insertions'''
import sys
import pysam
from hivwholeseq.utils.mapping import pair_generator
from hivwholeseq.utils.mapping import extract_mapped_reads_subsample_open
from collections import Counter
haplotypes = Counter()
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if maxreads == -1:
reads_iter = pair_generator(bamfile)
else:
reads_iter = extract_mapped_reads_subsample_open(bamfile,
maxreads,
VERBOSE=VERBOSE,
pairs=True)
for irp, reads in enumerate(reads_iter):
if VERBOSE >= 2:
if not ((irp + 1) % 10000):
if irp + 1 != 10000:
sys.stdout.write("\x1b[1A\n")
if label:
sys.stdout.write(label + '\t')
sys.stdout.write(str(irp + 1))
sys.stdout.flush()
# Sort fwd read first: this is important because with our insert
# size we know the fwd read starts <= the rev read
is_fwd = reads[0].is_reverse
reads = [reads[is_fwd], reads[not is_fwd]]
# Check for coverage of the region
start_fwd = reads[0].pos
end_fwd = start_fwd + sum(
bl for (bt, bl) in reads[0].cigar if bt in (0, 2))
start_rev = reads[1].pos
end_rev = start_rev + sum(
bl for (bt, bl) in reads[1].cigar if bt in (0, 2))
overlap_len = max(0, end_fwd - start_rev)
# Various scenarios possible
if start_fwd > start:
continue
if end_rev < end:
continue
# No single read covers the whole region AND (the insert has a whole
# OR a very short overlap)
if (end_fwd < end) and (start_rev > start) and (overlap_len < 20):
continue
# Now the good cases
if (start_fwd <= start) and (end_fwd >= end):
seq = trim_read_roi(reads[0], start, end)
elif (start_rev <= start) and (end_rev >= end):
seq = trim_read_roi(reads[1], start, end)
else:
seqs = [trim_read_roi(read, start, end) for read in reads]
seq = merge_read_pair(*seqs)
haplotypes[seq] += 1
if VERBOSE >= 4:
import ipdb
ipdb.set_trace()
if VERBOSE >= 2:
if irp >= 10000:
sys.stdout.write('\n')
sys.stdout.flush()
return haplotypes
def quality_score_along_reads_mapped(read_len, bamfilename,
insertsize_range=[400, 1000],
skipreads=0,
maxreads=-1,
randomreads=True,
VERBOSE=0):
'''Calculate the quality score along the reads'''
from hivwholeseq.utils.mapping import trim_read_pair_crossoverhangs as trim_coh
from hivwholeseq.utils.mapping import pair_generator
quality = [[[] for j in xrange(read_len)] for i in xrange(2)]
# Precompute conversion table
SANGER_SCORE_OFFSET = ord("!")
q_mapping = dict()
for letter in range(0, 255):
q_mapping[chr(letter)] = letter - SANGER_SCORE_OFFSET
# Iterate over all reads (using fast iterators)
with pysam.Samfile(bamfilename, 'rb') as bamfile:
if not randomreads:
reads_all = []
for i, read_pair in enumerate(pair_generator(bamfile)):
if i < skipreads:
continue
if i == skipreads + maxreads:
if VERBOSE:
print 'Maximal number of read pairs reached:', maxreads
break
if VERBOSE and (not ((i + 1) % 10000)):
print i + 1
reads_all.append(read_pair)
else:
reads_all = extract_mapped_reads_subsample_open(bamfile, maxreads,
VERBOSE=VERBOSE)
print len(reads_all)
for reads in reads_all:
# Check insert size
read = reads[reads[0].is_reverse]
if (read.is_unmapped or (not read.is_proper_pair) or \
(read.isize < insertsize_range[0]) or \
(read.isize >= insertsize_range[1])):
continue
trim_coh(reads, trim=5, include_tests=False)
pos_read = 0
for read in reads:
ip = read.is_read2
for (bt, bl) in read.cigar:
if bt == 1:
pos_read += bl
elif bt == 2:
pass
elif bt == 0:
qualb = read.qual[pos_read: pos_read + bl]
poss_read = np.arange(pos_read, pos_read + bl)
if read.is_reverse:
poss_read = len(read.seq) - 1 - poss_read
for j, qletter in izip(poss_read, qualb):
quality[ip][j].append(q_mapping[qletter])
for qual in quality:
for qpos in qual:
qpos.sort()
return quality
