def htseq_reader(align_file):
"""
returns a read-by-read sequence reader for a BAM or SAM file
"""
if bam.is_sam(align_file):
read_seq = HTSeq.SAM_Reader(align_file)
elif bam.is_bam(align_file):
read_seq = HTSeq.BAM_Reader(align_file)
else:
logger.error("%s is not a SAM or BAM file" % (align_file))
sys.exit(1)
return read_seq
def htseq_reader(align_file):
"""
returns a read-by-read sequence reader for a BAM or SAM file
"""
if bam.is_sam(align_file):
read_seq = HTSeq.SAM_Reader(align_file)
elif bam.is_bam(align_file):
read_seq = HTSeq.BAM_Reader(align_file)
else:
logger.error("%s is not a SAM or BAM file" % (align_file))
sys.exit(1)
return read_seq
def htseq_count(data):
""" adapted from Simon Anders htseq-count.py script
http://www-huber.embl.de/users/anders/HTSeq/doc/count.html
"""
sam_filename, gff_filename, out_file, stats_file = _get_files(data)
stranded = _get_stranded_flag(data["config"])
overlap_mode = "union"
feature_type = "exon"
id_attribute = "gene_id"
minaqual = 0
if file_exists(out_file):
return out_file
logger.info("Counting reads mapping to exons in %s using %s as the "
"annotation and strandedness as %s." % (os.path.basename(sam_filename),
os.path.basename(gff_filename), _get_strandedness(data["config"])))
features = HTSeq.GenomicArrayOfSets("auto", stranded != "no")
counts = {}
# Try to open samfile to fail early in case it is not there
open(sam_filename).close()
gff = HTSeq.GFF_Reader(gff_filename)
i = 0
try:
for f in gff:
if f.type == feature_type:
try:
feature_id = f.attr[id_attribute]
except KeyError:
sys.exit("Feature %s does not contain a '%s' attribute" %
(f.name, id_attribute))
if stranded != "no" and f.iv.strand == ".":
sys.exit("Feature %s at %s does not have strand "
"information but you are running htseq-count "
"in stranded mode. Use '--stranded=no'." %
(f.name, f.iv))
features[f.iv] += feature_id
counts[f.attr[id_attribute]] = 0
i += 1
if i % 100000 == 0:
sys.stderr.write("%d GFF lines processed.\n" % i)
except:
sys.stderr.write("Error occured in %s.\n"
% gff.get_line_number_string())
raise
sys.stderr.write("%d GFF lines processed.\n" % i)
if len(counts) == 0:
sys.stderr.write("Warning: No features of type '%s' found.\n"
% feature_type)
try:
if bam.is_sam(sam_filename):
read_seq = HTSeq.SAM_Reader(sam_filename)
elif bam.is_bam(sam_filename):
read_seq = HTSeq.BAM_Reader(sam_filename)
first_read = iter(read_seq).next()
pe_mode = first_read.paired_end
except:
sys.stderr.write("Error occured when reading first line of sam "
"file.\n")
raise
try:
if pe_mode:
read_seq_pe_file = read_seq
read_seq = HTSeq.pair_SAM_alignments(read_seq)
empty = 0
ambiguous = 0
notaligned = 0
lowqual = 0
nonunique = 0
i = 0
for r in read_seq:
i += 1
if not pe_mode:
if not r.aligned:
notaligned += 1
continue
try:
if r.optional_field("NH") > 1:
nonunique += 1
continue
except KeyError:
pass
if r.aQual < minaqual:
lowqual += 1
continue
if stranded != "reverse":
iv_seq = (co.ref_iv for co in r.cigar if co.type == "M"
and co.size > 0)
else:
iv_seq = (invert_strand(co.ref_iv) for co in r.cigar if
co.type == "M" and co.size > 0)
else:
if r[0] is not None and r[0].aligned:
if stranded != "reverse":
iv_seq = (co.ref_iv for co in r[0].cigar if
co.type == "M" and co.size > 0)
else:
iv_seq = (invert_strand(co.ref_iv) for co in r[0].cigar if
co.type == "M" and co.size > 0)
else:
iv_seq = tuple()
if r[1] is not None and r[1].aligned:
if stranded != "reverse":
iv_seq = itertools.chain(iv_seq,
(invert_strand(co.ref_iv) for co
in r[1].cigar if co.type == "M"
and co.size > 0))
else:
iv_seq = itertools.chain(iv_seq,
(co.ref_iv for co in r[1].cigar
if co.type == "M" and co.size
> 0))
else:
if (r[0] is None) or not (r[0].aligned):
notaligned += 1
continue
try:
if (r[0] is not None and r[0].optional_field("NH") > 1) or \
(r[1] is not None and r[1].optional_field("NH") > 1):
nonunique += 1
continue
except KeyError:
pass
if (r[0] and r[0].aQual < minaqual) or (r[1] and
r[1].aQual < minaqual):
lowqual += 1
continue
try:
if overlap_mode == "union":
fs = set()
for iv in iv_seq:
if iv.chrom not in features.chrom_vectors:
raise UnknownChrom
for iv2, fs2 in features[iv].steps():
fs = fs.union(fs2)
elif (overlap_mode == "intersection-strict" or
overlap_mode == "intersection-nonempty"):
fs = None
for iv in iv_seq:
if iv.chrom not in features.chrom_vectors:
raise UnknownChrom
for iv2, fs2 in features[iv].steps():
if (len(fs2) > 0 or overlap_mode == "intersection-strict"):
if fs is None:
fs = fs2.copy()
else:
fs = fs.intersection(fs2)
else:
sys.exit("Illegal overlap mode.")
if fs is None or len(fs) == 0:
empty += 1
elif len(fs) > 1:
ambiguous += 1
else:
counts[list(fs)[0]] += 1
except UnknownChrom:
if not pe_mode:
rr = r
else:
rr = r[0] if r[0] is not None else r[1]
empty += 1
if i % 100000 == 0:
sys.stderr.write("%d sam %s processed.\n" % ( i, "lines " if not pe_mode else "line pairs"))
except:
if not pe_mode:
sys.stderr.write("Error occured in %s.\n" % read_seq.get_line_number_string())
else:
sys.stderr.write("Error occured in %s.\n" % read_seq_pe_file.get_line_number_string() )
raise
sys.stderr.write("%d sam %s processed.\n" % (i, "lines " if not pe_mode else "line pairs"))
with file_transaction(out_file) as tmp_out_file:
with open(tmp_out_file, "w") as out_handle:
on_feature = 0
for fn in sorted(counts.keys()):
on_feature += counts[fn]
out_handle.write("%s\t%d\n" % (fn, counts[fn]))
with file_transaction(stats_file) as tmp_stats_file:
with open(tmp_stats_file, "w") as out_handle:
out_handle.write("on_feature\t%d\n" % on_feature)
out_handle.write("no_feature\t%d\n" % empty)
out_handle.write("ambiguous\t%d\n" % ambiguous)
out_handle.write("too_low_aQual\t%d\n" % lowqual)
out_handle.write("not_aligned\t%d\n" % notaligned)
out_handle.write("alignment_not_unique\t%d\n" % nonunique)
return out_file
