def _analyze_lifted_line(line, reads, precursors, database):
query_name = line[0]
sequence = line[1]
logger.debug(("READ::line name:{0}").format(line))
if sequence and sequence.find("N") > -1:
return reads
if query_name not in reads:
reads[query_name].set_sequence(sequence)
reads[query_name].counts = _get_freq(query_name)
reads[query_name].sequence = sequence
chrom = line[2]
start = line[3]
iso = isomir()
iso.align = line
iso.set_pos(start, len(reads[query_name].sequence))
logger.debug("READ::From BAM start %s end %s at chrom %s" %
(iso.start, iso.end, chrom))
if len(precursors[chrom]) < start + len(reads[query_name].sequence):
logger.debug(
"READ::%s start + %s sequence size are bigger than"
" size precursor %s" %
(chrom, len(reads[query_name].sequence), len(precursors[chrom])))
return reads
iso.subs, iso.add, iso.cigar = filter.tune(reads[query_name].sequence,
precursors[chrom], start, None)
logger.debug("READ::After tune start %s end %s" % (iso.start, iso.end))
logger.debug("READ::iso add %s iso subs %s" % (iso.add, iso.subs))
reads[query_name].set_precursor(chrom, iso)
return reads
def read_bam(bam_fn, args, clean=True):
"""
Read bam file and perform realignment of hits
Args:
*bam_fn*: a BAM file with alignments to the precursor
*precursors*: dict with keys being precursor names and values
being sequences. Come from mirtop.mirna.fasta.read_precursor().
*clean*: Use mirtop.filter.clean_hits() to remove lower score hits.
Returns:
*reads (dict)*:
keys are read_id and values are *mirtop.realign.hits*
"""
precursors = args.precursors
bam_fn = _sam_to_bam(bam_fn)
bam_fn = _bam_sort(bam_fn)
mode = "r" if bam_fn.endswith("sam") else "rb"
handle = pysam.Samfile(bam_fn, mode)
reads = defaultdict(hits)
for line in handle:
if line.reference_id < 0:
logger.debug("Sequence not mapped: %s" % line.reference_id)
continue
query_name = line.query_name
# if query_name not in reads and line.query_sequence:
# continue
if line.query_sequence and line.query_sequence.find("N") > -1:
continue
if query_name not in reads:
reads[query_name].set_sequence(line.query_sequence)
reads[query_name].counts = _get_freq(query_name)
if line.is_reverse:
logger.debug("Sequence is reverse: %s" % line.query_name)
continue
chrom = handle.getrname(line.reference_id)
cigar = line.cigartuples
iso = isomir()
iso.align = line
iso.set_pos(line.reference_start, len(reads[query_name].sequence))
logger.debug("READ::From BAM start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < line.reference_start + len(
reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(reads[query_name].sequence,
precursors[chrom],
line.reference_start, cigar)
logger.debug("READ::After tune start %s end %s" % (iso.start, iso.end))
if len(iso.subs) < 2:
reads[query_name].set_precursor(chrom, iso)
logger.info("Hits: %s" % len(reads))
if clean:
reads = filter.clean_hits(reads)
logger.info("Hits after clean: %s" % len(reads))
return reads
def read_bam(bam_fn, args, clean=True):
"""
Read bam file and perform realignment of hits
Args:
*bam_fn*: a BAM file with alignments to the precursor
*precursors*: dict with keys being precursor names and values
being sequences. Come from mirtop.mirna.fasta.read_precursor().
*clean*: Use mirtop.filter.clean_hits() to remove lower score hits.
Returns:
*reads (dict)*:
keys are read_id and values are *mirtop.realign.hits*
"""
precursors = args.precursors
bam_fn = _sam_to_bam(bam_fn)
bam_fn = _bam_sort(bam_fn)
mode = "r" if bam_fn.endswith("sam") else "rb"
handle = pysam.Samfile(bam_fn, mode)
reads = defaultdict(hits)
for line in handle:
if line.reference_id < 0:
logger.debug("Sequence not mapped: %s" % line.reference_id)
continue
query_name = line.query_name
# if query_name not in reads and line.query_sequence:
# continue
if line.query_sequence and line.query_sequence.find("N") > -1:
continue
if query_name not in reads:
reads[query_name].set_sequence(line.query_sequence)
reads[query_name].counts = _get_freq(query_name)
if line.is_reverse:
logger.debug("Sequence is reverse: %s" % line.query_name)
continue
chrom = handle.getrname(line.reference_id)
cigar = line.cigartuples
iso = isomir()
iso.align = line
iso.set_pos(line.reference_start, len(reads[query_name].sequence))
logger.debug("READ::From BAM start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < line.reference_start + len(reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(
reads[query_name].sequence, precursors[chrom],
line.reference_start, cigar)
logger.debug("READ::After tune start %s end %s" % (iso.start, iso.end))
if len(iso.subs) < 2:
reads[query_name].set_precursor(chrom, iso)
logger.info("Hits: %s" % len(reads))
if clean:
reads = filter.clean_hits(reads)
logger.info("Hits after clean: %s" % len(reads))
return reads
def read_file(fn, args):
"""
Read seqbuster file and convert to mirtop GFF format.
Args:
*fn(str)*: file name with seqbuster output information.
*database(str)*: database name.
*args(namedtuple)*: arguments from command line.
See *mirtop.libs.parse.add_subparser_gff()*.
Returns:
*reads*: dictionary where keys are read_id and values are *mirtop.realign.hits*
"""
precursors = args.precursors
reads = defaultdict(hits)
with open(fn) as handle:
handle.readline()
for line in handle:
cols = line.strip().split("\t")
query_name = cols[1]
query_sequence = cols[0]
reference_start = int(cols[4]) - 1
seqbuster_iso = ":".join(cols[6:10])
if query_name not in reads and query_sequence == None:
continue
if query_sequence and query_sequence.find("N") > -1:
continue
if query_name not in reads:
reads[query_name].set_sequence(query_sequence)
reads[query_name].counts = _get_freq(query_name)
chrom = cols[13]
logger.debug("\nSEQBUSTER::NEW::query: {query_sequence}\n"
" precursor {chrom}\n"
" name: {query_name}\n"
" start: {reference_start}\n"
" iso: {seqbuster_iso}".format(**locals()))
# logger.debug("SEQBUSTER:: cigar {cigar}".format(**locals()))
iso = isomir()
iso.align = line
iso.set_pos(reference_start, len(reads[query_name].sequence))
logger.debug("SEQBUSTER:: start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < reference_start + len(
reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(
reads[query_name].sequence, precursors[chrom], reference_start,
None)
logger.debug("SEQBUSTER::After tune start %s end %s" %
(iso.start, iso.end))
if len(iso.subs) < 2:
reads[query_name].set_precursor(chrom, iso)
logger.info("Hits: %s" % len(reads))
return reads
def read_file(fn, precursors):
"""
read srnabench file and perform realignment of hits
"""
reads = defaultdict(hits)
with open(fn) as handle:
for line in handle:
cols = line.strip().split("\t")
query_name = cols[0]
query_sequence = cols[0]
if query_name not in reads and query_sequence == None:
continue
if query_sequence and query_sequence.find("N") > -1:
continue
if cols[3].find("mature") == -1:
continue
if query_name not in reads:
reads[query_name].set_sequence(query_sequence)
reads[query_name].counts = _get_freq(int(cols[1]))
for hit in cols[4].split("$"):
logger.debug("SRNABENCH::line hit: %s" % hit)
hit_info = hit.split("#")
pos_info = hit_info[3].split(",")
reference_start = int(pos_info[1]) - 1
chrom = pos_info[0]
logger.debug("SRNABENCH::query: {query_sequence}\n"
" precursor {chrom}\n"
" name: {query_name}\n"
" start: {reference_start}\n"
" hit: {hit}".format(**locals()))
iso = isomir()
iso.align = line
iso.set_pos(reference_start, len(reads[query_name].sequence))
logger.debug("SRNABENCH:: start %s end %s" %
(iso.start, iso.end))
if len(precursors[chrom]) < reference_start + len(
reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(
reads[query_name].sequence, precursors[chrom],
reference_start, None)
logger.debug("SRNABENCH::After tune start %s end %s" %
(iso.start, iso.end))
if len(iso.subs) < 3:
reads[query_name].set_precursor(chrom, iso)
logger.info("Hits: %s" % len(reads))
return reads
def _analyze_line(line, reads, precursors, handle, args):
if line.reference_id < 0:
logger.debug("READ::Sequence not mapped: %s" % line.reference_id)
return reads
if not line.cigarstring:
logger.debug("READ::Sequence malformed: %s" % line)
return reads
query_name = line.query_name
if query_name not in reads and not line.query_sequence:
return reads
sequence = line.query_sequence if not line.is_reverse else reverse_complement(
line.query_sequence)
logger.debug(("READ::Read name:{0} and Read sequence:{1}").format(
line.query_name, sequence))
if line.query_sequence and line.query_sequence.find("N") > -1:
return reads
if query_name not in reads:
reads[query_name].set_sequence(sequence)
reads[query_name].counts = _get_freq(query_name)
# TODO if args.quant set to 0
# TODO if args.quant increase by 1
if line.is_reverse and not args.genomic:
logger.debug("READ::Sequence is reverse: %s" % line.query_name)
return reads
chrom = handle.getrname(line.reference_id)
start = line.reference_start
cigar = line.cigartuples
# if line.cigarstring.find("I") > -1:
# indels_skip += 1
iso = isomir()
iso.align = line
iso.set_pos(start, len(reads[query_name].sequence))
logger.debug("READ::From BAM start %s end %s at chrom %s" %
(iso.start, iso.end, chrom))
if len(precursors[chrom].replace("N", "")) + 3 < start + len(
reads[query_name].sequence):
logger.debug("READ::%s start + %s sequence size are bigger than"
" size precursor %s" %
(line.reference_id, len(
reads[query_name].sequence), len(precursors[chrom])))
return reads
iso.subs, iso.add, iso.cigar = filter.tune(reads[query_name].sequence,
precursors[chrom], start, cigar)
logger.debug("READ::After tune start %s end %s" % (iso.start, iso.end))
logger.debug("READ::iso add %s iso subs %s" % (iso.add, iso.subs))
reads[query_name].set_precursor(chrom, iso)
return reads
def read_bam(bam_fn, precursors, clean=True):
"""
read bam file and perform realignment of hits
"""
bam_fn = _sam_to_bam(bam_fn)
bam_fn = _bam_sort(bam_fn)
mode = "r" if bam_fn.endswith("sam") else "rb"
handle = pysam.Samfile(bam_fn, mode)
reads = defaultdict(hits)
for line in handle:
if line.reference_id < 0:
logger.debug("Sequence not mapped: %s" % line.reference_id)
continue
query_name = line.query_name
if query_name not in reads and line.query_sequence == None:
continue
if line.query_sequence and line.query_sequence.find("N") > -1:
continue
if query_name not in reads:
reads[query_name].set_sequence(line.query_sequence)
reads[query_name].counts = _get_freq(query_name)
if line.is_reverse:
logger.debug("Sequence is reverse: %s" % line.query_name)
continue
chrom = handle.getrname(line.reference_id)
# print "%s %s %s %s" % (line.query_name, line.reference_start, line.query_sequence, chrom)
cigar = line.cigartuples
iso = isomir()
iso.align = line
iso.set_pos(line.reference_start, len(reads[query_name].sequence))
logger.debug("READ::From BAM start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < line.reference_start + len(
reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(reads[query_name].sequence,
precursors[chrom],
line.reference_start, cigar)
logger.debug("READ::After tune start %s end %s" % (iso.start, iso.end))
if len(iso.subs) < 2:
reads[query_name].set_precursor(chrom, iso)
logger.info("Hits: %s" % len(reads))
if clean:
reads = filter.clean_hits(reads)
logger.info("Hits after clean: %s" % len(reads))
return reads
def read_file(fn, precursors):
"""
read bam file and perform realignment of hits
"""
reads = defaultdict(hits)
with open(fn) as handle:
handle.readline()
for line in handle:
cols = line.strip().split("\t")
query_name = cols[1]
query_sequence = cols[0]
reference_start = int(cols[4]) - 1
seqbuster_iso = ":".join(cols[6:10])
if query_name not in reads and query_sequence == None:
continue
if query_sequence and query_sequence.find("N") > -1:
continue
if query_name not in reads:
reads[query_name].set_sequence(query_sequence)
reads[query_name].counts = _get_freq(query_name)
chrom = cols[13]
logger.debug("\nSEQBUSTER::NEW::query: {query_sequence}\n"
" precursor {chrom}\n"
" name: {query_name}\n"
" start: {reference_start}\n"
" iso: {seqbuster_iso}".format(**locals()))
# logger.debug("SEQBUSTER:: cigar {cigar}".format(**locals()))
iso = isomir()
iso.align = line
iso.set_pos(reference_start, len(reads[query_name].sequence))
logger.debug("SEQBUSTER:: start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < reference_start + len(
reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(
reads[query_name].sequence, precursors[chrom], reference_start,
None)
logger.debug("SEQBUSTER::After tune start %s end %s" %
(iso.start, iso.end))
if len(iso.subs) < 2:
reads[query_name].set_precursor(chrom, iso)
logger.info("Hits: %s" % len(reads))
return reads
def _read_line(line, col_fix, precursors):
reads = defaultdict(hits)
cols = line.strip().split("\t")
query_name = cols[1]
query_sequence = cols[0]
reference_start = int(cols[4 - col_fix]) - 1
seqbuster_iso = ":".join(cols[6 - col_fix:10 - col_fix])
if query_sequence and query_sequence.find("N") > -1:
return reads
if query_name not in reads:
reads[query_name].set_sequence(query_sequence)
reads[query_name].counts = _get_freq(query_name)
chrom = cols[13 - col_fix]
logger.debug("\nSEQBUSTER::NEW::query: {query_sequence}\n"
" precursor: {chrom}\n"
" name: {query_name}\n"
" start: {reference_start}\n"
" iso: {seqbuster_iso}".format(**locals()))
# logger.debug("SEQBUSTER:: cigar {cigar}".format(**locals()))
iso = isomir()
iso.align = line
iso.set_pos(reference_start, len(reads[query_name].sequence))
logger.debug("\nSEQBUSTER:: start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < reference_start + len(
reads[query_name].sequence):
logger.debug("\nSEQBUSTER::len precursor" % len(precursors[chrom]))
return reads
iso.subs, iso.add, iso.cigar = filter.tune(reads[query_name].sequence,
precursors[chrom],
reference_start, None)
logger.debug("\nSEQBUSTER::After tune start %s end %s" %
(iso.start, iso.end))
if len(iso.subs) < 6:
logger.debug("\nSEQBUSTER::iso.subs %s - length %s" %
(iso.subs, len(iso.subs)))
reads[query_name].set_precursor(chrom, iso)
return reads
def read_file(fn, hairpins, database, mirna_gtf):
"""
Read PROST! file and convert to mirtop GFF format.
Args:
*fn(str)*: file name with PROST output information.
*database(str)*: database name.
*args(namedtuple)*: arguments from command line.
See *mirtop.libs.parse.add_subparser_gff()*.
Returns:
*reads*: dictionary where keys are read_id and values are *mirtop.realign.hits*
"""
reads = defaultdict(hits)
sample = os.path.splitext(os.path.basename(fn))[0]
genomics = mapper.read_gtf_to_mirna(mirna_gtf)
matures = mapper.read_gtf_to_precursor(mirna_gtf)
non_mirna = 0
non_chromosome_mirna = 0
outside_mirna = 0
lines_read = 0
ann, ann_type = _group_seqs_by_ann(fn)
with open(fn) as handle:
handle.readline()
for line in handle:
lines_read += 1
cols = line.strip().split("\t")
query_name = cols[0]
query_sequence = cols[0]
if not ann[query_sequence]:
non_mirna += 1
continue
miRNA = ann_type[ann[query_sequence]][1]
preNames = ann_type[ann[query_sequence]][0]
if query_name not in reads and query_sequence==None:
continue
if query_sequence and query_sequence.find("N") > -1:
continue
reads[query_name].set_sequence(query_sequence)
reads[query_name].counts = cols[9]
for preName in preNames.split(","):
if preName in reads[query_name].precursors:
continue
if preName not in hairpins:
non_chromosome_mirna += 1
continue
reference_start = _align_to_mature(query_sequence, hairpins[preName], matures[preName][miRNA])
logger.debug("\nPROST!::NEW::query: {query_sequence}\n"
" precursor {preName}\n"
" name: {query_name}\n"
" reference_start: {reference_start}\n"
" mirna: {miRNA}".format(**locals()))
iso = isomir()
iso.align = line
iso.set_pos(reference_start, len(reads[query_name].sequence))
logger.debug("PROST!:: start %s end %s" % (iso.start, iso.end))
if len(hairpins[preName]) < reference_start + len(reads[query_name].sequence):
continue
iso.subs, iso.add, iso.cigar = filter.tune(
reads[query_name].sequence,
hairpins[preName],
reference_start, None)
logger.debug("PROST!::After tune start %s end %s" % (
iso.start, iso.end))
if len(iso.subs) < 2:
reads[query_name].set_precursor(preName, iso)
logger.info("Lines loaded: %s" % lines_read)
logger.info("Skipped lines because non miRNA in line: %s" % non_mirna)
logger.info("Skipped lines because non chromosome in GTF:"
" %s" % non_chromosome_mirna)
logger.info("Skipped lines because outside precursor: %s" % outside_mirna)
logger.info("Hits: %s" % len(reads))
return reads
def read_file(folder, precursors):
"""
read srnabench file and perform realignment of hits
"""
n_out = 0
n_nonmature = 0
n_ns = 0
n_in = 0
n_non_precursor = 0
reads_anno = os.path.join(folder, "reads.annotation")
reads_iso = os.path.join(folder, "microRNAannotation.txt")
reads = defaultdict(hits)
source_iso = _read_iso(reads_iso)
logger.info("Reads with isomiR information %s" % len(source_iso))
with open(reads_anno) as handle:
for line in handle:
cols = line.strip().split("\t")
query_name = cols[0]
query_sequence = cols[0]
if query_name not in reads and query_sequence == None:
continue
if query_sequence and query_sequence.find("N") > -1:
n_ns += 1
continue
if cols[3].find("mature") == -1:
n_nonmature += 1
continue
if query_name not in reads:
reads[query_name].set_sequence(query_sequence)
reads[query_name].counts = int(cols[1])
for hit in cols[4].split("$"):
logger.debug("SRNABENCH::line hit: %s" % hit)
hit_info = hit.split("#")
pos_info = hit_info[3].split(",")
reference_start = int(pos_info[1]) - 1
chrom = pos_info[0]
iso = isomir()
iso.align = line
if (query_sequence, hit_info[1]) in source_iso:
iso.external = source_iso[(query_sequence, hit_info[1])]
external = iso.external
logger.debug("SRNABENCH::query: {query_sequence}\n"
" precursor {chrom}\n"
" name: {query_name}\n"
" start: {reference_start}\n"
" external: {external}\n"
" hit: {hit}".format(**locals()))
iso.set_pos(reference_start, len(reads[query_name].sequence))
logger.debug("SRNABENCH:: start %s end %s" % (iso.start, iso.end))
if len(precursors[chrom]) < reference_start + len(reads[query_name].sequence):
n_out += 1
continue
iso.subs, iso.add, iso.cigar = filter.tune(reads[query_name].sequence,
precursors[chrom],
reference_start, None)
logger.debug("SRNABENCH::After tune start %s end %s" % (iso.start, iso.end))
n_in += 1
reads[query_name].set_precursor(chrom, iso)
if len(reads[query_name].precursors) == 0:
n_non_precursor += 1
logger.info("Loaded %s reads with %s hits" % (len(reads), n_in))
logger.info("Reads without precursor information: %s" % n_non_precursor)
logger.info("Hit Filtered by having > 3 changes: %s" % n_out)
logger.info("Hit Filtered by being non-mature: %s" % n_nonmature)
return reads
def _analyze_line(line, precursors, database, sample, sep, args):
start_idx = 10
end_idx = 11
attr_idx = 15
query_name = line[3]
sequence = line[4]
if str(line).find(get_primary_transcript(guess_database(args))) < 0: # only working with mirbase
return None
logger.debug(("READ::line name:{0}").format(line))
if sequence and sequence.find("N") > -1:
return None
chrom = line[attr_idx].strip().split("Name=")[-1]
start = line[1]
end = line[2]
strand = line[5]
counts = float(line[6])
Filter = "Pass"
reads = dict()
if not start:
return None
if strand == "+":
start = int(start) - int(line[start_idx]) + 1
else:
start = int(line[end_idx]) - int(end)
iso = isomir()
iso.align = line
iso.set_pos(start, len(sequence))
logger.debug("READ::From BAM start %s end %s at chrom %s" % (iso.start, iso.end, chrom))
if len(precursors[chrom]) < start + len(sequence):
logger.debug("READ::%s start + %s sequence size are bigger than"
" size precursor %s" % (
chrom,
len(sequence),
len(precursors[chrom])))
iso.subs, iso.add, iso.cigar = filter.tune(
sequence, precursors[chrom],
start, None)
logger.debug("READ::After tune start %s end %s" % (iso.start, iso.end))
logger.debug("READ::iso add %s iso subs %s" % (iso.add, iso.subs))
idu = make_id(sequence)
reads[query_name] = hits()
reads[query_name].set_sequence(sequence)
reads[query_name].counts = counts
reads[query_name].sequence = sequence
reads[query_name].set_precursor(chrom, iso)
reads = annotate(reads, args.matures, args.precursors, quiet=True)
gff_line = body.create(reads, args.database, sample, args, quiet=True)
if start not in gff_line[chrom]:
return None
line = gff_line[chrom][start][0][4]
logger.debug("READ::line:%s" % line)
if args.add_extra:
extra = variant_with_nt(line, args.precursors,
args.matures)
line = "%s Changes %s;" % (line, extra)
line = paste_columns(feature(line), sep=sep)
return {'chrom': chrom,
'start': start,
'name': query_name,
'mirna': reads[query_name].precursors[chrom].mirna,
'line': [idu, chrom, counts, sample, line]}
