def ref_filter(ref, tophat_dir, cufflinks_dir):
'''
Extract isoform with at least two supported junction reads for each
junction
'''
print('Read gene annotations...')
# read junction information
junction_f = tophat_dir + '/junctions.bed'
junc = parse_junc(junction_f)
print('Filter gene annotations with junction information...')
# filter out gene annotations using junction reads
filtered_junction_f = '%s/filtered_junction.txt' % cufflinks_dir
with open(ref, 'r') as ref_f, open(filtered_junction_f, 'w') as out_f:
for line in ref_f:
chrom = line.split()[2]
starts = line.split()[10].rstrip(',').split(',')[:-1]
ends = line.split()[9].rstrip(',').split(',')[1:]
for s, e in zip(starts, ends):
junc_id = '%s\t%s\t%s' % (chrom, s, e)
if junc[junc_id] < 2:
break
else: # all the junctions have enough reads
out_f.write('\t'.join(line.split()[1:]) + '\n')
filtered_junction_gtf = '%s/filtered_junction.gtf' % cufflinks_dir
return_code = os.system('genePredToGtf file %s %s' %
(filtered_junction_f, filtered_junction_gtf)) >> 8
if return_code:
sys.exit('Error: cannot convert GenePred to GTF!')
def parse_splice_site(denovo_dir, tophat_dir, pAplus_dir, output_dir):
"""
Characterize all the alternative splice site selections.
"""
print('Start to parse alternative splice sites...')
splice_site_5 = set()
splice_site_3 = set()
# set path
fusion_f = '%s/circularRNA_full.txt' % denovo_dir
pAminus_junc_f = tophat_dir + '/junctions.bed'
(pAminus_junc,
pAminus_left_junc,
pAminus_right_junc) = parse_junc(pAminus_junc_f, 2)
pAplus_junc_f = '%s/junctions.bed' % pAplus_dir
(pAplus_junc,
pAplus_left_junc,
pAplus_right_junc) = parse_junc(pAplus_junc_f, 2)
with open(fusion_f, 'r') as f:
for line in f:
circ_type = line.split()[13]
if circ_type == 'ciRNA': # not check ciRNAs
continue
chrom = line.split()[0]
start = int(line.split()[1])
strand = line.split()[5]
sizes = [int(x) for x in line.split()[10].split(',')]
offsets = [int(x) for x in line.split()[11].split(',')]
starts, ends = [], []
for s, o in zip(sizes, offsets):
starts.append(start + o)
ends.append(start + o + s)
starts = starts[1:]
ends = ends[:-1]
for s, e in zip(ends, starts):
loc = '%s\t%d\t%d' % (chrom, s, e)
left_id = '%s\t%d' % (chrom, s)
right_id = '%s\t%d' % (chrom, e)
if loc in pAminus_junc:
pAminus_reads = pAminus_junc[loc]
else: # circ_pcu=0
continue
if loc in pAplus_junc:
pAplus_reads = pAplus_junc[loc]
else:
pAplus_reads = 0
if pAminus_left_junc[left_id] != 0:
pAminus_left_total = pAminus_left_junc[left_id]
pAminus_left_psu = (pAminus_reads * 100.0 /
pAminus_left_total)
pAplus_left_total = pAplus_left_junc[left_id]
if pAplus_left_total != 0:
pAplus_left_psu = (pAplus_reads * 100.0 /
pAplus_left_total)
else:
pAplus_left_psu = 0.0
else: # circ_left_total_reads=0
continue
if pAminus_right_junc[right_id] != 0:
pAminus_right_total = pAminus_right_junc[right_id]
pAminus_right_psu = (pAminus_reads * 100.0 /
pAminus_right_total)
pAplus_right_total = pAplus_right_junc[right_id]
if pAplus_right_total != 0:
pAplus_right_psu = (pAplus_reads * 100.0 /
pAplus_right_total)
else:
pAplus_right_psu = 0.0
else: # circ_right_total_reads=0
continue
f = '%s\t%s\t%d\t%d\t%f\t%d\t%d\t%f\n'
left_info = f % (loc, strand, pAminus_reads,
pAminus_left_total, pAminus_left_psu,
pAplus_reads, pAplus_left_total,
pAplus_left_psu)
right_info = f % (loc, strand, pAminus_reads,
pAminus_right_total, pAminus_right_psu,
pAplus_reads, pAplus_right_total,
pAplus_right_psu)
if strand == '+':
if pAminus_left_psu != 100:
splice_site_3.add(left_info)
if pAminus_right_psu != 100:
splice_site_5.add(right_info)
else:
if pAminus_left_psu != 100:
splice_site_5.add(left_info)
if pAminus_right_psu != 100:
splice_site_3.add(right_info)
output_f = '%s/all_A5SS_info.txt' % output_dir
with open(output_f, 'w') as output:
output.write(''.join(splice_site_5))
output_f = '%s/all_A3SS_info.txt' % output_dir
with open(output_f, 'w') as output:
output.write(''.join(splice_site_3))
print('Complete parsing alternative splice sites!')
def extract_retained_intron(denovo_dir, tophat_dir, pAplus_dir, output_dir):
"""
Check each intron and fetch PIR
Modified from Braunschweig et al., Genome Research, 2014, gr-177790.
"""
print('Start to parse circular RNA introns...')
# set path
fusion_f = '%s/circularRNA_full.txt' % denovo_dir
pAminus_junc_f = tophat_dir + '/junctions.bed'
pAminus_junc = parse_junc(pAminus_junc_f)
pAminus_bam_f = tophat_dir + '/accepted_hits.bam'
pAminus_bam = pysam.AlignmentFile(pAminus_bam_f, 'rb')
pAplus_junc_f = '%s/junctions.bed' % pAplus_dir
pAplus_junc = parse_junc(pAplus_junc_f)
pAplus_bam_f = '%s/accepted_hits.bam' % pAplus_dir
pAplus_bam = pysam.AlignmentFile(pAplus_bam_f, 'rb')
excluded_region = defaultdict(list)
novel_region = defaultdict(list)
intron = defaultdict(list)
intron_list = set()
intron_info_list = {}
with open(fusion_f, 'r') as f:
for line in f:
chrom, start, end = line.split()[:3]
start = int(start)
end = int(end)
strand = line.split()[5]
circ_type = line.split()[13]
if circ_type == 'ciRNA': # not check ciRNAs
excluded_region[chrom].append([start, end])
continue
sizes = [int(x) for x in line.split()[10].split(',')]
offsets = [int(x) for x in line.split()[11].split(',')]
reads = line.split()[12]
gene, iso = line.split()[14:16]
for s, o in zip(sizes, offsets):
if gene.startswith('CUFF'):
novel_region[chrom].append([start + o, start + o + s])
else:
excluded_region[chrom].append([start + o, start + o + s])
if gene.startswith('CUFF'): # only check annotated introns
continue
num = int(line.split()[9])
for i in range(num - 1):
sta = start + offsets[i] + sizes[i]
end = start + offsets[i + 1]
if end - sta == 0:
continue
intron_info = '%s\t%d\t%d\t%s' % (chrom, sta, end, strand)
if intron_info in intron_list:
if int(reads) > int(intron_info_list[intron_info][2]):
intron_info_list[intron_info] = [gene, iso, reads]
continue
intron[chrom].append([sta, end, intron_info])
intron_list.add(intron_info)
intron_info_list[intron_info] = [gene, iso, reads]
intron_set = set()
for chrom in excluded_region:
intron_region = []
# retain introns covered by novel assembled transcripts
# combined_region = Interval(novel_region[chrom]).interval
# for region in Interval.overlapwith(combined_region, intron[chrom]):
# retain all intron regions in this step
for region in intron[chrom]:
if len(region) >= 3:
for intron_info in region[2:]:
chrom, start, end = intron_info.split()[:3]
intron_region.append([int(start), int(end), intron_info])
intron_set.add(intron_info)
# remove introns overlapped with annotated exons
combined_region = Interval(excluded_region[chrom]).interval
for region in Interval.overlapwith(combined_region, intron_region):
if len(region) >= 3:
for intron_info in region[2:]:
intron_set.discard(intron_info)
output_f = '%s/all_intron_info.txt' % output_dir
# import pdb;pdb.set_trace()
with open(output_f, 'w') as output:
total_i_n = len(intron_set)
finished_n = 0
for intron in intron_set:
chrom, sta, end, strand = intron.split()
intron_info = '\t'.join([chrom, sta, end])
sta = int(sta)
end = int(end)
# fetch junctions for circular RNAs
circ_junc_read = pAminus_junc[intron_info]
circ_left_read = fetch_read(pAminus_bam, chrom, sta - 8, sta + 8)
circ_right_read = fetch_read(pAminus_bam, chrom, end - 8, end + 8)
circ_ri_read = circ_left_read + circ_right_read
circ_intron_read = fetch_read(pAminus_bam, chrom, sta, end, flag=0)
# calculate PIR for circular RNAs
if circ_ri_read == 0 and circ_junc_read == 0:
pir_circ = 0
else:
pir_circ = 100.0 * circ_ri_read / (circ_ri_read +
2 * circ_junc_read)
# exact binomial test for circular RNAs
m = min(circ_left_read, circ_right_read, circ_intron_read)
n = m + max(circ_left_read, circ_right_read, circ_intron_read)
p = 1 / 3.5
p1 = binom.cdf(m, n, p) # one-side binomial test
# fetch junctions for linear RNAs
linear_junc_read = pAplus_junc[intron_info]
linear_left_read = fetch_read(pAplus_bam, chrom, sta - 8, sta + 8)
linear_right_read = fetch_read(pAplus_bam, chrom, end - 8, end + 8)
linear_ri_read = linear_left_read + linear_right_read
linear_intron_read = fetch_read(pAplus_bam, chrom, sta, end,
flag=0)
# calculate PIR for linear RNAs
if linear_ri_read == 0 and linear_junc_read == 0:
pir_linear = 0
else:
pir_linear = 100.0 * linear_ri_read / (linear_ri_read +
linear_junc_read * 2)
# exact binomial test for linear RNAs
m = min(linear_left_read, linear_right_read,
linear_intron_read)
n = m + max(linear_left_read, linear_right_read,
linear_intron_read)
p = 1 / 3.5
p2 = binom.cdf(m, n, p) # one-side binomial test
info = '\t'.join(str(round(float(x), 3))
for x in (pir_circ, pir_linear, p1, p2,
circ_ri_read,
circ_junc_read,
circ_intron_read,
linear_ri_read,
linear_junc_read,
linear_intron_read))
other_info = '\t'.join(intron_info_list[intron])
output.write('\t'.join([chrom, str(sta), str(end), 'Intron', '0',
strand, other_info, info]))
output.write('\n')
finished_n += 1
sys.stdout.write("Progress: %d/%d \r" % (finished_n, total_i_n) )
sys.stdout.flush()
print('Complete parsing circular RNA introns!')
def extract_cassette_exon(denovo_dir, tophat_dir, pAplus_dir, output_dir,
rpkm_flag):
"""
1. Check each exon and fetch PSI
2. Calculate RPKM if needed
Modified from Han et al., Nature, 2013, 498:241-245.
"""
print('Start to parse circular RNA exons...')
exons = {}
# set path
fusion_f = '%s/circularRNA_full.txt' % denovo_dir
pAminus_junc_f = tophat_dir + '/junctions.bed'
(pAminus_junc,
pAminus_left_junc,
pAminus_right_junc) = parse_junc(pAminus_junc_f, 1)
pAplus_junc_f = '%s/junctions.bed' % pAplus_dir
(pAplus_junc,
pAplus_left_junc,
pAplus_right_junc) = parse_junc(pAplus_junc_f, 1)
if rpkm_flag:
pAminus_bam = Expression('%s/accepted_hits.bam' % tophat_dir)
pAplus_bam = Expression('%s/accepted_hits.bam' % pAplus_dir)
with open(fusion_f, 'r') as f:
for line in f:
circ_type = line.split()[13]
if circ_type == 'ciRNA': # not check ciRNAs
continue
reads = line.split()[12]
chrom = line.split()[0]
start = int(line.split()[1])
strand = line.split()[5]
sizes = [int(x) for x in line.split()[10].split(',')]
offsets = [int(x) for x in line.split()[11].split(',')]
gene, iso = line.split()[14:16]
exon_deque = deque(maxlen=3) # set exon sliding window
for s, o in zip(sizes, offsets):
sta = start + o
end = start + o + s
exon_id = [sta, end]
exon_deque.append(exon_id)
gene_info = '\t'.join([strand, gene, iso])
if len(exon_deque) == 3: # only check middle exon
exon_info = '%s\t%d\t%d' % (chrom, exon_deque[1][0],
exon_deque[1][1])
if exon_info in exons:
if exons[exon_info][0].find('CUFF'):
if not gene.startswith('CUFF'): # annotated exon
exons[exon_info][0] = gene_info
if int(reads) > int(exons[exon_info][1]): # more reads
exons[exon_info][1] = reads
else:
# fetch junctions for circular RNAs
(psi_circ,
inclusion_circ,
exclusion_circ,
max_left_circ,
max_right_circ) = fetch_psi(exon_info,
pAminus_junc,
pAminus_left_junc,
pAminus_right_junc)
if max_left_circ == 'None' or max_left_circ == 'None':
flag = []
else:
flag = [max_left_circ, max_right_circ]
# fetch junctions for linear RNAs
(psi_linear,
inclusion_linear,
exclusion_linear) = fetch_psi(exon_info,
pAplus_junc,
pAplus_left_junc,
pAplus_right_junc,
flag)
# fisher exact test (circular > linear)
odd1, p1 = fisher_exact([[inclusion_circ,
2 * exclusion_circ],
[inclusion_linear,
2 * exclusion_linear]],
alternative='greater')
# fisher exact test (circular < linear)
odd2, p2 = fisher_exact([[inclusion_circ,
2 * exclusion_circ],
[inclusion_linear,
2 * exclusion_linear]],
alternative='less')
info = '\t'.join(str(round(float(x), 3))
for x in (psi_circ, psi_linear, p1,
p2,
inclusion_circ,
exclusion_circ,
inclusion_linear,
exclusion_linear))
if rpkm_flag:
circ_exp = pAminus_bam.rpkm(chrom, *exon_deque[1])
linear_exp = pAplus_bam.rpkm(chrom, *exon_deque[1])
info += '\t%.3f\t%.3f' % (circ_exp, linear_exp)
exons[exon_info] = [gene_info, reads, info]
output_f = '%s/all_exon_info.txt' % output_dir
with open(output_f, 'w') as output:
for exon in exons:
chrom, start, end = exon.split()
output.write('\t'.join([chrom, start, end, 'Exon', '0']))
output.write('\t' + '\t'.join(exons[exon]))
output.write('\n')
print('Complete parsing circular RNA exons!')
def parse_splice_site(denovo_dir, tophat_dir, pAplus_dir):
"""
Characterize all the alternative splice site selections.
"""
print('Start to parse alternative splice sites...')
splice_site_5 = set()
splice_site_3 = set()
# set path
fusion_f = '%s/circ_fusion.txt' % denovo_dir
pAminus_junc_f = tophat_dir + '/junctions.bed'
(pAminus_junc,
pAminus_left_junc,
pAminus_right_junc) = parse_junc(pAminus_junc_f, 2)
pAplus_junc_f = '%s/junctions.bed' % pAplus_dir
(pAplus_junc,
pAplus_left_junc,
pAplus_right_junc) = parse_junc(pAplus_junc_f, 2)
with open(fusion_f, 'r') as f:
for line in f:
circ_type = line.split()[13]
if circ_type == 'ciRNA': # not check ciRNAs
continue
chrom = line.split()[0]
start = int(line.split()[1])
strand = line.split()[5]
sizes = [int(x) for x in line.split()[10].split(',')]
offsets = [int(x) for x in line.split()[11].split(',')]
starts, ends = [], []
for s, o in zip(sizes, offsets):
starts.append(start + o)
ends.append(start + o + s)
starts = starts[1:]
ends = ends[:-1]
for s, e in zip(ends, starts):
loc = '%s\t%d\t%d' % (chrom, s, e)
left_id = '%s\t%d' % (chrom, s)
right_id = '%s\t%d' % (chrom, e)
if loc in pAminus_junc:
pAminus_reads = pAminus_junc[loc]
else: # circ_pcu=0
continue
if loc in pAplus_junc:
pAplus_reads = pAplus_junc[loc]
else:
pAplus_reads = 0
if pAminus_left_junc[left_id] != 0:
pAminus_left_total = pAminus_left_junc[left_id]
pAminus_left_psu = (pAminus_reads * 100.0 /
pAminus_left_total)
pAplus_left_total = pAplus_left_junc[left_id]
if pAplus_left_total != 0:
pAplus_left_psu = (pAplus_reads * 100.0 /
pAplus_left_total)
else:
pAplus_left_psu = 0.0
else: # circ_left_total_reads=0
continue
if pAminus_right_junc[right_id] != 0:
pAminus_right_total = pAminus_right_junc[right_id]
pAminus_right_psu = (pAminus_reads * 100.0 /
pAminus_right_total)
pAplus_right_total = pAplus_right_junc[right_id]
if pAplus_right_total != 0:
pAplus_right_psu = (pAplus_reads * 100.0 /
pAplus_right_total)
else:
pAplus_right_psu = 0.0
else: # circ_right_total_reads=0
continue
f = '%s\t%s\t%d\t%d\t%f\t%d\t%d\t%f\n'
left_info = f % (loc, strand, pAminus_reads,
pAminus_left_total, pAminus_left_psu,
pAplus_reads, pAplus_left_total,
pAplus_left_psu)
right_info = f % (loc, strand, pAminus_reads,
pAminus_right_total, pAminus_right_psu,
pAplus_reads, pAplus_right_total,
pAplus_right_psu)
if strand == '+':
if pAminus_left_psu != 100:
splice_site_3.add(left_info)
if pAminus_right_psu != 100:
splice_site_5.add(right_info)
else:
if pAminus_left_psu != 100:
splice_site_5.add(left_info)
if pAminus_right_psu != 100:
splice_site_3.add(right_info)
output_f = '%s/all_A5SS_info.txt' % denovo_dir
with open(output_f, 'w') as output:
output.write(''.join(splice_site_5))
output_f = '%s/all_A3SS_info.txt' % denovo_dir
with open(output_f, 'w') as output:
output.write(''.join(splice_site_3))
print('Complete parsing alternative splice sites!')
def extract_retained_intron(denovo_dir, tophat_dir, pAplus_dir):
"""
Check each intron and fetch PIR
Modified from Braunschweig et al., Genome Research, 2014, gr-177790.
"""
print('Start to parse circular RNA introns...')
# set path
fusion_f = '%s/circ_fusion.txt' % denovo_dir
pAminus_junc_f = tophat_dir + '/junctions.bed'
pAminus_junc = parse_junc(pAminus_junc_f)
pAminus_bam_f = tophat_dir + '/accepted_hits.bam'
pAminus_bam = pysam.AlignmentFile(pAminus_bam_f, 'rb')
pAplus_junc_f = '%s/junctions.bed' % pAplus_dir
pAplus_junc = parse_junc(pAplus_junc_f)
pAplus_bam_f = '%s/accepted_hits.bam' % pAplus_dir
pAplus_bam = pysam.AlignmentFile(pAplus_bam_f, 'rb')
excluded_region = defaultdict(list)
novel_region = defaultdict(list)
intron = defaultdict(list)
intron_list = set()
intron_info_list = {}
with open(fusion_f, 'r') as f:
for line in f:
chrom, start, end = line.split()[:3]
start = int(start)
end = int(end)
strand = line.split()[5]
circ_type = line.split()[13]
if circ_type == 'ciRNA': # not check ciRNAs
excluded_region[chrom].append([start, end])
continue
sizes = [int(x) for x in line.split()[10].split(',')]
offsets = [int(x) for x in line.split()[11].split(',')]
reads = line.split()[12]
gene, iso = line.split()[14:16]
for s, o in zip(sizes, offsets):
if gene.startswith('CUFF'):
novel_region[chrom].append([start + o, start + o + s])
else:
excluded_region[chrom].append([start + o, start + o + s])
if gene.startswith('CUFF'): # only check annotated introns
continue
num = int(line.split()[9])
for i in range(num - 1):
sta = start + offsets[i] + sizes[i]
end = start + offsets[i + 1]
if end - sta == 0:
continue
intron_info = '%s\t%d\t%d\t%s' % (chrom, sta, end, strand)
if intron_info in intron_list:
if int(reads) > int(intron_info_list[intron_info][2]):
intron_info_list[intron_info] = [gene, iso, reads]
continue
intron[chrom].append([sta, end, intron_info])
intron_list.add(intron_info)
intron_info_list[intron_info] = [gene, iso, reads]
intron_set = set()
for chrom in excluded_region:
intron_region = []
# retain introns covered by novel assembled transcripts
combined_region = Interval(novel_region[chrom]).interval
for region in Interval.overlapwith(combined_region, intron[chrom]):
if len(region) >= 3:
for intron_info in region[2:]:
chrom, start, end = intron_info.split()[:3]
intron_region.append([int(start), int(end), intron_info])
intron_set.add(intron_info)
# remove introns overlapped with annotated exons
combined_region = Interval(excluded_region[chrom]).interval
for region in Interval.overlapwith(combined_region, intron_region):
if len(region) >= 3:
for intron_info in region[2:]:
intron_set.discard(intron_info)
output_f = '%s/all_intron_info.txt' % denovo_dir
with open(output_f, 'w') as output:
for intron in intron_set:
chrom, sta, end, strand = intron.split()
intron_info = '\t'.join([chrom, sta, end])
sta = int(sta)
end = int(end)
# fetch junctions for circular RNAs
circ_junc_read = pAminus_junc[intron_info]
circ_left_read = fetch_read(pAminus_bam, chrom, sta - 8, sta + 8)
circ_right_read = fetch_read(pAminus_bam, chrom, end - 8, end + 8)
circ_ri_read = circ_left_read + circ_right_read
circ_intron_read = fetch_read(pAminus_bam, chrom, sta, end, flag=0)
# calculate PIR for circular RNAs
if circ_ri_read == 0 and circ_junc_read == 0:
pir_circ = 0
else:
pir_circ = 100.0 * circ_ri_read / (circ_ri_read +
2 * circ_junc_read)
# exact binomial test for circular RNAs
m = min(circ_left_read, circ_right_read, circ_intron_read)
n = m + max(circ_left_read, circ_right_read, circ_intron_read)
p = 1 / 3.5
p1 = binom.cdf(m, n, p) # one-side binomial test
# fetch junctions for linear RNAs
linear_junc_read = pAplus_junc[intron_info]
linear_left_read = fetch_read(pAplus_bam, chrom, sta - 8, sta + 8)
linear_right_read = fetch_read(pAplus_bam, chrom, end - 8, end + 8)
linear_ri_read = linear_left_read + linear_right_read
linear_intron_read = fetch_read(pAplus_bam, chrom, sta, end,
flag=0)
# calculate PIR for linear RNAs
if linear_ri_read == 0 and linear_junc_read == 0:
pir_linear = 0
else:
pir_linear = 100.0 * linear_ri_read / (linear_ri_read +
linear_junc_read * 2)
# exact binomial test for linear RNAs
m = min(linear_left_read, linear_right_read,
linear_intron_read)
n = m + max(linear_left_read, linear_right_read,
linear_intron_read)
p = 1 / 3.5
p2 = binom.cdf(m, n, p) # one-side binomial test
info = '\t'.join(str(round(x, 3))
for x in (pir_circ, pir_linear, p1, p2,
circ_ri_read,
circ_junc_read,
circ_intron_read,
linear_ri_read,
linear_junc_read,
linear_intron_read))
other_info = '\t'.join(intron_info_list[intron])
output.write('\t'.join([chrom, str(sta), str(end), 'Intron', '0',
strand, other_info, info]))
output.write('\n')
print('Complete parsing circular RNA introns!')
def extract_cassette_exon(denovo_dir, tophat_dir, pAplus_dir, rpkm_flag):
"""
1. Check each exon and fetch PSI
2. Calculate RPKM if needed
Modified from Han et al., Nature, 2013, 498:241-245.
"""
print('Start to parse circular RNA exons...')
exons = {}
# set path
fusion_f = '%s/circ_fusion.txt' % denovo_dir
pAminus_junc_f = tophat_dir + '/junctions.bed'
(pAminus_junc,
pAminus_left_junc,
pAminus_right_junc) = parse_junc(pAminus_junc_f, 1)
pAplus_junc_f = '%s/junctions.bed' % pAplus_dir
(pAplus_junc,
pAplus_left_junc,
pAplus_right_junc) = parse_junc(pAplus_junc_f, 1)
if rpkm_flag:
pAminus_bam = Expression('%s/accepted_hits.bam' % tophat_dir)
pAplus_bam = Expression('%s/accepted_hits.bam' % pAplus_dir)
with open(fusion_f, 'r') as f:
for line in f:
circ_type = line.split()[13]
if circ_type == 'ciRNA': # not check ciRNAs
continue
reads = line.split()[12]
chrom = line.split()[0]
start = int(line.split()[1])
strand = line.split()[5]
sizes = [int(x) for x in line.split()[10].split(',')]
offsets = [int(x) for x in line.split()[11].split(',')]
gene, iso = line.split()[14:16]
exon_deque = deque(maxlen=3) # set exon sliding window
for s, o in zip(sizes, offsets):
sta = start + o
end = start + o + s
exon_id = [sta, end]
exon_deque.append(exon_id)
gene_info = '\t'.join([strand, gene, iso])
if len(exon_deque) == 3: # only check middle exon
exon_info = '%s\t%d\t%d' % (chrom, exon_deque[1][0],
exon_deque[1][1])
if exon_info in exons:
if exons[exon_info][0].find('CUFF'):
if not gene.startswith('CUFF'): # annotated exon
exons[exon_info][0] = gene_info
if int(reads) > int(exons[exon_info][1]): # more reads
exons[exon_info][1] = reads
else:
# fetch junctions for circular RNAs
(psi_circ,
inclusion_circ,
exclusion_circ,
max_left_circ,
max_right_circ) = fetch_psi(exon_info,
pAminus_junc,
pAminus_left_junc,
pAminus_right_junc)
if max_left_circ == 'None' or max_left_circ == 'None':
flag = []
else:
flag = [max_left_circ, max_right_circ]
# fetch junctions for linear RNAs
(psi_linear,
inclusion_linear,
exclusion_linear) = fetch_psi(exon_info,
pAplus_junc,
pAplus_left_junc,
pAplus_right_junc,
flag)
# fisher exact test (circular > linear)
odd1, p1 = fisher_exact([[inclusion_circ,
2 * exclusion_circ],
[inclusion_linear,
2 * exclusion_linear]],
alternative='greater')
# fisher exact test (circular < linear)
odd2, p2 = fisher_exact([[inclusion_circ,
2 * exclusion_circ],
[inclusion_linear,
2 * exclusion_linear]],
alternative='less')
info = '\t'.join(str(round(x, 3))
for x in (psi_circ, psi_linear, p1,
p2,
inclusion_circ,
exclusion_circ,
inclusion_linear,
exclusion_linear))
if rpkm_flag:
circ_exp = pAminus_bam.rpkm(chrom, *exon_deque[1])
linear_exp = pAplus_bam.rpkm(chrom, *exon_deque[1])
info += '\t%.3f\t%.3f' % (circ_exp, linear_exp)
exons[exon_info] = [gene_info, reads, info]
output_f = '%s/all_exon_info.txt' % denovo_dir
with open(output_f, 'w') as output:
for exon in exons:
chrom, start, end = exon.split()
output.write('\t'.join([chrom, start, end, 'Exon', '0']))
output.write('\t' + '\t'.join(exons[exon]))
output.write('\n')
print('Complete parsing circular RNA exons!')