def main():
usage="%prog [options]" + '\n' + __doc__ + "\n"
parser = OptionParser(usage,version="%prog " + __version__)
parser.add_option("-i","--input-file",action="store",type="string",dest="input_file",help="Alignment file in BAM or SAM format.")
parser.add_option("-r","--refgene",action="store",type="string",dest="ref_gene_model",help="Reference gene model in bed format.")
(options,args)=parser.parse_args()
if not (options.input_file and options.ref_gene_model):
parser.print_help()
sys.exit(0)
if not os.path.exists(options.ref_gene_model):
print >>sys.stderr, '\n\n' + options.ref_gene_model + " does NOT exists" + '\n'
#parser.print_help()
sys.exit(0)
if not os.path.exists(options.input_file):
print >>sys.stderr, '\n\n' + options.input_file + " does NOT exists" + '\n'
sys.exit(0)
#build bitset
(cds_exon_r, intron_r, utr_5_r, utr_3_r,\
intergenic_up_1kb_r,intergenic_up_5kb_r,intergenic_up_10kb_r,\
intergenic_down_1kb_r,intergenic_down_5kb_r,intergenic_down_10kb_r,\
cds_exon_base,intron_base,utr_5_base,utr_3_base,\
intergenic_up1kb_base,intergenic_up5kb_base,intergenic_up10kb_base,\
intergenic_down1kb_base,intergenic_down5kb_base,intergenic_down10kb_base) = process_gene_model(options.ref_gene_model)
intron_read=0
cds_exon_read=0
utr_5_read=0
utr_3_read=0
intergenic_up1kb_read=0
intergenic_down1kb_read=0
intergenic_up5kb_read=0
intergenic_down5kb_read=0
intergenic_up10kb_read=0
intergenic_down10kb_read=0
totalReads=0
totalFrags=0
unAssignFrags=0
obj = SAM.ParseBAM(options.input_file)
R_qc_fail=0
R_duplicate=0
R_nonprimary=0
R_unmap=0
print >>sys.stderr, "processing " + options.input_file + " ...",
try:
while(1):
aligned_read = obj.samfile.next()
if aligned_read.is_qcfail: #skip QC fail read
R_qc_fail +=1
continue
if aligned_read.is_duplicate: #skip duplicate read
R_duplicate +=1
continue
if aligned_read.is_secondary: #skip non primary hit
R_nonprimary +=1
continue
if aligned_read.is_unmapped: #skip unmap read
R_unmap +=1
continue
totalReads +=1
chrom = obj.samfile.getrname(aligned_read.tid)
chrom=chrom.upper()
exons = bam_cigar.fetch_exon(chrom, aligned_read.pos, aligned_read.cigar)
totalFrags += len(exons)
for exn in exons:
#print chrom + '\t' + str(exn[1]) + '\t' + str(exn[2])
mid = int(exn[1]) + int((int(exn[2]) - int(exn[1]))/2)
if foundone(chrom,cds_exon_r,mid,mid) > 0:
cds_exon_read += 1
continue
elif foundone(chrom,utr_5_r,mid,mid) >0 and foundone(chrom,utr_3_r,mid,mid) == 0:
utr_5_read += 1
continue
elif foundone(chrom,utr_3_r,mid,mid) >0 and foundone(chrom,utr_5_r,mid,mid) == 0:
utr_3_read += 1
continue
elif foundone(chrom,utr_3_r,mid,mid) >0 and foundone(chrom,utr_5_r,mid,mid) > 0:
unAssignFrags +=1
continue
elif foundone(chrom,intron_r,mid,mid) > 0:
intron_read += 1
continue
elif foundone(chrom,intergenic_up_10kb_r,mid,mid) >0 and foundone(chrom,intergenic_down_10kb_r,mid,mid) > 0:
unAssignFrags +=1
continue
elif foundone(chrom,intergenic_up_1kb_r,mid,mid) >0:
intergenic_up1kb_read += 1
intergenic_up5kb_read += 1
intergenic_up10kb_read += 1
elif foundone(chrom,intergenic_up_5kb_r,mid,mid) >0:
intergenic_up5kb_read += 1
intergenic_up10kb_read += 1
elif foundone(chrom,intergenic_up_10kb_r,mid,mid) >0:
intergenic_up10kb_read += 1
elif foundone(chrom,intergenic_down_1kb_r,mid,mid) >0:
intergenic_down1kb_read += 1
intergenic_down5kb_read += 1
intergenic_down10kb_read += 1
elif foundone(chrom,intergenic_down_5kb_r,mid,mid) >0:
intergenic_down5kb_read += 1
intergenic_down10kb_read += 1
elif foundone(chrom,intergenic_down_10kb_r,mid,mid) >0:
intergenic_down10kb_read += 1
else:
unAssignFrags +=1
except StopIteration:
print >>sys.stderr, "Finished\n"
print "%-30s%d" % ("Total Reads",totalReads)
print "%-30s%d" % ("Total Tags",totalFrags)
print "%-30s%d" % ("Total Assigned Tags",totalFrags-unAssignFrags)
print "====================================================================="
print "%-20s%-20s%-20s%-20s" % ('Group','Total_bases','Tag_count','Tags/Kb')
print "%-20s%-20d%-20d%-18.2f" % ('CDS_Exons',cds_exon_base,cds_exon_read,cds_exon_read*1000.0/(cds_exon_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("5'UTR_Exons",utr_5_base,utr_5_read, utr_5_read*1000.0/(utr_5_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("3'UTR_Exons",utr_3_base,utr_3_read, utr_3_read*1000.0/(utr_3_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("Introns",intron_base,intron_read,intron_read*1000.0/(intron_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TSS_up_1kb",intergenic_up1kb_base, intergenic_up1kb_read, intergenic_up1kb_read*1000.0/(intergenic_up1kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TSS_up_5kb",intergenic_up5kb_base, intergenic_up5kb_read, intergenic_up5kb_read*1000.0/(intergenic_up5kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TSS_up_10kb",intergenic_up10kb_base, intergenic_up10kb_read, intergenic_up10kb_read*1000.0/(intergenic_up10kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TES_down_1kb",intergenic_down1kb_base, intergenic_down1kb_read, intergenic_down1kb_read*1000.0/(intergenic_down1kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TES_down_5kb",intergenic_down5kb_base, intergenic_down5kb_read, intergenic_down5kb_read*1000.0/(intergenic_down5kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TES_down_10kb",intergenic_down10kb_base, intergenic_down10kb_read, intergenic_down10kb_read*1000.0/(intergenic_down10kb_base+1))
print "====================================================================="
def main():
usage = "%prog [options]" + '\n' + __doc__ + "\n"
parser = OptionParser(usage, version="%prog " + __version__)
parser.add_option(
"-i",
"--input-file",
action="store",
type="string",
dest="input_file",
help="Alignment file in BAM format (SAM is not supported). [required]")
parser.add_option("-o",
"--out-prefix",
action="store",
type="string",
dest="output_prefix",
help="Prefix of output files(s). [required]")
parser.add_option("-r",
"--refgene",
action="store",
type="string",
dest="refgene_bed",
help="Reference gene model in bed fomat. [required]")
parser.add_option(
"-d",
"--strand",
action="store",
type="string",
dest="strand_rule",
default=None,
help=
"How read(s) were stranded during sequencing. For example: --strand='1++,1--,2+-,2-+' means that this is a pair-end, strand-specific RNA-seq, and the strand rule is: read1 mapped to '+' => parental gene on '+'; read1 mapped to '-' => parental gene on '-'; read2 mapped to '+' => parental gene on '-'; read2 mapped to '-' => parental gene on '+'. If you are not sure about the strand rule, run \'infer_experiment.py' default=%default (Not a strand specific RNA-seq data)"
)
parser.add_option(
"-u",
"--skip-multi-hits",
action="store_true",
dest="skip_multi",
help=
"How to deal with multiple hit reads. Presence this option renders program to skip multiple hits reads."
)
parser.add_option(
"-e",
"--only-exonic",
action="store_true",
dest="only_exon",
help=
"How to count total reads. Presence of this option renders program only used exonic (UTR exons and CDS exons) reads, otherwise use all reads."
)
(options, args) = parser.parse_args()
if not (options.output_prefix and options.input_file
and options.refgene_bed):
parser.print_help()
sys.exit(0)
if not os.path.exists(options.input_file + '.bai'):
print >> sys.stderr, "cannot find index file of input BAM file"
print >> sys.stderr, options.input_file + '.bai' + " does not exists"
sys.exit(0)
for file in (options.input_file, options.refgene_bed):
if not os.path.exists(file):
print >> sys.stderr, file + " does NOT exists" + '\n'
sys.exit(0)
obj = SAM.ParseBAM(options.input_file)
OUT = open(options.output_prefix + '_read_count.xls', 'w')
#++++++++++++++++++++++++++++++++++++determine strand rule
strandRule = {}
if options.strand_rule is None: # Not strand-specific
pass
elif len(options.strand_rule.split(',')) == 4: #PairEnd, strand-specific
for i in options.strand_rule.split(','):
strandRule[i[0] + i[1]] = i[2]
elif len(options.strand_rule.split(',')) == 2: #singeEnd, strand-specific
for i in options.strand_rule.split(','):
strandRule[i[0]] = i[1]
else:
print >> sys.stderr, "Unknown value of option :'strand_rule' " + options.strand_rule
sys.exit(1)
#++++++++++++++++++++++++++++++++++++counting reads
print >> sys.stderr, "Retrieve exon regions from " + options.refgene_bed + '...'
gene_ranges = build_range(options.refgene_bed)
#print gene_ranges['ERCC-00002'].find(0,100)
print >> sys.stderr, "Counting total reads ... ",
total_reads = 0
total_tags = 0
total_exonic_tags = 0
try:
while (1):
flag = 0
aligned_read = obj.samfile.next()
if aligned_read.is_qcfail: continue #skip low quanlity
if aligned_read.is_duplicate: continue #skip duplicate read
if aligned_read.is_secondary: continue #skip non primary hit
if aligned_read.is_unmapped: continue #skip unmap read
if options.skip_multi:
if len(aligned_read.tags) > 0: #( ("NM", 1),("RG", "L1") )
for i in aligned_read.tags:
if i[0] in SAM.ParseBAM.multi_hit_tags and i[1] > 1:
flag = 1 #multiple hit read
break
if flag == 1: continue #skip multiple map read
total_reads += 1
chrom = obj.samfile.getrname(aligned_read.tid).upper()
hit_st = aligned_read.pos
exon_blocks = bam_cigar.fetch_exon(chrom, hit_st,
aligned_read.cigar)
total_tags += len(exon_blocks)
for exn in exon_blocks:
mid = exn[1] + int((exn[2] - exn[1]) / 2)
#print chrom,mid,mid+1
#print gene_ranges[chrom].find(mid,mid+1)
if (chrom in gene_ranges) and len(gene_ranges[chrom].find(
mid, mid + 1)) > 0:
total_exonic_tags += 1
except StopIteration:
print >> sys.stderr, "Done"
print >> sys.stderr, "Total Reads = %-20s" % (str(total_reads))
print >> sys.stderr, "Total Tags = %-20s" % (str(total_tags))
print >> sys.stderr, "Total Exon Tags = %-20s" % (str(total_exonic_tags))
if total_tags > 0 and total_exonic_tags > 0:
if options.only_exon:
denominator = total_exonic_tags
else:
denominator = total_tags
else:
print >> sys.stderr, "Total tags cannot be 0 or negative number"
sys.exit(1)
#++++++++++++++++++++++++++++++++++++++++++++++++
obj = SAM.ParseBAM(options.input_file)
if options.strand_rule is None:
OUT.write('#chrom' + '\t' + 'st' + '\t' + 'end' + '\t' + 'accession' +
'\t' + 'score' + '\t' + 'gene_strand' + '\t' + 'tag_count' +
'\t' + 'RPKM' + '\n')
else:
OUT.write('#chrom' + '\t' + 'st' + '\t' + 'end' + '\t' + 'accession' +
'\t' + 'score' + '\t' + 'gene_strand' + '\t' +
'tag_count_Forward' + '\t' + 'tag_count_Reverse' + '\t' +
'RPKM_Forward' + '\t' + 'RPKM_Reverse' + '\n')
genome_total_read = 0
genome_unique_read = 0
gene_finished = 0
#calculate raw count, RPKM for each gene
for line in open(options.refgene_bed, 'r'):
exon_range = Intersecter()
intron_range = Intersecter()
if line.startswith(('#', 'track', 'browser')): continue
fields = line.split()
chrom = fields[0]
tx_start = int(fields[1])
tx_end = int(fields[2])
geneName = fields[3]
gstrand = fields[5].replace(" ", "_")
cds_start = int(fields[6])
cds_end = int(fields[7])
exon_starts = map(int, fields[11].rstrip(',\n').split(','))
exon_starts = map((lambda x: x + tx_start), exon_starts)
exon_ends = map(int, fields[10].rstrip(',\n').split(','))
exon_ends = map((lambda x, y: x + y), exon_starts, exon_ends)
intron_starts = exon_ends[:-1]
intron_ends = exon_starts[1:]
plus_ranges = Intersecter()
minus_ranges = Intersecter()
unstrand_ranges = Intersecter()
try:
alignedReads = obj.samfile.fetch(chrom, tx_start, tx_end)
except:
print >> sys.stderr, "No alignments for " + geneName + ". Skip"
continue
for aligned_read in alignedReads:
flag = 0
if aligned_read.is_qcfail: continue #skip low quanlity
if aligned_read.is_duplicate: continue #skip duplicate read
if aligned_read.is_secondary: continue #skip non primary hit
if aligned_read.is_unmapped: continue #skip unmap read
if options.skip_multi:
if len(aligned_read.tags) > 0: #( ("NM", 1),("RG", "L1") )
for i in aligned_read.tags:
if i[0] in SAM.ParseBAM.multi_hit_tags and i[1] > 1:
flag = 1 #multiple hit read
break
if flag == 1: continue #skip multiple map read
if aligned_read.is_paired: #pair end
if aligned_read.is_read1: read_id = '1'
if aligned_read.is_read2: read_id = '2'
else: read_id = '' #single end
if aligned_read.is_reverse: map_strand = '-'
else: map_strand = '+'
strand_key = read_id + map_strand #used to determine if a read should assign to gene(+) or gene(-)
hit_st = aligned_read.pos
exon_blocks = bam_cigar.fetch_exon(chrom, hit_st,
aligned_read.cigar)
#construct bitset
if options.strand_rule is not None:
if strandRule[strand_key] == '+':
for block in exon_blocks:
mid = block[1] + int((block[2] - block[1]) / 2)
plus_ranges.add_interval(Interval(mid, mid + 1))
elif strandRule[strand_key] == '-':
for block in exon_blocks:
mid = block[1] + int((block[2] - block[1]) / 2)
minus_ranges.add_interval(Interval(mid, mid + 1))
elif options.strand_rule is None:
for block in exon_blocks:
mid = block[1] + int((block[2] - block[1]) / 2)
unstrand_ranges.add_interval(Interval(mid, mid + 1))
mRNA_plus_hits = 0
mRNA_plus_rpkm = 0.0
mRNA_minus_hits = 0
mRNA_minus_rpkm = 0.0
mRNA_hits = 0
mRNA_rpkm = 0.0
mRNA_length = 0
#assign reads to region:exon,intron,mRNA
if (options.strand_rule is not None): #this is strand specific
if gstrand == '-':
intronNum = len(intron_starts)
exonNum = len(exon_starts)
elif gstrand == '+':
intronNum = 1
exonNum = 1
#assign reads to intron regions
for st, end in zip(intron_starts, intron_ends):
if end > st:
size = end - st
elif end == st:
size = 1
hits_plus = len(plus_ranges.find(st, end))
hits_minus = len(minus_ranges.find(st, end))
hits_plus_rpkm = hits_plus * 1000000000.0 / (size *
denominator)
hits_minus_rpkm = hits_minus * 1000000000.0 / (size *
denominator)
print >> OUT, '\t'.join([
'%s', '%d', '%d', '%s', '%d', '%s', '%d', '%d', '%.3f',
'%.3f'
]) % (chrom, st, end, geneName + "_intron_" + str(intronNum),
0, gstrand, hits_plus, hits_minus, hits_plus_rpkm,
hits_minus_rpkm)
if gstrand == '-': intronNum -= 1
elif gstrand == '+': intronNum += 1
#assign reads to exon regions
for st, end in zip(exon_starts, exon_ends):
if end > st:
size = end - st
elif end == st:
size = 1
hits_plus = len(plus_ranges.find(st, end))
hits_minus = len(minus_ranges.find(st, end))
hits_plus_rpkm = hits_plus * 1000000000.0 / (size *
denominator)
hits_minus_rpkm = hits_minus * 1000000000.0 / (size *
denominator)
print >> OUT, '\t'.join([
'%s', '%d', '%d', '%s', '%d', '%s', '%d', '%d', '%.3f',
'%.3f'
]) % (chrom, st, end, geneName + "_exon_" + str(exonNum), 0,
gstrand, hits_plus, hits_minus, hits_plus_rpkm,
hits_minus_rpkm)
if gstrand == '-': exonNum -= 1
elif gstrand == '+': exonNum += 1
mRNA_plus_hits += hits_plus
mRNA_minus_hits += hits_minus
mRNA_length += size
mRNA_plus_rpkm = mRNA_plus_hits * 1000000000.0 / (mRNA_length *
denominator)
mRNA_minus_rpkm = mRNA_minus_hits * 1000000000.0 / (mRNA_length *
denominator)
print >> OUT, '\t'.join([
'%s', '%d', '%d', '%s', '%d', '%s', '%d', '%d', '%.3f', '%.3f'
]) % (chrom, tx_start, tx_end, geneName + "_mRNA", 0, gstrand,
mRNA_plus_hits, mRNA_minus_hits, mRNA_plus_rpkm,
mRNA_minus_rpkm)
elif (options.strand_rule is None): #this is NOT strand specific
if gstrand == '-':
intronNum = len(intron_starts)
exonNum = len(exon_starts)
elif gstrand == '+':
intronNum = 1
exonNum = 1
#assign reads to intron regions
for st, end in zip(intron_starts, intron_ends):
if end > st:
size = end - st
elif end == st:
size = 1
hits = len(unstrand_ranges.find(st, end))
hits_rpkm = hits * 1000000000.0 / (size * denominator)
print >> OUT, '\t'.join([
'%s', '%d', '%d', '%s', '%d', '%s', '%d', '%.3f'
]) % (chrom, st, end, geneName + "_intron_" + str(intronNum),
0, gstrand, hits, hits_rpkm)
if gstrand == '-': intronNum -= 1
elif gstrand == '+': intronNum += 1
#assign reads to exon regions
for st, end in zip(exon_starts, exon_ends):
if end > st:
size = end - st
elif end == st:
size = 1
hits = len(unstrand_ranges.find(st, end))
hits_rpkm = hits * 1000000000.0 / (size * denominator)
print >> OUT, '\t'.join([
'%s', '%d', '%d', '%s', '%d', '%s', '%d', '%.3f'
]) % (chrom, st, end, geneName + "_exon_" + str(exonNum), 0,
gstrand, hits, hits_rpkm)
if gstrand == '-': exonNum -= 1
elif gstrand == '+': exonNum += 1
mRNA_hits += hits
mRNA_length += size
mRNA_rpkm = mRNA_hits * 1000000000.0 / (mRNA_length * denominator)
print >> OUT, '\t'.join([
'%s', '%d', '%d', '%s', '%d', '%s', '%d', '%.3f'
]) % (chrom, tx_start, tx_end, geneName + "_mRNA", 0, gstrand,
mRNA_hits, mRNA_rpkm)
gene_finished += 1
print >> sys.stderr, " %d transcripts finished\r" % (gene_finished),
def main():
usage="%prog [options]" + '\n' + __doc__ + "\n"
parser = OptionParser(usage,version="%prog " + __version__)
parser.add_option("-i","--input-file",action="store",type="string",dest="input_file",help="Alignment file in BAM or SAM format.")
parser.add_option("-r","--refgene",action="store",type="string",dest="ref_gene_model",help="Reference gene model in bed format.")
(options,args)=parser.parse_args()
if not (options.input_file and options.ref_gene_model):
parser.print_help()
sys.exit(0)
if not os.path.exists(options.ref_gene_model):
print >>sys.stderr, '\n\n' + options.ref_gene_model + " does NOT exists" + '\n'
#parser.print_help()
sys.exit(0)
if not os.path.exists(options.input_file):
print >>sys.stderr, '\n\n' + options.input_file + " does NOT exists" + '\n'
sys.exit(0)
#build bitset
(cds_exon_r, intron_r, utr_5_r, utr_3_r,\
intergenic_up_1kb_r,intergenic_up_5kb_r,intergenic_up_10kb_r,\
intergenic_down_1kb_r,intergenic_down_5kb_r,intergenic_down_10kb_r,\
cds_exon_base,intron_base,utr_5_base,utr_3_base,\
intergenic_up1kb_base,intergenic_up5kb_base,intergenic_up10kb_base,\
intergenic_down1kb_base,intergenic_down5kb_base,intergenic_down10kb_base) = process_gene_model(options.ref_gene_model)
intron_read=0
cds_exon_read=0
utr_5_read=0
utr_3_read=0
intergenic_up1kb_read=0
intergenic_down1kb_read=0
intergenic_up5kb_read=0
intergenic_down5kb_read=0
intergenic_up10kb_read=0
intergenic_down10kb_read=0
totalReads=0
totalFrags=0
unAssignFrags=0
obj = SAM.ParseBAM(options.input_file)
R_qc_fail=0
R_duplicate=0
R_nonprimary=0
R_unmap=0
print >>sys.stderr, "processing " + options.input_file + " ...",
try:
while(1):
aligned_read = obj.samfile.next()
if aligned_read.is_qcfail: #skip QC fail read
R_qc_fail +=1
continue
if aligned_read.is_duplicate: #skip duplicate read
R_duplicate +=1
continue
if aligned_read.is_secondary: #skip non primary hit
R_nonprimary +=1
continue
if aligned_read.is_unmapped: #skip unmap read
R_unmap +=1
continue
totalReads +=1
chrom = obj.samfile.getrname(aligned_read.tid)
chrom=chrom.upper()
exons = bam_cigar.fetch_exon(chrom, aligned_read.pos, aligned_read.cigar)
totalFrags += len(exons)
for exn in exons:
#print chrom + '\t' + str(exn[1]) + '\t' + str(exn[2])
mid = int(exn[1]) + int((int(exn[2]) - int(exn[1]))/2)
if foundone(chrom,cds_exon_r,mid,mid) > 0:
cds_exon_read += 1
continue
elif foundone(chrom,utr_5_r,mid,mid) >0 and foundone(chrom,utr_3_r,mid,mid) == 0:
utr_5_read += 1
continue
elif foundone(chrom,utr_3_r,mid,mid) >0 and foundone(chrom,utr_5_r,mid,mid) == 0:
utr_3_read += 1
continue
elif foundone(chrom,utr_3_r,mid,mid) >0 and foundone(chrom,utr_5_r,mid,mid) > 0:
unAssignFrags +=1
continue
elif foundone(chrom,intron_r,mid,mid) > 0:
intron_read += 1
continue
elif foundone(chrom,intergenic_up_10kb_r,mid,mid) >0 and foundone(chrom,intergenic_down_10kb_r,mid,mid) > 0:
unAssignFrags +=1
continue
elif foundone(chrom,intergenic_up_1kb_r,mid,mid) >0:
intergenic_up1kb_read += 1
intergenic_up5kb_read += 1
intergenic_up10kb_read += 1
elif foundone(chrom,intergenic_up_5kb_r,mid,mid) >0:
intergenic_up5kb_read += 1
intergenic_up10kb_read += 1
elif foundone(chrom,intergenic_up_10kb_r,mid,mid) >0:
intergenic_up10kb_read += 1
elif foundone(chrom,intergenic_down_1kb_r,mid,mid) >0:
intergenic_down1kb_read += 1
intergenic_down5kb_read += 1
intergenic_down10kb_read += 1
elif foundone(chrom,intergenic_down_5kb_r,mid,mid) >0:
intergenic_down5kb_read += 1
intergenic_down10kb_read += 1
elif foundone(chrom,intergenic_down_10kb_r,mid,mid) >0:
intergenic_down10kb_read += 1
else:
unAssignFrags +=1
except StopIteration:
print >>sys.stderr, "Finished\n"
print "%-30s%d" % ("Total Reads",totalReads)
print "%-30s%d" % ("Total Tags",totalFrags)
print "%-30s%d" % ("Total Assigned Tags",totalFrags-unAssignFrags)
print "====================================================================="
print "%-20s%-20s%-20s%-20s" % ('Group','Total_bases','Tag_count','Tags/Kb')
print "%-20s%-20d%-20d%-18.2f" % ('CDS_Exons',cds_exon_base,cds_exon_read,cds_exon_read*1000.0/(cds_exon_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("5'UTR_Exons",utr_5_base,utr_5_read, utr_5_read*1000.0/(utr_5_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("3'UTR_Exons",utr_3_base,utr_3_read, utr_3_read*1000.0/(utr_3_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("Introns",intron_base,intron_read,intron_read*1000.0/(intron_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TSS_up_1kb",intergenic_up1kb_base, intergenic_up1kb_read, intergenic_up1kb_read*1000.0/(intergenic_up1kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TSS_up_5kb",intergenic_up5kb_base, intergenic_up5kb_read, intergenic_up5kb_read*1000.0/(intergenic_up5kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TSS_up_10kb",intergenic_up10kb_base, intergenic_up10kb_read, intergenic_up10kb_read*1000.0/(intergenic_up10kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TES_down_1kb",intergenic_down1kb_base, intergenic_down1kb_read, intergenic_down1kb_read*1000.0/(intergenic_down1kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TES_down_5kb",intergenic_down5kb_base, intergenic_down5kb_read, intergenic_down5kb_read*1000.0/(intergenic_down5kb_base+1))
print "%-20s%-20d%-20d%-18.2f" % ("TES_down_10kb",intergenic_down10kb_base, intergenic_down10kb_read, intergenic_down10kb_read*1000.0/(intergenic_down10kb_base+1))
print "====================================================================="
def main():
usage="%prog [options]" + '\n' + __doc__ + "\n"
parser = OptionParser(usage,version="%prog " + __version__)
parser.add_option("-i","--input-file",action="store",type="string",dest="input_file",help="Alignment file in BAM format (SAM is not supported). [required]")
parser.add_option("-o","--out-prefix",action="store",type="string",dest="output_prefix",help="Prefix of output files(s). [required]")
parser.add_option("-r","--refgene",action="store",type="string",dest="refgene_bed",help="Reference gene model in bed fomat. [required]")
parser.add_option("-d","--strand",action="store",type="string",dest="strand_rule",default=None,help="How read(s) were stranded during sequencing. For example: --strand='1++,1--,2+-,2-+' means that this is a pair-end, strand-specific RNA-seq, and the strand rule is: read1 mapped to '+' => parental gene on '+'; read1 mapped to '-' => parental gene on '-'; read2 mapped to '+' => parental gene on '-'; read2 mapped to '-' => parental gene on '+'. If you are not sure about the strand rule, run \'infer_experiment.py' default=%default (Not a strand specific RNA-seq data)")
parser.add_option("-u","--skip-multi-hits",action="store_true",dest="skip_multi",help="How to deal with multiple hit reads. Presence this option renders program to skip multiple hits reads.")
parser.add_option("-e","--only-exonic",action="store_true",dest="only_exon",help="How to count total reads. Presence of this option renders program only used exonic (UTR exons and CDS exons) reads, otherwise use all reads.")
parser.add_option("-q","--mapq",action="store",type="int",dest="map_qual",default=30,help="Minimum mapping quality (phred scaled) for an alignment to be called \"uniquely mapped\". default=%default")
(options,args)=parser.parse_args()
if not (options.output_prefix and options.input_file and options.refgene_bed):
parser.print_help()
sys.exit(0)
if not os.path.exists(options.input_file + '.bai'):
print >>sys.stderr, "cannot find index file of input BAM file"
print >>sys.stderr, options.input_file + '.bai' + " does not exists"
sys.exit(0)
for file in (options.input_file, options.refgene_bed):
if not os.path.exists(file):
print >>sys.stderr, file + " does NOT exists" + '\n'
sys.exit(0)
obj = SAM.ParseBAM(options.input_file)
OUT = open(options.output_prefix + '_read_count.xls','w')
#++++++++++++++++++++++++++++++++++++determine strand rule
strandRule={}
if options.strand_rule is None: # Not strand-specific
pass
elif len(options.strand_rule.split(',')) ==4: #PairEnd, strand-specific
for i in options.strand_rule.split(','):strandRule[i[0]+i[1]]=i[2]
elif len(options.strand_rule.split(',')) ==2: #singeEnd, strand-specific
for i in options.strand_rule.split(','):strandRule[i[0]]=i[1]
else:
print >>sys.stderr, "Unknown value of option :'strand_rule' " + options.strand_rule
sys.exit(1)
#++++++++++++++++++++++++++++++++++++counting reads
print >>sys.stderr, "Retrieve exon regions from "+ options.refgene_bed + '...'
gene_ranges = build_range( options.refgene_bed)
#print gene_ranges['ERCC-00002'].find(0,100)
print >>sys.stderr, "Counting total reads ... ",
total_reads =0
total_tags =0
total_exonic_tags =0
try:
while(1):
aligned_read = obj.samfile.next()
if aligned_read.is_qcfail:continue #skip low quanlity
if aligned_read.is_duplicate:continue #skip duplicate read
if aligned_read.is_secondary:continue #skip non primary hit
if aligned_read.is_unmapped:continue #skip unmap read
if options.skip_multi:
if aligned_read.mapq < options.map_qual:
continue
total_reads +=1
chrom = obj.samfile.getrname(aligned_read.tid).upper()
hit_st = aligned_read.pos
exon_blocks = bam_cigar.fetch_exon(chrom, hit_st, aligned_read.cigar)
total_tags += len(exon_blocks)
for exn in exon_blocks:
mid = exn[1] + int((exn[2]-exn[1])/2)
#print chrom,mid,mid+1
#print gene_ranges[chrom].find(mid,mid+1)
if (chrom in gene_ranges) and len(gene_ranges[chrom].find(mid,mid+1)) >0:
total_exonic_tags += 1
except StopIteration:
print >>sys.stderr, "Done"
print >>sys.stderr, "Total Reads = %-20s" % (str(total_reads))
print >>sys.stderr, "Total Tags = %-20s" % (str(total_tags))
print >>sys.stderr, "Total Exon Tags = %-20s" % (str(total_exonic_tags))
if total_tags >0 and total_exonic_tags>0:
if options.only_exon:
denominator = total_exonic_tags
else:
denominator = total_tags
else:
print >>sys.stderr, "Total tags cannot be 0 or negative number"
sys.exit(1)
#++++++++++++++++++++++++++++++++++++++++++++++++
obj = SAM.ParseBAM(options.input_file)
if options.strand_rule is None:
OUT.write('#chrom' + '\t' + 'st' + '\t' + 'end' + '\t' + 'accession' + '\t' + 'score' + '\t' + 'gene_strand' + '\t' + 'tag_count' + '\t' + 'RPKM' + '\n')
else:
OUT.write('#chrom' + '\t' + 'st' + '\t' + 'end' + '\t' + 'accession' + '\t' + 'score' + '\t' + 'gene_strand' + '\t' + 'tag_count_Forward' + '\t' + 'tag_count_Reverse' +'\t' + 'RPKM_Forward' + '\t' + 'RPKM_Reverse' + '\n')
genome_total_read=0
genome_unique_read=0
gene_finished=0
#calculate raw count, RPKM for each gene
for line in open(options.refgene_bed,'r'):
exon_range=Intersecter()
intron_range=Intersecter()
if line.startswith(('#','track','browser')):continue
fields = line.split()
chrom = fields[0]
tx_start = int( fields[1] )
tx_end = int( fields[2] )
geneName = fields[3]
gstrand = fields[5].replace(" ","_")
cds_start = int( fields[6] )
cds_end = int( fields[7] )
exon_starts = map( int, fields[11].rstrip( ',\n' ).split( ',' ) )
exon_starts = map((lambda x: x + tx_start ), exon_starts)
exon_ends = map( int, fields[10].rstrip( ',\n' ).split( ',' ) )
exon_ends = map((lambda x, y: x + y ), exon_starts, exon_ends);
intron_starts = exon_ends[:-1]
intron_ends = exon_starts[1:]
plus_ranges=Intersecter()
minus_ranges=Intersecter()
unstrand_ranges= Intersecter()
try:
alignedReads = obj.samfile.fetch(chrom,tx_start,tx_end)
except:
print >>sys.stderr, "No alignments for " + geneName + ". Skip"
continue
for aligned_read in alignedReads:
flag=0
if aligned_read.is_qcfail:continue #skip low quanlity
if aligned_read.is_duplicate:continue #skip duplicate read
if aligned_read.is_secondary:continue #skip non primary hit
if aligned_read.is_unmapped:continue #skip unmap read
if options.skip_multi:
if len(aligned_read.tags)>0: #( ("NM", 1),("RG", "L1") )
for i in aligned_read.tags:
if i[0] in SAM.ParseBAM.multi_hit_tags and i[1] >1:
flag=1 #multiple hit read
break
if flag==1:continue #skip multiple map read
if aligned_read.is_paired: #pair end
if aligned_read.is_read1:read_id = '1'
if aligned_read.is_read2:read_id = '2'
else:read_id = '' #single end
if aligned_read.is_reverse:map_strand = '-'
else:map_strand = '+'
strand_key = read_id + map_strand #used to determine if a read should assign to gene(+) or gene(-)
hit_st = aligned_read.pos
exon_blocks = bam_cigar.fetch_exon(chrom, hit_st, aligned_read.cigar)
#construct bitset
if options.strand_rule is not None:
if strandRule[strand_key] == '+':
for block in exon_blocks:
mid = block[1] + int((block[2] - block[1])/2)
plus_ranges.add_interval( Interval( mid,mid+1 ) )
elif strandRule[strand_key] == '-':
for block in exon_blocks:
mid = block[1] + int((block[2] - block[1])/2)
minus_ranges.add_interval( Interval( mid,mid+1 ) )
elif options.strand_rule is None:
for block in exon_blocks:
mid = block[1] + int((block[2] - block[1])/2)
unstrand_ranges.add_interval( Interval( mid,mid+1 ) )
mRNA_plus_hits =0
mRNA_plus_rpkm =0.0
mRNA_minus_hits =0
mRNA_minus_rpkm =0.0
mRNA_hits =0
mRNA_rpkm =0.0
mRNA_length=0
#assign reads to region:exon,intron,mRNA
if (options.strand_rule is not None): #this is strand specific
if gstrand == '-':
intronNum=len(intron_starts)
exonNum=len(exon_starts)
elif gstrand == '+':
intronNum=1
exonNum=1
#assign reads to intron regions
for st,end in zip(intron_starts,intron_ends):
if end >st:
size = end - st
elif end == st:
size = 1
hits_plus = len(plus_ranges.find(st,end))
hits_minus = len(minus_ranges.find(st,end))
hits_plus_rpkm = hits_plus*1000000000.0/(size*denominator)
hits_minus_rpkm = hits_minus*1000000000.0/(size*denominator)
print >>OUT, '\t'.join(['%s','%d','%d','%s','%d','%s','%d','%d','%.3f','%.3f']) % (chrom,st,end,geneName + "_intron_" + str(intronNum),0,gstrand,hits_plus,hits_minus,hits_plus_rpkm,hits_minus_rpkm)
if gstrand == '-':intronNum -= 1
elif gstrand == '+':intronNum +=1
#assign reads to exon regions
for st,end in zip(exon_starts,exon_ends):
if end >st:
size = end - st
elif end == st:
size = 1
hits_plus = len(plus_ranges.find(st,end))
hits_minus = len(minus_ranges.find(st,end))
hits_plus_rpkm = hits_plus*1000000000.0/(size*denominator)
hits_minus_rpkm = hits_minus*1000000000.0/(size*denominator)
print >>OUT, '\t'.join(['%s','%d','%d','%s','%d','%s','%d','%d','%.3f','%.3f']) % (chrom,st,end,geneName + "_exon_" + str(exonNum),0,gstrand,hits_plus,hits_minus,hits_plus_rpkm,hits_minus_rpkm)
if gstrand == '-':exonNum -= 1
elif gstrand == '+':exonNum += 1
mRNA_plus_hits += hits_plus
mRNA_minus_hits += hits_minus
mRNA_length += size
mRNA_plus_rpkm = mRNA_plus_hits*1000000000.0/(mRNA_length*denominator)
mRNA_minus_rpkm = mRNA_minus_hits*1000000000.0/(mRNA_length*denominator)
print >>OUT, '\t'.join(['%s','%d','%d','%s','%d','%s','%d','%d','%.3f','%.3f']) % (chrom,tx_start,tx_end,geneName + "_mRNA",0,gstrand,mRNA_plus_hits,mRNA_minus_hits,mRNA_plus_rpkm,mRNA_minus_rpkm)
elif (options.strand_rule is None): #this is NOT strand specific
if gstrand == '-':
intronNum=len(intron_starts)
exonNum=len(exon_starts)
elif gstrand == '+':
intronNum=1
exonNum=1
#assign reads to intron regions
for st,end in zip(intron_starts,intron_ends):
if end >st:
size = end - st
elif end == st:
size = 1
hits = len(unstrand_ranges.find(st,end))
hits_rpkm = hits*1000000000.0/(size*denominator)
print >>OUT, '\t'.join(['%s','%d','%d','%s','%d','%s','%d','%.3f']) % (chrom,st,end,geneName + "_intron_" + str(intronNum),0,gstrand,hits,hits_rpkm)
if gstrand == '-':intronNum -= 1
elif gstrand == '+':intronNum +=1
#assign reads to exon regions
for st,end in zip(exon_starts,exon_ends):
if end >st:
size = end - st
elif end == st:
size = 1
hits = len(unstrand_ranges.find(st,end))
hits_rpkm = hits*1000000000.0/(size*denominator)
print >>OUT, '\t'.join(['%s','%d','%d','%s','%d','%s','%d','%.3f']) % (chrom,st,end,geneName + "_exon_" + str(exonNum),0,gstrand,hits,hits_rpkm)
if gstrand == '-':exonNum -= 1
elif gstrand == '+':exonNum += 1
mRNA_hits += hits
mRNA_length += size
mRNA_rpkm = mRNA_hits*1000000000.0/(mRNA_length*denominator)
print >>OUT, '\t'.join(['%s','%d','%d','%s','%d','%s','%d','%.3f']) % (chrom,tx_start,tx_end,geneName + "_mRNA",0,gstrand,mRNA_hits,mRNA_rpkm)
gene_finished +=1
print >>sys.stderr, " %d transcripts finished\r" % (gene_finished),
