def statRead(self, read):
global WARNED_BZIP2_ERROR
seq = read[1]
qual = read[3]
seqlen = len(seq)
gc = 0
for i in xrange(seqlen):
self.totalNum[i] += 1
try:
qnum = util.qualNum(qual[i])
except Exception:
if self.filename.endswith(
"bz2") and WARNED_BZIP2_ERROR == False:
WARNED_BZIP2_ERROR = True
print(
"WARNING: Incompatible bzip2 format, please note that the file compressed with pbzip2 may have problem. Please compress it with bzip2 insteadly.\n"
)
continue
self.totalQual[i] += qnum
b = seq[i]
if b == 'G' or b == 'C':
gc += 1
if b in ALL_BASES:
self.baseCounts[b][i] += 1
self.baseTotalQual[b][i] += qnum
# calculate discontinuity
left = i - 2
right = i + 3
if left < 0:
left = 0
right = 5
elif right >= seqlen:
right = seqlen
left = seqlen - 5
discontinuity = 0
for j in xrange(left, right - 1):
if seq[j] != seq[j + 1]:
discontinuity += 1
self.totalDiscontinuity[i] += discontinuity
#gcPer = int(1000.0* float(gc)/seqlen)
self.gcHistogram[gc] += 1
for i in xrange(seqlen - self.kmerLen):
self.totalKmer += 1
kmer = seq[i:i + self.kmerLen]
if kmer in self.kmerCount:
self.kmerCount[kmer] += 1
else:
self.kmerCount[kmer] = 1
rcKmer = util.reverseComplement(kmer)
if rcKmer not in self.kmerCount:
self.kmerCount[rcKmer] = 0
def statRead(self, read):
seq = read[1]
qual = read[3]
for i in xrange(len(seq)):
self.totalNum[i] += 1
qnum = util.qualNum(qual[i])
self.totalQual[i] += qnum
b = seq[i]
if b in ALL_BASES:
self.baseCounts[b][i] += 1
self.baseTotalQual[b][i] += qnum
for i in xrange(len(seq) - self.kmerLen):
self.totalKmer += 1
kmer = seq[i:i+self.kmerLen]
if kmer in self.kmerCount:
self.kmerCount[kmer] += 1
else:
self.kmerCount[kmer] = 1
def statRead(self, read):
seq = read[1]
qual = read[3]
seqlen = len(seq)
gc = 0
for i in xrange(seqlen):
self.totalNum[i] += 1
qnum = util.qualNum(qual[i])
self.totalQual[i] += qnum
b = seq[i]
if b=='G' or b=='C':
gc += 1
if b in ALL_BASES:
self.baseCounts[b][i] += 1
self.baseTotalQual[b][i] += qnum
# calculate discontinuity
left = i-2
right = i+3
if left<0:
left = 0
right = 5
elif right >= seqlen:
right = seqlen
left = seqlen - 5
discontinuity = 0
for j in xrange(left, right-1):
if seq[j] != seq[j+1]:
discontinuity += 1
self.totalDiscontinuity[i] += discontinuity
#gcPer = int(1000.0* float(gc)/seqlen)
self.gcHistogram[gc] += 1
for i in xrange(seqlen - self.kmerLen):
self.totalKmer += 1
kmer = seq[i:i+self.kmerLen]
if kmer in self.kmerCount:
self.kmerCount[kmer] += 1
else:
self.kmerCount[kmer] = 1
rcKmer = util.reverseComplement(kmer)
if rcKmer not in self.kmerCount:
self.kmerCount[rcKmer] = 0
def statRead(self, read):
seq = read[1]
qual = read[3]
seqlen = len(seq)
gc = 0
for i in xrange(seqlen):
self.totalNum[i] += 1
qnum = util.qualNum(qual[i])
self.totalQual[i] += qnum
b = seq[i]
if b=='G' or b=='C':
gc += 1
if b in ALL_BASES:
self.baseCounts[b][i] += 1
self.baseTotalQual[b][i] += qnum
# calculate discontinuity
left = i-2
right = i+3
if left<0:
left = 0
right = 5
elif right >= seqlen:
right = seqlen
left = seqlen - 5
discontinuity = 0
for j in xrange(left, right-1):
if seq[j] != seq[j+1]:
discontinuity += 1
self.totalDiscontinuity[i] += discontinuity
#gcPer = int(1000.0* float(gc)/seqlen)
self.gcHistogram[gc] += 1
for i in xrange(seqlen - self.kmerLen):
self.totalKmer += 1
kmer = seq[i:i+self.kmerLen]
if kmer in self.kmerCount:
self.kmerCount[kmer] += 1
else:
self.kmerCount[kmer] = 1
rcKmer = util.reverseComplement(kmer)
if rcKmer not in self.kmerCount:
self.kmerCount[rcKmer] = 0
def run(self):
if self.options.debubble:
self.loadBubbleCircles()
#read1_file is required
read1_file = fastq.Reader(self.options.read1_file)
#no front trim if sequence is barcoded
if self.options.barcode:
self.options.trim_front = 0
reporter = QCReporter()
self.r1qc_prefilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
self.r2qc_prefilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
self.r1qc_prefilter.statFile(self.options.read1_file)
if self.options.read2_file != None:
self.r2qc_prefilter.statFile(self.options.read2_file)
self.r1qc_postfilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
self.r2qc_postfilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
readLen = self.r1qc_prefilter.readLen
overlap_histgram = [0 for x in xrange(readLen + 1)]
distance_histgram = [0 for x in xrange(readLen + 1)]
#auto detect trim front and trim tail
if self.options.trim_front == -1 or self.options.trim_tail == -1:
#auto trim for read1
trimFront, trimTail = self.r1qc_prefilter.autoTrim()
if self.options.trim_front == -1:
self.options.trim_front = trimFront
if self.options.trim_tail == -1:
self.options.trim_tail = trimTail
#auto trim for read2
if self.options.read2_file != None:
# check if we should keep same trimming for read1/read2 to keep their length identical
# this option is on by default because lots of dedup algorithms require this feature
if self.options.trim_pair_same:
self.options.trim_front2 = self.options.trim_front
self.options.trim_tail2 = self.options.trim_tail
else:
trimFront2, trimTail2 = self.r2qc_prefilter.autoTrim()
if self.options.trim_front2 == -1:
self.options.trim_front2 = trimFront2
if self.options.trim_tail2 == -1:
self.options.trim_tail2 = trimTail2
print(self.options.read1_file + " options:")
print(self.options)
#if good output folder not specified, set it as the same folder of read1 file
good_dir = self.options.good_output_folder
if good_dir == None:
good_dir = os.path.dirname(self.options.read1_file)
#if bad output folder not specified, set it as the same folder of read1 file
bad_dir = self.options.bad_output_folder
if bad_dir == None:
bad_dir = os.path.join(
os.path.dirname(os.path.dirname(good_dir + "/")), "bad")
#if overlap output folder not specified, set it as the same folder of read1 file
overlap_dir = self.options.overlap_output_folder
if overlap_dir == None:
# overlap_dir = os.path.dirname(self.options.read1_file)
overlap_dir = os.path.join(
os.path.dirname(os.path.dirname(good_dir + "/")), "overlap")
#save QC results at the same folder of good
qc_base_folder = self.options.report_output_folder
if qc_base_folder == None:
qc_base_folder = os.path.join(
os.path.dirname(os.path.dirname(good_dir + "/")), "QC")
if not os.path.exists(qc_base_folder):
os.makedirs(qc_base_folder)
qc_dir = qc_base_folder
if not os.path.exists(good_dir):
os.makedirs(good_dir)
if not os.path.exists(bad_dir):
os.makedirs(bad_dir)
if self.options.store_overlap and self.options.read2_file != None and (
not os.path.exists(overlap_dir)):
os.makedirs(overlap_dir)
gzip_out = self.options.gzip
gzip_comp = self.options.compression
if not gzip_out and self.options.read1_file.endswith(".gz"):
gzip_out = True
good_read1_file = None
bad_read1_file = None
overlap_read1_file = None
if not self.options.qc_only:
good_read1_file = fastq.Writer(
os.path.join(good_dir,
getMainName(self.options.read1_file) +
".good.fq"), gzip_out, gzip_comp)
bad_read1_file = fastq.Writer(
os.path.join(bad_dir,
getMainName(self.options.read1_file) + ".bad.fq"),
gzip_out, gzip_comp)
overlap_read1_file = None
if self.options.store_overlap:
overlap_read1_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.read1_file) + ".overlap.fq"),
gzip_out, gzip_comp)
#other files are optional
read2_file = None
good_read2_file = None
bad_read2_file = None
overlap_read2_file = None
index1_file = None
good_index1_file = None
bad_index1_file = None
overlap_index1_file = None
index2_file = None
good_index2_file = None
bad_index2_file = None
overlap_index2_file = None
#if other files are specified, then read them
if self.options.read2_file != None:
read2_file = fastq.Reader(self.options.read2_file)
if not self.options.qc_only:
good_read2_file = fastq.Writer(
os.path.join(
good_dir,
getMainName(self.options.read2_file) + ".good.fq"),
gzip_out, gzip_comp)
bad_read2_file = fastq.Writer(
os.path.join(
bad_dir,
getMainName(self.options.read2_file) + ".bad.fq"),
gzip_out, gzip_comp)
if self.options.store_overlap and self.options.read2_file != None:
overlap_read2_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.read2_file) +
".overlap.fq"), gzip_out, gzip_comp)
if self.options.index1_file != None:
index1_file = fastq.Reader(self.options.index1_file)
if not self.options.qc_only:
good_index1_file = fastq.Writer(
os.path.join(
good_dir,
getMainName(self.options.index1_file) + ".good.fq"),
gzip_out, gzip_comp)
bad_index1_file = fastq.Writer(
os.path.join(
bad_dir,
getMainName(self.options.index1_file) + ".bad.fq"),
gzip_out, gzip_comp)
if self.options.store_overlap and self.options.read2_file != None:
overlap_index1_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.index1_file) +
".overlap.fq"), gzip_out, gzip_comp)
if self.options.index2_file != None:
index2_file = fastq.Reader(self.options.index2_file)
if not self.options.qc_only:
good_index2_file = fastq.Writer(
os.path.join(
good_dir,
getMainName(self.options.index2_file) + ".good.fq"),
gzip_out, gzip_comp)
bad_index2_file = fastq.Writer(
os.path.join(
bad_dir,
getMainName(self.options.index2_file) + ".bad.fq"),
gzip_out, gzip_comp)
if self.options.store_overlap and self.options.read2_file != None:
overlap_index2_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.index2_file) +
".overlap.fq"), gzip_out, gzip_comp)
r1 = None
r2 = None
i1 = None
i2 = None
# stat numbers
TOTAL_BASES = 0
GOOD_BASES = 0
TOTAL_READS = 0
GOOD_READS = 0
BAD_READS = 0
BADBCD1 = 0
BADBCD2 = 0
BADTRIM1 = 0
BADTRIM2 = 0
BADBBL = 0
BADLEN = 0
BADPOL = 0
BADLQC = 0
BADNCT = 0
BADINDEL = 0
BADMISMATCH = 0
BADDIFF = 0
READ_CORRECTED = 0
BASE_CORRECTED = 0
BASE_SKIPPED_CORRECTION = 0
BASE_ZERO_QUAL_MASKED = 0
OVERLAPPED = 0
OVERLAP_LEN_SUM = 0
OVERLAP_BASE_SUM = 0
# error profiling by overlap analysis
OVERLAP_BASE_ERR = 0
OVERLAP_ERR_MATRIX = init_error_matrix()
#adapter trimming by overlap analysis
TRIMMED_ADAPTER_BASE = 0
TRIMMED_ADAPTER_READ = 0
while True:
r1 = read1_file.nextRead()
if r1 == None:
break
else:
TOTAL_BASES += len(r1[1])
if read2_file != None:
r2 = read2_file.nextRead()
if r2 == None:
break
if index1_file != None:
i1 = index1_file.nextRead()
if i1 == None:
break
if index2_file != None:
i2 = index2_file.nextRead()
if i2 == None:
break
else:
TOTAL_BASES += len(r2[1])
TOTAL_READS += 1
#barcode processing
if self.options.barcode:
barcodeLen1 = barcodeprocesser.detectBarcode(
r1[1], self.options.barcode_length,
self.options.barcode_verify)
if barcodeLen1 == 0:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADBCD1")
BADBCD1 += 1
continue
else:
if r2 == None:
barcodeprocesser.moveBarcodeToName(
r1, self.options.barcode_length,
self.options.barcode_verify)
else:
barcodeLen2 = barcodeprocesser.detectBarcode(
r2[1], self.options.barcode_length,
self.options.barcode_verify)
if barcodeLen2 == 0:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADBCD2")
BADBCD2 += 1
continue
else:
barcodeprocesser.moveAndTrimPair(
r1, r2, barcodeLen1, barcodeLen2,
self.options.barcode_verify)
#trim
if self.options.trim_front > 0 or self.options.trim_tail > 0:
r1 = trim(r1, self.options.trim_front, self.options.trim_tail)
if len(r1[1]) < 5:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADTRIM1")
BADTRIM1 += 1
continue
if r2 != None:
r2 = trim(r2, self.options.trim_front2,
self.options.trim_tail2)
if len(r2[1]) < 5:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADTRIM2")
BADTRIM2 += 1
continue
#filter debubble
if self.options.debubble:
if self.isInBubble(r1[0]):
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADBBL")
BADBBL += 1
continue
#filter sequence length
if len(r1[1]) < self.options.seq_len_req:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file,
bad_index1_file, bad_index2_file, "BADLEN")
BADLEN += 1
continue
#check polyX
if self.options.poly_size_limit > 0:
poly1 = hasPolyX(r1[1], self.options.poly_size_limit,
self.options.allow_mismatch_in_poly)
poly2 = None
if r2 != None:
poly2 = hasPolyX(r2[1], self.options.poly_size_limit,
self.options.allow_mismatch_in_poly)
if poly1 != None or poly2 != None:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADPOL")
BADPOL += 1
continue
#check low quality count
if self.options.unqualified_base_limit > 0:
lowQual1 = lowQualityNum(r1,
self.options.qualified_quality_phred)
lowQual2 = 0
if r2 != None:
lowQual2 = lowQualityNum(
r2, self.options.qualified_quality_phred)
if lowQual1 > self.options.unqualified_base_limit or lowQual1 > self.options.unqualified_base_limit:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADLQC")
BADLQC += 1
continue
#check N number
if self.options.n_base_limit > 0:
nNum1 = nNumber(r1)
nNum2 = 0
if r2 != None:
nNum2 = nNumber(r2)
if nNum1 > self.options.n_base_limit or nNum2 > self.options.n_base_limit:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADNCT")
BADNCT += 1
continue
#check overlap and do error correction
if r2 != None and (not self.options.no_overlap):
(offset, overlap_len, distance) = util.overlap(r1[1], r2[1])
overlap_histgram[overlap_len] += 1
# deal with the case insert DNA is shorter than read length and cause offset is negative
# in this case the adapter is sequenced and should be trimmed
if offset < 0 and overlap_len > 30:
# shift the junk bases
r1[1] = r1[1][0:overlap_len]
r1[3] = r1[3][0:overlap_len]
r2[1] = r2[1][0:overlap_len]
r2[3] = r2[3][0:overlap_len]
TRIMMED_ADAPTER_BASE += abs(offset) * 2
TRIMMED_ADAPTER_READ += 1
# check the sequence length again after adapter trimmed
if len(r1[1]) < self.options.seq_len_req:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADLEN")
BADLEN += 1
continue
# then calc overlap again
(offset, overlap_len,
distance) = util.overlap(r1[1], r2[1])
distance_histgram[distance] += 1
# if distance is too high, then set it as bad mismatch
if distance > 3:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADDIFF")
BADDIFF += 1
continue
if overlap_len > 30:
OVERLAPPED += 1
OVERLAP_LEN_SUM += overlap_len
# we consider the distance is caused by sequencing error
OVERLAP_BASE_SUM += overlap_len * 2
OVERLAP_BASE_ERR += distance
corrected = 0
zero_qual_masked = 0
skipped_mismatch = 0
if distance > 0:
#try to fix low quality base
#hamming = util.hammingDistance(r1[1][len(r1[1]) - overlap_len:], util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#if hamming != distance:
# self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADINDEL")
# BADINDEL += 1
# continue
#print(r1[1][len(r1[1]) - overlap_len:])
#print(util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#print(r1[3][len(r1[1]) - overlap_len:])
#print(util.reverse(r2[3][len(r2[1]) - overlap_len:]))
err_mtx = init_error_matrix()
for o in xrange(overlap_len):
b1 = r1[1][len(r1[1]) - overlap_len + o]
b2 = util.complement(r2[1][-o - 1])
q1 = r1[3][len(r1[3]) - overlap_len + o]
q2 = r2[3][-o - 1]
if b1 != b2:
# print(TOTAL_READS, o, b1, b2, q1, q2)
this_is_corrected = False
if util.qualNum(q1) >= 30 and util.qualNum(
q2) <= 14:
if b1 != 'N' and b2 != 'N':
err_mtx[util.complement(b1)][
util.complement(b2)] += 1
if not self.options.no_correction:
r2[1] = util.changeString(
r2[1], -o - 1, util.complement(b1))
r2[3] = util.changeString(
r2[3], -o - 1, q1)
corrected += 1
this_is_corrected = True
elif util.qualNum(q2) >= 30 and util.qualNum(
q1) <= 14:
if b1 != 'N' and b2 != 'N':
err_mtx[b2][b1] += 1
if not self.options.no_correction:
r1[1] = util.changeString(
r1[1],
len(r1[1]) - overlap_len + o, b2)
r1[3] = util.changeString(
r1[3],
len(r1[3]) - overlap_len + o, q2)
corrected += 1
this_is_corrected = True
if not this_is_corrected:
if self.options.mask_mismatch:
# mask them as zero qual if it is not corrected
zero_qual = '!'
r2[3] = util.changeString(
r2[3], -o - 1, zero_qual)
r1[3] = util.changeString(
r1[3],
len(r1[3]) - overlap_len + o,
zero_qual)
zero_qual_masked += 1
else:
skipped_mismatch += 1
if corrected + zero_qual_masked + skipped_mismatch >= distance:
break
#print(r1[1][len(r1[1]) - overlap_len:])
#print(util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#print(r1[3][len(r1[1]) - overlap_len:])
#print(util.reverse(r2[3][len(r2[1]) - overlap_len:]))
if corrected + zero_qual_masked + skipped_mismatch == distance:
merge_error_matrix(OVERLAP_ERR_MATRIX, err_mtx)
if corrected > 0:
READ_CORRECTED += 1
BASE_CORRECTED += corrected
# multiply by 2 since we mask bases by pair
BASE_ZERO_QUAL_MASKED += zero_qual_masked * 2
BASE_SKIPPED_CORRECTION += skipped_mismatch * 2
else:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADMISMATCH")
BADMISMATCH += 1
continue
if distance == 0 or distance == corrected:
if self.options.store_overlap:
self.writeReads(getOverlap(r1, overlap_len),
getOverlap(r2, overlap_len), i1,
i2, overlap_read1_file,
overlap_read2_file,
overlap_index1_file,
overlap_index2_file, None)
#write to good
self.writeReads(r1, r2, i1, i2, good_read1_file, good_read2_file,
good_index1_file, good_index2_file, None)
GOOD_BASES += len(r1[1])
if i2 != None:
GOOD_BASES += len(r2[1])
if self.options.qc_sample <= 0 or TOTAL_READS < self.options.qc_sample:
self.r1qc_postfilter.statRead(r1)
if r2 != None:
self.r2qc_postfilter.statRead(r2)
GOOD_READS += 1
if self.options.qc_only and TOTAL_READS >= self.options.qc_sample:
break
self.r1qc_postfilter.qc()
#self.r1qc_postfilter.plot(qc_dir, "R1-postfilter")
if self.options.read2_file != None:
self.r2qc_postfilter.qc()
#self.r2qc_postfilter.plot(qc_dir, "R2-postfilter")
#close all files
if not self.options.qc_only:
good_read1_file.close()
bad_read1_file.close()
if self.options.read2_file != None:
good_read2_file.close()
bad_read2_file.close()
if self.options.index1_file != None:
good_index1_file.close()
bad_index1_file.close()
if self.options.index2_file != None:
good_index2_file.close()
bad_index2_file.close()
# print stat numbers
BAD_READS = TOTAL_READS - GOOD_READS
result = {}
result['total_bases'] = TOTAL_BASES
result['good_bases'] = GOOD_BASES
result['total_reads'] = TOTAL_READS
result['good_reads'] = GOOD_READS
result['bad_reads'] = BAD_READS
result['bad_reads_with_bad_barcode'] = BADBCD1 + BADBCD2
result['bad_reads_with_reads_in_bubble'] = BADBBL
result['bad_reads_with_bad_read_length'] = BADLEN + BADTRIM1 + BADTRIM2
result['bad_reads_with_polyX'] = BADPOL
result['bad_reads_with_low_quality'] = BADLQC
result['bad_reads_with_too_many_N'] = BADNCT
result['bad_reads_with_bad_overlap'] = BADMISMATCH + BADINDEL + BADDIFF
result['readlen'] = readLen
# plot result bar figure
labels = [
'good reads', 'has_polyX', 'low_quality', 'too_short', 'too_many_N'
]
counts = [
GOOD_READS, BADPOL, BADLQC, BADLEN + BADTRIM1 + BADTRIM2, BADNCT
]
colors = ['#66BB11', '#FF33AF', '#FFD3F2', '#FFA322', '#FF8899']
if self.options.read2_file != None:
labels.append('bad_overlap')
counts.append(BADMISMATCH + BADINDEL + BADDIFF)
colors.append('#FF6600')
if self.options.debubble:
labels.append('in_bubble')
counts.append(BADBBL)
colors.append('#EEBB00')
if self.options.barcode:
labels.append('bad_barcode')
counts.append(BADBCD1 + BADBCD2)
colors.append('#CCDD22')
for i in xrange(len(counts)):
type_percent = 0.0
if TOTAL_READS > 0:
type_percent = 100.0 * float(counts[i]) / TOTAL_READS
labels[i] = labels[i] + ": " + str(
counts[i]) + "(" + str(type_percent) + "%)"
reporter.addFigure(
'Good reads and bad reads after filtering',
self.r1qc_prefilter.statPlotly(labels, counts, TOTAL_READS,
'filter_stat'), 'filter_stat', "")
#self.r1qc_prefilter.plotFilterStats(labels, counts, colors, TOTAL_READS, os.path.join(qc_dir, "filter-stat.png"))
#squeeze qc data for JSON output
self.r1qc_prefilter.squeeze()
self.r1qc_postfilter.squeeze()
if self.options.read2_file != None:
self.r2qc_prefilter.squeeze()
self.r2qc_postfilter.squeeze()
stat = {}
# stat["options"]=self.options
stat["afterqc_main_summary"] = result
stat["command"] = makeDict(self.options)
stat["kmer_content"] = {}
stat["kmer_content"][
"read1_prefilter"] = self.r1qc_prefilter.topKmerCount[0:10]
stat["kmer_content"][
"read1_postfilter"] = self.r1qc_postfilter.topKmerCount[0:10]
# output more data in JSON file for offline plotting directly from JSON
stat["base_quality"] = {}
stat["base_quality"][
"read1_prefilter"] = self.r1qc_prefilter.baseMeanQual
stat["base_quality"][
"read1_postfilter"] = self.r1qc_postfilter.baseMeanQual
stat["mean_quality"] = {}
stat["mean_quality"]["read1_prefilter"] = self.r1qc_prefilter.meanQual
stat["mean_quality"][
"read1_postfilter"] = self.r1qc_postfilter.meanQual
stat["base_content"] = {}
stat["base_content"]["read1_prefilter"] = self.r1qc_prefilter.percents
stat["base_content"][
"read1_postfilter"] = self.r1qc_postfilter.percents
stat["gc_content"] = {}
stat["gc_content"]["read1_prefilter"] = self.r1qc_prefilter.gcPercents
stat["gc_content"][
"read1_postfilter"] = self.r1qc_postfilter.gcPercents
if self.options.read2_file != None:
stat["kmer_content"][
"read2_prefilter"] = self.r2qc_prefilter.topKmerCount[0:10]
stat["kmer_content"][
"read2_postfilter"] = self.r2qc_postfilter.topKmerCount[0:10]
stat["base_quality"][
"read2_prefilter"] = self.r2qc_prefilter.baseMeanQual
stat["base_quality"][
"read2_postfilter"] = self.r2qc_postfilter.baseMeanQual
stat["mean_quality"][
"read2_prefilter"] = self.r2qc_prefilter.meanQual
stat["mean_quality"][
"read2_postfilter"] = self.r2qc_postfilter.meanQual
stat["base_content"][
"read2_prefilter"] = self.r2qc_prefilter.percents
stat["base_content"][
"read2_postfilter"] = self.r2qc_postfilter.percents
stat["gc_content"][
"read2_prefilter"] = self.r2qc_prefilter.gcPercents
stat["gc_content"][
"read2_postfilter"] = self.r2qc_postfilter.gcPercents
stat["afterqc_overlap"] = {}
stat["afterqc_overlap"]['overlapped_pairs'] = OVERLAPPED
if OVERLAPPED > 0:
stat["afterqc_overlap"]['average_overlap_length'] = float(
OVERLAP_LEN_SUM / OVERLAPPED)
else:
stat["afterqc_overlap"]['average_overlap_length'] = 0.0
stat["afterqc_overlap"]['bad_mismatch_reads'] = BADMISMATCH
stat["afterqc_overlap"]['bad_diff'] = BADDIFF
stat["afterqc_overlap"]['bad_indel_reads'] = BADINDEL
stat["afterqc_overlap"]['corrected_reads'] = READ_CORRECTED
stat["afterqc_overlap"]['corrected_bases'] = BASE_CORRECTED
stat["afterqc_overlap"][
'skipped_correction_bases'] = BASE_SKIPPED_CORRECTION
stat["afterqc_overlap"]['zero_qual_masked'] = BASE_ZERO_QUAL_MASKED
stat["afterqc_overlap"][
'zero_qual_skipped'] = BASE_ZERO_QUAL_MASKED
stat["afterqc_overlap"][
'trimmed_adapter_bases'] = TRIMMED_ADAPTER_BASE
stat["afterqc_overlap"][
'trimmed_adapter_reads'] = TRIMMED_ADAPTER_READ
if OVERLAP_BASE_SUM > 0:
stat["afterqc_overlap"]['error_rate'] = float(
OVERLAP_BASE_ERR) / float(OVERLAP_BASE_SUM)
else:
stat["afterqc_overlap"]['error_rate'] = 0.0
stat["afterqc_overlap"]['error_matrix'] = OVERLAP_ERR_MATRIX
stat["afterqc_overlap"][
'edit_distance_histogram'] = distance_histgram[0:10]
reporter.addFigure(
'Sequence error distribution',
self.r1qc_prefilter.errorPlotly(OVERLAP_ERR_MATRIX,
'error_matrix'),
'error_matrix', "")
reporter.addFigure(
'Overlap length distribution',
self.r1qc_prefilter.overlapPlotly(overlap_histgram, readLen,
TOTAL_READS, 'overlap_stat'),
'overlap_stat', "")
#self.r1qc_prefilter.plotOverlapHistgram(overlap_histgram, readLen, TOTAL_READS, os.path.join(qc_dir, "overlap.png"))
stat_file = open(
os.path.join(qc_dir,
os.path.basename(self.options.read1_file) + ".json"),
"w")
stat_json = json.dumps(stat,
sort_keys=True,
indent=4,
separators=(',', ': '))
stat_file.write(stat_json)
stat_file.close()
self.addFiguresToReport(reporter)
reporter.setStat(stat)
reporter.setVersion(self.options.version)
reporter.output(
os.path.join(qc_dir,
os.path.basename(self.options.read1_file) + ".html"))
def run(self):
if self.options.debubble:
self.loadBubbleCircles()
#read1_file is required
read1_file = fastq.Reader(self.options.read1_file)
#create a QC folder to contains QC results
qc_base_folder = os.path.join(os.path.dirname(self.options.read1_file),
"QC")
if not os.path.exists(qc_base_folder):
os.makedirs(qc_base_folder)
#QC result of this file/pair
qc_dir = os.path.join(qc_base_folder,
os.path.basename(self.options.read1_file))
if not os.path.exists(qc_dir):
os.makedirs(qc_dir)
#no front trim if sequence is barcoded
if self.options.barcode:
self.options.trim_front = 0
reporter = QCReporter()
r1qc_prefilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
r2qc_prefilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
r1qc_prefilter.statFile(self.options.read1_file)
r1qc_prefilter.plot(qc_dir, "R1-prefilter")
if self.options.read2_file != None:
r2qc_prefilter.statFile(self.options.read2_file)
r2qc_prefilter.plot(qc_dir, "R2-prefilter")
r1qc_postfilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
r2qc_postfilter = QualityControl(self.options.qc_sample,
self.options.qc_kmer)
readLen = r1qc_prefilter.readLen
overlap_histgram = [0 for x in xrange(readLen + 1)]
distance_histgram = [0 for x in xrange(readLen + 1)]
#auto detect trim front and trim tail
if self.options.trim_front == -1 or self.options.trim_tail == -1:
#auto trim for read1
trimFront, trimTail = r1qc_prefilter.autoTrim()
if self.options.trim_front == -1:
self.options.trim_front = trimFront
if self.options.trim_tail == -1:
self.options.trim_tail = trimTail
#auto trim for read2
if self.options.read2_file != None:
# check if we should keep same trimming for read1/read2 to keep their length identical
# this option is on by default because lots of dedup algorithms require this feature
if self.options.trim_pair_same:
self.options.trim_front2 = self.options.trim_front
self.options.trim_tail2 = self.options.trim_tail
else:
trimFront2, trimTail2 = r2qc_prefilter.autoTrim()
if self.options.trim_front2 == -1:
self.options.trim_front2 = trimFront2
if self.options.trim_tail2 == -1:
self.options.trim_tail2 = trimTail2
print(self.options.read1_file + " options:")
print(self.options)
#if good output folder not specified, set it as the same folder of read1 file
good_dir = self.options.good_output_folder
if good_dir == None:
good_dir = os.path.dirname(self.options.read1_file)
#if bad output folder not specified, set it as the same folder of read1 file
bad_dir = self.options.bad_output_folder
if bad_dir == None:
bad_dir = os.path.dirname(self.options.read1_file)
#if overlap output folder not specified, set it as the same folder of read1 file
overlap_dir = self.options.overlap_output_folder
if overlap_dir == None:
overlap_dir = os.path.dirname(self.options.read1_file)
if not os.path.exists(good_dir):
os.makedirs(good_dir)
if not os.path.exists(bad_dir):
os.makedirs(bad_dir)
if self.options.store_overlap and self.options.read2_file != None and (
not os.path.exists(overlap_dir)):
os.makedirs(overlap_dir)
good_read1_file = None
bad_read1_file = None
overlap_read1_file = None
if not self.options.qc_only:
good_read1_file = fastq.Writer(
os.path.join(good_dir,
getMainName(self.options.read1_file) +
".good.fq"))
bad_read1_file = fastq.Writer(
os.path.join(bad_dir,
getMainName(self.options.read1_file) + ".bad.fq"))
overlap_read1_file = None
if self.options.store_overlap:
overlap_read1_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.read1_file) + ".overlap.fq"))
#other files are optional
read2_file = None
good_read2_file = None
bad_read2_file = None
overlap_read2_file = None
index1_file = None
good_index1_file = None
bad_index1_file = None
overlap_index1_file = None
index2_file = None
good_index2_file = None
bad_index2_file = None
overlap_index2_file = None
#if other files are specified, then read them
if self.options.read2_file != None:
read2_file = fastq.Reader(self.options.read2_file)
if not self.options.qc_only:
good_read2_file = fastq.Writer(
os.path.join(
good_dir,
getMainName(self.options.read2_file) + ".good.fq"))
bad_read2_file = fastq.Writer(
os.path.join(
bad_dir,
getMainName(self.options.read2_file) + ".bad.fq"))
if self.options.store_overlap and self.options.read2_file != None:
overlap_read2_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.read2_file) +
".overlap.fq"))
if self.options.index1_file != None:
index1_file = fastq.Reader(self.options.index1_file)
if not self.options.qc_only:
good_index1_file = fastq.Writer(
os.path.join(
good_dir,
getMainName(self.options.index1_file) + ".good.fq"))
bad_index1_file = fastq.Writer(
os.path.join(
bad_dir,
getMainName(self.options.index1_file) + ".bad.fq"))
if self.options.store_overlap and self.options.read2_file != None:
overlap_index1_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.index1_file) +
".overlap.fq"))
if self.options.index2_file != None:
index2_file = fastq.Reader(self.options.index2_file)
if not self.options.qc_only:
good_index2_file = fastq.Writer(
os.path.join(
good_dir,
getMainName(self.options.index2_file) + ".good.fq"))
bad_index2_file = fastq.Writer(
os.path.join(
bad_dir,
getMainName(self.options.index2_file) + ".bad.fq"))
if self.options.store_overlap and self.options.read2_file != None:
overlap_index2_file = fastq.Writer(
os.path.join(
overlap_dir,
getMainName(self.options.index2_file) +
".overlap.fq"))
r1 = None
r2 = None
i1 = None
i2 = None
# stat numbers
TOTAL = 0
GOOD = 0
BAD = 0
BADBCD1 = 0
BADBCD2 = 0
BADTRIM1 = 0
BADTRIM2 = 0
BADBBL = 0
BADLEN = 0
BADPOL = 0
BADLQC = 0
BADNCT = 0
BADOL = 0
BADINDEL = 0
BADMISMATCH = 0
BASE_CORRECTED = 0
OVERLAPPED = 0
OVERLAP_LEN_SUM = 0
while True:
r1 = read1_file.nextRead()
if r1 == None:
break
if read2_file != None:
r2 = read2_file.nextRead()
if r2 == None:
break
if index1_file != None:
i1 = index1_file.nextRead()
if i1 == None:
break
if index2_file != None:
i2 = index2_file.nextRead()
if i2 == None:
break
TOTAL += 1
#barcode processing
if self.options.barcode:
barcodeLen1 = barcodeprocesser.detectBarcode(
r1[1], self.options.barcode_length,
self.options.barcode_verify)
if barcodeLen1 == 0:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADBCD1")
BADBCD1 += 1
continue
else:
if r2 == None:
barcodeprocesser.moveBarcodeToName(
r1, self.options.barcode_length,
self.options.barcode_verify)
else:
barcodeLen2 = barcodeprocesser.detectBarcode(
r2[1], self.options.barcode_length,
self.options.barcode_verify)
if barcodeLen2 == 0:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADBCD2")
BADBCD2 += 1
continue
else:
barcodeprocesser.moveAndTrimPair(
r1, r2, barcodeLen1, barcodeLen2,
self.options.barcode_verify)
#trim
if self.options.trim_front > 0 or self.options.trim_tail > 0:
r1 = trim(r1, self.options.trim_front, self.options.trim_tail)
if len(r1[1]) < 5:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADTRIM1")
BADTRIM1 += 1
continue
if r2 != None:
r2 = trim(r2, self.options.trim_front2,
self.options.trim_tail2)
if len(r2[1]) < 5:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADTRIM2")
BADTRIM2 += 1
continue
#filter debubble
if self.options.debubble:
if self.isInBubble(r1[0]):
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADBBL")
BADBBL += 1
continue
#filter sequence length
if len(r1[1]) < self.options.seq_len_req:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file,
bad_index1_file, bad_index2_file, "BADLEN")
BADLEN += 1
continue
#check polyX
if self.options.poly_size_limit > 0:
poly1 = hasPolyX(r1[1], self.options.poly_size_limit,
self.options.allow_mismatch_in_poly)
poly2 = None
if r2 != None:
poly2 = hasPolyX(r2[1], self.options.poly_size_limit,
self.options.allow_mismatch_in_poly)
if poly1 != None or poly2 != None:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADPOL")
BADPOL += 1
continue
#check low quality count
if self.options.unqualified_base_limit > 0:
lowQual1 = lowQualityNum(r1,
self.options.qualified_quality_phred)
lowQual2 = 0
if r2 != None:
lowQual2 = lowQualityNum(
r2, self.options.qualified_quality_phred)
if lowQual1 > self.options.unqualified_base_limit or lowQual1 > self.options.unqualified_base_limit:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADLQC")
BADLQC += 1
continue
#check N number
if self.options.n_base_limit > 0:
nNum1 = nNumber(r1)
nNum2 = 0
if r2 != None:
nNum2 = nNumber(r2)
if nNum1 > self.options.n_base_limit or nNum2 > self.options.n_base_limit:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADNCT")
BADNCT += 1
continue
#check overlap and do error correction
if r2 != None:
(offset, overlap_len, distance) = util.overlap(r1[1], r2[1])
overlap_histgram[overlap_len] += 1
# deal with the case insert DNA is shorter than read length and cause offset is negative
if offset < 0 and overlap_len > 30:
# shift the junk bases
r1[1] = r1[1][0:overlap_len]
r1[3] = r1[3][0:overlap_len]
r2[1] = r2[1][-offset:-offset + overlap_len]
r2[3] = r2[3][-offset:-offset + overlap_len]
# then calc overlap again
(offset, overlap_len,
distance) = util.overlap(r1[1], r2[1])
if overlap_len > 30:
OVERLAPPED += 1
distance_histgram[distance] += 1
OVERLAP_LEN_SUM += overlap_len
corrected = 0
if distance > 2:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADOL")
BADOL += 1
continue
elif distance > 0:
#try to fix low quality base
hamming = util.hammingDistance(
r1[1][len(r1[1]) - overlap_len:],
util.reverseComplement(r2[1][len(r2[1]) -
overlap_len:]))
if hamming != distance:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADINDEL")
BADINDEL += 1
continue
#print(r1[1][len(r1[1]) - overlap_len:])
#print(util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#print(r1[3][len(r1[1]) - overlap_len:])
#print(util.reverse(r2[3][len(r2[1]) - overlap_len:]))
for o in xrange(overlap_len):
b1 = r1[1][len(r1[1]) - overlap_len + o]
b2 = util.complement(r2[1][-o - 1])
q1 = r1[3][len(r1[3]) - overlap_len + o]
q2 = r2[3][-o - 1]
if b1 != b2:
# print(TOTAL, o, b1, b2, q1, q2)
if util.qualNum(q1) >= 27 and util.qualNum(
q2) <= 16:
r2[1] = util.changeString(
r2[1], -o - 1, util.complement(b1))
r2[3] = util.changeString(
r2[3], -o - 1, q1)
corrected += 1
elif util.qualNum(q2) >= 27 and util.qualNum(
q1) <= 16:
r1[1] = util.changeString(
r1[1],
len(r1[1]) - overlap_len + o, b2)
r1[3] = util.changeString(
r1[3],
len(r1[3]) - overlap_len + o, q2)
corrected += 1
if corrected >= distance:
break
#print(r1[1][len(r1[1]) - overlap_len:])
#print(util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#print(r1[3][len(r1[1]) - overlap_len:])
#print(util.reverse(r2[3][len(r2[1]) - overlap_len:]))
if corrected == distance:
BASE_CORRECTED += 1
else:
self.writeReads(r1, r2, i1, i2, bad_read1_file,
bad_read2_file, bad_index1_file,
bad_index2_file, "BADMISMATCH")
BADMISMATCH += 1
continue
if distance == 0 or distance == corrected:
if self.options.store_overlap:
self.writeReads(getOverlap(r1, overlap_len),
getOverlap(r2, overlap_len), i1,
i2, overlap_read1_file,
overlap_read2_file,
overlap_index1_file,
overlap_index2_file, None)
#write to good
self.writeReads(r1, r2, i1, i2, good_read1_file, good_read2_file,
good_index1_file, good_index2_file, None)
if self.options.qc_sample <= 0 or TOTAL < self.options.qc_sample:
r1qc_postfilter.statRead(r1)
if r2 != None:
r2qc_postfilter.statRead(r2)
GOOD += 1
if self.options.qc_only and TOTAL >= self.options.qc_sample:
break
r1qc_postfilter.qc()
r1qc_postfilter.plot(qc_dir, "R1-postfilter")
if self.options.read2_file != None:
r2qc_postfilter.qc()
r2qc_postfilter.plot(qc_dir, "R2-postfilter")
#close all files
if not self.options.qc_only:
good_read1_file.flush()
bad_read1_file.flush()
if self.options.read2_file != None:
good_read2_file.flush()
bad_read2_file.flush()
if self.options.index1_file != None:
good_index1_file.flush()
bad_index1_file.flush()
if self.options.index2_file != None:
good_index2_file.flush()
bad_index2_file.flush()
# print stat numbers
BAD = TOTAL - GOOD
result = {}
result['total_reads'] = TOTAL
result['good_reads'] = GOOD
result['bad_reads'] = BAD
result['bad_reads_with_bad_barcode'] = BADBCD1 + BADBCD2
result['bad_reads_with_reads_in_bubble'] = BADBBL
result['bad_reads_with_bad_read_length'] = BADLEN + BADTRIM1 + BADTRIM2
result['bad_reads_with_polyX'] = BADPOL
result['bad_reads_with_low_quality'] = BADLQC
result['bad_reads_with_too_many_N'] = BADNCT
result['bad_reads_with_bad_overlap'] = BADOL + BADMISMATCH + BADINDEL
# plot result bar figure
labels = [
'good reads', 'has_polyX', 'low_quality', 'too_short', 'too_many_N'
]
counts = [GOOD, BADPOL, BADLQC, BADLEN + BADTRIM1 + BADTRIM2, BADNCT]
colors = ['#66BB11', '#FF33AF', '#FFD3F2', '#FFA322', '#FF8899']
if self.options.read2_file != None:
labels.append('bad_overlap')
counts.append(BADOL + BADMISMATCH + BADINDEL)
colors.append('#FF6600')
if self.options.debubble:
labels.append('in_bubble')
counts.append(BADBBL)
colors.append('#EEBB00')
if self.options.barcode:
labels.append('bad_barcode')
counts.append(BADBCD1 + BADBCD2)
colors.append('#CCDD22')
for i in xrange(len(counts)):
labels[i] = labels[i] + ": " + str(counts[i]) + "(" + str(
100.0 * float(counts[i]) / TOTAL) + "%)"
fig = plt.figure(1)
plt.title("Filtering statistics of sampled " + str(TOTAL) + " reads",
fontsize=12,
color='#666666')
plt.axis('equal')
patches, texts = plt.pie(counts, colors=colors, radius=0.7)
patches, labels, dummy = zip(*sorted(
zip(patches, labels, counts), key=lambda x: x[2], reverse=True))
plt.legend(patches, labels, loc='upper left', fontsize=9)
plt.savefig(os.path.join(qc_dir, "filter-stat.png"),
bbox_inches='tight')
plt.close(1)
stat = {}
# stat["options"]=self.options
stat["summary"] = result
stat["command"] = makeDict(self.options)
stat["kmer_content"] = {}
stat["kmer_content"]["read1_prefilter"] = r1qc_prefilter.topKmerCount[
0:10]
stat["kmer_content"][
"read1_postfilter"] = r1qc_postfilter.topKmerCount[0:10]
if self.options.read2_file != None:
stat["kmer_content"][
"read2_prefilter"] = r2qc_prefilter.topKmerCount[0:10]
stat["kmer_content"][
"read2_postfilter"] = r2qc_postfilter.topKmerCount[0:10]
stat["overlap"] = {}
stat["overlap"]['overlapped_pairs'] = OVERLAPPED
if OVERLAPPED > 0:
stat["overlap"]['average_overlap_length'] = float(
OVERLAP_LEN_SUM / OVERLAPPED)
else:
stat["overlap"]['average_overlap_length'] = 0.0
stat["overlap"]['bad_edit_distance'] = BADOL
stat["overlap"]['bad_mismatch_bases'] = BADMISMATCH
stat["overlap"]['bad_indel'] = BADINDEL
stat["overlap"][
'reads_with_corrected_mismatch_bases'] = BASE_CORRECTED
stat["overlap"][
'overlapped_area_edit_distance_histogram'] = distance_histgram[
0:10]
plotOverlapHistgram(overlap_histgram, readLen, TOTAL,
os.path.join(qc_dir, "overlap.png"))
stat_file = open(os.path.join(qc_dir, "after.json"), "w")
stat_json = json.dumps(stat,
sort_keys=True,
indent=4,
separators=(',', ': '))
stat_file.write(stat_json)
stat_file.close()
self.addFiguresToReport(reporter)
reporter.output(os.path.join(qc_dir, "report.html"))
def run(self):
if self.options.debubble:
self.loadBubbleCircles()
#read1_file is required
read1_file = fastq.Reader(self.options.read1_file)
#create a QC folder to contains QC results
qc_base_folder = os.path.join(os.path.dirname(self.options.read1_file), "QC")
if not os.path.exists(qc_base_folder):
os.makedirs(qc_base_folder)
#QC result of this file/pair
qc_dir = os.path.join(qc_base_folder, os.path.basename(self.options.read1_file))
if not os.path.exists(qc_dir):
os.makedirs(qc_dir)
#no front trim if sequence is barcoded
if self.options.barcode:
self.options.trim_front = 0
reporter = QCReporter()
r1qc_prefilter = QualityControl(self.options.qc_sample, self.options.qc_kmer)
r2qc_prefilter = QualityControl(self.options.qc_sample, self.options.qc_kmer)
r1qc_prefilter.statFile(self.options.read1_file)
r1qc_prefilter.plot(qc_dir, "R1-prefilter")
if self.options.read2_file != None:
r2qc_prefilter.statFile(self.options.read2_file)
r2qc_prefilter.plot(qc_dir, "R2-prefilter")
r1qc_postfilter = QualityControl(self.options.qc_sample, self.options.qc_kmer)
r2qc_postfilter = QualityControl(self.options.qc_sample, self.options.qc_kmer)
readLen = r1qc_prefilter.readLen
overlap_histgram = [0 for x in xrange(readLen+1)]
distance_histgram = [0 for x in xrange(readLen+1)]
#auto detect trim front and trim tail
if self.options.trim_front == -1 or self.options.trim_tail == -1:
#auto trim for read1
trimFront, trimTail = r1qc_prefilter.autoTrim()
if self.options.trim_front == -1:
self.options.trim_front = trimFront
if self.options.trim_tail == -1:
self.options.trim_tail = trimTail
#auto trim for read2
if self.options.read2_file != None:
# check if we should keep same trimming for read1/read2 to keep their length identical
# this option is on by default because lots of dedup algorithms require this feature
if self.options.trim_pair_same:
self.options.trim_front2 = self.options.trim_front
self.options.trim_tail2 = self.options.trim_tail
else:
trimFront2, trimTail2 = r2qc_prefilter.autoTrim()
if self.options.trim_front2 == -1:
self.options.trim_front2 = trimFront2
if self.options.trim_tail2 == -1:
self.options.trim_tail2 = trimTail2
print(self.options.read1_file + " options:")
print(self.options)
#if good output folder not specified, set it as the same folder of read1 file
good_dir = self.options.good_output_folder
if good_dir == None:
good_dir = os.path.dirname(self.options.read1_file)
#if bad output folder not specified, set it as the same folder of read1 file
bad_dir = self.options.bad_output_folder
if bad_dir == None:
bad_dir = os.path.dirname(self.options.read1_file)
#if overlap output folder not specified, set it as the same folder of read1 file
overlap_dir = self.options.overlap_output_folder
if overlap_dir == None:
overlap_dir = os.path.dirname(self.options.read1_file)
if not os.path.exists(good_dir):
os.makedirs(good_dir)
if not os.path.exists(bad_dir):
os.makedirs(bad_dir)
if self.options.store_overlap and self.options.read2_file != None and (not os.path.exists(overlap_dir)):
os.makedirs(overlap_dir)
good_read1_file = None
bad_read1_file = None
overlap_read1_file = None
if not self.options.qc_only:
good_read1_file = fastq.Writer(os.path.join(good_dir, getMainName(self.options.read1_file)+".good.fq"))
bad_read1_file = fastq.Writer(os.path.join(bad_dir, getMainName(self.options.read1_file)+".bad.fq"))
overlap_read1_file = None
if self.options.store_overlap:
overlap_read1_file = fastq.Writer(os.path.join(overlap_dir, getMainName(self.options.read1_file)+".overlap.fq"))
#other files are optional
read2_file = None
good_read2_file = None
bad_read2_file = None
overlap_read2_file = None
index1_file = None
good_index1_file = None
bad_index1_file = None
overlap_index1_file = None
index2_file = None
good_index2_file = None
bad_index2_file = None
overlap_index2_file = None
#if other files are specified, then read them
if self.options.read2_file != None:
read2_file = fastq.Reader(self.options.read2_file)
if not self.options.qc_only:
good_read2_file = fastq.Writer(os.path.join(good_dir, getMainName(self.options.read2_file)+".good.fq"))
bad_read2_file = fastq.Writer(os.path.join(bad_dir, getMainName(self.options.read2_file)+".bad.fq"))
if self.options.store_overlap and self.options.read2_file != None:
overlap_read2_file = fastq.Writer(os.path.join(overlap_dir, getMainName(self.options.read2_file)+".overlap.fq"))
if self.options.index1_file != None:
index1_file = fastq.Reader(self.options.index1_file)
if not self.options.qc_only:
good_index1_file = fastq.Writer(os.path.join(good_dir, getMainName(self.options.index1_file)+".good.fq"))
bad_index1_file = fastq.Writer(os.path.join(bad_dir, getMainName(self.options.index1_file)+".bad.fq"))
if self.options.store_overlap and self.options.read2_file != None:
overlap_index1_file = fastq.Writer(os.path.join(overlap_dir, getMainName(self.options.index1_file)+".overlap.fq"))
if self.options.index2_file != None:
index2_file = fastq.Reader(self.options.index2_file)
if not self.options.qc_only:
good_index2_file = fastq.Writer(os.path.join(good_dir, getMainName(self.options.index2_file)+".good.fq"))
bad_index2_file = fastq.Writer(os.path.join(bad_dir, getMainName(self.options.index2_file)+".bad.fq"))
if self.options.store_overlap and self.options.read2_file != None:
overlap_index2_file = fastq.Writer(os.path.join(overlap_dir, getMainName(self.options.index2_file)+".overlap.fq"))
r1 = None
r2 = None
i1 = None
i2 = None
# stat numbers
TOTAL = 0
GOOD = 0
BAD = 0
BADBCD1 = 0
BADBCD2 = 0
BADTRIM1 = 0
BADTRIM2 = 0
BADBBL = 0
BADLEN = 0
BADPOL = 0
BADLQC = 0
BADNCT = 0
BADOL = 0
BADINDEL = 0
BADMISMATCH = 0
BASE_CORRECTED = 0
OVERLAPPED = 0
OVERLAP_LEN_SUM = 0
while True:
r1 = read1_file.nextRead()
if r1==None:
break
if read2_file != None:
r2 = read2_file.nextRead()
if r2==None:
break
if index1_file != None:
i1 = index1_file.nextRead()
if i1==None:
break
if index2_file != None:
i2 = index2_file.nextRead()
if i2==None:
break
TOTAL += 1
#barcode processing
if self.options.barcode:
barcodeLen1 = barcodeprocesser.detectBarcode(r1[1], self.options.barcode_length, self.options.barcode_verify)
if barcodeLen1 == 0:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADBCD1")
BADBCD1 += 1
continue
else:
if r2 == None:
barcodeprocesser.moveBarcodeToName(r1, self.options.barcode_length, self.options.barcode_verify)
else:
barcodeLen2 = barcodeprocesser.detectBarcode(r2[1], self.options.barcode_length, self.options.barcode_verify)
if barcodeLen2 == 0:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADBCD2")
BADBCD2 += 1
continue
else:
barcodeprocesser.moveAndTrimPair(r1, r2, barcodeLen1, barcodeLen2, self.options.barcode_verify)
#trim
if self.options.trim_front > 0 or self.options.trim_tail > 0:
r1 = trim(r1, self.options.trim_front, self.options.trim_tail)
if len(r1[1]) < 5:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADTRIM1")
BADTRIM1 += 1
continue
if r2 != None:
r2 = trim(r2, self.options.trim_front2, self.options.trim_tail2)
if len(r2[1]) < 5:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADTRIM2")
BADTRIM2 += 1
continue
#filter debubble
if self.options.debubble:
if self.isInBubble(r1[0]):
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADBBL")
BADBBL += 1
continue
#filter sequence length
if len(r1[1])<self.options.seq_len_req:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADLEN")
BADLEN += 1
continue
#check polyX
if self.options.poly_size_limit > 0:
poly1 = hasPolyX(r1[1], self.options.poly_size_limit, self.options.allow_mismatch_in_poly)
poly2 = None
if r2!=None:
poly2 = hasPolyX(r2[1], self.options.poly_size_limit, self.options.allow_mismatch_in_poly)
if poly1!=None or poly2!=None:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADPOL")
BADPOL += 1
continue
#check low quality count
if self.options.unqualified_base_limit > 0:
lowQual1 = lowQualityNum(r1, self.options.qualified_quality_phred)
lowQual2 = 0
if r2!=None:
lowQual2 = lowQualityNum(r2, self.options.qualified_quality_phred)
if lowQual1 > self.options.unqualified_base_limit or lowQual1 > self.options.unqualified_base_limit:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADLQC")
BADLQC += 1
continue
#check N number
if self.options.n_base_limit > 0:
nNum1 = nNumber(r1)
nNum2 = 0
if r2!=None:
nNum2 = nNumber(r2)
if nNum1 > self.options.n_base_limit or nNum2 > self.options.n_base_limit:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADNCT")
BADNCT += 1
continue
#check overlap and do error correction
if r2!=None:
(offset, overlap_len, distance) = util.overlap(r1[1], r2[1])
overlap_histgram[overlap_len] += 1
# deal with the case insert DNA is shorter than read length and cause offset is negative
if offset <0 and overlap_len > 30:
# shift the junk bases
r1[1] = r1[1][0:overlap_len]
r1[3] = r1[3][0:overlap_len]
r2[1] = r2[1][-offset:-offset+overlap_len]
r2[3] = r2[3][-offset:-offset+overlap_len]
# then calc overlap again
(offset, overlap_len, distance) = util.overlap(r1[1], r2[1])
if overlap_len>30:
OVERLAPPED += 1
distance_histgram[distance] += 1
OVERLAP_LEN_SUM += overlap_len
corrected = 0
if distance > 2:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADOL")
BADOL += 1
continue
elif distance>0:
#try to fix low quality base
hamming = util.hammingDistance(r1[1][len(r1[1]) - overlap_len:], util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
if hamming != distance:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADINDEL")
BADINDEL += 1
continue
#print(r1[1][len(r1[1]) - overlap_len:])
#print(util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#print(r1[3][len(r1[1]) - overlap_len:])
#print(util.reverse(r2[3][len(r2[1]) - overlap_len:]))
for o in xrange(overlap_len):
b1 = r1[1][len(r1[1]) - overlap_len + o]
b2 = util.complement(r2[1][-o-1])
q1 = r1[3][len(r1[3]) - overlap_len + o]
q2 = r2[3][-o-1]
if b1 != b2:
# print(TOTAL, o, b1, b2, q1, q2)
if util.qualNum(q1) >= 27 and util.qualNum(q2) <= 16:
r2[1] = util.changeString(r2[1], -o-1, util.complement(b1))
r2[3] = util.changeString(r2[3], -o-1, q1)
corrected += 1
elif util.qualNum(q2) >= 27 and util.qualNum(q1) <= 16:
r1[1]= util.changeString(r1[1], len(r1[1]) - overlap_len + o, b2)
r1[3] = util.changeString(r1[3], len(r1[3]) - overlap_len + o, q2)
corrected += 1
if corrected >= distance:
break
#print(r1[1][len(r1[1]) - overlap_len:])
#print(util.reverseComplement(r2[1][len(r2[1]) - overlap_len:]))
#print(r1[3][len(r1[1]) - overlap_len:])
#print(util.reverse(r2[3][len(r2[1]) - overlap_len:]))
if corrected == distance:
BASE_CORRECTED += 1
else:
self.writeReads(r1, r2, i1, i2, bad_read1_file, bad_read2_file, bad_index1_file, bad_index2_file, "BADMISMATCH")
BADMISMATCH += 1
continue
if distance == 0 or distance == corrected:
if self.options.store_overlap:
self.writeReads(getOverlap(r1, overlap_len), getOverlap(r2, overlap_len), i1, i2, overlap_read1_file, overlap_read2_file, overlap_index1_file, overlap_index2_file, None)
#write to good
self.writeReads(r1, r2, i1, i2, good_read1_file, good_read2_file, good_index1_file, good_index2_file, None)
r1qc_postfilter.statRead(r1)
if r2 != None:
r2qc_postfilter.statRead(r2)
GOOD += 1
if self.options.qc_only and TOTAL >= self.options.qc_sample:
break
r1qc_postfilter.qc()
r1qc_postfilter.plot(qc_dir, "R1-postfilter")
if self.options.read2_file != None:
r2qc_postfilter.qc()
r2qc_postfilter.plot(qc_dir, "R2-postfilter")
#close all files
if not self.options.qc_only:
good_read1_file.flush()
bad_read1_file.flush()
if self.options.read2_file != None:
good_read2_file.flush()
bad_read2_file.flush()
if self.options.index1_file != None:
good_index1_file.flush()
bad_index1_file.flush()
if self.options.index2_file != None:
good_index2_file.flush()
bad_index2_file.flush()
# print stat numbers
BAD = TOTAL - GOOD
result = {}
result['total_reads']=TOTAL
result['good_reads']=GOOD
result['bad_reads']=BAD
result['bad_reads_with_bad_barcode']= BADBCD1 + BADBCD2
result['bad_reads_with_reads_in_bubble']= BADBBL
result['bad_reads_with_bad_read_length']= BADLEN + BADTRIM1 + BADTRIM2
result['bad_reads_with_polyX']= BADPOL
result['bad_reads_with_low_quality']=BADLQC
result['bad_reads_with_too_many_N']= BADNCT
result['bad_reads_with_bad_overlap']= BADOL + BADMISMATCH + BADINDEL
# plot result bar figure
labels = ['good reads', 'has_polyX', 'low_quality', 'too_short', 'too_many_N']
counts = [GOOD, BADPOL, BADLQC, BADLEN + BADTRIM1 + BADTRIM2, BADNCT]
colors = ['green', '#FF1111', '#FF3333', '#FF5555', '#FF7777']
if self.options.read2_file != None:
labels.append('bad_overlap')
counts.append(BADOL + BADMISMATCH + BADINDEL)
colors.append('#FF9999')
if self.options.debubble:
labels.append('in_bubble')
counts.append(BADBBL)
colors.append('#FFBBBB')
if self.options.barcode:
labels.append('bad_barcode')
counts.append(BADBCD1 + BADBCD2)
colors.append('#FFDDDD')
fig = plt.figure(1)
plt.title("Good reads (green) and bad reads (red) of total " + str(TOTAL))
fig.subplots_adjust(left = 0.14)
lefts = xrange(len(counts))
plt.yticks(lefts, labels)
plt.ylim(-0.5, len(counts)-0.5)
plt.barh(lefts, counts, align='center', height=0.5, alpha=0.8, color=colors)
plt.savefig(os.path.join(qc_dir, "filter-stat.png"))
plt.close(1)
stat={}
# stat["options"]=self.options
stat["summary"]=result
stat["command"]=makeDict(self.options)
stat["kmer_content"] = {}
stat["kmer_content"]["read1_prefilter"] = r1qc_prefilter.topKmerCount[0:10]
stat["kmer_content"]["read1_postfilter"] = r1qc_postfilter.topKmerCount[0:10]
if self.options.read2_file != None:
stat["kmer_content"]["read2_prefilter"] = r2qc_prefilter.topKmerCount[0:10]
stat["kmer_content"]["read2_postfilter"] = r2qc_postfilter.topKmerCount[0:10]
stat["overlap"]={}
stat["overlap"]['overlapped_pairs']=OVERLAPPED
if OVERLAPPED > 0:
stat["overlap"]['average_overlap_length']=float(OVERLAP_LEN_SUM/OVERLAPPED)
else:
stat["overlap"]['average_overlap_length']=0.0
stat["overlap"]['bad_edit_distance']=BADOL
stat["overlap"]['bad_mismatch_bases']=BADMISMATCH
stat["overlap"]['bad_indel']=BADINDEL
stat["overlap"]['reads_with_corrected_mismatch_bases']=BASE_CORRECTED
stat["overlap"]['overlapped_area_edit_distance_histogram']=distance_histgram[0:10]
plotOverlapHistgram(overlap_histgram, readLen, TOTAL, os.path.join(qc_dir, "overlap.png"))
stat_file = open(os.path.join(qc_dir, "after.json"), "w")
stat_json = json.dumps(stat, sort_keys=True,indent=4, separators=(',', ': '))
stat_file.write(stat_json)
stat_file.close()
self.addFiguresToReport(reporter)
reporter.output(os.path.join(qc_dir, "report.html"))