def primerStrip(file, GoodOut, BadOut, fwdprimer, revprimer):
PL = len(fwdprimer)
with open(GoodOut, 'w') as good:
with open(BadOut, 'w') as bad:
for title, seq, qual in FastqGeneralIterator(open(file)):
Diffs = primer.MatchPrefix(seq, fwdprimer)
if Diffs <= args.primer_mismatch:
Seq = seq[PL:]
Qual = qual[PL:]
if revprimer:#now need to look for reverse primer
BestPosRev, BestDiffsRev = primer.BestMatch2(Seq, revcomp_lib.RevComp(revprimer), args.primer_mismatch)
if BestPosRev > 0: #reverse primer was found
Seq = Seq[:BestPosRev]
Qual = Qual[:BestPosRev]
good.write("@%s\n%s\n+\n%s\n" % (title, Seq, Qual))
else:
bad.write("@%s\n%s\n+\n%s\n" % (title, seq, qual))
if Barcode == "": #if not found, move onto next record
continue
BarcodeLength = len(Barcode)
seq = seq[BarcodeLength:]
qual = qual[BarcodeLength:]
#look for forward primer
if args.require_primer != 'off': #means we only want ones with forward primer and or reverse
Diffs = primer.MatchPrefix(seq, FwdPrimer)
if Diffs > args.primer_mismatch:
continue
#if found, trim away primer
seq = seq[trim:]
qual = qual[trim:]
if args.require_primer == 'both':
#look for reverse primer, strip if found
BestPosRev, BestDiffsRev = primer.BestMatch2(
seq, ReverseCompRev, args.primer_mismatch)
if BestPosRev > 0:
seq = seq[:BestPosRev]
qual = qual[:BestPosRev]
else:
continue
#check size
if len(
seq
) < args.min_len: #filter out sequences less than minimum length.
continue
runningTotal += 1
fileout = os.path.join(args.out, BarcodeLabel)
with open(fileout, 'ab') as output:
output.write("@%s\n%s\n+\n%s\n" % (title, seq, qual))
if args.require_primer == 'off':
def processRead(input):
base = os.path.basename(input).split('.')[0]
PL = len(FwdPrimer)
RL = len(RevPrimer)
DemuxOut = os.path.join(tmpdir, base+'.demux.fq')
StatsOut = os.path.join(tmpdir, base+'.stats')
Total = 0
NoBarcode = 0
NoRevBarcode = 0
NoPrimer = 0
TooShort = 0
RevPrimerFound = 0
ValidSeqs = 0
with open(StatsOut, 'w') as counts:
with open(DemuxOut, 'w') as out:
for title, seq, qual in FastqGeneralIterator(open(input)):
Total += 1
#look for barcode, trim it off
Barcode, BarcodeLabel = FindBarcode(seq, Barcodes)
if Barcode == "":
NoBarcode += 1
continue
BarcodeLength = len(Barcode)
Seq = seq[BarcodeLength:]
Qual = qual[BarcodeLength:]
#now search for forward primer
Diffs = primer.MatchPrefix(Seq, FwdPrimer)
if Diffs > args.primer_mismatch:
NoPrimer += 1
continue
ForTrim = PL
#now search for reverse primer
BestPosRev, BestDiffsRev = primer.BestMatch2(Seq, RevPrimer, args.primer_mismatch)
if BestPosRev > 0: #reverse primer was found
RevPrimerFound += 1
#location to trim sequences
RevTrim = BestPosRev
#determine reverse barcode
if args.reverse_barcode:
RevBCdiffs = 0
BCcut = BestPosRev + RL
CutSeq = Seq[BCcut:]
RevBarcode, RevBarcodeLabel = FindBarcode(CutSeq, RevBarcodes)
if RevBarcode == "":
NoRevBarcode += 1
continue
BarcodeLabel = BarcodeLabel+'_'+RevBarcodeLabel
#now trim record remove forward and reverse reads
Seq = Seq[ForTrim:RevTrim]
Qual = Qual[ForTrim:RevTrim]
#since found reverse primer, now also need to pad/trim
if not args.full_length:
#check minimum length here or primer dimer type sequences will get padded with Ns
if len(Seq) < int(args.min_len):
TooShort += 1
continue
if len(Seq) < args.trim_len and args.pad == 'on':
pad = args.trim_len - len(Seq)
Seq = Seq + pad*'N'
Qual = Qual +pad*'J'
else: #len(Seq) > args.trim_len:
Seq = Seq[:args.trim_len]
Qual = Qual[:args.trim_len]
else:
#trim record, did not find reverse primer
if args.full_length: #if full length then move to next record
continue
#trim away forward primer
Seq = Seq[ForTrim:]
Qual = Qual[ForTrim:]
#check length and trim, throw away if too short as it was bad read
if len(Seq) < args.trim_len:
TooShort += 1
continue
Seq = Seq[:args.trim_len]
Qual = Qual[:args.trim_len]
#check minimum length
if len(Seq) < int(args.min_len):
TooShort += 1
continue
ValidSeqs += 1
#rename header
Name = 'R_'+str(ValidSeqs)+';barcodelabel='+BarcodeLabel+';'
out.write("@%s\n%s\n+\n%s\n" % (Name, Seq, Qual))
counts.write('%i,%i,%i,%i,%i,%i,%i\n' % (Total, NoBarcode, NoPrimer, RevPrimerFound, NoRevBarcode, TooShort, ValidSeqs))
def ProcessReads(records):
global OutCount
for rec in records:
#convert to string for processing
Seq = str(rec.seq)
#look for barcodes
Barcode, BarcodeLabel = FindBarcode(Seq, Barcodes)
if Barcode == "": #if not found, try to find with mismatches
if args.barcode_mismatch > 0:
hit = [None, None, 0, None, None]
for k, v in Barcodes.items():
alignment = amptklib.fuzzymatch(v, Seq,
args.barcode_mismatch)
if alignment:
if alignment[0] > hit[2]:
hit = [
k, v, alignment[0], alignment[1], alignment[2]
]
if hit[0] != None:
BarcodeLength = hit[4] - hit[
3] #might be shorter than actual barcode
BarcodeLabel = hit[0]
Barcode = hit[1]
else:
continue
else:
continue
else: #barcode was found from dictionary
BarcodeLength = len(Barcode)
#now look for primer, if not found, move onto next record
BestPosFor, BestDiffsFor = primer.BestMatch2(Seq, FwdPrimer,
MAX_PRIMER_MISMATCHES)
if BestPosFor > 0 and BestPosFor <= BarcodeLength + 2: #if found will be > 0, and should be found after barcode
ForTrim = BestPosFor + PL
else:
continue
#counter for numbering reads
OutCount += 1
#look for reverse primer
BestPosRev, BestDiffsRev = primer.BestMatch2(Seq, RevPrimer,
MAX_PRIMER_MISMATCHES)
if BestPosRev > 0: #reverse primer was found
#location to trim sequences
RevTrim = BestPosRev
#determine reverse barcode
if args.reverse_barcode:
BCcut = BestPosRev + RL
CutSeq = Seq[BCcut:]
if not CutSeq in RevBarcodes:
if args.barcode_mismatch > 0:
hit = [None, None, 0, None, None]
for k, v in RevBarcodes.items():
alignment = amptklib.fuzzymatch(
k, CutSeq, args.barcode_mismatch)
if alignment:
if alignment[0] > hit[2]:
hit = [
v, k, alignment[0], alignment[1],
alignment[2]
]
if hit[0] != None:
BCname = hit[0]
else:
continue
else:
continue
else:
BCname = RevBarcodes.get(CutSeq)
#update name
BarcodeLabel = BarcodeLabel + '_' + BCname
#trim record
rec = TrimRead(rec, ForTrim, RevTrim, BarcodeLabel, OutCount)
#check length
L = len(rec.seq)
if L < MinLen:
continue
if not args.full_length:
#now check trim length, pad if necessary
if L < TrimLen:
pad = TrimLen - L
Seq = str(rec.seq)
Seq = Seq + pad * 'N'
Qual = rec.letter_annotations["phred_quality"]
pad = TrimLen - L
add = [40] * pad
Qual.extend(add)
del rec.letter_annotations["phred_quality"]
rec.seq = Seq
rec.letter_annotations["phred_quality"] = Qual
yield rec
elif L >= TrimLen:
rec = rec[:TrimLen]
yield rec
else:
yield rec
else: #if it is full length, we did not find reverse primer, so drop read
if not args.full_length:
#trim record
rec = TrimRead(rec, ForTrim, False, BarcodeLabel, OutCount)
#check length
L = len(rec.seq)
if L < MinLen: #remove if shorter than minimum length
continue
#truncate down to trim length
if L >= TrimLen:
rec = rec[:TrimLen]
yield rec
def OnRec(Label, Seq, Qual):
global PL, LabelPrefix, SeqCount, OutCount, TooShortCount, PadCount
global FwdPrimerMismatchCount, RevPrimerStrippedCount
global FwdPrimer, RevPrimer
if SeqCount == 0:
progress.InitFile(fastq.File)
progress.File("%u reads, %u outupt, %u bad fwd primer, %u rev primer stripped, %u too short. %u padded" % \
(SeqCount, OutCount, FwdPrimerMismatchCount, RevPrimerStrippedCount, TooShortCount, PadCount))
SeqCount += 1
Seq = Seq
Qual = Qual
Diffs = MatchesPrimer(Seq, FwdPrimer)
if Diffs > MAX_PRIMER_MISMATCHES:
FwdPrimerMismatchCount += 1
return
OutCount += 1
Label = LabelPrefix + str(OutCount) + ";barcodelabel=" + SampleLabel + ";"
# Strip fwd primer
Seq = Seq[PL:]
Qual = Qual[PL:]
BestPosRev, BestDiffsRev = primer.BestMatch2(Seq, RevPrimer,
MAX_PRIMER_MISMATCHES)
if BestPosRev > 0:
# Strip rev primer
RevPrimerStrippedCount += 1
StrippedSeq = Seq[:BestPosRev]
StrippedQual = Qual[:BestPosRev]
# correctness checks
if 1:
Tail = Seq[BestPosRev:]
Diffs2 = primer.MatchPrefix(Tail, RevPrimer)
if Diffs2 != BestDiffsRev:
print >> sys.stderr
print >> sys.stderr, " Seq=" + Seq
print >> sys.stderr, "Tail=" + Tail
print >> sys.stderr, "RevP=" + RevPrimer
die.Die("BestPosRev %u Diffs2 %u BestDiffsRev %u" %
(BestPosRev, Diffs2, BestDiffsRev))
assert StrippedSeq + Tail == Seq
Seq = StrippedSeq
Qual = StrippedQual
L = len(Seq)
assert len(Qual) == L
if L < MinLen:
return
if L < TrimLen:
PadCount += 1
Seq = Seq + (TrimLen - L) * 'N'
Qual = Qual + (TrimLen - L) * 'I'
L = len(Seq)
assert L == TrimLen
assert len(Qual) == TrimLen
L = len(Seq)
if L < TrimLen:
TooShortCount += 1
return
if L > TrimLen:
Seq = Seq[:TrimLen]
Qual = Qual[:TrimLen]
L = len(Seq)
assert L == TrimLen
assert len(Qual) == TrimLen
fastq.WriteRec(out_file, Label, Seq, Qual)
def stripPrimer(records):
for rec in records:
if args.utax == 'unite2utax':
latin = unicode(rec.description, 'utf-8')
test = latin.encode('ascii', 'latin2ascii')
fields = test.split("|")
for i in fields:
if i.startswith("k__"):
tax = i
elif i.startswith("SH"):
unite = i
elif i.startswith("re"):
reps = i
else:
gbID = i
taxonomy = re.sub(";", ",", tax)
taxonomy = re.sub("__", ":", taxonomy)
tf = taxonomy.split(",")
k = tf[0]
k = re.sub('_', ' ', k)
p = tf[1]
p = re.sub('_', ' ', p)
c = tf[2]
c = re.sub('_', ' ', c)
o = tf[3]
o = re.sub('_', ' ', o)
f = tf[4]
f = re.sub('_', ' ', f)
g = tf[5]
g = re.sub('_', ' ', g)
s = tf[6]
s = re.sub('[(].*$', '', s)
s = re.sub('_', ' ', s)
s = re.sub('\.', '', s)
test_species = s.split(' ')
if len(test_species) < 2:
s = 's:'
reformat_tax = []
removal = ("unidentified", "Incertae", "uncultured", "Group",
"incertae")
sp_removal = (" sp", "_sp", "uncultured", "isolate", "mycorrhizae",
"vouchered", "fungal", "basidiomycete", "ascomycete",
"fungus", "symbiont")
if not any(x in k for x in removal):
reformat_tax.append(k)
if not any(x in p for x in removal):
reformat_tax.append(p)
if not any(x in c for x in removal):
reformat_tax.append(c)
if not any(x in o for x in removal):
reformat_tax.append(o)
if not any(x in f for x in removal):
reformat_tax.append(f)
if not any(x in g for x in removal):
reformat_tax.append(g)
if not any(x in s for x in sp_removal):
reformat_tax.append(s)
rec.id = gbID + ";tax=" + ",".join(reformat_tax)
rec.id = re.sub(",s:$", "", rec.id)
rec.id = re.sub("=s:$", "=", rec.id)
if rec.id.endswith(
";tax="): #if there is no taxonomy, get rid of it
rec.id = ""
rec.name = ""
rec.description = ""
elif args.utax == 'rdp2utax':
latin = unicode(rec.description, 'utf-8')
test = latin.encode('ascii', 'latin2ascii')
temp = test.split("\t")
taxLevels = temp[-1]
split_temp = temp[0].split(";")
ID = split_temp[0].split(" ")[0]
s = "s:" + split_temp[0].split(" ", 1)[-1]
s = re.sub('[(].*$', '', s)
s = re.sub(',', '_', s)
s = re.sub('\.', '', s)
test_species = s.split(' ')
if len(test_species) < 2:
s = 's:'
split_tax = taxLevels.split(";")
if "domain" in split_tax:
ki = split_tax.index("domain") - 1
k = "k:" + split_tax[ki]
k = k.replace('"', '')
k = k.split(" ")[0]
else:
k = ""
if "phylum" in split_tax:
pi = split_tax.index("phylum") - 1
p = "p:" + split_tax[pi]
p = p.replace('"', '')
p = p.split(" ")[0]
else:
p = ""
if "class" in split_tax:
ci = split_tax.index("class") - 1
c = "c:" + split_tax[ci]
c = c.replace('"', '')
c = c.split(" ")[0]
else:
c = ""
if "order" in split_tax:
oi = split_tax.index("order") - 1
o = "o:" + split_tax[oi]
o = o.replace('"', '')
o = o.split(" ")[0]
else:
o = ""
if "family" in split_tax:
fi = split_tax.index("family") - 1
f = "f:" + split_tax[fi]
f = f.replace('"', '')
f = f.split(" ")[0]
else:
f = ""
if "genus" in split_tax:
gi = split_tax.index("genus") - 1
g = "g:" + split_tax[gi]
g = g.replace('"', '')
g = g.split(" ")[0]
else:
g = ""
reformat_tax = []
removal = ("unidentified", "Incertae", "uncultured", "Group",
"incertae", "Chloroplast", "unclassified", "Family")
sp_removal = (" sp", "_sp", "uncultured", "isolate", "mycorrhizae",
"vouchered", "fungal", "basidiomycete", "ascomycete",
"fungus", "symbiont", "unclassified", "unidentified",
"bacterium", "phytoplasma")
if not any(x in k for x in removal) and k != "":
reformat_tax.append(k)
if not any(x in p for x in removal) and p != "":
reformat_tax.append(p)
if not any(x in c for x in removal) and c != "":
reformat_tax.append(c)
if not any(x in o for x in removal) and o != "":
reformat_tax.append(o)
if not any(x in f for x in removal) and f != "":
reformat_tax.append(f)
if not any(x in g for x in removal) and g != "":
reformat_tax.append(g)
if not any(x in s for x in sp_removal):
reformat_tax.append(s)
rec.id = ID + ";tax=" + ",".join(reformat_tax)
rec.id = re.sub(",s:$", "", rec.id)
if rec.id.endswith(
";tax="): #if there is no taxonomy, get rid of it
rec.id = ""
rec.name = ""
rec.description = ""
if not args.trimming:
Seq = rec.seq
MAX_PRIMER_MISMATCHES = int(args.primer_mismatch)
revPrimer = revcomp_lib.RevComp(RevPrimer)
BestPosFor, BestDiffsFor = primer.BestMatch2(
Seq, FwdPrimer, MAX_PRIMER_MISMATCHES)
if BestDiffsFor < MAX_PRIMER_MISMATCHES:
if BestPosFor > 0:
stripfwdlen = fwdLen + BestPosFor
StripSeq = Seq[stripfwdlen:]
#now look for reverse
BestPosRev, BestDiffsRev = primer.BestMatch2(
StripSeq, revPrimer, MAX_PRIMER_MISMATCHES)
if BestDiffsRev < MAX_PRIMER_MISMATCHES:
StrippedSeq = StripSeq[:BestPosRev]
else:
StrippedSeq = StripSeq
#after stripping primers, check for ambig bases
if args.drop_ns != 0 and 'N' * args.drop_ns in StrippedSeq:
continue
rec.seq = StrippedSeq
if rec.id != "" and rec.seq != "" and len(rec.seq) > 50:
yield rec
else: #if can't find forward primer, try to reverse complement and look again
RevSeq = revcomp_lib.RevComp(Seq)
BestPosFor, BestDiffsFor = primer.BestMatch2(
RevSeq, FwdPrimer, MAX_PRIMER_MISMATCHES)
if BestDiffsFor < MAX_PRIMER_MISMATCHES:
if BestPosFor > 0:
stripfwdlen = fwdLen + BestPosFor
StripSeq = Seq[stripfwdlen:]
#now look for reverse
BestPosRev, BestDiffsRev = primer.BestMatch2(
StripSeq, revPrimer, MAX_PRIMER_MISMATCHES)
if BestDiffsRev < MAX_PRIMER_MISMATCHES:
StrippedSeq = StripSeq[:BestPosRev]
else:
StrippedSeq = StripSeq
#after stripping primers, check for ambig bases
if args.drop_ns != 0 and 'N' * args.drop_ns in StrippedSeq:
continue
rec.seq = StrippedSeq
if rec.id != "" and rec.seq != "" and len(
rec.seq) > 50:
yield rec
else:
if args.keep_all:
StripSeq = Seq
#now look for reverse
BestPosRev, BestDiffsRev = primer.BestMatch2(
StripSeq, revPrimer, MAX_PRIMER_MISMATCHES)
if BestDiffsRev < MAX_PRIMER_MISMATCHES:
StrippedSeq = StripSeq[:BestPosRev]
else:
StrippedSeq = StripSeq
#after stripping primers, check for ambig bases
if args.drop_ns != 0 and 'N' * args.drop_ns in StrippedSeq:
continue
rec.seq = StrippedSeq
if rec.id != "" and rec.seq != "" and len(
rec.seq) > 50:
yield rec
else:
#check for ambig bases
Seq = str(rec.seq)
if args.drop_ns != 0 and 'N' * args.drop_ns in Seq:
continue
if rec.id != "" and rec.seq != "" and len(rec.seq) > 50:
yield rec
def processRead(input):
#input is expected to be a FASTQ file
#local variables that need to be previously declared: ForPrimer, RevPrimer
Name = os.path.basename(input).split(".fq",-1)[0]
DemuxOut = os.path.join(args.out, Name + '.demux.fq')
Sample = Name.split('_')[0]
StatsOut = os.path.join(args.out, Name+'.stats')
Total = 0
NoPrimer = 0
TooShort = 0
RevPrimerFound = 0
ValidSeqs = 0
PL = len(FwdPrimer)
with open(StatsOut, 'w') as counts:
with open(DemuxOut, 'w') as out:
for title, seq, qual in FastqGeneralIterator(open(input)):
Total += 1
#first thing is look for forward primer, if found trim it off
Diffs = primer.MatchPrefix(seq, FwdPrimer)
#if require primer is on make finding primer in amplicon required if amplicon is larger than read length
#if less than read length, can't enforce primer because could have been trimmed via staggered trim in fastq_mergepairs
if args.primer == 'on' and len(seq) > ReadLen:
if Diffs > args.primer_mismatch:
NoPrimer += 1
continue
Seq = seq[PL:]
Qual = qual[PL:]
else:
if Diffs <= args.primer_mismatch:
Seq = seq[PL:]
Qual = qual[PL:]
else:
NoPrimer += 1
Seq = seq
Qual = qual
#now look for reverse primer
BestPosRev, BestDiffsRev = primer.BestMatch2(Seq, RevPrimer, args.primer_mismatch)
if BestPosRev > 0: #reverse primer was found
RevPrimerFound += 1
#location to trim sequences, trim seqs
Seq = Seq[:BestPosRev]
Qual = Qual[:BestPosRev]
else:
if args.full_length and len(Seq) > ReadLen: #if full length and no primer found, exit, except when length is less than read length
continue
#if full_length is passed, then only trim primers
if not args.full_length:
#got here if primers were found they were trimmed
#now check seq length, pad if too short, trim if too long
if len(Seq) < args.min_len: #need this check here or primer dimers will get through
TooShort += 1
continue
if len(Seq) < args.trim_len and args.pad == 'on':
pad = args.trim_len - len(Seq)
Seq = Seq + pad*'N'
Qual = Qual +pad*'J'
else: #len(Seq) > args.trim_len:
Seq = Seq[:args.trim_len]
Qual = Qual[:args.trim_len]
#got here, reads are primers trimmed and trim/padded, check length
if len(Seq) < args.min_len:
TooShort += 1
continue
ValidSeqs += 1
#now fix header
Title = 'R_'+str(ValidSeqs)+';barcodelabel='+Sample+';'
#now write to file
out.write("@%s\n%s\n+\n%s\n" % (Title, Seq, Qual))
counts.write('%i,%i,%i,%i,%i\n' % (Total, NoPrimer, RevPrimerFound, TooShort, ValidSeqs))
def ProcessReads(records):
OutCount = 0
MAX_PRIMER_MISMATCHES = int(args.primer_mismatch)
LabelPrefix = args.prefix
MinLen = int(args.min_len)
TrimLen = int(args.trim_len)
PL = len(FwdPrimer)
revPrimer = revcomp_lib.RevComp(RevPrimer)
for rec in records:
OutCount += 1
rec.id = LabelPrefix + str(OutCount) + ";barcodelabel=" + name + ";"
rec.name = ""
rec.description = ""
#turn sequence into string for matching
Seq = str(rec.seq)
Diffs = MatchesPrimer(Seq, FwdPrimer)
if args.primer == "on":
if Diffs > MAX_PRIMER_MISMATCHES:
continue
# Strip fwd primer from rec
rec = rec[PL:]
elif args.primer == "off":
if Diffs < MAX_PRIMER_MISMATCHES:
# Strip fwd primer from rec
rec = rec[PL:]
#turn seq into str again
Seq = str(rec.seq)
#look for reverse primer
BestPosRev, BestDiffsRev = primer.BestMatch2(Seq, revPrimer,
MAX_PRIMER_MISMATCHES)
if BestPosRev > 0:
# Strip rev primer from rec.seq
rec = rec[:BestPosRev]
#check length
L = len(rec.seq)
if L < MinLen:
continue
if not args.full_length:
#now check trim length, pad if necessary
if L < TrimLen:
pad = TrimLen - L
Seq = str(rec.seq)
Seq = Seq + pad * 'N'
Qual = rec.letter_annotations["phred_quality"]
pad = TrimLen - L
add = [40] * pad
Qual.extend(add)
del rec.letter_annotations["phred_quality"]
rec.seq = Seq
rec.letter_annotations["phred_quality"] = Qual
yield rec
elif L >= TrimLen:
rec = rec[:TrimLen]
yield rec
else:
yield rec
else:
#check length
L = len(rec.seq)
if not args.full_length:
if args.primer == 'off': #if custom primer used, then need to pad from end not only if rev primer found
if L < MinLen: #but for quality control, need to cull reads that are really short as they are likely garbage
continue
if L < TrimLen:
pad = TrimLen - L
Seq = str(rec.seq)
Seq = Seq + pad * 'N'
Qual = rec.letter_annotations["phred_quality"]
pad = TrimLen - L
add = [40] * pad
Qual.extend(add)
del rec.letter_annotations["phred_quality"]
rec.seq = Seq
rec.letter_annotations["phred_quality"] = Qual
yield rec
elif L >= TrimLen:
rec = rec[:TrimLen]
yield rec
elif args.primer == 'on':
#truncate down to trim length
if L >= TrimLen:
rec = rec[:TrimLen]
yield rec
else:
if L >= MinLen:
yield rec
