def _processPrimers(self, primer_fn_forward, primer_fn_reverse, window_size, primer_out_fn,
revcmp_primers=False):
"""
Do basic sanity checks that:
(1) all primers in forward start with f_xxx and are unique
(2) all primers in reverse start with r_xxx and are unique
(3) check that no forward primers appear in reverse primers (no symmetry)
(4) write the primers (f_xxx, f_xxx_revcmp, r_xxx, r_xxx_revcmp) all to one primer file
"""
def sanity_check_primers(reader, prefix):
"""
Go through the primers, check that the prefix exists and all seqs are unique
"""
primers = {} # primer -> sequence, but can also contain the revcmp version with _revcmp suffix
for r in reader:
if not r.name.startswith(prefix):
errMsg = "Forward primer should start with f_, but saw:", r.name
raise ClassifierException(errMsg)
if len(r.sequence) > window_size:
errMsg = "Primer {n} has length {l} which is longer than {k}.".\
format(n=r.name, l=len(r.sequence), k=window_size)
logging.error(errMsg)
raise ClassifierException(errMsg)
ss = r.sequence.upper()
if ss in primers.itervalues():
errMsg = "Duplicate sequences found for", ss
raise ClassifierException(errMsg)
primers[r.name.strip()] = r.sequence
# revcmp not needed becuz phmmer does both strands apparently...
#primers[r.name.strip() + "_revcmp"] = revcmp(r.sequence)
return primers
logging.info("Process primers for {case}.".
format(case=("finding primers" if not revcmp_primers
else "detecting chimeras")))
reader_f = FastaReader(primer_fn_forward)
reader_r = FastaReader(primer_fn_reverse)
primers_f = sanity_check_primers(reader_f, prefix="f_")
primers_r = sanity_check_primers(reader_r, prefix="r_")
reader_f.close()
reader_r.close()
same_seqs = set(primers_f.values()).intersection(primers_r.values())
if len(same_seqs) > 0:
errMsg = "Identical sequences found in both Forward/Reverse!\n"
errMsg += "\n".join(same_seqs)
raise ClassifierException(errMsg)
# Write Fi and reverse-complemented Ri to primer_out_fn
with open(primer_out_fn, 'w') as f:
for (name, seq) in primers_f.iteritems():
f.write(">{n}\n{s}\n".format(n=name, s=seq))
for (name, seq) in primers_r.iteritems():
f.write(">{n}\n{s}\n".format(n=name, s=revcmp(seq)))
return primers_f.keys() + primers_r.keys()
primers.append(['R{n}_revcmp'.format(n=primerComboId),
revcmpR + "T" * 4])
else: # Ri and Fi are not revcmp identical
if revcmp_primers is False:
# Save >Ri and revcmp(Ri_sequence)
primers.append([expectedName, revcmpR])
else:
# Save >Ri and Ri_sequence
primers.append([expectedName, fwdR])
# Save >Fi_revcmp and revcmp(Fi_sequence)
primers.append(['F{n}_revcmp'.format(n=primerComboId),
revcmpF])
# Save >Ri_revcmp and revcmp(Ri_sequence)
primers.append(['R{n}_revcmp'.format(n=primerComboId),
revcmpR])
freader.close()
# Write Fi and reverse-complemented Ri to primer_out_fn
f = open(primer_out_fn, 'w')
for (name, seq) in primers:
f.write(">{n}\n{s}\n".format(n=name, s=seq))
f.close()
return range(0, primerComboId + 1)
@property
def numReads(self):
"""Return the number of reads in reads_fn."""
cmd = "grep -c '>' {r}".format(r=real_upath(self.reads_fn))
output, errCode, errMsg = backticks(cmd)
if errCode != 0:
raise ClassifierException(
raise ClassifierException(errMsg)
primers[r.name.strip()] = r.sequence
# revcmp not needed becuz phmmer does both strands apparently...
#primers[r.name.strip() + "_revcmp"] = revcmp(r.sequence)
return primers
logging.info("Process primers for {case}.".format(case=(
"finding primers" if not revcmp_primers else "detecting chimeras"
)))
reader_f = FastaReader(primer_fn_forward)
reader_r = FastaReader(primer_fn_reverse)
primers_f = sanity_check_primers(reader_f, prefix="f_")
primers_r = sanity_check_primers(reader_r, prefix="r_")
reader_f.close()
reader_r.close()
same_seqs = set(primers_f.values()).intersection(primers_r.values())
if len(same_seqs) > 0:
errMsg = "Identical sequences found in both Forward/Reverse!\n"
errMsg += "\n".join(same_seqs)
raise ClassifierException(errMsg)
# Write Fi and reverse-complemented Ri to primer_out_fn
with open(primer_out_fn, 'w') as f:
for (name, seq) in primers_f.iteritems():
f.write(">{n}\n{s}\n".format(n=name, s=seq))
for (name, seq) in primers_r.iteritems():
f.write(">{n}\n{s}\n".format(n=name, s=revcmp(seq)))
return primers_f.keys() + primers_r.keys()
def _processPrimers(self, primer_fn, window_size, primer_out_fn,
revcmp_primers=False):
"""
Check and generate primers.
1. Check primers in primer_fn are in order F0, R0, F1, R1, ...
Fn, Rn, and lengths are all < k, where k is the primer search
window length.
F0 5' NNNNNNNNNN 3'
R0 3' NNNNNNNNNN 5'
2. If Ri and Fi are revers complementarily identical,
add a polyA tail to 3' of Ri.
3. For each combo of primers Fi and Ri, save the following to
primer_out_fn.
3.1 If revcmp_primers is False,
>Fi
Fi_sequence
>Ri
revcmp(Ri_sequence)
3.2 If revcmp_primers is True,
>Fi
Fi_sequence
>Ri
Ri_sequence
>Fi_revcmp
revcmp(Fi_sqeuence)
>Ri_revcmp
revcmp(Ri_sqeuence)
4. return primers range(0, n)
"""
logging.info("Process primers for {case}.".
format(case=("finding primers" if not revcmp_primers
else "detecting chimeras")))
freader = FastaReader(primer_fn)
primers = []
primerComboId = -1
for i, r in enumerate(freader):
if i % 2 == 0:
direction = "F"
primerComboId += 1
else:
direction = "R"
expectedName = "{d}{n}".format(d=direction, n=primerComboId)
if r.name != expectedName:
errMsg = "Primers should be placed in order F0, R0, F1, R1..."
logging.error(errMsg)
raise ClassifierException(errMsg)
if len(r.sequence) > window_size:
errMsg = "Primer {n} has length {l} which is longer than {k}.".\
format(n=expectedName, l=len(r.sequence), k=window_size)
logging.error(errMsg)
raise ClassifierException(errMsg)
if direction == "F":
# Save >Fi and Fi_sequence.
primers.append([expectedName, r.sequence])
else: # direction is "R"
# fwdF/fwdR is the forward sequence of Fi/Ri
fwdF, fwdR = primers[-1][1], r.sequence
# revcmpF/revcmpR is the reverse complement of Fi/Ri
revcmpF, revcmpR = revcmp(fwdF), revcmp(fwdR)
# If Fi and Ri are reverse complementariliy identical, bail out,
# because we need Poly A tail to distinguish Fi and Ri.
if fwdF.find(revcmpR) >= 0 or revcmpR.find(fwdF) >= 0:
infoMsg = "Primer F{n}, R{n} ".format(n=primerComboId) + \
"are reverse complementarily identical. " + \
"Need to add 'AAAA' to 3' to distinguish them."
logging.info(infoMsg)
if revcmp_primers is False:
# Save primer Ri and revcmp(Ri_sequence) + TTTT
primers.append([expectedName, revcmpR + "T" * 4])
else: # revcmp_primers is True
primers.append([expectedName, "A" * 4 + fwdR])
primers.append(['F{n}_revcmp'.format(n=primerComboId),
revcmpF])
primers.append(['R{n}_revcmp'.format(n=primerComboId),
revcmpR + "T" * 4])
else: # Ri and Fi are not revcmp identical
if revcmp_primers is False:
# Save >Ri and revcmp(Ri_sequence)
primers.append([expectedName, revcmpR])
else:
# Save >Ri and Ri_sequence
primers.append([expectedName, fwdR])
# Save >Fi_revcmp and revcmp(Fi_sequence)
primers.append(['F{n}_revcmp'.format(n=primerComboId),
revcmpF])
# Save >Ri_revcmp and revcmp(Ri_sequence)
primers.append(['R{n}_revcmp'.format(n=primerComboId),
revcmpR])
freader.close()
# Write Fi and reverse-complemented Ri to primer_out_fn
f = open(primer_out_fn, 'w')
for (name, seq) in primers:
f.write(">{n}\n{s}\n".format(n=name, s=seq))
f.close()
return range(0, primerComboId + 1)
