def test_fileBased(self):
test_file_roots = [
'primer_must_use_th', 'primer_task_th', 'primer_thal_args',
'primer_thal_max_seq_error', 'primer_first_base_index',
'test_compl_error', 'test_left_to_right_of_right',
'dv_conc_vs_dntp_conc', 'primer_internal', 'primer_tm_lc_masking',
'primer_ok_regions', 'primer_start_codon', 'primer_task',
'primer_renewed_tasks', 'primer_must_overlap_point',
'primer_overlap_junction', 'primer_all_settingsfiles',
'primer_high_tm_load_set', 'primer_high_gc_load_set',
'primer_gc_end', 'primer_num_best', 'primer_check',
'primer_end_pathology', 'long_seq', 'p3-tmpl-mispriming'
]
print()
failures = []
for fn_root in test_file_roots:
base_fp = os.path.join(LOCAL_DIR, 'input_files', fn_root)
input_fp = base_fp + '_input'
with open(input_fp) as input_fd:
input_raw = input_fd.read()
input_dicts = self._convertBoulderInput(input_raw)
sys.stdout.write('->Testing file {:<40}\r'.format(fn_root))
sys.stdout.flush()
current_global_args = {}
for global_args, seq_args, p3_args in input_dicts:
test_id = str(seq_args.get('SEQUENCE_ID', ''))
current_global_args.update(global_args)
simulated_binding_res = simulatedbindings.designPrimers(
seq_args, current_global_args)
wrapper_error = simulated_binding_res.get('PRIMER_ERROR')
if wrapper_error is not None:
with self.assertRaises(IOError):
binding_res = bindings.designPrimers(
seq_args, current_global_args)
else:
try:
binding_res = bindings.designPrimers(
seq_args, current_global_args)
except IOError:
if max([
x in p3_args.get('P3_COMMENT', '')
for x in ('complain', 'fail')
]):
pass
disagreements = self._compareResults(
binding_res, simulated_binding_res)
if disagreements is not None:
failures.append((fn_root, test_id, disagreements))
print(' ' * 60, end='\r')
if len(failures):
err_msg = ('Failures occured during file testing:\n' +
'\n'.join(['->{}\t{}\n{}'.format(*f)
for f in failures]))
raise RuntimeError(err_msg)
def test_memoryLeaks(self):
sm = _getMemUsage()
for x in range(100):
# bindings.runP3Design()
bindings.designPrimers(
{
"SEQUENCE_ID": "MH1000",
"SEQUENCE_TEMPLATE": "GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG",
"SEQUENCE_INCLUDED_REGION": [36, 342],
},
{
"PRIMER_OPT_SIZE": 20,
"PRIMER_PICK_INTERNAL_OLIGO": 1,
"PRIMER_INTERNAL_MAX_SELF_END": 8,
"PRIMER_MIN_SIZE": 18,
"PRIMER_MAX_SIZE": 25,
"PRIMER_OPT_TM": 60.0,
"PRIMER_MIN_TM": 57.0,
"PRIMER_MAX_TM": 63.0,
"PRIMER_MIN_GC": 20.0,
"PRIMER_MAX_GC": 80.0,
"PRIMER_MAX_POLY_X": 100,
"PRIMER_INTERNAL_MAX_POLY_X": 100,
"PRIMER_SALT_MONOVALENT": 50.0,
"PRIMER_DNA_CONC": 50.0,
"PRIMER_MAX_NS_ACCEPTED": 0,
"PRIMER_MAX_SELF_ANY": 12,
"PRIMER_MAX_SELF_END": 8,
"PRIMER_PAIR_MAX_COMPL_ANY": 12,
"PRIMER_PAIR_MAX_COMPL_END": 8,
"PRIMER_PRODUCT_SIZE_RANGE": [
[75, 100],
[100, 125],
[125, 150],
[150, 175],
[175, 200],
[200, 225],
],
},
)
sleep(0.1) # Pause for any GC
em = _getMemUsage()
print("\n\tMemory usage before 1k runs of designPrimers: ", sm)
print("\tMemory usage after 1k runs of designPrimers: ", em)
print("\t\t\t\t\tDifference: \t", em - sm)
if em - sm > 1000:
raise AssertionError(
"Memory usage increase after 1k runs of \n\t"
"designPrimers > 1000 bytes -- potential \n\t"
"memory leak (mem increase: {})".format(em - sm)
)
def makeprimers(name, template, minsize, maxsize):
try:
binding_res = bindings.designPrimers(
{
'SEQUENCE_ID': name,
'SEQUENCE_TEMPLATE': template,
},
{
'PRIMER_OPT_SIZE': 24,
'PRIMER_MIN_SIZE': 21,
'PRIMER_MAX_SIZE': 27,
'PRIMER_OPT_TM': 58.0,
'PRIMER_MIN_TM': 56.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_GC_CLAMP': 1,
'PRIMER_PRODUCT_SIZE_RANGE': [[minsize,maxsize]],
}
)
leftprimers = {}
rightprimers = {}
for k, v in binding_res.items():
if k.endswith('SEQUENCE'):
if k.startswith('PRIMER_LEFT'):
leftprimers[int(k.split('_')[2])] = v
if k.startswith('PRIMER_RIGHT'):
rightprimers[int(k.split('_')[2])] = v
return leftprimers, rightprimers
except:
sys.stderr.write("warning: primer design failed for " + name + "\n")
return {}, {}
def test_compareSim(self):
sequence_template = 'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG'
quality_list = [random.randint(20,90) for i in range(len(sequence_template))]
seq_args = {
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': sequence_template,
'SEQUENCE_QUALITY': quality_list,
'SEQUENCE_INCLUDED_REGION': [36,342]
}
global_args = {
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': [[75,100],[100,125],[125,150],[150,175],[175,200],[200,225]],
}
simulated_binding_res = simulatedbindings.designPrimers(seq_args, global_args)
binding_res = bindings.designPrimers(seq_args, global_args)
self._compareResults(binding_res, simulated_binding_res)
def test_CompareSim(self):
sequence_template = 'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG'
quality_list = [random.randint(20,90) for i in range(len(sequence_template))]
seq_args = {
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': sequence_template,
'SEQUENCE_QUALITY': quality_list,
'SEQUENCE_INCLUDED_REGION': [36,342]
}
global_args = {
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': [[75,100],[100,125],[125,150],[150,175],[175,200],[200,225]],
}
simulated_binding_res = simulatedbindings.designPrimers(seq_args, global_args)
binding_res = bindings.designPrimers(seq_args, global_args)
self._compareResults(binding_res, simulated_binding_res)
def for_record(cls, record: SeqRecord) -> "Primers":
"""Create Primers to amplify a SeqRecord.
Args:
record: the sequence to amplify via primers
Returns:
to PCR the record
"""
p3_output = designPrimers(
{
"SEQUENCE_TEMPLATE": str(record.seq),
"SEQUENCE_INCLUDED_REGION": [0, len(record.seq)],
},
{
"PRIMER_PRODUCT_SIZE_RANGE": [len(record.seq), len(record.seq)],
"PRIMER_TASK": "pick_cloning_primers",
"PRIMER_NUM_RETURN": 1,
"PRIMER_PICK_ANYWAY": 1,
"PRIMER_MIN_SIZE": MIN_PRIMER_LEN,
},
)
return cls.from_p3(p3_output)
def findPrimers(inputData, resultFormat="better"):
""" return primer3 result with given format
Parameters
----------
inputData : input data
asd
resultFormat : result format (raw/better),optional
sadsa
Returns
----------
result : sad
"""
inputData = transformInput(inputData)
result = {}
try:
result = designPrimers(inputData['seq_args'], inputData['global_args'])
except: # input data is broken
raise Exception('input data is broken')
if resultFormat == "better":
return createBetterResult(result);
return result
def make_primers(region):
# Designs primers to span a 500 bp region
contig, start, stop = re.search('(^.+?)\[(\d+), (\d+)\)', region).groups()
start = int(start)
stop = int(stop)
seq = contigs_dict[contig]
index = [
'PRIMER_LEFT_0_SEQUENCE', 'PRIMER_RIGHT_0_SEQUENCE',
'PRIMER_PAIR_0_PENALTY', 'PRIMER_PAIR_0_PRODUCT_SIZE',
'amplicon_start', 'amplicon_end'
]
try:
primers = bindings.designPrimers(
{
'SEQUENCE_ID': region,
'SEQUENCE_TEMPLATE': str(seq.seq),
'SEQUENCE_INCLUDED_REGION': [start - 500, 2100],
'SEQUENCE_TARGET': [[start - 200, 400]],
}, {
'PRIMER_PRODUCT_SIZE_RANGE': [[75, 2100]],
'PRIMER_EXPLAIN_FLAG': 1,
'PRIMER_MAX_TM': 68,
'PRIMER_MIN_TM': 52,
'PRIMER_PICK_INTERNAL_OLIGO': 0
})
return pd.Series([
primers['PRIMER_LEFT_0_SEQUENCE'],
primers['PRIMER_RIGHT_0_SEQUENCE'],
primers['PRIMER_PAIR_0_PENALTY'],
primers['PRIMER_PAIR_0_PRODUCT_SIZE'], primers['PRIMER_LEFT_0'][0],
primers['PRIMER_RIGHT_0'][0] + primers['PRIMER_RIGHT_0'][1]
],
index=index)
except:
return pd.Series([np.nan] * 6, index=index)
def _primer3py(
self,
seq_args: dict = None,
global_args: dict = None,
) -> dict:
"""Return dictionary of primer3-py results for sequence flanked by iP and iS sequences.
Notes:
Refer to the primer3-py documentation for further information.
Args:
seq_args: Primer3 sequence arguments. If left blank chooses suitable defaults.
global_args: Primer3 global arguments If left blank chooses suitable defaults.
"""
template = str(self.basic_slice().seq)
input_seq_args = {
"SEQUENCE_TEMPLATE": template,
}
if seq_args:
input_seq_args.update(seq_args)
input_global_args = {
"PRIMER_TASK": "generic",
"PRIMER_PICK_LEFT_PRIMER": 1,
"PRIMER_PICK_RIGHT_PRIMER": 1,
"PRIMER_PRODUCT_SIZE_RANGE": [[len(template),
len(template)]],
}
if global_args:
input_global_args.update(global_args)
return designPrimers(
input_seq_args,
input_global_args,
)
def primer_main(args):
from genomonsv import realignmentFunction
from primer3 import bindings
# make directory for output if necessary
if os.path.dirname(args.output) != "" and not os.path.exists(os.path.dirname(args.output)):
os.makedirs(os.path.dirname(args.output))
# yaml input
param = {"reference_genome": args.reference, "split_refernece_thres": 1000, "validate_sequence_length": 250}
hout = open(args.output, 'w')
with open(args.result_file, 'r') as hin:
for line in hin:
if line.startswith("Chr_1" + '\t' + "Pos_1"):
line = line.rstrip('\n')
header_info.read(line)
print >> hout, line + '\t' + "Primer1" + '\t' + "Primer2" + '\t' + "Primer3" + '\t' + "Primer4" + '\t' + "Primer5"
continue
F = line.rstrip('\n').split('\t')
chr1, pos1, dir1, chr2, pos2, dir2, junc_seq = F[header_info.chr_1], F[header_info.pos_1], F[header_info.dir_1], \
F[header_info.chr_2], F[header_info.pos_2], F[header_info.dir_2], F[header_info.inserted_seq]
junc_seq_len = 0 if junc_seq == "---" else len(junc_seq)
realignmentFunction.getRefAltForSV(args.output + ".contig.tmp.fa", param, chr1, pos1, dir1, chr2, pos2, dir2, junc_seq)
with open(args.output + ".contig.tmp.fa") as hin2:
lines2 = hin2.readlines()
for i in range(len(lines2)):
lines2[i] = lines2[i].rstrip('\n')
if lines2[i].startswith('>') and lines2[i].endswith("alt"):
seq = lines2[i + 1].rstrip('\n')
primer = bindings.designPrimers(
{
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': seq,
'SEQUENCE_TARGET': [225,50 + junc_seq_len],
'SEQUENCE_INCLUDED_REGION': [10, len(seq) - 20]
},
{
'PRIMER_PRODUCT_SIZE_RANGE': [[150,250],[100,300],[301,400],[401,500]],
})
primer_left_right = ["---"] * 5
for i in range(5):
if "PRIMER_LEFT_" + str(i) + "_SEQUENCE" in primer and "PRIMER_RIGHT_" + str(i) + "_SEQUENCE" in primer:
primer_left_right[i] = primer["PRIMER_LEFT_" + str(i) + "_SEQUENCE"] + ";" + primer["PRIMER_RIGHT_" + str(i) + "_SEQUENCE"]
print >> hout, '\t'.join(F) + '\t' + '\t'.join(primer_left_right)
hout.close()
subprocess.call(["rm", "-rf", args.output + ".contig.tmp.fa"])
def CheckingPrimersWidth_(self, crFasta, start, end, width):
prev = crFasta.sequence[start - width:start]
next = crFasta.sequence[end:end + width]
#build dictionaries
primerDict = {
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': prev + next,
'SEQUENCE_INCLUDED_REGION': [0, 2 * width],
'SEQUENCE_EXCLUDED_REGION': [width - 80, 160]
}
primerDict2 = {
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': [[width - 75, 2 * width]],
}
ans = primer3.designPrimers(primerDict, primerDict2)
return_values = [
'PRIMER_LEFT_%s_SEQUENCE', 'PRIMER_LEFT_%s_SEQUENCE',
'PRIMER_RIGHT_%s_SEQUENCE', 'PRIMER_LEFT_%s_TM',
'PRIMER_RIGHT_%s_TM', 'PRIMER_LEFT_%s_GC_PERCENT',
'PRIMER_RIGHT_%s_GC_PERCENT', 'PRIMER_PAIR_%s_PRODUCT_SIZE',
'PRIMER_LEFT_%s_TM', 'PRIMER_RIGHT_%s_TM'
]
primerDesingCheck = []
for x in range(self._numAlternativeCheckings):
auxDict = {}
for elem in return_values:
designPrimer_key = elem % x
auxDict[designPrimer_key] = ans[designPrimer_key]
auxDict['negative_result'] = ans['PRIMER_PAIR_%s_PRODUCT_SIZE' %
x] + (end - start)
primerDesingCheck.append(auxDict)
return primerDesingCheck
def test_memoryLeaks(self):
sm = _getMemUsage()
for x in range(100):
# bindings.runP3Design()
bindings.designPrimers(
{
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': 'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG',
'SEQUENCE_INCLUDED_REGION': [36,342]
},
{
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': [[75,100],[100,125],[125,150],[150,175],[175,200],[200,225]],
})
sleep(0.1) # Pause for any GC
em = _getMemUsage()
print('\n\tMemory usage before 1k runs of designPrimers: ', sm)
print('\tMemory usage after 1k runs of designPrimers: ', em)
print('\t\t\t\t\tDifference: \t', em-sm)
if em-sm > 1000:
raise AssertionError('Memory usage increase after 1k runs of \n\t'
'designPrimers > 1000 bytes -- potential \n\t'
'memory leak (mem increase: {})'.format(em-sm))
def test_memoryLeaks(self):
sm = _getMemUsage()
for x in range(100):
bindings.designPrimers(
{
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': 'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG',
'SEQUENCE_INCLUDED_REGION': [36,342]
},
{
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': [[75,100],[100,125],[125,150],[150,175],[175,200],[200,225]],
})
sleep(0.1) # Pause for any GC
em = _getMemUsage()
print('\n\tMemory usage before 1k runs of designPrimers: ', sm)
print('\tMemory usage after 1k runs of designPrimers: ', em)
print('\t\t\t\t\tDifference: \t', em-sm)
if em-sm > 1000:
raise AssertionError('Memory usage increase after 1k runs of \n\t'
'designPrimers > 1000 bytes -- potential \n\t'
'memory leak (mem increase: {})'.format(em-sm))
def make_primers(sequence):
'''default iSECT values:
size: optimal=21 min=18 max=28
Tm: opt=61 Min=53 Max=71
%GC: min=20 max=80
clamp=1
maxN=300 ext5=300 ext3=300
primer_zone=200
BPos=110 (distance from LB primer to insertion site)
'''
primer3_seq_args['SEQUENCE_TEMPLATE'] = sequence
a = designPrimers(primer3_seq_args, primer3_primer_args)
return a
primer = bindings.designPrimers(
{
'SEQUENCE_ID': seq_id,
'SEQUENCE_TEMPLATE': seq,
'SEQUENCE_FORCE_LEFT_END': 249
}, {
'PRIMER_OPT_SIZE':
22,
'PRIMER_NUM_RETURN':
1,
'PRIMER_PICK_INTERNAL_OLIGO':
0,
'PRIMER_INTERNAL_MAX_SELF_END':
8,
'PRIMER_MIN_SIZE':
18,
'PRIMER_MAX_SIZE':
25,
'PRIMER_OPT_TM':
60.0,
'PRIMER_MIN_TM':
57.0,
'PRIMER_MAX_TM':
63.0,
'PRIMER_MIN_GC':
20.0,
'PRIMER_MAX_GC':
80.0,
'PRIMER_MAX_POLY_X':
100,
'PRIMER_INTERNAL_MAX_POLY_X':
100,
'PRIMER_SALT_MONOVALENT':
50.0,
'PRIMER_DNA_CONC':
50.0,
'PRIMER_MAX_NS_ACCEPTED':
0,
'PRIMER_MAX_SELF_ANY':
12,
'PRIMER_MAX_SELF_END':
8,
'PRIMER_PAIR_MAX_COMPL_ANY':
12,
'PRIMER_PAIR_MAX_COMPL_END':
8,
'PRIMER_PRODUCT_SIZE_RANGE': [[100, 125], [125, 150], [
150, 175
], [200, 225], [225, 250], [250, 275], [275, 300]],
})
def primer3(request):
params = request.json_body
p_keys = params.keys()
if 'gene_name' in p_keys:
gene_name = params.get('gene_name')
if 'sequence' in p_keys:
sequence = params.get('sequence')
if gene_name is not None and sequence is not None:
return HTTPBadRequest(
body=json.dumps({'error': 'Both gene name AND sequence provided'}))
if gene_name is None and sequence is None:
return HTTPBadRequest(
body=json.dumps({'error': 'No gene name OR sequence provided'}))
if gene_name is None:
decodeseq = sequence
sequence = str(sequence.replace('\r', '').replace('\n', ''))
input = 'seq'
else:
gene_name = gene_name.upper()
locus = DBSession.query(Locusdbentity).filter(
or_(Locusdbentity.gene_name == gene_name,
Locusdbentity.systematic_name == gene_name)).one_or_none()
if locus is None:
return HTTPBadRequest(body=json.dumps({
'error':
'Gene name provided does not exist in the database: ' +
gene_name
}))
tax_id = DBSession.query(Straindbentity.taxonomy_id).filter(
Straindbentity.strain_type == 'Reference').one_or_none()
dna = DBSession.query(Dnasequenceannotation.residues).filter(
and_(Dnasequenceannotation.taxonomy_id == tax_id,
Dnasequenceannotation.dbentity_id == locus.dbentity_id,
Dnasequenceannotation.dna_type == '1KB')).one_or_none()
if dna is None:
return HTTPBadRequest(body=json.dumps({
'error':
'Sequence for provided gene name does not exist in the database: '
+ gene_name
}))
else:
decodeseq = dna
sequence = str(dna)
sequence = sequence[3:-3]
input = 'name'
if 'maximum_tm' in p_keys:
maximum_tm = params.get('maximum_tm')
if 'minimum_tm' in p_keys:
minimum_tm = params.get('minimum_tm')
if 'optimum_tm' in p_keys:
optimum_tm = params.get('optimum_tm')
if 'maximum_gc' in p_keys:
maximum_gc = params.get('maximum_gc')
if 'minimum_gc' in p_keys:
minimum_gc = params.get('minimum_gc')
if 'optimum_gc' in p_keys:
optimum_gc = params.get('optimum_gc')
if 'maximum_length' in p_keys:
maximum_length = params.get('maximum_length')
if 'minimum_length' in p_keys:
minimum_length = params.get('minimum_length')
if 'optimum_primer_length' in p_keys:
optimum_primer_length = params.get('optimum_primer_length')
if 'max_three_prime_pair_complementarity' in p_keys:
max_three_prime_pair_complementarity = params.get(
'max_three_prime_pair_complementarity')
if 'max_pair_complementarity' in p_keys:
max_pair_complementarity = params.get('max_pair_complementarity')
if 'max_three_prime_self_complementarity' in p_keys:
max_three_prime_self_complementarity = params.get(
'max_three_prime_self_complementarity')
if 'max_self_complementarity' in p_keys:
max_self_complementarity = params.get('max_self_complementarity')
if 'input_end' in p_keys:
input_end = params.get('input_end')
if 'input_start' in p_keys:
input_start = params.get('input_start')
if 'maximum_product_size' in p_keys:
maximum_product_size = params.get('maximum_product_size')
if 'end_point' in p_keys:
end_point = params.get('end_point')
if gene_name is None:
target_start = input_start
target_extend_by = input_end - input_start
else:
if input_start < 0:
target_start = 1000 - abs(input_start)
target_extend_by = (1000 + input_end) - target_start
else:
target_start = 1000 + input_start
target_extend_by = input_end - input_start
interval_range = [[100, 150], [150, 250], [100, 300], [301, 400],
[401, 500], [501, 600], [601, 700], [701, 850],
[851, 1000]]
if maximum_product_size:
range_start = target_extend_by
range_stop = maximum_product_size
if maximum_product_size < target_extend_by:
return HTTPBadRequest(body=json.dumps({
'error':
'Maximum product size cannot be less than target size.'
}))
interval_range = [[range_start, range_stop]]
elif (target_extend_by > 800):
interval_range = [[target_extend_by, target_extend_by + 300]]
sequence_target = [[target_start, target_extend_by]]
if end_point == 'YES':
force_left_start = target_start
force_right_start = target_start + target_extend_by
sequence_target = []
elif end_point == 'NO':
force_left_start = -1000000
force_right_start = -1000000
try:
result = bindings.designPrimers(
{
'SEQUENCE_ID': str(gene_name),
'SEQUENCE_TEMPLATE': sequence,
'SEQUENCE_TARGET': sequence_target,
'SEQUENCE_FORCE_LEFT_START': force_left_start,
'SEQUENCE_FORCE_RIGHT_START': force_right_start
}, {
'PRIMER_FIRST_BASE_INDEX': 1,
'PRIMER_THERMODYNAMIC_OLIGO_ALIGNMENT': 1,
'PRIMER_THERMODYNAMIC_TEMPLATE_ALIGNMENT': 0,
'PRIMER_PICK_LEFT_PRIMER': 1,
'PRIMER_PICK_INTERNAL_OLIGO': 0,
'PRIMER_PICK_RIGHT_PRIMER': 1,
'PRIMER_LIBERAL_BASE': 1,
'PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS': 0,
'PRIMER_LOWERCASE_MASKING': 0,
'PRIMER_PICK_ANYWAY': 0,
'PRIMER_EXPLAIN_FLAG': 1,
'PRIMER_MASK_TEMPLATE': 0,
'PRIMER_TASK': 'generic',
'PRIMER_MASK_FAILURE_RATE': 0.1,
'PRIMER_MASK_5P_DIRECTION': 1,
'PRIMER_MASK_3P_DIRECTION': 0,
'PRIMER_MIN_QUALITY': 0,
'PRIMER_MIN_END_QUALITY': 0,
'PRIMER_QUALITY_RANGE_MIN': 0,
'PRIMER_QUALITY_RANGE_MAX': 100,
'PRIMER_MIN_SIZE': minimum_length,
'PRIMER_OPT_SIZE': optimum_primer_length,
'PRIMER_MAX_SIZE': maximum_length,
'PRIMER_MIN_TM': minimum_tm,
'PRIMER_OPT_TM': optimum_tm,
'PRIMER_MAX_TM': maximum_tm,
'PRIMER_PAIR_MAX_DIFF_TM': 5.0,
'PRIMER_TM_FORMULA': 1,
'PRIMER_PRODUCT_MIN_TM': -1000000.0,
'PRIMER_PRODUCT_OPT_TM': 0.0,
'PRIMER_PRODUCT_MAX_TM': 1000000.0,
'PRIMER_MIN_GC': minimum_gc,
'PRIMER_OPT_GC_PERCENT': optimum_gc,
'PRIMER_MAX_GC': maximum_gc,
'PRIMER_PRODUCT_SIZE_RANGE': interval_range,
'PRIMER_NUM_RETURN': 5,
'PRIMER_MAX_END_STABILITY': 9.0,
'PRIMER_MAX_LIBRARY_MISPRIMING': 12.00,
'PRIMER_PAIR_MAX_LIBRARY_MISPRIMING': 20.00,
'PRIMER_MAX_SELF_ANY_TH': 45.0,
'PRIMER_MAX_SELF_END_TH': 35.0,
'PRIMER_PAIR_MAX_COMPL_ANY_TH': 45.0,
'PRIMER_PAIR_MAX_COMPL_END_TH': 35.0,
'PRIMER_MAX_HAIRPIN_TH': 24.0,
'PRIMER_MAX_SELF_ANY': max_self_complementarity,
'PRIMER_MAX_SELF_END': max_three_prime_self_complementarity,
'PRIMER_PAIR_MAX_COMPL_ANY': max_pair_complementarity,
'PRIMER_PAIR_MAX_COMPL_END':
max_three_prime_pair_complementarity,
'PRIMER_MAX_TEMPLATE_MISPRIMING_TH': 40.00,
'PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH': 70.00,
'PRIMER_MAX_TEMPLATE_MISPRIMING': 12.00,
'PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING': 24.00,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_POLY_X': 4,
'PRIMER_INSIDE_PENALTY': -1.0,
'PRIMER_OUTSIDE_PENALTY': 0,
'PRIMER_GC_CLAMP': 0,
'PRIMER_MAX_END_GC': 5,
'PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE': 3,
'PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE': 3,
'PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION': 7,
'PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION': 4,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_SALT_CORRECTIONS': 1,
'PRIMER_SALT_DIVALENT': 1.5,
'PRIMER_DNTP_CONC': 0.6,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_SEQUENCING_SPACING': 500,
'PRIMER_SEQUENCING_INTERVAL': 250,
'PRIMER_SEQUENCING_LEAD': 50,
'PRIMER_SEQUENCING_ACCURACY': 20,
'PRIMER_WT_SIZE_LT': 1.0,
'PRIMER_WT_SIZE_GT': 1.0,
'PRIMER_WT_TM_LT': 1.0,
'PRIMER_WT_TM_GT': 1.0,
'PRIMER_WT_GC_PERCENT_LT': 0.0,
'PRIMER_WT_GC_PERCENT_GT': 0.0,
'PRIMER_WT_SELF_ANY_TH': 0.0,
'PRIMER_WT_SELF_END_TH': 0.0,
'PRIMER_WT_HAIRPIN_TH': 0.0,
'PRIMER_WT_TEMPLATE_MISPRIMING_TH': 0.0,
'PRIMER_WT_SELF_ANY': 0.0,
'PRIMER_WT_SELF_END': 0.0,
'PRIMER_WT_TEMPLATE_MISPRIMING': 0.0,
'PRIMER_WT_NUM_NS': 0.0,
'PRIMER_WT_LIBRARY_MISPRIMING': 0.0,
'PRIMER_WT_SEQ_QUAL': 0.0,
'PRIMER_WT_END_QUAL': 0.0,
'PRIMER_WT_POS_PENALTY': 0.0,
'PRIMER_WT_END_STABILITY': 0.0,
'PRIMER_WT_MASK_FAILURE_RATE': 0.0,
'PRIMER_PAIR_WT_PRODUCT_SIZE_LT': 0.0,
'PRIMER_PAIR_WT_PRODUCT_SIZE_GT': 0.0,
'PRIMER_PAIR_WT_PRODUCT_TM_LT': 0.0,
'PRIMER_PAIR_WT_PRODUCT_TM_GT': 0.0,
'PRIMER_PAIR_WT_COMPL_ANY_TH': 0.0,
'PRIMER_PAIR_WT_COMPL_END_TH': 0.0,
'PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH': 0.0,
'PRIMER_PAIR_WT_COMPL_ANY': 0.0,
'PRIMER_PAIR_WT_COMPL_END': 0.0,
'PRIMER_PAIR_WT_TEMPLATE_MISPRIMING': 0.0,
'PRIMER_PAIR_WT_DIFF_TM': 0.0,
'PRIMER_PAIR_WT_LIBRARY_MISPRIMING': 0.0,
'PRIMER_PAIR_WT_PR_PENALTY': 1.0,
'PRIMER_PAIR_WT_IO_PENALTY': 0.0,
'PRIMER_INTERNAL_MIN_SIZE': 18,
'PRIMER_INTERNAL_OPT_SIZE': 20,
'PRIMER_INTERNAL_MAX_SIZE': 27,
'PRIMER_INTERNAL_MIN_TM': 57.0,
'PRIMER_INTERNAL_OPT_TM': 60.0,
'PRIMER_INTERNAL_MAX_TM': 63.0,
'PRIMER_INTERNAL_MIN_GC': 20.0,
'PRIMER_INTERNAL_OPT_GC_PERCENT': 50.0,
'PRIMER_INTERNAL_MAX_GC': 80.0,
'PRIMER_INTERNAL_MAX_SELF_ANY_TH': 47.00,
'PRIMER_INTERNAL_MAX_SELF_END_TH': 47.00,
'PRIMER_INTERNAL_MAX_HAIRPIN_TH': 47.00,
'PRIMER_INTERNAL_MAX_SELF_ANY': 12.00,
'PRIMER_INTERNAL_MAX_SELF_END': 12.00,
'PRIMER_INTERNAL_MIN_QUALITY': 0,
'PRIMER_INTERNAL_MAX_NS_ACCEPTED': 0,
'PRIMER_INTERNAL_MAX_POLY_X': 5,
'PRIMER_INTERNAL_MAX_LIBRARY_MISHYB': 12.00,
'PRIMER_INTERNAL_SALT_MONOVALENT': 50.0,
'PRIMER_INTERNAL_DNA_CONC': 50.0,
'PRIMER_INTERNAL_SALT_DIVALENT': 1.5,
'PRIMER_INTERNAL_DNTP_CONC': 0.0,
'PRIMER_INTERNAL_WT_SIZE_LT': 1.0,
'PRIMER_INTERNAL_WT_SIZE_GT': 1.0,
'PRIMER_INTERNAL_WT_TM_LT': 1.0,
'PRIMER_INTERNAL_WT_TM_GT': 1.0,
'PRIMER_INTERNAL_WT_GC_PERCENT_LT': 0.0,
'PRIMER_INTERNAL_WT_GC_PERCENT_GT': 0.0,
'PRIMER_INTERNAL_WT_SELF_ANY_TH': 0.0,
'PRIMER_INTERNAL_WT_SELF_END_TH': 0.0,
'PRIMER_INTERNAL_WT_HAIRPIN_TH': 0.0,
'PRIMER_INTERNAL_WT_SELF_ANY': 0.0,
'PRIMER_INTERNAL_WT_SELF_END': 0.0,
'PRIMER_INTERNAL_WT_NUM_NS': 0.0,
'PRIMER_INTERNAL_WT_LIBRARY_MISHYB': 0.0,
'PRIMER_INTERNAL_WT_SEQ_QUAL': 0.0,
'PRIMER_INTERNAL_WT_END_QUAL': 0.0
},
debug=False)
presult, notes = primer3_parser(result)
obj = {
'result': presult,
'gene_name': gene_name,
'seq': decodeseq,
'input': input
}
return obj
except Exception as e:
return HTTPBadRequest(body=json.dumps({'error': str(e)}))
def testHuman(self):
binding_res = bindings.designPrimers(
{
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': 'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG',
'SEQUENCE_INCLUDED_REGION': [36,342]
},
{
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': [[75,100],[100,125],[125,150],[150,175],[175,200],[200,225]],
}
)
wrapper_res = wrappers.designPrimers(
{
'PRIMER_OPT_SIZE': 20,
'PRIMER_PICK_INTERNAL_OLIGO': 1,
'PRIMER_INTERNAL_MAX_SELF_END': 8,
'PRIMER_MIN_SIZE': 18,
'PRIMER_MAX_SIZE': 25,
'PRIMER_OPT_TM': 60.0,
'PRIMER_MIN_TM': 57.0,
'PRIMER_MAX_TM': 63.0,
'PRIMER_MIN_GC': 20.0,
'PRIMER_MAX_GC': 80.0,
'PRIMER_MAX_POLY_X': 100,
'PRIMER_INTERNAL_MAX_POLY_X': 100,
'PRIMER_SALT_MONOVALENT': 50.0,
'PRIMER_DNA_CONC': 50.0,
'PRIMER_MAX_NS_ACCEPTED': 0,
'PRIMER_MAX_SELF_ANY': 12,
'PRIMER_MAX_SELF_END': 8,
'PRIMER_PAIR_MAX_COMPL_ANY': 12,
'PRIMER_PAIR_MAX_COMPL_END': 8,
'PRIMER_PRODUCT_SIZE_RANGE': '75-100 100-125 125-150 150-175 175-200 200-225',
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': 'GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG',
'SEQUENCE_INCLUDED_REGION': '36,342'
}
)
print('\n\n\n{:<30} {:<25} {:<25}'.format('Output Key', 'Wrapper Result', 'Binding Result'))
print('-'*80)
for k, v in binding_res.items():
print('{:<30} {:<25} {:<25}'.format(k, repr(wrapper_res.get(k)), repr(v)))
forward_primer, reverse_primer = 'SO:0000121', 'SO:0000132'
genes_with_errors = []
with open(genome_name+'_KO_primers_pyrG_selection.csv', 'w') as pyrg_f, open(genome_name+'_KO_primers_ptrA_selection.csv', 'w') as ptra_f, \
open('genes_with_errors', 'w') as genes_with_errors_f, open('primers.gff', 'w') as gff_f:
files = (pyrg_f, ptra_f)
for f in files:
f.write(','.join(['primer name', 'primer sequence', 'penalty', 'product size'])+'\n')
for gene in coord:
try:
print(gene.name)
if gene.min_coord - 1400 > 0 and gene.max_coord+1400 < len(fasta_dict[gene.chrom]) and gene.max_coord - gene.min_coord > 200:
left = bindings.designPrimers({
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': str(fasta_dict[gene.chrom].seq),
'SEQUENCE_INCLUDED_REGION': [gene.min_coord-1400, 1500],
'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST': [-1, -1, gene.min_coord, 100],
},
{'PRIMER_PRODUCT_SIZE_RANGE': [[1100, 1300]]})
right = bindings.designPrimers({
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': str(fasta_dict[gene.chrom].seq),
'SEQUENCE_INCLUDED_REGION': [gene.max_coord-100, 1500],
'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST': [gene.max_coord-100, 100, -1, -1],
},
{'PRIMER_PRODUCT_SIZE_RANGE': [[1100, 1300]]})
left_to_right_dist = (right['PRIMER_RIGHT_0'][0] - right['PRIMER_RIGHT_0'][1]) - (left['PRIMER_LEFT_0'][0] + left['PRIMER_LEFT_0'][1])
nested_range = [left_to_right_dist-200, left_to_right_dist]
nest = bindings.designPrimers({
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': str(fasta_dict[gene.chrom].seq),
def test_fileBased(self):
test_file_roots = [
'primer_must_use_th',
'primer_task_th',
'primer_thal_args',
'primer_thal_max_seq_error',
'primer_first_base_index',
'test_compl_error',
'test_left_to_right_of_right',
'dv_conc_vs_dntp_conc',
'primer_internal',
'primer_tm_lc_masking',
'primer_ok_regions',
'primer_start_codon',
'primer_task',
'primer_renewed_tasks',
'primer_must_overlap_point',
'primer_overlap_junction',
'primer_all_settingsfiles',
'primer_high_tm_load_set',
'primer_high_gc_load_set',
'primer_gc_end',
'primer_num_best',
'primer_check',
'primer_end_pathology',
'long_seq',
'p3-tmpl-mispriming'
]
print()
failures = []
for fn_root in test_file_roots:
base_fp = os.path.join(LOCAL_DIR, 'input_files', fn_root)
input_fp = base_fp + '_input'
with open(input_fp) as input_fd:
input_raw = input_fd.read()
input_dicts = self._convertBoulderInput(input_raw)
sys.stdout.write('->Testing file {:<40}\r'.format(fn_root))
sys.stdout.flush()
current_global_args = {}
for global_args, seq_args, p3_args in input_dicts:
test_id = str(seq_args.get('SEQUENCE_ID', ''))
current_global_args.update(global_args)
simulated_binding_res = simulatedbindings.designPrimers(
seq_args, current_global_args)
wrapper_error = simulated_binding_res.get('PRIMER_ERROR')
if wrapper_error is not None:
with self.assertRaises(IOError):
binding_res = bindings.designPrimers(seq_args,
current_global_args)
else:
try:
binding_res = bindings.designPrimers(seq_args,
current_global_args)
except IOError:
if max([x in p3_args.get('P3_COMMENT', '') for x in
('complain', 'fail')]):
pass
disagreements = self._compareResults(binding_res,
simulated_binding_res)
if disagreements is not None:
failures.append((fn_root, test_id, disagreements))
print(' '* 60, end='\r')
if len(failures):
err_msg = ('Failures occured during file testing:\n' +
'\n'.join(['->{}\t{}\n{}'.format(*f) for f in
failures]))
raise RuntimeError(err_msg)
r_primer_map = {'+': 'R', '-': 'F'}
with open('Af3357_qPCR_primers.csv','w') as f, \
open('genes_with_errors','w') as genes_with_errors_f, open('primers.gff','w') as gff_f:
temp = []
for idx, gene in enumerate(coord, 1):
if len(gene.seq) < 100:
continue
if len(gene.exons) > 2:
print(gene.name)
goi = bindings.designPrimers(
{
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': gene.cdna,
'SEQUENCE_OVERLAP_JUNCTION_LIST': gene.exon_juncs,
}, {
'PRIMER_PRODUCT_SIZE_RANGE': [[75, 150]],
'PRIMER_EXPLAIN_FLAG': 1,
'PRIMER_MAX_TM': 68,
'PRIMER_MIN_TM': 52,
'PRIMER_PICK_INTERNAL_OLIGO': 0,
overlap_map[gene.strand]: 9
})
else:
print(gene.name)
goi = bindings.designPrimers(
{
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': gene.cdna
}, {
'PRIMER_PRODUCT_SIZE_RANGE': [[75, 150]],
'PRIMER_EXPLAIN_FLAG': 1,
for f in files:
f.write(','.join(
['primer name', 'primer sequence', 'penalty', 'product size']) +
'\n')
for gene in coord:
try:
print(gene.name)
if gene.min_coord - 1400 > 0 and gene.max_coord + 1400 < len(
fasta_dict[
gene.chrom]) and gene.max_coord - gene.min_coord > 200:
left = bindings.designPrimers(
{
'SEQUENCE_ID':
gene.name,
'SEQUENCE_TEMPLATE':
str(fasta_dict[gene.chrom].seq),
'SEQUENCE_INCLUDED_REGION':
[gene.min_coord - 1400, 1500],
'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST':
[-1, -1, gene.min_coord, 100],
}, {'PRIMER_PRODUCT_SIZE_RANGE': [[1100, 1300]]})
right = bindings.designPrimers(
{
'SEQUENCE_ID':
gene.name,
'SEQUENCE_TEMPLATE':
str(fasta_dict[gene.chrom].seq),
'SEQUENCE_INCLUDED_REGION':
[gene.max_coord - 100, 1500],
'SEQUENCE_PRIMER_PAIR_OK_REGION_LIST':
[gene.max_coord - 100, 100, -1, -1],
def primer_main(args):
from genomon_sv import realignmentFunction
from primer3 import bindings
# make directory for output if necessary
if os.path.dirname(args.output) != "" and not os.path.exists(os.path.dirname(args.output)):
os.makedirs(os.path.dirname(args.output))
param = {"reference_genome": args.reference, "split_refernece_thres": 1000, "validate_sequence_length": 250}
hout = open(args.output, 'w')
with open(args.result_file, 'r') as hin:
for line in hin:
if line.startswith("#"): continue
if utils.header_check(line.rstrip('\n')):
line = line.rstrip('\n')
header_info.read(line)
print(line + '\t' + "Primer1" + '\t' + "Primer2" + '\t' + "Primer3" + '\t' + "Primer4" + '\t' + "Primer5", file = hout)
continue
F = line.rstrip('\n').split('\t')
chr1, pos1, dir1, chr2, pos2, dir2, junc_seq = F[header_info.chr_1], F[header_info.pos_1], F[header_info.dir_1], \
F[header_info.chr_2], F[header_info.pos_2], F[header_info.dir_2], F[header_info.inserted_seq]
if utils.check_atypical_chromosomes(chr1, chr2):
print("Skip a SV incolving atypical chromosomes: %s,%s,%s,%s,%s,%s" % \
(chr1, pos1, dir1, chr2, pos2, dir2), file = sys.stderr)
continue
junc_seq_len = 0 if junc_seq == "---" else len(junc_seq)
realignmentFunction.getRefAltForSV(args.output + ".contig.tmp.fa", chr1, pos1, dir1, chr2, pos2, dir2, junc_seq, args.reference, 1000, 250)
with open(args.output + ".contig.tmp.fa") as hin2:
lines2 = hin2.readlines()
for i in range(len(lines2)):
lines2[i] = lines2[i].rstrip('\n')
if lines2[i].startswith('>') and lines2[i].endswith("alt"):
seq = lines2[i + 1].rstrip('\n')
primer = bindings.designPrimers(
{
'SEQUENCE_ID': 'MH1000',
'SEQUENCE_TEMPLATE': seq,
'SEQUENCE_TARGET': [225,50 + junc_seq_len],
'SEQUENCE_INCLUDED_REGION': [10, len(seq) - 20]
},
{
'PRIMER_PRODUCT_SIZE_RANGE': [[150,250],[100,300],[301,400],[401,500]],
})
primer_left_right = ["---"] * 5
for i in range(5):
if "PRIMER_LEFT_" + str(i) + "_SEQUENCE" in primer and "PRIMER_RIGHT_" + str(i) + "_SEQUENCE" in primer and \
"PRIMER_LEFT_" + str(i) + "_TM" in primer and "PRIMER_RIGHT_" + str(i) + "_TM" in primer and \
"PRIMER_PAIR_" + str(i) + "_PRODUCT_SIZE" in primer:
primer_left_right[i] = primer["PRIMER_LEFT_" + str(i) + "_SEQUENCE"] + ";" + primer["PRIMER_RIGHT_" + str(i) + "_SEQUENCE"] + ';' + \
str(round(primer["PRIMER_LEFT_" + str(i) + "_TM"], 3)) + ";" + str(round(primer["PRIMER_RIGHT_" + str(i) + "_TM"], 3)) + ';' + \
str(primer["PRIMER_PAIR_" + str(i) + "_PRODUCT_SIZE"])
print('\t'.join(F) + '\t' + '\t'.join(primer_left_right), file = hout)
hout.close()
subprocess.check_call(["rm", "-rf", args.output + ".contig.tmp.fa"])
f_primer_map = {'+': 'F', '-': 'R'}
r_primer_map = {'+': 'R', '-': 'F'}
with open('Af3357_qPCR_primers.csv','w') as f, \
open('genes_with_errors','w') as genes_with_errors_f, open('primers.gff','w') as gff_f:
temp = []
for idx, gene in enumerate(coord,1):
if len(gene.seq) < 100:
continue
if len(gene.exons) > 2:
print(gene.name)
goi = bindings.designPrimers({
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': gene.cdna,
'SEQUENCE_OVERLAP_JUNCTION_LIST': gene.exon_juncs,
},
{'PRIMER_PRODUCT_SIZE_RANGE': [[75, 150]],
'PRIMER_EXPLAIN_FLAG':1,
'PRIMER_MAX_TM':68,
'PRIMER_MIN_TM':52,
'PRIMER_PICK_INTERNAL_OLIGO':0,
overlap_map[gene.strand]: 9})
else:
print(gene.name)
goi = bindings.designPrimers({
'SEQUENCE_ID': gene.name,
'SEQUENCE_TEMPLATE': gene.cdna
},
{'PRIMER_PRODUCT_SIZE_RANGE': [[75, 150]],
'PRIMER_EXPLAIN_FLAG':1,
'PRIMER_MAX_TM':68,
'PRIMER_MIN_TM':52,
def testHuman(self):
binding_res = bindings.designPrimers(
{
"SEQUENCE_ID": "MH1000",
"SEQUENCE_TEMPLATE": "GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG",
"SEQUENCE_INCLUDED_REGION": [36, 342],
},
{
"PRIMER_OPT_SIZE": 20,
"PRIMER_PICK_INTERNAL_OLIGO": 1,
"PRIMER_INTERNAL_MAX_SELF_END": 8,
"PRIMER_MIN_SIZE": 18,
"PRIMER_MAX_SIZE": 25,
"PRIMER_OPT_TM": 60.0,
"PRIMER_MIN_TM": 57.0,
"PRIMER_MAX_TM": 63.0,
"PRIMER_MIN_GC": 20.0,
"PRIMER_MAX_GC": 80.0,
"PRIMER_MAX_POLY_X": 100,
"PRIMER_INTERNAL_MAX_POLY_X": 100,
"PRIMER_SALT_MONOVALENT": 50.0,
"PRIMER_DNA_CONC": 50.0,
"PRIMER_MAX_NS_ACCEPTED": 0,
"PRIMER_MAX_SELF_ANY": 12,
"PRIMER_MAX_SELF_END": 8,
"PRIMER_PAIR_MAX_COMPL_ANY": 12,
"PRIMER_PAIR_MAX_COMPL_END": 8,
"PRIMER_PRODUCT_SIZE_RANGE": [[75, 100], [100, 125], [125, 150], [150, 175], [175, 200], [200, 225]],
},
)
wrapper_res = wrappers.designPrimers(
{
"PRIMER_OPT_SIZE": 20,
"PRIMER_PICK_INTERNAL_OLIGO": 1,
"PRIMER_INTERNAL_MAX_SELF_END": 8,
"PRIMER_MIN_SIZE": 18,
"PRIMER_MAX_SIZE": 25,
"PRIMER_OPT_TM": 60.0,
"PRIMER_MIN_TM": 57.0,
"PRIMER_MAX_TM": 63.0,
"PRIMER_MIN_GC": 20.0,
"PRIMER_MAX_GC": 80.0,
"PRIMER_MAX_POLY_X": 100,
"PRIMER_INTERNAL_MAX_POLY_X": 100,
"PRIMER_SALT_MONOVALENT": 50.0,
"PRIMER_DNA_CONC": 50.0,
"PRIMER_MAX_NS_ACCEPTED": 0,
"PRIMER_MAX_SELF_ANY": 12,
"PRIMER_MAX_SELF_END": 8,
"PRIMER_PAIR_MAX_COMPL_ANY": 12,
"PRIMER_PAIR_MAX_COMPL_END": 8,
"PRIMER_PRODUCT_SIZE_RANGE": "75-100 100-125 125-150 150-175 175-200 200-225",
"SEQUENCE_ID": "MH1000",
"SEQUENCE_TEMPLATE": "GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCCTACATTTTAGCATCAGTGAGTACAGCATGCTTACTGGAAGAGAGGGTCATGCAACAGATTAGGAGGTAAGTTTGCAAAGGCAGGCTAAGGAGGAGACGCACTGAATGCCATGGTAAGAACTCTGGACATAAAAATATTGGAAGTTGTTGAGCAAGTNAAAAAAATGTTTGGAAGTGTTACTTTAGCAATGGCAAGAATGATAGTATGGAATAGATTGGCAGAATGAAGGCAAAATGATTAGACATATTGCATTAAGGTAAAAAATGATAACTGAAGAATTATGTGCCACACTTATTAATAAGAAAGAATATGTGAACCTTGCAGATGTTTCCCTCTAGTAG",
"SEQUENCE_INCLUDED_REGION": "36,342",
}
)
print("\n\n\n{:<30} {:<25} {:<25}".format("Output Key", "Wrapper Result", "Binding Result"))
print("-" * 80)
for k, v in binding_res.items():
print("{:<30} {:<25} {:<25}".format(k, repr(wrapper_res.get(k)), repr(v)))