def score(seq):
s1, s2 = seq[int(len(seq) / 2):], seq[:int(len(seq) / 2)]
if abs(melting.temp(s1) -
72.5) < 7.5 and abs(melting.temp(s2) - 72.5) < 7.5 and abs(
gc(s1) - 0.5) < 0.1 and abs(gc(s2) - 0.5) and (s2[0] == "A"
or s2[0] == "T"):
ssc = sec(s1, s2)
return ssc
return 0
def test_low_cation_ratio():
expected_tm = 82.2
given_tm = melting.temp("GCGCGCGCGCGCGCGC",
DNA_c=200,
Na_c=500,
Mg_c=3,
dNTPs_c=0.8)
assert within_margin(given_tm, expected_tm, margin=1)
def test_melting():
# Temps taken from https://www.idtdna.com/calc/analyzer
# on 2015-10-29 using qPCR parameters:
# - Oligo Conc: 0.2 uM
# - Na+ Conc: 50mM
# - Mg++ Conc: 3mM
# - dNTPs Conc: 0.8mM
idt_temps = {
"ATGCATGC": 26.2,
"CCCCTTTT": 21.7,
"GCGCGCGCGCGCGCGC": 76.6,
}
for seq, expected_tm in idt_temps.items():
given_tm = melting.temp(seq, DNA_c=200, Na_c=50, Mg_c=3, dNTPs_c=0.8)
assert within_margin(given_tm, expected_tm)
def test_melting():
# Temps taken from https://www.idtdna.com/calc/analyzer
# on 2015-10-29 using qPCR parameters:
# - Oligo Conc: 0.2 uM
# - Na+ Conc: 50mM
# - Mg++ Conc: 3mM
# - dNTPs Conc: 0.8mM
idt_temps = {
"ATGCATGC": 26.2,
"CCCCTTTT": 21.7,
"GCGCGCGCGCGCGCGC": 76.6,
}
for seq, expected_tm in idt_temps.items():
given_tm = melting.temp(seq, DNA_c=200, Na_c=50, Mg_c=3, dNTPs_c=0.8)
assert within_margin(given_tm, expected_tm)
def test_low_cation_ratio():
expected_tm = 82.2
given_tm = melting.temp("GCGCGCGCGCGCGCGC", DNA_c=200, Na_c=500, Mg_c=3, dNTPs_c=0.8)
assert within_margin(given_tm, expected_tm, margin=1)
def test_uncorrected_tm():
corrected = melting.temp("ATGCATGC")
uncorrected = melting.temp("ATGCATGC", uncorrected=True)
assert corrected != uncorrected
def update_tm(self):
self.tm = melting.temp(self.seq)
def design(sense_oligo, antisense_oligo, guide_context, orientation):
#identify strand for Tm comparison and tagging
orient_error = 'Orientation not correctly specified.\n'
if orientation == 'sense':
i = 0
j = 1
elif orientation == 'antisense':
i = 1
j = 0
else:
print(orient_error)
return
#convert sequences to Seq objects for manipulation
sense = Seq(sense_oligo)
antisense = Seq(antisense_oligo)
context = Seq(guide_context)
primers = (sense, antisense)
#check guide sequence + context is big enough for design
size_error = 'Guide and context is not big enough for design'
if len(context) < 35:
print(size_error)
return
#calculate Tm for primers
MT = []
for primer in primers:
temptm = melting.temp(primer, DNA_c=1000, Mg_c=1.5, dNTPs_c=0.2)
MT.append(temptm)
#design headloop tags
frame = 0
poss_rc = [] #initialise lists
poss_offset = []
master_rc = [] #initialise master lists
master_offset = []
while frame < 3:
#create list of possible reverse complement tags
temp_rc = context[frame:(frame + 20)] #extract sequence
rc = temp_rc.reverse_complement() #reverse complement temp_rc
rc_temp_tm = melting.temp(rc, DNA_c=1000, Mg_c=1.5,
dNTPs_c=0.2) #calculate Tm melting method
rc_temp_diff = abs(
MT[i] - rc_temp_tm) #calculate Tm difference with sense primer
if rc_temp_diff < 3:
rc_flag = 0
else:
rc_flag = 1
poss_rc = (rc, rc_temp_tm, rc_temp_diff, frame, rc_flag) #create list
master_rc.append(poss_rc) #append to master list
#create list of possible offset tags
offset = context[(frame + 12):(frame + 32)] #extract offset sequence
off_temp_tm = melting.temp(offset, DNA_c=1000, Mg_c=1.5,
dNTPs_c=0.2) #calculate Tm melting method
off_temp_diff = abs(
MT[j] -
off_temp_tm) #calculate Tm difference with antisense primer
if off_temp_diff < 3:
off_flag = 0
else:
off_flag = 1
poss_offset = (offset, off_temp_tm, off_temp_diff, frame, off_flag
) #create list
master_offset.append(poss_offset) #append to master list
frame = frame + 1 #frameshift by 1
#sort lists of optional tags by Tm difference with primer and, in tiebreaks, position
master_rc.sort(key=lambda x: (x[2], x[3]))
master_offset.sort(key=lambda x: (x[2], -x[3]))
#concatenate headloop tags with correct primer, depending on strand orientation
if orientation == 'sense':
temp_sense_hl = master_rc[0][
0] + sense #create headloop sense primer sequence
temp_antisense_hl = master_offset[0][
0] + antisense #create headloop antisense primer sequence
#formats seq records
sense_hl = SeqRecord(temp_sense_hl, id='Sense HL')
if master_rc[0][4] == 1:
sense_hl.description = 'WARNING: Could not optimise sense headloop tag (Tm difference > 3\u00b0C)'
else:
sense_hl.description = 'Tm difference < 3\u00b0C'
antisense_hl = SeqRecord(temp_antisense_hl, id='Antisense HL')
if master_offset[0][4] == 1:
antisense_hl.description = 'WARNING: Could not optimise antisense headloop tag (Tm difference > 3\u00b0C)'
else:
antisense_hl.description = 'Tm difference < 3\u00b0C'
else:
temp_antisense_hl = master_rc[0][
0] + antisense #create headloop antisense primer sequence
temp_sense_hl = master_offset[0][0] + sense
#formats seq records
sense_hl = SeqRecord(temp_sense_hl, id='Sense HL')
if master_offset[0][4] == 1:
sense_hl.description = 'WARNING: Could not optimise sense headloop tag (Tm difference > 3\u00b0C)'
else:
sense_hl.description = 'Tm difference < 3\u00b0C'
antisense_hl = SeqRecord(temp_antisense_hl, id='Antisense HL')
if master_rc[0][4] == 1:
antisense_hl.description = 'WARNING: Could not optimise antisense headloop tag (Tm difference > 3\u00b0C)'
else:
antisense_hl.description = 'Tm difference < 3\u00b0C'
return (sense_hl, antisense_hl)
def calc_Tm(s, digits=3): ''' calculate Tm using the method in melting, as the primer3.calcTm produces -999999.9999 for some sequences ''' tm = melting.temp(s, DNA_c=50, Na_c=50, Mg_c=0, dNTPs_c=0.8); return(round(tm,digits));
def test_uncorrected_tm():
corrected = melting.temp("ATGCATGC")
uncorrected = melting.temp("ATGCATGC", uncorrected=True)
assert corrected != uncorrected