def AssembleNAnneal(graph, nodes, edges, start, end):
dseq_list = enc.toDSEQ(graph, edges, nodes)
p1 = Dseqrecord(nodes[start])
p2 = Dseqrecord(enc.getSeqComplement(nodes[end]))
assembly = Assembly(dseq_list, limit=10, only_terminal_overlaps=True)
print("\n" + str(assembly) + "\n")
candidates = []
for i in range(len(assembly.linear_products)):
product = assembly.linear_products[i]
template = Dseqrecord(product)
pcr = Anneal([p1, p2], template, limit=10)
gel = len(nodes) * enc.SEQ_LEN
if len(pcr.products) != 0:
print(product.detailed_figure())
print(product.figure())
for p in pcr.products:
if len(p.seq) == gel:
p.seq = p.seq[10:]
p.seq = p.seq[:-10]
candidates.append(p)
# print("\n" +str(nodes))
# print(str(edges) +"\n")
return candidates
def test_amplicon_dbd_low_gc():
from pydna.amplify import Anneal
from pydna.dseqrecord import Dseqrecord
from pydna.primer import Primer
from textwrap import dedent
template = Dseqrecord("AAAATATTTTTATACATAATACAATTGTATATTCTTAAATAAAAAATACGTCATC")
p1 = Primer("AAAATATTTTTATACAT")
p2 = Primer("GATGACGTATTTTTTAT")
ann = Anneal((p1,p2),template)
prod = ann.products[0]
assert repr(prod) == 'Amplicon(55)'
fig =( r'''
Pfu-Sso7d (rate 15s/kb) |55bp
Three-step| 30 cycles | |Tm formula: Pydna tmbresluc
98.0°C |98.0°C | |SaltC 50mM
__________|_____ 72.0°C |72.0°C|Primer1C 1.0µM
00min30s |10s \ 39.0°C ________|______|Primer2C 1.0µM
| \______/ 0min 0s|10min |GC 14%
| 10s | |4-12°C
'''[1:])
fig = dedent(fig)
assert str(prod.pfu_sso7d_program()) == fig
def test_amplicon_dbd():
from pydna.amplify import Anneal
from pydna.dseqrecord import Dseqrecord
from pydna.primer import Primer
from textwrap import dedent
template = Dseqrecord("GCGTCCAGCGGCTGCCCGAGGCGCCAAGTGCCCGGGCCGAGCCCGCATCTGAGGCCGCCGCGGGC")
p1 = Primer("GCGTCCAGCGGCTGCCCGAGG")
p2 = Primer("GCCCGCGGCGGCCTCAGATGCGG")
ann = Anneal((p1,p2),template)
prod = ann.products[0]
assert repr(prod) == 'Amplicon(65)'
fig =( r'''
Pfu-Sso7d (rate 15s/kb)
Two-step| 30 cycles | |65bp
98.0°C |98.0C | |Tm formula: Pydna tmbresluc
_____ __|_____ | |SaltC 50mM
00min30s|10s \ | |Primer1C 1.0µM
| \ 72.0°C|72.0°C|Primer2C 1.0µM
| \______|______|GC 81%
| 0min 0s|10min |4-12°C
'''[1:])
fig = dedent(fig)
assert str(prod.pfu_sso7d_program()) == fig
def test_amplicon():
from pydna.amplify import Anneal
from pydna.dseqrecord import Dseqrecord
from pydna.primer import Primer
template = Dseqrecord("AAAtacactcaccgtctatcattatctactatcgactgtatcatctgatagcacTTT")
p1 = Primer("CCCtacactcaccgtctatcattatc")
p2 = Primer("GGGgtgctatcagatgatacagtcg")
ann = Anneal((p1,p2),template)
prod = ann.products[0]
assert repr(prod) == 'Amplicon(57)'
assert prod._repr_html_() == 'Amplicon(57)'
from unittest.mock import MagicMock
pp = MagicMock()
prod._repr_pretty_(pp, None)
#assert pp.text.assert_called_with('Amplicon(57)')
fig=''' 5tacactcaccgtctatcattatc...cgactgtatcatctgatagcac3
|||||||||||||||||||||| tm 55.9 (dbd) 60.5
3gctgacatagtagactatcgtgGGG5
5CCCtacactcaccgtctatcattatc3
||||||||||||||||||||||| tm 54.6 (dbd) 58.8
3atgtgagtggcagatagtaatag...gctgacatagtagactatcgtg5'''
import textwrap
assert prod.figure() == textwrap.dedent(fig)
assert prod.program() == prod.taq_program()
assert prod.pfu_sso7d_program() == prod.dbd_program()
from pydna.amplicon import Amplicon
from Bio.Seq import Seq
from Bio.Alphabet.IUPAC import IUPACAmbiguousDNA
from pydna.seqrecord import SeqRecord
arg = SeqRecord(Seq("aaa", IUPACAmbiguousDNA()))
x = Amplicon.from_SeqRecord(arg)
def test_too_short_primers():
f, r = parse_primers('''>ForwardPrimer
gctactacacacgtactgactg
>ReversePrimer
tgtggttactgactctatcttg''')
t = Dseqrecord("gctactacacacgtactgactgcctccaagatagagtcagtaaccaca")
ann = Anneal((f, r), t, limit=22)
assert ann.report() == (
"Template name 48 nt linear:\n"
"Primer ForwardPrimer anneals forward at position 22\n"
"\n"
"Primer ReversePrimer anneals reverse at position 26")
assert repr(ann) == "Reaction(products = 1)"
p = ann.products[0]
assert str(p.seq) == str(t.seq)
ann = Anneal((f, r), t, limit=23)
assert ann.products == []
assert ann.report() == ("Template name 48 nt linear:\n"
"No forward primers anneal...\n"
"\n"
"No reverse primers anneal...")
assert repr(ann) == "Reaction(products = 0)"
def cloning_primers(template,
minlength=16,
maxlength=29,
fp=None,
rp=None,
fp_tail='',
rp_tail='',
target_tm=55.0,
primerc=1000.0,
saltc=50.0,
formula=tmbresluc):
'''This function can design primers for PCR amplification of a given sequence.
This function accepts a Dseqrecord object containing the template sequence and
returns a tuple cntaining two ::mod`Bio.SeqRecord.SeqRecord` objects describing
the primers.
Primer tails can optionally be given in the form of strings.
An predesigned primer can be given, either the forward or reverse primers. In this
case this function tries to design a primer with a Tm to match the given primer.
Parameters
----------
template : Dseqrecord
a Dseqrecord object.
minlength : int, optional
Minimum length of the annealing part of the primer
maxlength : int, optional
Maximum length (including tail) for designed primers.
fp, rp : SeqRecord, optional
optional Biopython SeqRecord objects containing one primer each.
fp_tail, rp_tail : string, optional
optional tails to be added to the forwars or reverse primers
target_tm : float, optional
target tm for the primers
primerc : float, optional
Concentration of each primer in nM, set to 1000.0 nM by default
saltc : float, optional
Salt concentration (monovalet cations) :mod:`tmbresluc` set to 50.0 mM by default
formula : function
formula used for tm calculation
this is the name of a function.
built in options are:
1. :func:`pydna.amplify.tmbresluc` (default)
2. :func:`pydna.amplify.basictm`
3. :func:`pydna.amplify.tmstaluc98`
4. :func:`pydna.amplify.tmbreslauer86`
These functions are imported from the :mod:`pydna.amplify` module, but can be
substituted for some other custom made function.
Returns
-------
fp, rp : tuple
fp is a :mod:Bio.SeqRecord object describing the forward primer
rp is a :mod:Bio.SeqRecord object describing the reverse primer
Examples
--------
>>> import pydna
>>> t=pydna.Dseqrecord("atgactgctaacccttccttggtgttgaacaagatcgacgacatttcgttcgaaacttacgatg")
>>> t
Dseqrecord(-64)
>>> pf,pr = pydna.cloning_primers(t)
>>> pf
Primer(seq=Seq('atgactgctaacccttc', IUPACAmbiguousDNA()), id='pfw64', name='pfw64', description='pfw64', dbxrefs=[])
>>> pr
Primer(seq=Seq('catcgtaagtttcgaac', IUPACAmbiguousDNA()), id='prv64', name='prv64', description='prv64', dbxrefs=[])
>>> pcr_prod = pydna.pcr(pf, pr, t)
>>> pcr_prod
Amplicon(64)
>>>
>>> print pcr_prod.figure()
5atgactgctaacccttc...gttcgaaacttacgatg3
||||||||||||||||| tm 42.4 (dbd) 52.9
3caagctttgaatgctac5
5atgactgctaacccttc3
||||||||||||||||| tm 44.5 (dbd) 54.0
3tactgacgattgggaag...caagctttgaatgctac5
>>> pf,pr = pydna.cloning_primers(t, fp_tail="GGATCC", rp_tail="GAATTC")
>>> pf
Primer(seq=Seq('GGATCCatgactgctaacccttc', IUPACAmbiguousDNA()), id='pfw64', name='pfw64', description='pfw64', dbxrefs=[])
>>> pr
Primer(seq=Seq('GAATTCcatcgtaagtttcgaac', IUPACAmbiguousDNA()), id='prv64', name='prv64', description='prv64', dbxrefs=[])
>>> pcr_prod = pydna.pcr(pf, pr, t)
>>> print pcr_prod.figure()
5atgactgctaacccttc...gttcgaaacttacgatg3
||||||||||||||||| tm 42.4 (dbd) 52.9
3caagctttgaatgctacCTTAAG5
5GGATCCatgactgctaacccttc3
||||||||||||||||| tm 44.5 (dbd) 54.0
3tactgacgattgggaag...caagctttgaatgctac5
>>> print pcr_prod.seq
GGATCCatgactgctaacccttccttggtgttgaacaagatcgacgacatttcgttcgaaacttacgatgGAATTC
>>>
>>> from Bio.Seq import Seq
>>> from Bio.SeqRecord import SeqRecord
>>> pf = SeqRecord(Seq("atgactgctaacccttccttggtgttg"))
>>> pf,pr = pydna.cloning_primers(t, fp = pf, fp_tail="GGATCC", rp_tail="GAATTC")
>>> pf
Primer(seq=Seq('GGATCCatgactgctaacccttccttggtgttg', Alphabet()), id='pfw64', name='pfw64', description='pfw64', dbxrefs=[])
>>> pr
Primer(seq=Seq('GAATTCcatcgtaagtttcgaacgaaatgtcgtc', IUPACAmbiguousDNA()), id='prv64', name='prv64', description='prv64', dbxrefs=[])
>>> ampl = pydna.pcr(pf,pr,t)
>>> print ampl.figure()
5atgactgctaacccttccttggtgttg...gacgacatttcgttcgaaacttacgatg3
|||||||||||||||||||||||||||| tm 57.5 (dbd) 72.2
3ctgctgtaaagcaagctttgaatgctacCTTAAG5
5GGATCCatgactgctaacccttccttggtgttg3
||||||||||||||||||||||||||| tm 59.0 (dbd) 72.3
3tactgacgattgggaaggaaccacaac...ctgctgtaaagcaagctttgaatgctac5
>>>
'''
if fp and not rp:
fp = Primer(Seq(fp_tail, IUPACAmbiguousDNA())) + fp
p = Anneal([fp], template).fwd_primers.pop()
fp = Primer(p.footprint)
fp_tail = Primer(p.tail)
rp = Primer(
Seq(
str(template[-(maxlength * 3 -
len(rp_tail)):].reverse_complement().seq),
IUPACAmbiguousDNA()))
target_tm = formula(str(fp.seq).upper(), primerc=primerc, saltc=saltc)
elif not fp and rp:
rp = Primer(Seq(rp_tail, IUPACAmbiguousDNA())) + rp
p = Anneal([rp], template).rev_primers.pop()
rp = Primer(p.footprint)
rp_tail = Primer(p.tail)
fp = Primer(
Seq(str(template[:maxlength * 3 - len(fp_tail)].seq),
IUPACAmbiguousDNA()))
target_tm = formula(str(rp.seq).upper(), primerc=primerc, saltc=saltc)
elif not fp and not rp:
fp = Primer(
Seq(str(template[:maxlength - len(fp_tail)].seq),
IUPACAmbiguousDNA()))
rp = Primer(
Seq(
str(template[-maxlength +
len(rp_tail):].reverse_complement().seq),
IUPACAmbiguousDNA()))
else:
raise Exception("Specify one or none of the primers, not both.")
lowtm, hightm = sorted([(formula(str(fp.seq), primerc, saltc), fp, "f"),
(formula(str(rp.seq), primerc, saltc), rp, "r")])
while lowtm[0] > target_tm and len(lowtm[1]) > minlength:
shorter = lowtm[1][:-1]
tm = formula(str(shorter.seq).upper(), primerc=primerc, saltc=saltc)
lowtm = (tm, shorter, lowtm[2])
while hightm[0] > lowtm[0] + 2.0 and len(hightm[1]) > minlength:
shorter = hightm[1][:-1]
tm = formula(str(shorter.seq).upper(), primerc=primerc, saltc=saltc)
hightm = (tm, shorter, hightm[2])
fp, rp = sorted((lowtm, hightm), key=itemgetter(2))
fp = fp_tail + fp[1]
rp = rp_tail + rp[1]
fp.description = "pfw{}".format(len(template))
rp.description = "prv{}".format(len(template))
fp.name = fp.description[:15]
rp.name = rp.description[:15]
fp.id = fp.name
rp.id = rp.name
#assert minlength<=len(fp)<=maxlength
#assert minlength<=len(rp)<=maxlength
if fp.seq.alphabet == Alphabet():
fp.seq.alphabet = IUPACAmbiguousDNA()
if rp.seq.alphabet == Alphabet():
rp.seq.alphabet = IUPACAmbiguousDNA()
return fp, rp
def cloning_primers(template,
minlength=16,
maxlength=29,
fp=None,
rp=None,
fp_tail='',
rp_tail='',
target_tm=55.0,
primerc=1000.0,
saltc=50.0,
formula=tmbresluc,
path=u""):
'''This function can design primers for PCR amplification of a given sequence.
This function accepts a Dseqrecord object containing the template sequence and
returns a tuple cntaining two ::mod`Bio.SeqRecord.SeqRecord` objects describing
the primers.
Primer tails can optionally be given in the form of strings.
An predesigned primer can be given, either the forward or reverse primers. In this
case this function tries to design a primer with a Tm to match the given primer.
Parameters
----------
template : Dseqrecord
a Dseqrecord object.
minlength : int, optional
Minimum length of the annealing part of the primer
maxlength : int, optional
Maximum length (including tail) for designed primers.
fp, rp : SeqRecord, optional
optional Biopython SeqRecord objects containing one primer each.
fp_tail, rp_tail : string, optional
optional tails to be added to the forwars or reverse primers
target_tm : float, optional
target tm for the primers
primerc : float, optional
Concentration of each primer in nM, set to 1000.0 nM by default
saltc : float, optional
Salt concentration (monovalet cations) :mod:`tmbresluc` set to 50.0 mM by default
formula : function
formula used for tm calculation
this is the name of a function.
built in options are:
1. :func:`pydna.amplify.tmbresluc` (default)
2. :func:`pydna.amplify.basictm`
3. :func:`pydna.amplify.tmstaluc98`
4. :func:`pydna.amplify.tmbreslauer86`
These functions are imported from the :mod:`pydna.amplify` module, but can be
substituted for some other custom made function.
path : unicode, optional
This variable can be set to a path to a text file, which will be created
if it does not exist.
This file (if it exists) will be parsed for sequences in fasta or
genbank format and a Biopython SeqRecord object will be created for
each sequence.
If a SeqRecord object is found with the same description as any of the
primers designed, the SeqRecord object parsed from the file will be
returned by this function instead of the newly designed primer.
If no sequence with the same description can be found, the primer(s)
will be appended to the file in fasta format.
Returns
-------
fp, rp : tuple
fp is a :mod:Bio.SeqRecord object describing the forward primer
rp is a :mod:Bio.SeqRecord object describing the reverse primer
Examples
--------
>>> import pydna
>>> t=pydna.Dseqrecord("atgactgctaacccttccttggtgttgaacaagatcgacgacatttcgttcgaaacttacgatg")
>>> t
Dseqrecord(-64)
>>> pf,pr = pydna.cloning_primers(t)
>>> pf
Primer(seq=Seq('atgactgctaacccttc', IUPACAmbiguousDNA()), id='fw64', name='fw64', description='fw64 -', dbxrefs=[])
>>> pr
Primer(seq=Seq('catcgtaagtttcgaac', IUPACAmbiguousDNA()), id='rv64', name='rv64', description='rv64 -', dbxrefs=[])
>>> pcr_prod = pydna.pcr(pf, pr, t)
>>> pcr_prod
Amplicon(64)
>>>
>>> print(pcr_prod.figure())
5atgactgctaacccttc...gttcgaaacttacgatg3
||||||||||||||||| tm 42.4 (dbd) 52.9
3caagctttgaatgctac5
5atgactgctaacccttc3
||||||||||||||||| tm 44.5 (dbd) 54.0
3tactgacgattgggaag...caagctttgaatgctac5
>>> pf,pr = pydna.cloning_primers(t, fp_tail="GGATCC", rp_tail="GAATTC")
>>> pf
Primer(seq=Seq('GGATCCatgactgctaacccttc', IUPACAmbiguousDNA()), id='fw64', name='fw64', description='fw64 -', dbxrefs=[])
>>> pr
Primer(seq=Seq('GAATTCcatcgtaagtttcgaac', IUPACAmbiguousDNA()), id='rv64', name='rv64', description='rv64 -', dbxrefs=[])
>>> pcr_prod = pydna.pcr(pf, pr, t)
>>> print(pcr_prod.figure())
5atgactgctaacccttc...gttcgaaacttacgatg3
||||||||||||||||| tm 42.4 (dbd) 52.9
3caagctttgaatgctacCTTAAG5
5GGATCCatgactgctaacccttc3
||||||||||||||||| tm 44.5 (dbd) 54.0
3tactgacgattgggaag...caagctttgaatgctac5
>>> print(pcr_prod.seq)
GGATCCatgactgctaacccttccttggtgttgaacaagatcgacgacatttcgttcgaaacttacgatgGAATTC
>>>
>>> from Bio.Seq import Seq
>>> from Bio.SeqRecord import SeqRecord
>>> pf = SeqRecord(Seq("atgactgctaacccttccttggtgttg"))
>>> pf,pr = pydna.cloning_primers(t, fp = pf, fp_tail="GGATCC", rp_tail="GAATTC")
>>> pf
Primer(seq=Seq('GGATCCatgactgctaacccttccttggtgttg', Alphabet()), id='fw64', name='fw64', description='fw64 -', dbxrefs=[])
>>> pr
Primer(seq=Seq('GAATTCcatcgtaagtttcgaacgaaatgtcgtc', IUPACAmbiguousDNA()), id='rv64', name='rv64', description='rv64 -', dbxrefs=[])
>>> ampl = pydna.pcr(pf,pr,t)
>>> print(ampl.figure())
5atgactgctaacccttccttggtgttg...gacgacatttcgttcgaaacttacgatg3
|||||||||||||||||||||||||||| tm 57.5 (dbd) 72.2
3ctgctgtaaagcaagctttgaatgctacCTTAAG5
5GGATCCatgactgctaacccttccttggtgttg3
||||||||||||||||||||||||||| tm 59.0 (dbd) 72.3
3tactgacgattgggaaggaaccacaac...ctgctgtaaagcaagctttgaatgctac5
>>>
'''
if fp and not rp:
fp = Primer(Seq(fp_tail, IUPACAmbiguousDNA())) + fp
p = Anneal([fp], template).fwd_primers.pop()
fp = Primer(p.footprint)
fp_tail = Primer(p.tail)
rp = Primer(
Seq(
str(template[-(maxlength * 3 -
len(rp_tail)):].reverse_complement().seq),
IUPACAmbiguousDNA()))
target_tm = formula(str(fp.seq).upper(), primerc=primerc, saltc=saltc)
elif not fp and rp:
rp = Primer(Seq(rp_tail, IUPACAmbiguousDNA())) + rp
p = Anneal([rp], template).rev_primers.pop()
rp = Primer(p.footprint)
rp_tail = Primer(p.tail)
fp = Primer(
Seq(str(template[:maxlength * 3 - len(fp_tail)].seq),
IUPACAmbiguousDNA()))
target_tm = formula(str(rp.seq).upper(), primerc=primerc, saltc=saltc)
elif not fp and not rp:
fp = Primer(
Seq(str(template[:maxlength - len(fp_tail)].seq),
IUPACAmbiguousDNA()))
rp = Primer(
Seq(
str(template[-maxlength +
len(rp_tail):].reverse_complement().seq),
IUPACAmbiguousDNA()))
else:
raise Exception("Specify maximum one of the two primers, not both.")
lowtm, hightm = sorted([(formula(str(fp.seq), primerc, saltc), fp, "f"),
(formula(str(rp.seq), primerc, saltc), rp, "r")])
while lowtm[0] > target_tm and len(lowtm[1]) > minlength:
shorter = lowtm[1][:-1]
tm = formula(str(shorter.seq).upper(), primerc=primerc, saltc=saltc)
lowtm = (tm, shorter, lowtm[2])
while hightm[0] > lowtm[0] + 2.0 and len(hightm[1]) > minlength:
shorter = hightm[1][:-1]
tm = formula(str(shorter.seq).upper(), primerc=primerc, saltc=saltc)
hightm = (tm, shorter, hightm[2])
fp, rp = sorted((lowtm, hightm), key=itemgetter(2))
fp = fp_tail + fp[1]
rp = rp_tail + rp[1]
#fp.description = "fw{}".format(len(template))
#rp.description = "rv{}".format(len(template))
#fp.name = "fw{}".format(len(template))[:15]
#rp.name = "rv{}".format(len(template))[:15]
fp.description = "fw{}".format(len(template)) + ' ' + template.accession
rp.description = "rv{}".format(len(template)) + ' ' + template.accession
fp.id = "fw{}".format(len(template))
rp.id = "rv{}".format(len(template))
fp.name = fp.id
rp.name = rp.id
if fp.seq.alphabet == Alphabet():
fp.seq.alphabet = IUPACAmbiguousDNA()
if rp.seq.alphabet == Alphabet():
rp.seq.alphabet = IUPACAmbiguousDNA()
# If the path argument is supplied primers will be written to a file with that
# path. If the file does not exist, it will be created and both primers will be
# written to it. If the file exists, the file will be parsed for sequences in
# fasta or genbank into .
if path:
try:
with open(path, 'rU') as f:
raw = f.read()
except IOError:
raw = u""
with open(path, 'w') as f:
f.write(fp.format("fasta"))
f.write(rp.format("fasta"))
else:
primer_dict = {x.description: x for x in parse(raw, ds=False)}
try:
fp = primer_dict[fp.description]
except KeyError:
with open(path, 'a') as f:
f.write(u"\n" + fp.format("fasta").strip())
try:
rp = primer_dict[rp.description]
except KeyError:
with open(path, 'a') as f:
f.write(u"\n" + rp.format("fasta").strip())
return fp, rp