def test_computes_primer_distance_to_junction(self):
upstream = "cagtacgatcgttggtatgctgactactagcgtagctagcacgtcgtgtccaggcttgagcgacgt"
product = "cagctggtaatcgtactcgtactagcatcgtacgtgtctgatcatctgacgtatcatctga"
downstream = (
"agtgacgtcgtgtgtagcgtactgtatcgtgtgtcgcgcgtagtcatctgatcgtacgtactgaat"
)
template = "".join([upstream, product, downstream])
WIN = 10
junctions = [len(upstream) + 1, len(upstream) + len(product) - 1]
res = design_primers_from_template(template,
len(upstream) + 1 - WIN,
len(product) + WIN * 2, junctions,
{})
self.assertEquals(len(res), 5)
for r in res:
left = r["PRIMER_LEFT_SEQUENCE"]
self.assertEquals(
r["PRIMER_LEFT_SEQUENCE_DISTANCE_TO_JUNCTION"] >= WIN, True)
self.assertEquals(
template.lower().index(left.lower()) + len(left) +
r["PRIMER_LEFT_SEQUENCE_DISTANCE_TO_JUNCTION"],
junctions[0],
)
right = str(Seq(r["PRIMER_RIGHT_SEQUENCE"]).reverse_complement())
self.assertEquals(
r["PRIMER_RIGHT_SEQUENCE_DISTANCE_TO_JUNCTION"] >= WIN, True)
self.assertEquals(
template.lower().index(right.lower()) -
r["PRIMER_RIGHT_SEQUENCE_DISTANCE_TO_JUNCTION"],
junctions[1],
)
def test_computes_primer_distance_to_junction(self):
upstream = "cagtacgatcgttggtatgctgactactagcgtagctagcacgtcgtgtccaggcttgagcgacgt"
product = "cagctggtaatcgtactcgtactagcatcgtacgtgtctgatcatctgacgtatcatctga"
downstream = "agtgacgtcgtgtgtagcgtactgtatcgtgtgtcgcgcgtagtcatctgatcgtacgtactgaat"
template = ''.join([upstream, product, downstream])
WIN = 10
junctions = [len(upstream)+1, len(upstream)+len(product)-1]
res = design_primers_from_template(template, len(upstream)+1-WIN, len(product)+WIN*2,
junctions, {})
self.assertEquals(len(res), 5)
for r in res:
left = r['PRIMER_LEFT_SEQUENCE']
self.assertEquals(r['PRIMER_LEFT_SEQUENCE_DISTANCE_TO_JUNCTION'] >= WIN, True)
self.assertEquals(template.lower().index(left.lower()) +
len(left) +
r['PRIMER_LEFT_SEQUENCE_DISTANCE_TO_JUNCTION'], junctions[0])
right = str(Seq(r['PRIMER_RIGHT_SEQUENCE']).reverse_complement())
self.assertEquals(r['PRIMER_RIGHT_SEQUENCE_DISTANCE_TO_JUNCTION'] >= WIN, True)
self.assertEquals(template.lower().index(right.lower()) -
r['PRIMER_RIGHT_SEQUENCE_DISTANCE_TO_JUNCTION'], junctions[1])
def get_verification_primers(genome, region, primer3_opts):
"""
Design primers to verify replacing region with cassette.
"""
fragment = Fragment.objects.get(pk=region.fragment_id).indexed_fragment()
cassette = region.cassette
check_junction_lu = CHECK_JUNCTION_LEFT_UP
check_junction_ld = CHECK_JUNCTION_LEFT_DN
check_junction_ru = CHECK_JUNCTION_RIGHT_UP
check_junction_rd = CHECK_JUNCTION_RIGHT_DN
if primer3_opts:
if 'EDGE_CHECK_JUNCTION_LEFT_HA' in primer3_opts:
check_junction_ld = int(primer3_opts['EDGE_CHECK_JUNCTION_LEFT_HA'])
if 'EDGE_CHECK_JUNCTION_RIGHT_HA' in primer3_opts:
check_junction_ru = int(primer3_opts['EDGE_CHECK_JUNCTION_RIGHT_HA'])
#
# front junction
#
upstream_window = CHECK_JUNCTION_PRIMER_WINDOW+check_junction_lu
downstream_window = min(len(cassette), CHECK_JUNCTION_PRIMER_WINDOW+check_junction_ld)
back = cassette[0:downstream_window]
# don't bother looking for primers if front part of cassette is same as
# region to be replaced
if region.start > 1 and back.lower() != region.sequence[0:downstream_window].lower():
front = fragment.get_sequence(region.start-upstream_window, region.start-1)
template = front+back
junction = [len(front)]
roi_start = len(front)-check_junction_lu if len(front) > check_junction_lu else 0
roi_len = min(check_junction_lu+check_junction_ld,
min(len(front), check_junction_ld)+len(cassette))
# remove primers that works on un-modified genome for creating a PCR product
region.verification_front =\
remove_working_primers(genome,
design_primers_from_template(template, roi_start, roi_len,
junction, primer3_opts))
#
# back junction
#
upstream_window = min(len(cassette), CHECK_JUNCTION_PRIMER_WINDOW+check_junction_ru)
downstream_window = CHECK_JUNCTION_PRIMER_WINDOW+check_junction_rd
front = cassette[-upstream_window:]
# don't bother looking for primers if front part of cassette is same as
# region to be replaced
if region.start+len(region.sequence) < fragment.length and\
front.lower() != region.sequence[-upstream_window:].lower():
back = fragment.get_sequence(region.start+len(region.sequence),
region.start+len(region.sequence)+downstream_window-1)
template = front+back
junction = [len(front)]
roi_start = len(front)-check_junction_ru if len(front) > check_junction_ru else 0
roi_len = min(check_junction_ru+check_junction_rd,
min(len(back), check_junction_rd)+len(cassette))
# remove primers that works on un-modified genome for creating a PCR product
region.verification_back =\
remove_working_primers(genome,
design_primers_from_template(template, roi_start, roi_len,
junction, primer3_opts))
#
# cassette
#
if region.start > 1 and region.start+len(region.sequence) < fragment.length:
front = fragment.get_sequence(region.start-CHECK_JUNCTION_PRIMER_WINDOW, region.start-1)
back = fragment.get_sequence(region.start+len(region.sequence),
region.start+len(region.sequence) +
CHECK_JUNCTION_PRIMER_WINDOW-1)
template = front+cassette+back
junctions = [len(front), len(front+cassette)-1]
roi_start = template.index(cassette)
roi_len = len(cassette)
region.verification_cassette =\
design_primers_from_template(template, roi_start, roi_len,
junctions, primer3_opts)
def get_verification_primers(genome, region, primer3_opts):
"""
Design primers to verify replacing region with cassette.
"""
fragment = Fragment.objects.get(pk=region.fragment_id).indexed_fragment()
cassette = region.cassette
check_junction_lu = CHECK_JUNCTION_LEFT_UP
check_junction_ld = CHECK_JUNCTION_LEFT_DN
check_junction_ru = CHECK_JUNCTION_RIGHT_UP
check_junction_rd = CHECK_JUNCTION_RIGHT_DN
if primer3_opts:
if 'EDGE_CHECK_JUNCTION_LEFT_HA' in primer3_opts:
check_junction_ld = int(primer3_opts['EDGE_CHECK_JUNCTION_LEFT_HA'])
if 'EDGE_CHECK_JUNCTION_RIGHT_HA' in primer3_opts:
check_junction_ru = int(primer3_opts['EDGE_CHECK_JUNCTION_RIGHT_HA'])
#
# front junction
#
upstream_window = CHECK_JUNCTION_PRIMER_WINDOW+check_junction_lu
downstream_window = min(len(cassette), CHECK_JUNCTION_PRIMER_WINDOW+check_junction_ld)
back = cassette[0:downstream_window]
# don't bother looking for primers if front part of cassette is same as
# region to be replaced
if region.start > 1 and back.lower() != region.sequence[0:downstream_window].lower():
front = fragment.get_sequence(region.start-upstream_window, region.start-1)
template = front+back
junction = [len(front)]
roi_start = len(front)-check_junction_lu if len(front) > check_junction_lu else 0
roi_len = min(check_junction_lu+check_junction_ld,
min(len(front), check_junction_ld)+len(cassette))
# remove primers that works on un-modified genome for creating a PCR product
region.verification_front =\
remove_working_primers(genome,
design_primers_from_template(template, roi_start, roi_len,
junction, primer3_opts))
#
# back junction
#
upstream_window = min(len(cassette), CHECK_JUNCTION_PRIMER_WINDOW+check_junction_ru)
downstream_window = CHECK_JUNCTION_PRIMER_WINDOW+check_junction_rd
front = cassette[-upstream_window:]
# don't bother looking for primers if front part of cassette is same as
# region to be replaced
if region.start+len(region.sequence) < fragment.length and\
front.lower() != region.sequence[-upstream_window:].lower():
back = fragment.get_sequence(region.start+len(region.sequence),
region.start+len(region.sequence)+downstream_window-1)
template = front+back
junction = [len(front)]
roi_start = len(front)-check_junction_ru if len(front) > check_junction_ru else 0
roi_len = min(check_junction_ru+check_junction_rd,
min(len(back), check_junction_rd)+len(cassette))
# remove primers that works on un-modified genome for creating a PCR product
region.verification_back =\
remove_working_primers(genome,
design_primers_from_template(template, roi_start, roi_len,
junction, primer3_opts))
#
# cassette
#
if region.start > 1 and region.start+len(region.sequence) < fragment.length:
front = fragment.get_sequence(region.start-CHECK_JUNCTION_PRIMER_WINDOW, region.start-1)
back = fragment.get_sequence(region.start+len(region.sequence),
region.start+len(region.sequence) +
CHECK_JUNCTION_PRIMER_WINDOW-1)
template = front+cassette+back
junctions = [len(front), len(front+cassette)-1]
roi_start = template.index(cassette)
roi_len = len(cassette)
region.verification_cassette =\
design_primers_from_template(template, roi_start, roi_len,
junctions, primer3_opts)