def filterStreubel(binding_site):
seq2 = reverseComplement(binding_site.seq2_seq)
if float(binding_site.seq1_seq.count("C") + binding_site.seq1_seq.count("G")) / len(binding_site.seq1_seq) < 0.25:
return True
if len(streubel_at_streak_re.findall(binding_site.seq1_seq)) > 0:
return True
if float(seq2.count("C") + seq2.count("G")) / len(seq2) < 0.25:
return True
if len(streubel_at_streak_re.findall(seq2)) > 0:
return True
return False
def RunFindTALTask(options):
logger = create_logger(options.logFilepath)
logger("Beginning")
if options.check_offtargets and options.offtargets_ncbi != "NA":
logger(
"Retrieving NCBI off-target sequence. This could take a while if this sequence hasn't been used recently and needs to be downloaded from NCBI."
)
with Conditional(
options.check_offtargets and options.offtargets_ncbi != "NA", CachedEntrezFile(logger, options.offtargets_ncbi)
) as maybe_entrez_file:
if options.check_offtargets:
if not tfcount_found:
raise TaskError("Non off-target counting worker attempted to process off-target counting task.")
if options.offtargets_ncbi != "NA":
logger("Finished retrieving NCBI off-target sequence.")
# Validate downloaded sequence
check_fasta_pasta(maybe_entrez_file.file)
for record in FastaIterator(maybe_entrez_file.file, alphabet=generic_dna):
if len(record.seq) > OFFTARGET_COUNTING_SIZE_LIMIT:
raise TaskError(
"Off-Target counting is only supported for NCBI records where all individual sequences are under %d megabases in size"
% (OFFTARGET_COUNTING_SIZE_LIMIT / 1000000)
)
offtarget_seq_filename = ""
if options.offtargets_fasta != "NA":
offtarget_seq_filename = options.offtargets_fasta
elif options.offtargets_ncbi != "NA":
offtarget_seq_filename = maybe_entrez_file.filepath
elif options.genome:
offtarget_seq_filename = GENOME_FILE % options.organism
elif options.promoterome:
offtarget_seq_filename = PROMOTEROME_FILE % options.organism
else:
offtarget_seq_filename = options.fasta
strong_binding_RVDs = {"A": "NI", "C": "HD", "G": "NN", "T": "NG"}
if options.gspec:
strong_binding_RVDs["G"] = "NH"
seq_file = open(options.fasta, "r")
if options.outpath == "NA":
output_filepath = options.outdir + options.job + options.outfile
else:
output_filepath = options.outpath
out = open(output_filepath, "w")
table_ignores = ["TAL1 length", "TAL2 length", "Spacer length"]
out.write("table_ignores:" + ",".join(table_ignores) + "\n")
strand_min = 15 if options.arraymin is None else options.arraymin
strand_max = 20 if options.arraymax is None else options.arraymax
spacer_min = 15 if options.min is None else options.min
spacer_max = 30 if options.max is None else options.max
u_bases = []
if options.cupstream != 1:
u_bases.append("T")
if options.cupstream != 0:
u_bases.append("C")
out.write(
"options_used:"
+ ", ".join(
[
"array_min = " + str(strand_min),
"array_max = " + str(strand_max),
"spacer_min = " + str(spacer_min),
"spacer_max = " + str(spacer_max),
"upstream_base = " + (" or ".join(u_bases)),
]
)
+ "\n"
)
offtarget_header = "\tOff-Target Counts" if options.check_offtargets else ""
out.write(
"Sequence Name\tCut Site\tTAL1 start\tTAL2 start\tTAL1 length\tTAL2 length\tSpacer length\tSpacer range\tTAL1 RVDs\tTAL2 RVDs\tPlus strand sequence\tUnique RE sites in spacer\t% RVDs HD or NN/NH"
+ offtarget_header
+ "\n"
)
binding_sites = []
for gene in FastaIterator(seq_file, alphabet=generic_dna):
sequence = str(gene.seq).upper()
site_entry_counts = {}
if options.filter == 1:
if options.filterbase > len(sequence):
logger("Skipped %s as the provided cut site was greater than the sequence length" % (gene.id))
continue
cut_site_positions = [options.filterbase]
else:
cut_site_positions = range(len(sequence))
logger("Scanning %s for binding sites" % (gene.id))
for i in cut_site_positions:
cut_site_potential_sites = []
for spacer_size in range(spacer_min, spacer_max + 1):
spacer_potential_sites = []
spacer_size_left = int(math.floor(float(spacer_size) / 2))
spacer_size_right = int(math.ceil(float(spacer_size) / 2))
if i < (strand_min + spacer_size_left + 1) or i > (
len(sequence) - (strand_min + spacer_size_right) - 1
):
continue
for u_base in u_bases:
if u_base == "T":
d_base = "A"
elif u_base == "C":
d_base = "G"
u_pos_search_start = i - (strand_max + spacer_size_left) - 1
if u_pos_search_start < 0:
u_pos_search_start = 0
u_pos_search_end = i - (strand_min + spacer_size_left)
d_pos_search_start = i + (strand_min + spacer_size_right)
d_pos_search_end = i + (strand_max + spacer_size_right) + 1
u_positions = []
u_pos = 0
while True:
u_pos = sequence.rfind(u_base, u_pos_search_start, u_pos_search_end)
if u_pos == -1:
break
else:
u_pos_search_end = u_pos
u_positions.append(u_pos)
d_positions = []
d_pos = 0
while True:
d_pos = sequence.find(d_base, d_pos_search_start, d_pos_search_end)
if d_pos == -1:
break
else:
d_pos_search_start = d_pos + 1
d_positions.append(d_pos)
break_out = False
for u_pos in reversed(u_positions):
for d_pos in reversed(d_positions):
# uses inclusive start, exclusive end
tal1_start = u_pos + 1
tal1_end = i - spacer_size_left
tal1_seq = sequence[tal1_start:tal1_end]
tal2_start = i + spacer_size_right
tal2_end = d_pos
tal2_seq = sequence[tal2_start:tal2_end]
if not (
(tal1_seq in site_entry_counts and tal2_seq in site_entry_counts[tal1_seq])
or (tal1_seq in site_entry_counts and tal1_seq in site_entry_counts[tal1_seq])
or (tal2_seq in site_entry_counts and tal1_seq in site_entry_counts[tal2_seq])
or (tal2_seq in site_entry_counts and tal2_seq in site_entry_counts[tal2_seq])
):
bad_site = False
cg_count = 0
tal1_rvd = []
for c in tal1_seq:
if c not in strong_binding_RVDs:
bad_site = True
break
if c == "C" or c == "G":
cg_count += 1
tal1_rvd.append(strong_binding_RVDs[c])
if bad_site:
continue
tal1_rvd = " ".join(tal1_rvd)
tal2_rvd = []
for c in reverseComplement(tal2_seq):
if c not in strong_binding_RVDs:
bad_site = True
break
if c == "C" or c == "G":
cg_count += 1
tal2_rvd.append(strong_binding_RVDs[c])
if bad_site:
continue
tal2_rvd = " ".join(tal2_rvd)
if options.filter == 0:
break_out = True
binding_site = BindingSite(
seq_id=gene.id,
cutsite=i,
seq1_start=tal1_start,
seq1_end=tal1_end,
seq1_seq=tal1_seq,
seq1_rvd=tal1_rvd,
spacer_start=tal1_end,
spacer_end=tal2_start,
spacer_seq=sequence[tal1_end:tal2_start],
seq2_start=tal2_start,
seq2_end=tal2_end,
seq2_seq=tal2_seq,
seq2_rvd=tal2_rvd,
upstream=u_base,
cg_percent=int(
round(float(cg_count) / (len(tal1_seq) + len(tal2_seq)), 2) * 100
),
)
findRESitesInSpacer(sequence, binding_site)
if binding_site.seq1_seq not in site_entry_counts:
site_entry_counts[binding_site.seq1_seq] = {}
if binding_site.seq2_seq not in site_entry_counts[tal1_seq]:
site_entry_counts[binding_site.seq1_seq][binding_site.seq2_seq] = []
site_entry_counts[binding_site.seq1_seq][binding_site.seq2_seq].append(binding_site)
spacer_potential_sites.append(binding_site)
if break_out:
break
if break_out:
break
if len(spacer_potential_sites) > 0:
if options.filter == 0:
cut_site_potential_sites.append(reduce(filterByTALSize, spacer_potential_sites))
else:
cut_site_potential_sites.extend(spacer_potential_sites)
if len(cut_site_potential_sites) > 0:
if options.filter == 0:
binding_sites.append(reduce(filterByTALSize, cut_site_potential_sites))
else:
binding_sites.extend(cut_site_potential_sites)
if options.streubel:
binding_sites[:] = list(ifilterfalse(filterStreubel, binding_sites))
if options.check_offtargets:
if len(binding_sites) > 0:
off_target_pairs = []
for i, binding_site in enumerate(binding_sites):
off_target_pairs.append([binding_site.seq1_rvd, binding_site.seq2_rvd])
off_target_counts = PairedTargetFinderCountTask(
offtarget_seq_filename,
options.logFilepath,
options.cupstream,
3.0,
spacer_min,
spacer_max,
off_target_pairs,
)
for i, binding_site in enumerate(binding_sites):
binding_site.offtarget_counts = off_target_counts[i]
for i, binding_site in enumerate(binding_sites):
output_items = [
str(binding_site.seq_id),
str(binding_site.cutsite),
str(binding_site.seq1_start),
str(binding_site.seq2_end - 1),
str(binding_site.seq1_end - binding_site.seq1_start),
str(binding_site.seq2_end - binding_site.seq2_start),
str(binding_site.spacer_end - binding_site.spacer_start),
str(binding_site.spacer_start) + "-" + str(binding_site.spacer_end - 1),
binding_site.seq1_rvd,
binding_site.seq2_rvd,
binding_site.upstream
+ " "
+ binding_site.seq1_seq
+ " "
+ binding_site.spacer_seq.lower()
+ " "
+ binding_site.seq2_seq
+ " "
+ ("A" if binding_site.upstream == "T" else "G"),
binding_site.re_sites,
str(binding_site.cg_percent),
]
if options.check_offtargets:
output_items.append(" ".join(str(binding_site.offtarget_counts[x]) for x in range(5)))
out.write("\t".join(output_items) + "\n")
out.close()
seq_file.close()
logger("Finished")
