def add_proximal_variants(self, somatic_variant_index,
wildtype_subsequence, mutation_position,
original_position, germline_variants_only):
mutation_offset = original_position - mutation_position
wildtype_subsequence_with_proximal_variants = wildtype_subsequence
if somatic_variant_index in self.proximal_variants.keys():
for (protein_position, lines
) in self.proximal_variants[somatic_variant_index].items():
if protein_position == original_position:
continue
if germline_variants_only:
filtered_lines = [
line for line in lines if line['type'] == 'germline'
]
else:
filtered_lines = lines
if len(filtered_lines) == 0:
continue
elif len(filtered_lines) == 1:
line = filtered_lines[0]
proximal_variant_wildtype_amino_acid, proximal_variant_mutant_amino_acid = line[
'amino_acid_change'].split('/')
else:
line = filtered_lines[0]
proximal_variant_wildtype_amino_acid = line[
'amino_acid_change'].split('/')[0]
codon_changes = [
item['codon_change'] for item in filtered_lines
]
proximal_variant_mutant_amino_acid = ProximalVariant.combine_conflicting_variants(
codon_changes)
proximal_variant_position = int(
protein_position) - 1 - mutation_offset
if proximal_variant_position <= 0 or proximal_variant_position >= len(
wildtype_subsequence):
continue
if len(proximal_variant_wildtype_amino_acid) != len(
proximal_variant_mutant_amino_acid):
print(
"Nearby variant is not a missense mutation. Skipping.")
continue
if wildtype_subsequence[
proximal_variant_position] != proximal_variant_wildtype_amino_acid:
sys.exit(
"Error when processing proximal variant.\n" +
"The wildtype amino acid for variant %s with substring %s is different than expected.\n"
% (somatic_variant_index, wildtype_subsequence) +
"Actual wildtype amino acid: %s\n" %
wildtype_subsequence[proximal_variant_position] +
"Wildtype amino acid of the proximal_variant: %s" %
proximal_variant_wildtype_amino_acid)
wildtype_subsequence_with_proximal_variants = wildtype_subsequence_with_proximal_variants[:proximal_variant_position] + proximal_variant_mutant_amino_acid + wildtype_subsequence_with_proximal_variants[
proximal_variant_position + 1:]
return wildtype_subsequence_with_proximal_variants
def execute(self):
peptide_sequence_length = self.peptide_sequence_length
reader = open(self.input_file, 'r')
tsvin = csv.DictReader(reader, delimiter='\t')
fasta_sequences = OrderedDict()
for line in tsvin:
variant_type = line['variant_type']
full_wildtype_sequence = line['wildtype_amino_acid_sequence']
if variant_type == 'FS':
position = int(line['protein_position'].split('-', 1)[0]) - 1
if line['amino_acid_change'] is not None and line[
'amino_acid_change'].split('/')[0] == '-':
if line['wildtype_amino_acid_sequence'][position] != line[
'downstream_amino_acid_sequence'][0]:
raise Exception(
"Leading amino acid of the Downstream protein sequence ({}) expected to match the wildtype amino acid at postion {} ({}). " \
"You may need to reannotate your VCF with a newer version of VEP." \
.format(line['downstream_amino_acid_sequence'], position, line['wildtype_amino_acid_sequence'][position])
)
elif variant_type == 'missense' or variant_type == 'inframe_ins':
if '/' not in line['amino_acid_change']:
continue
wildtype_amino_acid, mutant_amino_acid = line[
'amino_acid_change'].split('/')
if '*' in wildtype_amino_acid:
wildtype_amino_acid = wildtype_amino_acid.split('*')[0]
elif 'X' in wildtype_amino_acid:
wildtype_amino_acid = wildtype_amino_acid.split('X')[0]
if '*' in mutant_amino_acid:
mutant_amino_acid = mutant_amino_acid.split('*')[0]
stop_codon_added = True
elif 'X' in mutant_amino_acid:
mutant_amino_acid = mutant_amino_acid.split('X')[0]
stop_codon_added = True
else:
stop_codon_added = False
if wildtype_amino_acid == '-':
position = int(line['protein_position'].split('-', 1)[0])
wildtype_amino_acid_length = 0
else:
if '-' in line['protein_position']:
position = int(line['protein_position'].split(
'-', 1)[0]) - 1
wildtype_amino_acid_length = len(wildtype_amino_acid)
else:
position = int(line['protein_position']) - 1
wildtype_amino_acid_length = len(wildtype_amino_acid)
elif variant_type == 'inframe_del':
variant_type = 'inframe_del'
wildtype_amino_acid, mutant_amino_acid = line[
'amino_acid_change'].split('/')
if '*' in wildtype_amino_acid:
wildtype_amino_acid = wildtype_amino_acid.split('*')[0]
elif 'X' in wildtype_amino_acid:
wildtype_amino_acid = wildtype_amino_acid.split('X')[0]
if '*' in mutant_amino_acid:
mutant_amino_acid = mutant_amino_acid.split('*')[0]
stop_codon_added = True
elif 'X' in mutant_amino_acid:
mutant_amino_acid = mutant_amino_acid.split('X')[0]
stop_codon_added = True
else:
stop_codon_added = False
position = int(line['protein_position'].split('-', 1)[0]) - 1
wildtype_amino_acid_length = len(wildtype_amino_acid)
if mutant_amino_acid == '-':
mutant_amino_acid = ''
else:
continue
if self.position_out_of_bounds(position, full_wildtype_sequence):
continue
if variant_type == 'missense' and line[
'index'] in self.proximal_variants and line[
'protein_position'] in self.proximal_variants[
line['index']]:
codon_changes = [
item['codon_change'] for item in self.proximal_variants[
line['index']][line['protein_position']]
]
codon_changes.append(line['codon_change'])
mutant_amino_acid_with_proximal_variants = ProximalVariant.combine_conflicting_variants(
codon_changes)
elif variant_type != 'FS':
mutant_amino_acid_with_proximal_variants = mutant_amino_acid
if variant_type == 'FS':
wildtype_subsequence, left_flanking_subsequence = self.get_frameshift_subsequences(
position, full_wildtype_sequence, peptide_sequence_length,
line)
downstream_sequence = line['downstream_amino_acid_sequence']
if self.downstream_sequence_length and len(
downstream_sequence) > self.downstream_sequence_length:
downstream_sequence = downstream_sequence[
0:self.downstream_sequence_length]
mutation_start_position = len(left_flanking_subsequence)
wildtype_subsequence = self.add_proximal_variants(
line['index'], wildtype_subsequence,
mutation_start_position, position, True)
left_flanking_subsequence_with_proximal_variants = self.add_proximal_variants(
line['index'], left_flanking_subsequence,
mutation_start_position, position, False)
#The caveat here is that if a nearby variant is in the downstream sequence, the protein sequence would be further altered, which we aren't taking into account.
#we would need to recalculate the downstream protein sequence taking all downstream variants into account.
mutant_subsequence = left_flanking_subsequence_with_proximal_variants + downstream_sequence
else:
mutation_start_position, wildtype_subsequence = self.get_wildtype_subsequence(
position, full_wildtype_sequence,
wildtype_amino_acid_length, peptide_sequence_length, line)
mutation_end_position = mutation_start_position + wildtype_amino_acid_length
if wildtype_amino_acid != '-' and wildtype_amino_acid != wildtype_subsequence[
mutation_start_position:mutation_end_position]:
if line['amino_acid_change'].split('/')[0].count('*') > 1:
print(
"Warning: Amino acid change is not sane - contains multiple stops. Skipping entry {}"
.format(line['index']))
continue
else:
sys.exit(
"ERROR: There was a mismatch between the actual wildtype amino acid sequence ({}) and the expected amino acid sequence ({}). Did you use the same reference build version for VEP that you used for creating the VCF?\n{}"
.format(
wildtype_subsequence[mutation_start_position:
mutation_end_position],
wildtype_amino_acid, line))
wildtype_subsequence_with_proximal_variants = self.add_proximal_variants(
line['index'], wildtype_subsequence,
mutation_start_position, position, False)
wildtype_subsequence = self.add_proximal_variants(
line['index'], wildtype_subsequence,
mutation_start_position, position, True)
if stop_codon_added:
mutant_subsequence = wildtype_subsequence_with_proximal_variants[:
mutation_start_position] + mutant_amino_acid_with_proximal_variants
else:
mutant_subsequence = wildtype_subsequence_with_proximal_variants[:mutation_start_position] + mutant_amino_acid_with_proximal_variants + wildtype_subsequence_with_proximal_variants[
mutation_end_position:]
if '*' in wildtype_subsequence or '*' in mutant_subsequence:
continue
if 'X' in wildtype_subsequence or 'X' in mutant_subsequence:
continue
if 'U' in wildtype_subsequence or 'U' in mutant_subsequence:
print(
"Warning. Sequence contains unsupported amino acid U. Skipping entry {}"
.format(line['index']))
continue
if mutant_subsequence in wildtype_subsequence:
#This is not a novel peptide
continue
if len(wildtype_subsequence) < self.epitope_length or len(
mutant_subsequence) < self.epitope_length:
continue
variant_id = line['index']
for designation, subsequence in zip(
['WT', 'MT'], [wildtype_subsequence, mutant_subsequence]):
key = '%s.%s' % (designation, variant_id)
fasta_sequences.setdefault(subsequence, []).append(key)
writer = open(self.output_file, 'w')
key_writer = open(self.output_key_file, 'w')
count = 1
for (subsequence, keys) in fasta_sequences.items():
writer.writelines('>%s\n' % count)
writer.writelines('%s\n' % subsequence)
yaml.dump({count: keys}, key_writer, default_flow_style=False)
count += 1
reader.close()
writer.close()
key_writer.close()