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UTRAnnotator.pm
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UTRAnnotator.pm
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=head1 LICENSE
Copyright [1999-2015] Wellcome Trust Sanger Institute and the EMBL-European Bioinformatics Institute
Copyright [2016-2024] EMBL-European Bioinformatics Institute
Licensed under the Apache License, Version 2.0 (the "License");
you may not use this file except in compliance with the License.
You may obtain a copy of the License at
http://www.apache.org/licenses/LICENSE-2.0
Unless required by applicable law or agreed to in writing, software
distributed under the License is distributed on an "AS IS" BASIS,
WITHOUT WARRANTIES OR CONDITIONS OF ANY KIND, either express or implied.
See the License for the specific language governing permissions and
limitations under the License.
=head1 CONTACT
Xiaolei Zhang
Ensembl <http://www.ensembl.org/info/about/contact/index.html>
=cut
=head1 NAME
UTRAnnotator
=head1 SYNOPSIS
mv UTRAnnotator.pm ~/.vep/Plugins
vep -i variations.vcf --plugin UTRAnnotator,file=/path/to/uORF_starts_ends_GRCh38_PUBLIC.txt
# skip annotation for variants with a 80% or higher overlap of the UTR
vep -i variations.vcf --plugin UTRAnnotator,file=/path/to/uORF_starts_ends_GRCh38_PUBLIC.txt,max_overlap=80
=head1 DESCRIPTION
A VEP plugin that annotates the effect of 5' UTR variant especially for variant creating/disrupting upstream ORFs.
Available for both GRCh37 and GRCh38.
Options are passed to the plugin as key=value pairs:
file : (Required) Path to UTRAnnotator data file:
- Download 'uORF_5UTR_GRCh37_PUBLIC.txt' or 'uORF_5UTR_GRCh38_PUBLIC.txt'
from https://github.com/Ensembl/UTRannotator
- Download from http://sorfs.org
max_overlap : (Optional) Maximum percentage of overlap between variant and
UTR for UTR annotation (default: 100)
Citation
About the role of 5'UTR variants in human genetic disease:
Whiffin, N., Karczewski, K.J., Zhang, X. et al. Characterising the loss-of-function impact of 5’ untranslated region variants in 15,708 individuals. Nat Commun 11, 2523 (2020). https://doi.org/10.1038/s41467-019-10717-9
About UTRAnnotator:
The original UTRAnnotator plugin is written by Xiaolei Zhang et al. Later adopted by Ensembl VEP plugins with some changes.
You can find the original plugin here -
https://github.com/ImperialCardioGenetics/UTRannotator
Please cite the UTRannotator publication alongside the Ensembl VEP if you use this resource -
Annotating high-impact 5'untranslated region variants with the UTRannotator Zhang, X., Wakeling, M.N., Ware, J.S, Whiffin, N. Bioinformatics; doi: https://academic.oup.com/bioinformatics/advance-article/doi/10.1093/bioinformatics/btaa783/5905476
=cut
package UTRAnnotator;
use base qw(Bio::EnsEMBL::Variation::Utils::BaseVepPlugin);
use Bio::EnsEMBL::Utils::Sequence qw(reverse_comp);
use List::Util qw(min max);
use Scalar::Util qw(looks_like_number);
use strict;
sub feature_types {
return ['Transcript'];
}
sub variant_feature_types {
return ['VariationFeature', 'StructuralVariationFeature'];
}
sub new {
my $class = shift;
my $self = $class->SUPER::new(@_);
printf "\nWarning: no FASTA file specified (--fasta). VEP running will take a long time." if ($self->config->{fasta} eq "");
my $param_hash = $self->params_to_hash();
if (-e $param_hash->{file}){
open my $fh, "<", $param_hash->{file} or die $!;
my %uORF_evidence;
while (<$fh>) {
chomp;
my ($chr, $pos, $gene, $strand, $type, $stop_pos) = split /\t/;
next if $chr eq "chr" && $pos eq "pos";
die "$chr does not exist; Check your reference file." unless ($chr =~ m/^(chr*)/);
die "Position $pos is not a number; Check your reference file." unless looks_like_number($pos);
my $key = $chr . ":" . $pos; # chr has 'chr' prefix
$uORF_evidence{$key} = 1;
}
close $fh;
$self->{uORF_evidence} = \%uORF_evidence;
} else {
printf "Warning: small ORF file not found. For human, you could use the curated list of uORFs found in the repository (https://github.com/Ensembl/UTRannotator):\n" .
"'uORF_starts_ends_GRCh37_PUBLIC.txt' for GRCh37 or 'uORF_starts_ends_GRCh38_PUBLIC.txt' for GRCh38.\n";
}
# maximum percentage UTR overlap
$self->{max_overlap} = defined $param_hash->{max_overlap} ? $param_hash->{max_overlap} : 100;
# Kozak-Strength Class
my %kozak_strength;
$kozak_strength{1}='Weak';
$kozak_strength{2}='Moderate';
$kozak_strength{3}='Strong';
$self->{kozak_strength} = \%kozak_strength;
return $self;
}
sub get_header_info {
my $self->{_header_info} = {
'5UTR_consequence' => 'Variant consequence from UTRAnnotator',
'5UTR_annotation' => 'Variant annotation from UTRAnnotator',
Existing_uORFs => 'The number of existing uORFs with a stop codon within the 5 prime UTR',
Existing_OutOfFrame_oORFs => 'The number of existing out-of-frame overlapping ORFs (OutOfFrame oORF) at the 5 prime UTR',
Existing_InFrame_oORFs => 'The number of existing inFrame overlapping ORFs (inFrame oORF) at the 5 prime UTR',
};
return $self->{_header_info};
}
sub run {
my ($self, $tva) = @_;
#only annotate the effect if the variant is 5_prime_UTR_variant
return {} unless grep {$_->SO_term eq '5_prime_UTR_variant'} @{$tva->get_all_OverlapConsequences};
#retrieve the variant info
my $vf = $tva->can('variation_feature') ? $tva->variation_feature : $tva->structural_variation_feature;
my $chr = ($vf->{chr} || $vf->seq_region_name);
my $pos = ($vf->{start} || $vf->seq_region_start);
my $pos_end = ($vf->{end} || $vf->seq_region_end);
my @alleles = split /\//, $vf->allele_string;
my $ref = shift @alleles;
my $alt = $tva->can('variation_feature_seq') ? $tva->variation_feature_seq : '';
my %variant = (
"chr" => $chr,
"pos" => $pos,
"ref" => $ref,
"alt" => $alt,
);
#retrieve the UTR info: transcript id, strand, five prime UTR sequence, start and end genomic coordinates.
my $t = $tva->transcript;
my $transcript_id = (defined $t? $t->stable_id: undef);
#retrieve the gene symbol of the transcript
my $symbol = $t->{_gene_symbol} || $t->{_gene_hgnc};
#retrieve the strand of the transcript
my $tr_strand = $t->strand + 0;
my $cds = $t->translateable_seq();
#retrieve the five prime utr sequence
my $five_prime_seq = (defined $t->five_prime_utr? $t->five_prime_utr->seq(): undef);
#Type: five_prime_feature - Bio::EnsEMBL::Feature
my $UTRs = $t->get_all_five_prime_UTRs();
my @five_utr_starts;
my @five_utr_ends;
foreach my $utr (@$UTRs){
my $UTR_start = $utr->start(); # this will return the absolute starting positions in chromosome of the UTR exons
my $UTR_end = $utr->end(); # this will return the absolute ending positions in chromosome of the UTR exons
#avoid storing UTRs if variant goes over the maximum allowed UTR overlap percentage
my $UTR_length = $UTR_end - $UTR_start + 1;
my $UTR_overlap_start = max($UTR_start, $pos);
my $UTR_overlap_end = min($UTR_end, $pos_end);
my $UTR_overlap_perc = 100 * ($UTR_overlap_end - $UTR_overlap_start) / $UTR_length;
next if $UTR_overlap_perc >= $self->{max_overlap};
push(@five_utr_starts, $UTR_start);
push(@five_utr_ends, $UTR_end);
}
return {} unless (@five_utr_starts || @five_utr_ends);
my @sorted_starts = sort {$a <=> $b} @five_utr_starts;
my @sorted_ends = sort {$a <=> $b} @five_utr_ends;
my %UTR_info = (
"gene" => $symbol,
"start" => \@sorted_starts,
"end" => \@sorted_ends,
"seq" => $five_prime_seq, #the exon sequences in 5'UTR
"strand" => $tr_strand,
"cds_seq" => $cds,
);
$self->{ref_coding} = $self->get_ref_coding($ref);
$self->{alt_coding} = $self->get_alt_coding($alt,$tr_strand);
my %uAUG_gained = {};
my %uSTOP_lost = {};
my %uAUG_lost = {};
my %uSTOP_gained = {};
my %uFrameshift = {};
my ($mut_pos, $end_pos) = $self->get_allele_exon_pos($tr_strand, $pos, $self->{ref_coding}, \%UTR_info);
if(defined($mut_pos) && defined($end_pos)) {
my @sequence = split //, $five_prime_seq;
my $mut_utr_seq = $self->mut_utr_sequence(
\@sequence,$mut_pos,
$self->{ref_coding},
$self->{alt_coding},
$tr_strand
);
my @utr_sequence = split //, $UTR_info{seq};
my %existing_uORF = %{$self->existing_uORF(\@utr_sequence)};
my @mut_utr_seq = split //,$mut_utr_seq;
my %existing_utr_uORF = %{$self->existing_uORF(\@mut_utr_seq)};
my @start = @{$self->get_ATG_pos(\@utr_sequence)};
%uAUG_gained = %{$self->uAUG_gained(\%variant,\%UTR_info, $mut_pos, $mut_utr_seq, \%existing_utr_uORF)};
%uSTOP_lost = %{$self->uSTOP_lost(\%variant,\%UTR_info, $mut_pos, $mut_utr_seq, \%existing_uORF, \%existing_utr_uORF)};
%uAUG_lost = %{$self->uAUG_lost(\%variant,\%UTR_info, $mut_pos, $mut_utr_seq, \%existing_uORF, \@start)};
%uSTOP_gained = %{$self->uSTOP_gained(\%variant,\%UTR_info, $mut_pos, $end_pos, $mut_utr_seq, \%existing_uORF, \%existing_utr_uORF, \@start)};
%uFrameshift = %{$self->uFrameshift(\%variant,\%UTR_info, $mut_pos, $end_pos, $mut_utr_seq, \%existing_uORF, \%existing_utr_uORF, \@start)};
}
my %five_prime_flag = (
"uAUG_gained" => $uAUG_gained{'uAUG_gained_flag'},
"uSTOP_lost" => $uSTOP_lost{'uSTOP_lost_flag'},
"uAUG_lost" => $uAUG_lost{'uAUG_lost_flag'},
"uSTOP_gained" => $uSTOP_gained{'uSTOP_gained_flag'},
"uFrameshift" =>$uFrameshift{'uFrameShift_flag'},
);
my %five_prime_annotation = (
"uAUG_gained" => $uAUG_gained{"uAUG_gained_effect"},
"uSTOP_lost" => $uSTOP_lost{"uSTOP_lost_effect"},
"uAUG_lost" => $uAUG_lost{"uAUG_lost_effect"},
"uSTOP_gained" => $uSTOP_gained{'uSTOP_gained_effect'},
"uFrameshift" =>$uFrameshift{'uFrameShift_effect'},
);
my $output_five_prime_flag;
my $output_five_prime_annotation;
foreach my $flag (keys %five_prime_flag){
if($five_prime_flag{$flag}){
$output_five_prime_flag = $five_prime_flag{$flag};
$output_five_prime_annotation = $five_prime_annotation{$flag};
}
};
my %utr_effect;
if ($self->{config}->{output_format} eq "json" || $self->{config}->{rest}){
%utr_effect = (
"5UTR_consequence" => $output_five_prime_flag,
"5UTR_annotation" => $output_five_prime_annotation,
);
} else {
my @consequences;
foreach my $value (keys %{$output_five_prime_annotation}){
push @consequences, join(":", map { "$_=$output_five_prime_annotation->{$value}{$_}" } keys %{$output_five_prime_annotation->{$value}});
};
my $consequence;
$consequence = join(",", @consequences);
%utr_effect = (
"5UTR_consequence" => defined($output_five_prime_flag) ? $output_five_prime_flag : "-",
"5UTR_annotation" => defined($consequence) && $consequence ne '' ? $consequence : "-",
);
}
my $existing_uORF_num = $self->count_number_ATG($five_prime_seq);
my $output ={%utr_effect, %$existing_uORF_num};
return $output? $output: {};
}
##################
# Main functions #
##################
sub uAUG_gained {
# Description: annotate if a five_prime_UTR_variant creates ATG
my ($self, $variant_info,$UTR_info, $mut_pos, $mut_utr_seq, $existing_utr_uorf) = @_;
my $pos = $variant_info->{pos};
my $ref = $variant_info->{ref};
my $alt = $variant_info->{alt};
my @sequence = split //, $UTR_info->{seq};
my $strand = $UTR_info->{strand};
#return annotators
my $uAUG_gained_DistanceToCDS = ""; # the distance between the gained uAUG to CDS
my $uAUG_gained_KozakContext = ""; # the Kozak context sequence of the gained uAUG
my $uAUG_gained_KozakStrength = ""; # the Kozak strength of the gained uAUG
my $uAUG_gained_type = ""; # the type of uORF created - any of the following: uORF, inframe_oORF,OutOfFrame_oORF
my $uAUG_gained_DistanceFromCap = ""; # the distance between the gained uAUG to the start of the five prime UTR
my $uAUG_gained_DistanceToStop = ""; #the distance between the gained uAUG to stop codon (could be in CDS)
#indicate whether the variant creates a ATG
my $flag = 0;
my $current_kozak = "";
my $current_kozak_strength ="";
#the relative position of input variant in the UTR sequence
my %result = (); #result is a hash table with two elements: $flag and $output_effects
my $output_flag = "";
my %output_effects;
my $ref_coding = $self->{ref_coding};
my $alt_coding = $self->{ref_coding};
my @mut_utr_seq = split //,$mut_utr_seq;
my $mut_utr_length = @mut_utr_seq;
#get the nt sequence that might have the pos_A for a new ATG codon
my @mut_seq = @mut_utr_seq[$mut_pos-2..$mut_pos+length($alt_coding)+1];
my @mut_atg_pos = @{$self->get_ATG_pos(\@mut_seq)};
# check whether there is a ATG codon is to check the length of the array
#if there is a ATG codon, flag it and output the relative positive of A
my $pos_A;
if (@mut_atg_pos){
#get the pos of A (in ATG) in the UTR sequence
$pos_A=$mut_pos-2+$mut_atg_pos[0];
$flag=1;
}
if ($flag){
################################################################################
#annotator 1: get the distance to the start codon of the main ORF
################################################################################
$uAUG_gained_DistanceToCDS = $mut_utr_length-$pos_A;
$uAUG_gained_DistanceFromCap = $pos_A;
################################################################################
#annotator 2: determine kozak context;
################################################################################
if ((($pos_A-3)>=0)&&($mut_utr_seq[($pos_A+3)])){
$current_kozak = $mut_utr_seq[($pos_A-3)].$mut_utr_seq[($pos_A-2)].$mut_utr_seq[$pos_A-1]."ATG".$mut_utr_seq[$pos_A+3];
}
else{
$current_kozak = '-';
}
#get the strength of kozak context
if ($current_kozak !~ /-/){
my @split_kozak = split //, $current_kozak;
$current_kozak_strength = 1;
if ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))&&($split_kozak[6] eq 'G')){
$current_kozak_strength = 3;
}
elsif ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))||($split_kozak[6] eq 'G')){
$current_kozak_strength = 2;
}
}
$uAUG_gained_KozakContext=$current_kozak;
$uAUG_gained_KozakStrength=$self->{kozak_strength}{$current_kozak_strength}? $self->{kozak_strength}{$current_kozak_strength}:$current_kozak_strength;
################################################################################
#annotator 3: Type of new ORF with respect the main ORF: uORF/Overlapping_inFrame/Overlapping_outofframe
################################################################################
if(exists($existing_utr_uorf->{$pos_A})){ #if there is stop codon within 5'UTR
$uAUG_gained_type = "uORF";
}
else{
if(($mut_utr_length - $pos_A) % 3){
$uAUG_gained_type = "OutOfFrame_oORF";
}
else{
$uAUG_gained_type = "inFrame_oORF";
}
}
my @overlapping_seq = split //, $mut_utr_seq.$UTR_info->{cds_seq};
my %existing_uORF = %{$self->existing_uORF(\@overlapping_seq)};
if(exists($existing_uORF{$pos_A})){
my @stop_pos_array = sort{$a<=>$b}@{$existing_uORF{$pos_A}};
my $stop_pos = $stop_pos_array[0];
$uAUG_gained_DistanceToStop = $stop_pos-$pos_A;
} else {
$uAUG_gained_DistanceToStop = "NA";
}
my %uORF_effect = (
"KozakContext" => $uAUG_gained_KozakContext,
"KozakStrength" => $uAUG_gained_KozakStrength,
"DistanceToCDS" => $uAUG_gained_DistanceToCDS,
"type" => $uAUG_gained_type,
"DistanceToStop" => $uAUG_gained_DistanceToStop,
"CapDistanceToStart" => $uAUG_gained_DistanceFromCap,
);
$output_flag = "5_prime_UTR_premature_start_codon_gain_variant";
my $size = (keys %output_effects) + 1;
$output_effects{$size} = \%uORF_effect;
}
$result{'uAUG_gained_flag'} = $output_flag;
$result{'uAUG_gained_effect'} = \%output_effects;
return \%result;
}
sub uSTOP_gained {
# Description: annotate whether a five_prime_UTR_variant creates new stop codon. It only evaluate SNVs.
my ($self, $variant_info,$UTR_info, $mut_pos, $end_pos, $mut_utr_seq, $existing_ref_uORF, $mut_uORF, $start) = @_;
my $chr = $variant_info->{chr};
my $pos = $variant_info->{pos};
my $ref = $variant_info->{ref};
my $alt = $variant_info->{alt};
my @sequence = split //, $UTR_info->{seq};
my $strand = $UTR_info->{strand};
my $utr_length = @sequence;
#return annotators
my $uSTOP_gained_ref_StartDistanceToCDS = ""; # the distance between the uAUG of the disrupting uORF to CDS
my $uSTOP_gained_KozakContext = ""; # the Kozak context sequence of the disrupting uORF
my $uSTOP_gained_KozakStrength = ""; # the Kozak strength of the the disrupting uORF
my $uSTOP_gained_ref_type = ""; # the type of uORF being disrupted - any of the following: uORF, inframe_oORF,OutOfFrame_oORF
my $uSTOP_gained_newSTOPDistanceToCDS = ""; # the distance between the gained uSTOP to the start of the CDS
my $uSTOP_gained_evidence = ""; # the translation evidence of the disrupted uORF
#indicate whether the variant creates a stop codon
my $flag = 0;
my $current_kozak = "";
my $current_kozak_strength ="";
#the relative position of input variant in the UTR sequence
my %result = (); #result is a hash table with two elements: $flag and $output_effects
my $output_flag = 0;
my %output_effects;
my $ref_coding = $self->{ref_coding};
my $alt_coding = $self->{alt_coding};
#only evaluate SNVs and MNVs, for deletion and insertion it would be evaluated in the uframeshift
return{} unless(length($ref_coding) eq length($alt_coding));
# the length of the 5'UTR won't change so as the coordinates of the nts
my @mut_utr_seq = split //,$mut_utr_seq;
my $mut_utr_length = @mut_utr_seq;
my @start = @{$start};
if(@start){
for (my $i=0;$i<@start;$i++){
$flag=0;
my $start_pos = $start[$i];
# to set the range for searching new STOP codon: not including start_codon, (1) for uORFs - start_codon...stop_codon (2) for oORFs - start_codon...end of 5'UTR
my $check_point = $start_pos;
# the checking end point of eligible area
#For uORF: start_pos .. check_point
#For overlapping ORF: start_pos .. 3' end of 5'UTR sequence
#if the variant is entirely in the uORF (within 5'UTR
if(exists($existing_ref_uORF->{$start_pos})) {
my @stops = sort {$a <=> $b} @{$existing_ref_uORF->{$start_pos}};
$check_point = $stops[0]-1;
} else{
#if the existing uORF is an oORF
$check_point = $utr_length-1;
}
# only check the ones between start and stop codons
# ignore the cases at the boundary of start and stop codon since the effect on start/stop would be evaluated anyway
next if(($mut_pos<$start_pos+3)|($end_pos>$check_point));
#check whether there are new stop codon induced by this mutation
my @mut_stops;
next unless(exists($mut_uORF->{$start_pos}));
@mut_stops = sort {$a <=> $b} @{$mut_uORF->{$start_pos}};
my $mut_stop = $mut_stops[0];
if($mut_stop<$check_point){
$flag=1;
}
if($flag){
#getting the Kozak context and Kozak strength of the start codon
if ((($start_pos-3)>=0)&&($sequence[($start_pos+3)])){
$current_kozak = $sequence[($start_pos-3)].$sequence[($start_pos-2)].$sequence[$start_pos-1]."ATG".$sequence[$start_pos+3];
}
else{
$current_kozak = '-';
}
if ($current_kozak !~ /-/){
my @split_kozak = split //, $current_kozak;
$current_kozak_strength = 1;
if ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))&&($split_kozak[6] eq 'G')){
$current_kozak_strength = 3;
}
elsif ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))||($split_kozak[6] eq 'G')){
$current_kozak_strength = 2;
}
}
$uSTOP_gained_KozakContext=$current_kozak;
$uSTOP_gained_KozakStrength=$self->{kozak_strength}{$current_kozak_strength}? $self->{kozak_strength}{$current_kozak_strength}:$current_kozak_strength;
#the annotation of the original uORF
if (exists($existing_ref_uORF->{$start_pos})){ #if there is stop codon within 5'UTR
$uSTOP_gained_ref_type = "uORF"
} elsif (($utr_length-$start_pos) % 3){
$uSTOP_gained_ref_type = "OutOfFrame_oORF";
} else {
$uSTOP_gained_ref_type = "InFrame_oORF";
}
$uSTOP_gained_ref_StartDistanceToCDS = $utr_length - $start_pos;
$uSTOP_gained_newSTOPDistanceToCDS = $mut_utr_length - $mut_stop;
#find evidence in added reference file
$uSTOP_gained_evidence = (exists $self->{uORF_evidence})? $self->find_uorf_evidence($UTR_info,$chr,$start_pos): "NA";
my %uORF_effect = (
"ref_type" => $uSTOP_gained_ref_type,
"ref_StartDistanceToCDS" => $uSTOP_gained_ref_StartDistanceToCDS,
"newSTOPDistanceToCDS" => $uSTOP_gained_newSTOPDistanceToCDS,
"KozakContext" => $uSTOP_gained_KozakContext,
"KozakStrength" => $uSTOP_gained_KozakStrength,
"Evidence" => $uSTOP_gained_evidence,
);
$output_flag = "5_prime_UTR_uORF_stop_codon_gain_variant";
my $size = (keys %output_effects) + 1;
$output_effects{$size} = \%uORF_effect;
}
}
}
$result{'uSTOP_gained_flag'} = $output_flag;
$result{'uSTOP_gained_effect'} = \%output_effects;
return \%result;
}
sub uSTOP_lost {
# Description: annotate if a five_prime_UTR_varint removes a stop codon of an existing uORF (given that uORF does not not change)
my ($self, $variant_info, $UTR_info, $mut_pos, $mut_utr_seq, $existing_uORF, $mut_uORF) = @_;
my $chr = $variant_info->{chr};
my $pos = $variant_info->{pos};
my $ref = $variant_info->{ref};
my $alt = $variant_info->{alt};
my @sequence = split //, $UTR_info->{seq};
my $strand = $UTR_info->{strand};
#return annotators
my $uSTOP_lost_AltStop = ""; #whether there is an alternative stop codon downstream
my $uSTOP_lost_AltStopDistanceToCDS = ""; # the distance between alternative stop codon and the CDS
my $uSTOP_lost_FrameWithCDS = ""; # the frame of the uORF with respect to CDS
my $uSTOP_lost_KozakContext = ""; # the Kozak context sequence of the uORF with the lost stop codon
my $uSTOP_lost_KozakStrength = ""; # the Kozak strength of the uORF with the lost stop codon
my $uSTOP_lost_evidence = ""; # whether this uORF has any evidence of being translated
###
my $current_kozak = "";
my $current_kozak_strength = "";
#indicate whether the variant ever disrupts a stop codon
my $output_flag = "";
#indicate whether the variant ever disrupts the uORF that being analyzed
my $flag_uORF=0;
#the relative position of input variant in the UTR sequence
my %result = (); #result is a hash table with two elements: $flag and $output_effects
my %output_effects;
my @stop_codons = ("TAA","TGA","TAG");
my $ref_coding = $self->{ref_coding};
my $alt_coding = $self->{alt_coding};
my @mut_utr_seq = split //,$mut_utr_seq;
my $length = @mut_utr_seq;
my @start = sort {$a <=> $b} (keys %{$existing_uORF});
if(%{$existing_uORF}){
for (my $i=0;$i<@start;$i++){
$flag_uORF=0;
my $start_pos = $start[$i];
my @stops = sort {$a <=> $b} @{$existing_uORF->{$start_pos}};
my $stop_pos=$stops[0];
next if ($mut_pos-$stop_pos>2);
#for snps and deletion
next if (defined($ref_coding) & $mut_pos+length($ref_coding)-1<$stop_pos);
next if ($mut_pos<$stop_pos & !defined($ref_coding)); #for insertion
#for deletion, it definitely disrupting the stop codon.
if (length($alt_coding) eq 0) {
$flag_uORF=1;
} else {
my $mut_codon = $mut_utr_seq[$stop_pos].$mut_utr_seq[$stop_pos+1].$mut_utr_seq[$stop_pos+2];
next if (grep( /^$mut_codon$/, @stop_codons));
$flag_uORF=1;
}
if($flag_uORF){
#getting the Kozak context and Kozak strength of the start codon
if ((($start_pos-3)>=0)&&($sequence[($start_pos+3)])){
$current_kozak = $sequence[($start_pos-3)].$sequence[($start_pos-2)].$sequence[$start_pos-1]."ATG".$sequence[$start_pos+3];
}
else{
$current_kozak = '-';
}
if ($current_kozak !~ /-/){
my @split_kozak = split //, $current_kozak;
$current_kozak_strength = 1;
if ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))&&($split_kozak[6] eq 'G')){
$current_kozak_strength = 3;
}
elsif ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))||($split_kozak[6] eq 'G')){
$current_kozak_strength = 2;
}
}
$uSTOP_lost_KozakContext=$current_kozak;
$uSTOP_lost_KozakStrength=$self->{kozak_strength}{$current_kozak_strength}? $self->{kozak_strength}{$current_kozak_strength}:$current_kozak_strength;
# the sequence before mut_pos should not be changed. Thus start_pos shall still correspond to a start codon;
# if the sequence is indeed very short as such ATGTGA
my @mut_stops;
if(exists($mut_uORF->{$start_pos})){
@mut_stops = sort {$a <=> $b} @{$mut_uORF->{$start_pos}}
}
if (@mut_stops>0){
$uSTOP_lost_AltStop = "True";
$uSTOP_lost_AltStopDistanceToCDS = $length-$mut_stops[0];
} #if there is no alternative stop codon
else{
$uSTOP_lost_AltStop = "False";
$uSTOP_lost_AltStopDistanceToCDS = "NA";
}
if (($length-$start_pos) % 3){
$uSTOP_lost_FrameWithCDS = "outOfFrame";
}
else {
$uSTOP_lost_FrameWithCDS = "inFrame";
}
#find evidence from sorf
$uSTOP_lost_evidence= (exists $self->{uORF_evidence})? $self->find_uorf_evidence($UTR_info,$chr,$start_pos): "NA";
my %uORF_effect = (
"AltStop" => $uSTOP_lost_AltStop,
"AltStopDistanceToCDS" => $uSTOP_lost_AltStopDistanceToCDS,
"FrameWithCDS" => $uSTOP_lost_FrameWithCDS,
"KozakContext" => $uSTOP_lost_KozakContext,
"KozakStrength" => $uSTOP_lost_KozakStrength,
"Evidence" => $uSTOP_lost_evidence,
);
$output_flag = "5_prime_UTR_uORF_stop_codon_loss_variant";
my $size = (keys %output_effects) + 1;
$output_effects{$size} = \%uORF_effect;
}
}
}
$result{'uSTOP_lost_flag'} = $output_flag;
$result{'uSTOP_lost_effect'} = \%output_effects;
return \%result;
}
sub uAUG_lost {
# Description: annotate if a five_prime_UTR_varint removes a start codon of an existing uORF
my ($self, $variant_info, $UTR_info, $mut_pos, $mut_utr_seq, $existing_ref_uORF, $start) = @_;
my $chr = $variant_info->{chr};
my $pos = $variant_info->{pos};
my $ref = $variant_info->{ref};
my $alt = $variant_info->{alt};
my @sorted_starts = @{$UTR_info->{start}};
my @sequence = split //, $UTR_info->{seq};
my $utr_length = @sequence;
my $strand = $UTR_info->{strand};
#return annotators
my $uAUG_lost_type = ""; # the uORF type with the reference allele - uORF, inframe_oORF, outOfFrame_oORF
my $uAUG_lost_DistanceToCDS = ""; # the distance of the lost uAUG to CDS
my $uAUG_lost_DistanceToSTOP = ""; # the distance of the lost uAUG to stop codon (could be in CDS)
my $uAUG_lost_KozakContext = ""; # the Kozak context sequence of the lost uAUG
my $uAUG_lost_KozakStrength = ""; # the strength of KozakContext of the lost uAUG
my $uAUG_lost_evidence = ""; # whether this uAUG has any evidence of being translated
my $current_kozak = "";
my $current_kozak_strength = "";
#indicate whether the variant ever disrupts a stop codon
my $output_flag = "";
#indicate whether the variant ever disrupts the uORF that being analyzed
my $flag_uORF=0;
#the relative position of input variant in the UTR sequence
my %result = (); #result is a hash table with two elements: $flag and $output_effects
my %output_effects;
my $ref_coding = $self->{ref_coding};
my $alt_coding = $self->{alt_coding};
my @mut_utr_seq = split //,$mut_utr_seq;
my @start = @{$start};
for (my $i=0;$i<@start;$i++){
$flag_uORF=0;
my $start_pos = $start[$i];
next if ($mut_pos-$start_pos>2);
if (length($ref_coding) ne 0){
next if ($mut_pos+length($ref_coding)-1<$start_pos);
}
next if ($mut_pos<$start_pos); #for insertion
#for deletion, it definitely disrupting the uORF.
if (length($alt_coding) eq 0) {
$flag_uORF=1;
} else {
my $mut_codon = $mut_utr_seq[$start_pos].$mut_utr_seq[$start_pos+1].$mut_utr_seq[$start_pos+2];
next if ($mut_codon eq "ATG");
$flag_uORF=1;
}
if($flag_uORF){
#getting the Kozak context and Kozak strength of the lost start codon
if ((($start_pos-3)>=0)&&($sequence[($start_pos+3)])){
$current_kozak = $sequence[($start_pos-3)].$sequence[($start_pos-2)].$sequence[$start_pos-1]."ATG".$sequence[$start_pos+3];
} else {
$current_kozak = '-';
}
if ($current_kozak !~ /-/){
my @split_kozak = split //, $current_kozak;
$current_kozak_strength = 1;
if ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))&&($split_kozak[6] eq 'G')){
$current_kozak_strength = 3;
} elsif ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))||($split_kozak[6] eq 'G')){
$current_kozak_strength = 2;
}
}
$uAUG_lost_KozakContext=$current_kozak;
$uAUG_lost_KozakStrength=$self->{kozak_strength}{$current_kozak_strength}? $self->{kozak_strength}{$current_kozak_strength}:$current_kozak_strength;
#check what kind of uORF does that correspond to?
#first check whether it's overlapping with CDS
if (exists($existing_ref_uORF->{$start_pos})){ #if there is stop codon within 5'UTR
$uAUG_lost_type = "uORF"
} elsif(($utr_length-$start_pos) % 3){
$uAUG_lost_type = "OutOfFrame_oORF";
} else {
$uAUG_lost_type = "InFrame_oORF";
}
$uAUG_lost_DistanceToCDS = $utr_length - $start_pos;
my @overlapping_seq = split //, $UTR_info->{seq}.$UTR_info->{cds_seq};
my %existing_overlapping_uORF = %{$self->existing_uORF(\@overlapping_seq)};
if(exists($existing_overlapping_uORF{$start_pos})){
my @stop_pos_array = sort{$a<=>$b}@{$existing_overlapping_uORF{$start_pos}};
my $stop_pos = $stop_pos_array[0];
$uAUG_lost_DistanceToSTOP = $stop_pos-$start_pos;
} else {
$uAUG_lost_DistanceToSTOP = "NA"
}
$uAUG_lost_evidence= (exists $self->{uORF_evidence})? $self->find_uorf_evidence($UTR_info,$chr,$start_pos): "NA";
my %uORF_effect = (
"type" => $uAUG_lost_type,
"CapDistanceToStart" =>$start_pos,
"DistanceToCDS" => $uAUG_lost_DistanceToCDS,
"DistanceToStop" => $uAUG_lost_DistanceToSTOP,
"KozakContext" => $uAUG_lost_KozakContext,
"KozakStrength" => $uAUG_lost_KozakStrength,
"Evidence" => $uAUG_lost_evidence,
);
$output_flag = "5_prime_UTR_premature_start_codon_loss_variant";
my $size = (keys %output_effects) + 1;
$output_effects{$size} = \%uORF_effect;
}
}
$result{'uAUG_lost_flag'} = $output_flag;
$result{'uAUG_lost_effect'} = \%output_effects;
return \%result;
}
sub uFrameshift {
# Description: annotate if a five_prime_UTR_varint create a frameshift in existing uORFs
my ($self, $variant_info, $UTR_info, $mut_pos, $end_pos, $mut_utr_seq, $existing_uORF, $mut_uORF, $start) = @_;
my $chr = $variant_info->{chr};
my $pos = $variant_info->{pos};
my $ref = $variant_info->{ref};
my $alt = $variant_info->{alt};
my @sequence = split //, $UTR_info->{seq};
my $strand = $UTR_info->{strand};
my $utr_length = @sequence;
#return annotators
my $uFrameshift_ref_type = ""; # the type of uORF with the reference allele
my $uFrameshift_ref_type_length = ""; # the length of the uORF with of the reference allele.
my $uFrameshift_StartDistanceToCDS = ""; # the distance between the start codon of the disrupted uORF and CDS
my $uFrameshift_alt_type = ""; # the type of uORF with the alternative allele
my $uFrameshift_alt_type_length = ""; # the length of the uORF with of the alternative allele
my $uFrameshift_KozakContext = ""; # the Kozak context sequence of the disrupted uORF
my $uFrameshift_KozakStrength = ""; # the Kozak strength of the disrupted uORF
my $uFrameshift_evidence = ""; # whehter there is translation evidence for the disrupted uORF
#indicate whether the variant ever introduce a frameshift variant
my $flag_uORF;
my $output_flag = "";
my $current_kozak="";
my $current_kozak_strength="";
my %result = (); #result is a hash table with two elements: $flag and $output_effects
my %output_effects;
my $ref_coding = $self->{ref_coding};
my $alt_coding = $self->{alt_coding};
#skip alleles with same length
return {} unless(length($ref_coding) ne length($alt_coding));
#if it's a deletion at the boundary of exon and intron, we would skip the annotation
my @mut_utr_seq = split //,$mut_utr_seq;
my $length = @mut_utr_seq;
my @start = @{$start};
#check for each uORF
if(@start){
for (my $i=0;$i<@start;$i++){
$flag_uORF=0;
my $start_pos = $start[$i];
my $check_point = $start_pos;
# the checking end point of eligible area
#For uORF: start_pos .. check_point
#For overlapping ORF: start_pos .. 3' end of 5'UTR sequence
#if the variant is entirely in the uORF (within 5'UTR)
if(exists($existing_uORF->{$start_pos})) {
my @stops = sort {$a <=> $b} @{$existing_uORF->{$start_pos}};
$check_point = $stops[0]-1;
} else {
#if the existing uORF is an oORF
$check_point = $utr_length-1;
}
# only check the ones between start and stop codons and
# ignore the cases at the boundary of start and stop codon since the effect on start/stop would be evaluated anyway
if(($mut_pos>=$start_pos+3)&($end_pos<=$check_point)){
#snp and insertion could only be annotated here
$flag_uORF = abs(length($ref_coding)-length($alt_coding))%3;
}
if($flag_uORF){
#getting the Kozak context and Kozak strength of the start codon
if ((($start_pos-3)>=0)&&($sequence[($start_pos+3)])){
$current_kozak = $sequence[($start_pos-3)].$sequence[($start_pos-2)].$sequence[$start_pos-1]."ATG".$sequence[$start_pos+3];
}
else{
$current_kozak = '-';
}
if ($current_kozak !~ /-/){
my @split_kozak = split //, $current_kozak;
$current_kozak_strength = 1;
if ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))&&($split_kozak[6] eq 'G')){
$current_kozak_strength = 3;
}
elsif ((($split_kozak[0] eq 'A')||($split_kozak[0] eq 'G'))||($split_kozak[6] eq 'G')){
$current_kozak_strength = 2;
}
}
$uFrameshift_KozakContext=$current_kozak;
$uFrameshift_KozakStrength=$self->{kozak_strength}{$current_kozak_strength}? $self->{kozak_strength}{$current_kozak_strength}:$current_kozak_strength;
#the annotation of the original uORF
my @ref_overlapping_seq = split //, $UTR_info->{seq}.$UTR_info->{cds_seq};
my %ref_existing_oORF = %{$self->existing_uORF(\@ref_overlapping_seq)};
if (exists($existing_uORF->{$start_pos})){ #if there is stop codon within 5'UTR
$uFrameshift_ref_type = "uORF";
} elsif (($utr_length-$start_pos) % 3) {
$uFrameshift_ref_type = "OutOfFrame_oORF";
} else{
$uFrameshift_ref_type = "InFrame_oORF";
}
if (exists($ref_existing_oORF{$start_pos})){
my @stops = sort {$a <=> $b} @{$ref_existing_oORF{$start_pos}};
$uFrameshift_ref_type_length = $stops[0]-$start_pos+3;
} else {
$uFrameshift_ref_type_length = "NA";
}
$uFrameshift_StartDistanceToCDS = $utr_length - $start_pos;
my @alt_overlapping_seq = split //, $mut_utr_seq.$UTR_info->{cds_seq};
my %alt_existing_oORF = %{$self->existing_uORF(\@alt_overlapping_seq)};
if (exists($mut_uORF->{$start_pos})){
$uFrameshift_alt_type = "uORF";
} elsif(($length-$start_pos)%3){
$uFrameshift_alt_type = "OutOfFrame_oORF";
} else {
$uFrameshift_alt_type = "InFrame_oORF";
}
#get the length
if (exists($alt_existing_oORF{$start_pos})){
my @stops = sort {$a <=> $b} @{$alt_existing_oORF{$start_pos}};
$uFrameshift_alt_type_length = $stops[0]-$start_pos+3;
} else {
$uFrameshift_alt_type_length = "NA";
}
#find evidence in added reference file
$uFrameshift_evidence= (exists $self->{uORF_evidence})? $self->find_uorf_evidence($UTR_info,$chr,$start_pos): "NA";
my %uORF_effect = (
"ref_type" => $uFrameshift_ref_type,
"ref_type_length" => $uFrameshift_ref_type_length,
"ref_StartDistanceToCDS" => $uFrameshift_StartDistanceToCDS,
"alt_type" => $uFrameshift_alt_type,
"alt_type_length" => $uFrameshift_alt_type_length,
"KozakContext" => $uFrameshift_KozakContext,
"KozakStrength" => $uFrameshift_KozakStrength,
"Evidence" => $uFrameshift_evidence,