package NGS::VEP;
# wrapper around Ensembl Variant Effect Predictor stand-alone script;
# http://www.ensembl.org/info/docs/variation/vep/vep_script.html
use Moo;
use IO::All;
use IO::File;
use Text::CSV;
use Data::Dumper;
use FindBin qw($Bin); # warn $Bin;
use MooX::Types::MooseLike::Base qw(:all);
use NGS::MooX::Types qw(HashReference);
use NGS::DB;
# required to be passed on object contruction:
has src_data => ( is => 'ro', isa => Str, required => 1 );
has form_opts => ( is => 'ro', isa => HashReference, required => 1 );
# called internally by result():
has ref_table => ( is => 'lazy' ); # _build_ref_table()
my %text_csv_args = ( sep_char => "\t", binary => 1 ); # global args for Text::CSV
# location of variable_effect_predictor script:
my $vep_script = "$Bin/../script/variant_effect_predictor.pl";
#===============================================================================
sub result {
my $self = shift; # warn Dumper $self->database;
my $data = $self->_text_csv_slurp(); # warn Dumper $data; # arrayref
# remove 1st row (headers):
my $header_row = shift @$data; # warn Dumper $header_row;
# get hmds refs - in cols starting at col #6:
my $hmds_refs = _get_hmds_refs($header_row);
# get reference table (chromosome start & end points for sequences):
my $ref_table = $self->ref_table; # warn Dumper $ref_table;
# create vep file for variant_effect_predictor.pl
my $vep_file = "$Bin/../results.vep";
io($vep_file)->unlink if -e ($vep_file); # clear previous
my @vep_fields = qw(chromosome start_point end_point allele strand exon);
my @vep_input; # will hold pre-processed data = VEP script input
my %vep_data; # will hold ref data for merging with VEP script output
my %hmds_ref; # will hold read data (% positive and read number) for samples
my $i; # will hold total count of 'accepted' rows
ROW: for my $row (@$data) { # cols = exon name, base/variant, status, ....
next ROW unless lc $row->[2] eq 'accepted'; # 3rd col is status field
$i++; # increment 'accepted' row counter
my $exon = $row->[0]; # warn $exon;
# get data from reference table, or skip record:
my $ref = $ref_table->{$exon}; # warn Dumper $ref;
if (! $ref) {
warn "no ref table entry for $exon"; next ROW;
}
my $chromosome = $ref->{chromosome};
# split 2nd col to get location & variant (eg 136:A/T, 26-27:TG/--)
my ($location, $allele) = split ':', $row->[1]; # warn Dumper [$location, $allele];
# define initial start & end point as ref table start position minus 1:
my $start_point = my $end_point = $ref->{start_base} - 1; # eg 125436276
# if location > 1 base (eg 26-27, 134-137, etc):
if ( $location =~ /(\d+)-(\d+)/ ) { # warn $location; # eg 26-27
my $span = $2 - $1; # eg 26-27 will give span = 1
$start_point += $1; # ref tbl start pos + 1st capture (26 in above example)
$end_point = $start_point + $span;
}
else {
$end_point = $start_point += $location;
}
{ # manipulate exon name to provide unique ID for vep input
# eg TET2 exon 4 [891], TET2 exon 4 [1031], etc:
$exon =~ s/\s/%%/g; # vep can't deal with spaces in any col
$exon .= '%%[' . $location . ']'; # exon + location gives unique ID
}
{ # data structure for vep script input file:
my %h = (
chromosome => $chromosome,
start_point => $start_point,
end_point => $end_point,
allele => $allele, # eg T/C, A/G, etc
strand => '+', # required 5th col in VEP file
exon => $exon, # unique identifier eg "TET2 exon 4 [891]"
); # warn Dumper \%h;
push @vep_input, \%h;
{ # data for use with vep output (use chr:start_location as unique ID):
# vep returns eg 2:12345 or 2:12345-12347 in 'Location' col
my $base_num = ( $start_point == $end_point )
? $start_point # eg 2:12345
: $start_point .'-'. $end_point; # eg 2:12345-12347
my $unique_id = join ':', $chromosome, $base_num;
$vep_data{$unique_id} = \%h;
# get patient results in cols 6 onwards:
my $n = 0; # reset counter to 0
for my $ref ( @$hmds_refs ) { # warn $ref;
# result in 1st col of pair, read number in 2nd:
my $result = $row->[5 + $n];
if ( $result > 0 ) { # skip zero's
my $read_no = $row->[5 + $n + 1]; # next col
# add to hmds ref
$hmds_ref{$ref}{$unique_id} = {
result => $result,
read => $read_no,
};
}
$n += 2; # move 2 cols along
}
}
}
} # warn Dumper \%vep_data; # warn Dumper \%hmds_ref;
# sort data into chromosome order to (possibly) speed up vep processing:
for ( sort by_chr_location @vep_input ) {
my $line = join "\t", @{$_}{@vep_fields};
io($vep_file)->append($line . "\n");
}
{ # send to function running VEP script:
my %data = ( vep_data => \%vep_data, sample_data => \%hmds_ref );
my $result = $self->_run_vep(\%data); # hashref of data & orphaned rows
$result->{row_count} = $i; # add row count of accepted rows
return $result;
}
}
#-------------------------------------------------------------------------------
sub _run_vep {
my ($self, $data) = @_;
my $src_data = $data->{vep_data}; # "chr:start" => (start_point, end_point, allele, etc)
my $hmds_ref = $data->{sample_data}; # hmds_ref => { unique_id => { read results } }
my @results; # to hold tab-delimited results array from vep output
# load opts from vep.ini + user-selected options to augment:
my @opts = ( "-config=$Bin/../script/vep.ini" );
my $user_opts = $self->_get_form_options;
push @opts, @$user_opts;
# open pipe to vep script (input = $vep_file; output = stdout):
open my $fh, "-|", $vep_script, @opts;
while ( my $line = readline($fh) ) { # warn $line;
push @results, $line;
} # warn Dumper \@results;
my @input_fields = qw(exon chromosome start_point end_point allele);
my @data; # to hold composite of vep input & vep output
my %seen; # running tally of vep inputs with 1 or more results
for (@results) {
next if /^#/; # skip comment lines
my @cols = split /\t/;
my $chr_location = $cols[1]; # in same format as %$src_data key (eg 2:123456)
if ( my $ref = $src_data->{$chr_location} ) { # warn Dumper $ref;
# capture vep cols: consequence, amino_acids, existing_variation, extra:
my $extra = $cols[13]; # contains sift, polyphen, etc results
# extract sift & polyphen into separate cols:
my $results = $self->_prediction_and_score($extra);
my @output_data = ( @cols[6,10,12], @$results );
push @data, [ @{$ref}{@input_fields}, @output_data ]; # combine data
$seen{$chr_location}++; # vep input file has at least one result
# add vep data to $hmds_ref data if it's useful:
if ( _want_vep_result(\@cols) ) { # examines consequence & existing cols
REF: while ( my ($hmds, $d) = each %$hmds_ref ) {
ID: while ( my ($unique_id, $result) = each %$d ) {
next ID unless $unique_id eq $chr_location;
my %h = (
consequence => $cols[6],
existing => $cols[12],
polyphen => $results->[1],
sift => $results->[0],
exon => $ref->{exon},
);
# merge with $result:
map $result->{$_} = $h{$_}, keys %h;
}
}
}
}
else { warn "no match in src file for $_" } # no match in src file
}
# has no vep result:
my @orphans = sort by_chr_location
map $src_data->{$_}, grep { ! $seen{$_} } keys %$src_data;
my $samples = _sort_sample_results($hmds_ref); # hashref
return { data => \@data, orphans => \@orphans, samples => $samples }
}
#-------------------------------------------------------------------------------
sub _prediction_and_score {
my ($self, $str) = @_; # warn $str;
# regex to capture prediction and/or score eg SIFT=deleterious(0.02);
# PolyPhen=probably_damaging; SIFT=0.002: / (\w+)? ( \(? [\d\.]+ \)? )? /x
my $re = qr/
(\w+)? # optional 'prediction' (eg benign, deleterious, etc)
( # start of optional 'score'
\(? # optional open bracket (only if prediction AND score selected)
[\d\.]+ # any number of 0-9 & decimal point chars (eg 0.012)
\)? # optional close bracket (only if prediction AND score selected)
)? # end of optional 'score'
/x;
my @results; # capture in order 'sift', 'polyphen' to match expected order in tt:
if ( $str =~ /sift=($re)/i ) {
push @results, $1; # warn $sift;
}
if ( $str =~ /polyphen=($re)/i ) {
push @results, $1; # warn $poly;
}
=begin # combined, but doesn't work - gets killed
while ( $str =~ /(?:sift|polyphen)=($re)/ig ) { # don't capture words
push @results, $1; # warn $1;
}
=cut
map { # remove captured items within $re from $str:
my $quoted = quotemeta $_;
$str =~ s/(sift|polyphen)=$quoted(;?)//i; # remove trailing semi-colon
} @results; # warn $str;
push @results, $str; # what's left of it (non-sift, non-polyphen 'extra')
return \@results;
}
#-------------------------------------------------------------------------------
sub _sort_sample_results {
my $data = shift; # hashref of hmds_ref => { chr:location => data }
my %h;
REF: while ( my ($hmds_ref, $d ) = each %$data ) { # warn Dumper $d;
LOC: while ( my ($location, $results) = each %$d ) {
my $exon_name = $results->{exon} || next LOC;
# transform $hmds_ref slash to underscore for automatic tt sorting:
my $lab_no = join '_', reverse split '/', $hmds_ref; # warn $lab_no;
# add hmds_ref to $result:
$results->{hmds_ref} = $hmds_ref;
$h{$lab_no}{$exon_name} = $results;
}
} # warn Dumper \%h;
return \%h;
}
#-------------------------------------------------------------------------------
sub by_hmds_ref {
my ($r1, $y1) = split '/', $a; # warn Dumper [$r1, $y1];
my ($r2, $y2) = split '/', $b; # warn Dumper [$r2, $y2];
return $y1 <=> $y2 || $r1 <=> $r2;
}
#-------------------------------------------------------------------------------
sub _want_vep_result {
my $cols = shift; # arrayref
my $consequence = $cols->[6];
my $existing_variation = $cols->[12];
return 1 if
$consequence ne 'synonymous_variant' ||
$existing_variation eq '-' ||
$existing_variation =~ /COSM|TMP/;
return 0; # don't want VEP result
}
#-------------------------------------------------------------------------------
sub _get_hmds_refs {
my $header = shift; # arrayref of hmds refs (in pairs)
my $i = 0; # counter
my @refs;
for ( @$header[5 .. $#{$header} - 1] ) {
next unless $i++ % 2; # increment then test for modulus 2 - get every other col
my ($ref) = $_ =~ m!(\d+/\d{2})!;
push @refs, $ref;
} # warn Dumper \@refs;
return \@refs;
}
#-------------------------------------------------------------------------------
sub _get_form_options { # user submitted opts (eg sift, polyphen, etc)
my $self = shift;
my $params = $self->form_opts; # warn Dumper $form_opts;
my @opts;
# polyphen & sift take args (s, p or b)
push @opts, '--polyphen=' . $params->{polyphen} if $params->{polyphen};
push @opts, '--sift=' . $params->{sift} if $params->{sift};
# check_existing, regulatory & coding_only take no args:
for ( qw/check_existing coding_only regulatory/ ) {
push @opts, "--$_" if $params->{$_};
} # warn Dumper \@opts;
return \@opts;
}
#-------------------------------------------------------------------------------
sub by_chr_location { # alphanumeric sort on chromosome 1-22, X, and start_point
return ( # 1st sort on chromosome, then start_point:
( $a->{chromosome} !~ /^\d+/ || $b->{chromosome} !~ /^\d+/ )
? $a->{chromosome} cmp $b->{chromosome} # non-digit eg X
: $a->{chromosome} <=> $b->{chromosome} # digit eg 1 - 22
) || $a->{start_point} <=> $b->{start_point};
}
#-------------------------------------------------------------------------------
# extract tab-delimited data from csv source file:
sub _text_csv_slurp {
my $self = shift;
my $data = $self->src_data; # string
my $csv = Text::CSV->new(\%text_csv_args);
my @results;
for ( split "\n", $data ) { # warn $_;
if ( $csv->parse($_) ) {
my @row = $csv->fields();
push @results, \@row;
}
}
return \@results;
}
#-------------------------------------------------------------------------------
# get reference table as hashref of gene => { chromosome, start_base }:
sub _build_ref_table {
my $self = shift;
my $db = NGS::DB->new(); # warn $db->dbix;
my $ref_tbl = $db->dbix->select('ref_seq', '*')
->map_hashes('gene'); # warn Dumper $ref_tbl;
return $ref_tbl;
}
=begin _build_ref_table() from ref.csv
my $ref = "$Bin/../ref.csv";
my $io = new IO::File;
open( $io, '<', $ref ) || die $!;
my $csv = Text::CSV->new(\%text_csv_args);
my %h;
while ( my $row = $csv->getline( $io ) ) {
my ($ref_seq, $chr, $start) = @$row;
$h{$ref_seq} = {
chromosome => $chr,
start_base => $start,
};
} # warn Dumper \%h;
return \%h;
=cut
1;
