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trap_annotator.pl
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trap_annotator.pl
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#! /usr/bin/perl -w
#
# Guglielmo Roma
#
# This script retrieves the relevant annotation of the trapped region from the Ensembl database
#
$|=1;
use strict;
BEGIN {
require "/home/roma/src/scripts/unitrap2/unitrap_conf.pl";
};
use DBI;
use Getopt::Long;
use Data::Dumper;
use Bio::SeqFeature::Generic;
use Bio::SeqIO;
use Bio::Seq;
use Bio::Root::IO;
use Bio::SearchIO;
use Bio::Tools::RepeatMasker;
use Bio::SeqFeature::Collection;
use Bio::EnsEMBL::Registry;
use Bio::Unitrap::File;
my ( $enshost, $ensuser, $enspass, $ensdbname, $ensestdbname,
$traphost, $trapuser, $trappass, $trapdbname,
$fantomhost, $fantomuser, $fantompass, $fantomdbname,
$blastnexec, $blastdb, $hit_db, $idcount, $id, $tmp_dir, $mysql_path, $repeatMasker_dir, $debug, $debugSQL);
GetOptions(
"enshost=s", \$enshost,
"ensuser=s", \$ensuser,
"enspass=s", \$enspass,
"ensdbname=s", \$ensdbname,
"ensestdbname=s", \$ensestdbname,
"traphost=s", \$traphost,
"trapuser=s", \$trapuser,
"trapdbname=s", \$trapdbname,
"trappass=s", \$trappass,
"fantomhost=s", \$fantomhost,
"fantomuser=s", \$fantomuser,
"fantomdbname=s", \$fantomdbname,
"fantompass=s", \$fantompass,
"blastdb=s", \$blastdb,
"hit_db=s", \$hit_db,
"id=s", \$id,
"idcount=s", \$idcount,
"debug", \$debug,
"debugSQL", \$debugSQL
);
#### Configuration options
my %conf = %::conf;
$enshost = $conf{'enshost'};
$ensuser = $conf{'ensuser'};
$ensdbname = $conf{'ensdbname'};
$ensestdbname = $conf{'ensestdbname'};
$enspass = $conf{'enspass'};
$traphost = $conf{'traphost'};
$trapuser = $conf{'trapuser'};
$trapdbname = $conf{'trapdbname'};
$trappass = $conf{'trappass'};
$fantomhost = $conf{'fantom3host'};
$fantomuser = $conf{'fantom3user'};
$fantomdbname = $conf{'fantom3dbname'};
$fantompass = $conf{'fantom3pass'};
$blastdb = $conf{'blastdb'};
$hit_db = $conf{'hit_db'};
$blastnexec ||= $::conf{'blastnexec'};
$tmp_dir = $conf{'tmp_dir'};
$mysql_path = $conf{'mysql_path'};
$repeatMasker_dir = $conf{'repeatMasker_dir'};
$debug = $conf{'debug'};
$debugSQL = $conf{'debugSQL'};
my $registry = 'Bio::EnsEMBL::Registry';
$registry->load_registry_from_db(
-host => $enshost,
-user => $ensuser,
-pass => $enspass,
-database => $ensdbname
);
my $registry_est = 'Bio::EnsEMBL::Registry';
$registry_est->load_registry_from_db(
-host => $enshost,
-user => $ensuser,
-pass => $enspass,
-database => $ensestdbname
);
########################
#### Get BATCHARG params
#########################
die print "Must provide id and idcount as BATCHARG" if scalar(@ARGV) == 0;
chomp $ARGV[0];
chomp $ARGV[1];
$id = $ARGV[0];
$idcount = $ARGV[1];
unless ($id && $idcount) {
print STDERR " no trap id \n";
exit;
}
print STDOUT "ID $id\tID count $idcount\n";
#### Connecting to unitrap_db
my $trapdb = DBI->connect("DBI:mysql:database=$trapdbname;host=$traphost;port=3306", $trapuser, $trappass) || die "Can't connect: ", $DBI::errst;
my $ensdb = DBI->connect("DBI:mysql:database=$ensdbname;host=$enshost;port=3306", $ensuser, $enspass) || die "Can't connect: ", $DBI::errst;
my $ensestdb = DBI->connect("DBI:mysql:database=$ensestdbname;host=$enshost;port=3306", $ensuser, $enspass) || die "Can't connect: ", $DBI::errst;
#my $fantom3db = DBI->connect("DBI:mysql:database=$fantomdbname;host=$fantomhost;port=3306", $fantomuser, $fantompass) || die "Can't connect: ", $DBI::errst;
my $fantom3db;
$trapdb->{RaiseError}=1;
$trapdb->{RaiseError}=1;
$trapdb->{RaiseError}=1;
my $error;
my $status;
my $tid;
my $lastid = $id + $idcount - 1;
foreach ($tid = $id; $tid <= $lastid; $tid++) {
my $sth_map = $trapdb->prepare("select tm.trapmap_id, tm.start, tm.end, tm.hit_id, tm.chosen from trapmap tm where tm.trap_id = $tid");
$debug && print STDOUT "SQL CODE: select tm.trapmap_id, tm.start, tm.end, tm.hit_id, tm.chosen from trapmap tm where tm.trap_id = $tid\n";
my $nres = $sth_map->execute;
### If this trap id is in the database continue
if ($nres >= 1) {
while (my $rhref = $sth_map->fetchrow_hashref) {
my $trapmap_id = $rhref->{'trapmap_id'};
my $start = $rhref->{'start'};
my $end = $rhref->{'end'};
my $hit_id = $rhref->{'hit_id'};
my $chosen = $rhref->{'chosen'};
my $strand;
my @features;
print STDOUT "### TRAP: $tid\tTRAPMAP $trapmap_id\n";
my $sth_block = $trapdb->prepare("select tb.trapblock_id, tb.start, tb.end, tb.strand from trapblock tb where tb.trapmap_id=$trapmap_id");
$debug && print STDOUT "SQL CODE: select tb.trapblock_id, tb.start, tb.end, tb.strand from trapblock tb where tb.trapmap_id=$trapmap_id\n";
my $nres_block = $sth_block->execute;
if ($nres_block >= 1) {
while (my $rhref_block = $sth_block->fetchrow_hashref) {
my $trapblock_id = $rhref_block->{'trapblock_id'};
my $tbstart = $rhref_block->{'start'};
my $tbend = $rhref_block->{'end'};
$strand = $rhref_block->{'strand'};
### Create a feature for each trap_block
my $feature = Bio::SeqFeature::Generic->new( -display_name => $trapblock_id,
-start => $tbstart,
-end => $tbend,
-strand => $strand );
push @features,$feature;
$debug && print STDOUT "Trapblock $trapblock_id \n";
}
} else {
print STDERR "This trapmap ($trapmap_id) has no blocks!\n";
}
###############################
##### Retrieve Annotation #####
### Querying the Ensembl database
my $t2e_status = &annotate_with_ensembl (\@features, $hit_id, $start, $end, $tid);
my $sql_update = "update trapmap set checked=1 where trapmap_id = $trapmap_id;";
$debug && print STDOUT "SQL CODE: $sql_update\n";
my $sth_update = $trapdb->prepare($sql_update);
$sth_update->execute;
}
$status = 1;
} else {
print STDERR "not mapped trap ($tid) \n";
my $trap_id = $tid;
$error = "not mapped trap";
}
}
#######################
#
# SUBS
#
#######################
sub annotate_with_ensembl {
my ($fs, $chr, $start, $end, $trap_id) = @_;
### Ensembl genes parameters (included RefSeq genes)
my $do_ensgene = 1;
### Estgenes parameters
my $do_ensestgene = 0;
### Genescan parameters
my $do_genescan = 0;
### FANTOM3 parameters
my $do_fantom = 0;
my $fantom_coverage = 0;
my $fantom_perc_id = 96;
### UniGene parameters
my $do_unigene = 1;
my $unigene_coverage = 0;
my $unigene_perc_id = 96;
### cDNA parameters
my $do_mouse_cdna = 1;
my $cdna_coverage = 66;
my $cdna_perc_id = 96;
### Ests parameters
my $do_ensest = 1;
my $est_coverage = 66;
my $est_perc_id = 96;
### TCLG parameters
my $do_tclg = 0;
my $tclg_coverage = 0;
### Repeats parameters
my $do_ensrepeats = 0;
my $dodumper;
my @features = @{$fs};
my $region;
if ($chr =~ /NT/) {
$region = "supercontig";
} else {
$region = "chromosome";
}
$debug && print STDOUT "Got ".scalar @features." blocks to analyze\n";
### Reset coordinates to be local
foreach my $f (@features) {
my $s = $f->start();
$f->start($s-$start+1);
my $e = $f->end();
$f->end($e-$start+1);
}
### Get overlap with ensembl genes (both RefSeq and ENSEMBL)
if ($do_ensgene) {
my $hashref;
#my $slice = $ensdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry->get_adaptor( 'Mouse', 'Core', 'Slice' );
my $slice = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
$debug && print STDOUT "Annotating with $ensdbname \n";
$debug && print STDOUT "Got slice for chr ".$slice->seq_region_name." start ".$slice->start." to ".$slice->end."\n";
$debug && print STDOUT "Trap from 1 to ".$slice->length."\n";
foreach my $f (@features) {
$debug && print STDOUT "Trapblock ".$f->start." end ".$f->end."\n";
$hashref->{$f->display_name} = &annotate_with_ensembl_gene ($f, $slice);
}
$dodumper && print Dumper $hashref;
$hashref = &resolve_ensembl_conflicts($hashref);
### Add strand to the hash
foreach my $f (keys %{$hashref}) {
foreach my $g (keys %{$hashref->{$f}}){
if ($g =~ "ENSMUS") {
#my $gene = $ensdb->get_GeneAdaptor->fetch_by_stable_id ($g);
my $gene_adaptor = $registry->get_adaptor( 'Mouse', 'Core', 'Gene' );
my $gene = $gene_adaptor->fetch_by_stable_id ($g);
$hashref->{$f}{$g}{'strand'} = $gene->strand;
}
}
}
$dodumper && print Dumper $hashref;
my $stmt = &prepare_sql($trap_id, $hashref,'trapblock_ensg_annotation');
}
### Get overlap with ensembl est-genes
if ($do_ensestgene) {
my $hashref;
$debug && print STDOUT "Annotating with $ensestdbname \n";
#my $slestgene= $ensestdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry->get_adaptor( 'Mouse', 'otherfeatures', 'Slice' );
my $slestgene = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
foreach my $f (@features) {
$debug && print STDOUT "Trapblock ".$f->start." end ".$f->end."\n";
$hashref->{$f->display_name} = &annotate_with_ensembl_gene ($f, $slestgene);
}
$dodumper && print Dumper $hashref;
$hashref = &resolve_ensembl_conflicts($hashref);
### Add strand to the hash
# foreach my $f (keys %{$hashref}) {
# foreach my $g (keys %{$hashref->{$f}}){
# if ($g =~ "ENSMUS") {
# #my $gene = $ensestdb->get_GeneAdaptor->fetch_by_stable_id ($g);
# my $gene_adaptor = $registry_est->get_adaptor( 'Mouse', 'Core', 'Gene' );
# my $gene = $gene_adaptor->fetch_by_stable_id ($g);
#
# $hashref->{$f}{$g}{'strand'} = $gene->strand;
# }
# }
# }
$dodumper && print Dumper $hashref;
my $stmt = &prepare_sql ($trap_id, $hashref, 'trapblock_ensestg_annotation');
}
### Get overlap with genescan gene predictions
if ($do_genescan) {
#my $slice = $ensdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry->get_adaptor( 'Mouse', 'Core', 'Slice' );
my $slice = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
$debug && print STDOUT "Annotating with $ensdbname using prediction_transcripts (i.e., genescan)\n";
$debug && print STDOUT "Got slice for chr ".$slice->seq_region_name." start ".$slice->start." to ".$slice->end."\n";
$debug && print STDOUT "Trap from 1 to ".$slice->length."\n";
foreach my $f (@features) {
$debug && print STDOUT "Trapblock ".$f->start." end ".$f->end."\n";
&annotate_with_ensembl_genescan ($f, $slice, 'trapblock_abinitio_annotation', $trap_id);
}
}
### Get overlap with Fantom3 (previoulsy mapped with our in-house pipeline with a cut-off of 96% identity)
if ($do_fantom) {
my $sql_fantom = "select f.name, fb.start, fb.end, fb.strand, fb.perc_ident from feature f, featuremap fm, featureblock fb where f.feature_id=fm.feature_id and fm.featuremap_id=fb.featuremap_id and fb.start <= $end and fb.end >= $start and fm.hit_id='$chr'";
my $sth_fantom = $fantom3db->prepare($sql_fantom);
$debug && print STDOUT "SQL CODE: $sql_fantom\n";
$sth_fantom->execute;
$debug && print STDOUT "Annotating with features from $fantomdbname using Fantom\n";
my @dnadnafeat;
while (my $fantom = $sth_fantom->fetchrow_hashref) {
if ($fantom->{'perc_ident'} >= $fantom_perc_id) {
### Handle coordinates to be local
my $feature = Bio::SeqFeature::Generic->new ( -display_name => $fantom->{'name'},
-start => $fantom->{'start'}-$start+1,
-end => $fantom->{'end'}-$start+1,
-strand => $fantom->{'strand'});
push @dnadnafeat, $feature;
}
}
my $col = new Bio::SeqFeature::Collection();
my $totaladded = $col->add_features(\@dnadnafeat);
foreach my $f (@features) {
$debug && print STDOUT "Feature: ".$f->display_name."\t".$f->start."\t".$f->end."\n";
my @subset = $col->features_in_range (-range=>$f, -contain=> 0);
foreach my $s (@subset) {
$debug && print STDOUT "In range: ".$s->display_name."\t".$s->start."\t".$s->end."\n";
my $posrelative = &relative_positioning ($s->display_name, $s->start, $s->end, $f->display_name, $f->start, $f->end);
my $inter = $f->intersection($s);
$debug && print STDOUT "Intersection ".$inter->length."\n";
my $coverage = ($inter->length/$s->length)*100;
$debug && print STDOUT "Coverage ".$coverage."\n";
if ($coverage > $fantom_coverage) {
my %fantom_ins;
$fantom_ins{'display_name'} = $s->display_name;
$fantom_ins{'dS_s'} = $posrelative->{'dS_s'};
$fantom_ins{'dE_e'} = $posrelative->{'dE_e'};
$fantom_ins{'label'} = $posrelative->{'label'};
$fantom_ins{'comment'} = $posrelative->{'comment'};
$fantom_ins{'coverage'} = ($inter->length/$s->length)*100;
$fantom_ins{'trapblock_id'} = $f->display_name;
$fantom_ins{'trap_id'} = $trap_id;
$fantom_ins{'strand'} = $s->strand;
my $st = &prepare_stmt ($trapdb, \%fantom_ins);
my $insid = &insert_set ($trapdb, $st, 'trapblock_fantom_annotation');
$debug && print STDOUT "Inserted trapblock_fantom_annotation with id $insid\n";
}
}
}
}
### Get overlap with ensembl UniGene
if ($do_unigene) {
#my $slice = $ensdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry->get_adaptor( 'Mouse', 'Core', 'Slice' );
my $slice = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
$debug && print STDOUT "Annotating with dnadna align features from $ensdbname using UniGene\n";
my @dna_dna_align_feats = @{$slice->get_all_DnaAlignFeatures ('Unigene')};
my @dnadnafeat;
#ensembl dnaalign features is not compatible with Bio::RangeI
foreach my $dnadna (@dna_dna_align_feats) {
if ($dnadna->percent_id >= $unigene_perc_id && $dnadna->hseqname =~ /Mm/) {
# print $dnadna->hseqname . "\t".$dnadna->hstart ."\t". $dnadna->hend . "\t". $dnadna->hstrand . "\t". $dnadna->percent_id. "\n";
# print $dnadna->seqname . "\t". $dnadna->start ."\t". $dnadna->end . "\t". $dnadna->strand . "\t". $dnadna->score. "\n";
my $feature = Bio::SeqFeature::Generic->new ( -display_name => $dnadna->hseqname,
-start => $dnadna->hstart,
-end => $dnadna->hend,
-strand => $dnadna->hstrand);
push @dnadnafeat ,$feature;
}
}
my $col = new Bio::SeqFeature::Collection();
my $totaladded = $col->add_features(\@dnadnafeat);
foreach my $f (@features) {
$debug && print STDOUT "Feature: ".$f->display_name."\t".$f->start."\t".$f->end."\n";
my @subset = $col->features_in_range (-range=>$f, -contain=> 0);
foreach my $s (@subset) {
$debug && print STDOUT "In range: ".$s->display_name."\t".$s->start."\t".$s->end."\n";
my $posrelative = &relative_positioning ($s->display_name, $s->start, $s->end, $f->display_name, $f->start, $f->end);
my $inter = $f->intersection($s);
$debug && print STDOUT "Intersection ".$inter->length."\n";
my $coverage = ($inter->length/$s->length)*100;
$debug && print STDOUT "Coverage ".$coverage."\n";
if ($coverage > $unigene_coverage) {
my %unigene_ins;
$unigene_ins{'display_name'} = $s->display_name;
$unigene_ins{'dS_s'} = $posrelative->{'dS_s'};
$unigene_ins{'dE_e'} = $posrelative->{'dE_e'};
$unigene_ins{'label'} = $posrelative->{'label'};
$unigene_ins{'comment'} = $posrelative->{'comment'};
$unigene_ins{'coverage'} = ($inter->length/$s->length)*100;
$unigene_ins{'trapblock_id'} = $f->display_name;
$unigene_ins{'trap_id'} = $trap_id;
$unigene_ins{'strand'} = $s->strand;
my $st = &prepare_stmt ($trapdb, \%unigene_ins);
my $insid = &insert_set ($trapdb, $st, 'trapblock_unigene_annotation');
$debug && print STDOUT "Inserted trapblock_unigene_annotation with id $insid\n";
}
}
}
}
### Get overlap with ensembl mouse-cdnas
if ($do_mouse_cdna) {
#my $slice = $ensdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry->get_adaptor( 'Mouse', 'Core', 'Slice' );
my $slice = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
$debug && print STDOUT "Annotating with dnadna align features from $ensdbname using cDNA\n";
my @dna_dna_align_feats = @{$slice->get_all_DnaAlignFeatures ('mouse_cdna')};
my @dnadnafeat;
#ensembl dnaalign features is not compatible with Bio::RangeI
foreach my $dnadna (@dna_dna_align_feats) {
if ($dnadna->percent_id >= $cdna_perc_id) {
# print $dnadna->hseqname . "\t".$dnadna->hstart ."\t". $dnadna->hend . "\t". $dnadna->hstrand . "\t". $dnadna->percent_id. "\n";
# print $dnadna->seqname . "\t". $dnadna->start ."\t". $dnadna->end . "\t". $dnadna->strand . "\t". $dnadna->score. "\n";
my $feature = Bio::SeqFeature::Generic->new ( -display_name => $dnadna->hseqname,
-start => $dnadna->hstart,
-end => $dnadna->hend,
-strand => $dnadna->hstrand);
push @dnadnafeat ,$feature;
}
}
my $col = new Bio::SeqFeature::Collection();
my $totaladded = $col->add_features(\@dnadnafeat);
foreach my $f (@features) {
$debug && print STDERR "Feature: ".$f->display_name."\t".$f->start."\t".$f->end."\n";
my @subset = $col->features_in_range (-range=>$f, -contain=> 0);
foreach my $s (@subset) {
$debug && print STDOUT "In range: ".$s->display_name."\t".$s->start."\t".$s->end."\n";
my $posrelative = &relative_positioning ($s->display_name, $s->start, $s->end, $f->display_name, $f->start, $f->end);
my $inter = $f->intersection($s);
$debug && print STDOUT "Intersection ".$inter->length."\n";
my $coverage = ($inter->length/$s->length)*100;
$debug && print STDOUT "Coverage ".$coverage."\n";
if ($coverage > $cdna_coverage) {
my %cdna_ins;
$cdna_ins{'display_name'} = $s->display_name;
$cdna_ins{'dS_s'} = $posrelative->{'dS_s'};
$cdna_ins{'dE_e'} = $posrelative->{'dE_e'};
$cdna_ins{'label'} = $posrelative->{'label'};
$cdna_ins{'comment'} = $posrelative->{'comment'};
$cdna_ins{'coverage'} = ($inter->length/$s->length)*100;
$cdna_ins{'trapblock_id'} = $f->display_name;
$cdna_ins{'trap_id'} = $trap_id;
$cdna_ins{'strand'} = $s->strand;
my $st = &prepare_stmt ($trapdb, \%cdna_ins);
my $insid = &insert_set ($trapdb, $st, 'trapblock_cdna_annotation');
$debug && print STDOUT "Inserted trapblock_cdna_annotation with id $insid\n";
}
}
}
}
### Get overlap with ensembl mouse-ests
if ($do_ensest) {
$debug && print STDOUT "Annotating with dnadna align features from $ensestdbname using Ests\n";
#my $slest= $ensestdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry_est->get_adaptor( 'Mouse', 'otherfeatures', 'Slice' );
my $slest = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
my @dna_dna_align_feats = @{$slest->get_all_DnaAlignFeatures ('mouse_est')};
my @dnadnafeat;
### Ensembl dnaalign features is not compatible with Bio::RangeI
foreach my $dnadna (@dna_dna_align_feats) {
if ($dnadna->percent_id >= $est_perc_id) {
my $feature = Bio::SeqFeature::Generic->new ( -display_name => $dnadna->hseqname,
-start => $dnadna->hstart,
-end => $dnadna->hend,
-strand => $dnadna->hstrand);
push @dnadnafeat ,$feature;
}
}
my $col = new Bio::SeqFeature::Collection();
my $totaladded = $col->add_features(\@dnadnafeat);
foreach my $f (@features) {
$debug && print STDOUT "FEATURE: ".$f->display_name."\t".$f->start."\t".$f->end."\n";
my @subset = $col->features_in_range(-range=>$f, -contain=> 0);
foreach my $s (@subset) {
$debug && print STDOUT "in range: ".$s->display_name."\t".$s->start."\t".$s->end."\n";
my $posrelative = &relative_positioning ($s->display_name, $s->start, $s->end, $f->display_name, $f->start, $f->end);
my $inter = $f->intersection($s);
$debug && print STDOUT "intersection ".$inter->length."\n";
my $coverage = ($inter->length/$s->length)*100;
$debug && print STDOUT "Coverage ".$coverage."\n";
if ($coverage > $est_coverage) {
my %est_ins;
$est_ins{'display_name'} = $s->display_name;
$est_ins{'dS_s'} = $posrelative->{'dS_s'};
$est_ins{'dE_e'} = $posrelative->{'dE_e'};
$est_ins{'label'} = $posrelative->{'label'};
$est_ins{'comment'} = $posrelative->{'comment'};
$est_ins{'coverage'} = ($inter->length/$s->length)*100;;
$est_ins{'trapblock_id'} = $f->display_name;
$est_ins{'trap_id'} = $trap_id;
$est_ins{'strand'} = $s->strand;
my $st = &prepare_stmt ($trapdb, \%est_ins);
my $insid= &insert_set ($trapdb, $st, 'trapblock_ensest_annotation');
$debug && print STDOUT "inserted trapblock_ensest_annotation with id $insid\n";
}
}
}
}
### Get overlap with TrapclusterGene (build from our in-house pipeline)
if ($do_tclg) {
my $sql_tclg = "select n.accession, nb.start, nb.end, nb.strand from novel_gene n, novel_genemap nm, novel_geneblock nb where n.novel_gene_id=nm.novel_gene_id and nm.novel_genemap_id=nb.novel_genemap_id and nb.start <= $end and nb.end >= $start and nm.hit_id='$chr';";
my $sth_tclg = $trapdb->prepare($sql_tclg);
$debug && print STDOUT "SQL CODE: $sql_tclg\n";
$sth_tclg->execute;
$debug && print STDOUT "Annotating with features from $trapdbname using TrapCLusterGene\n";
my @dnadnafeat;
while (my $tclg = $sth_tclg->fetchrow_hashref) {
### Handle coordinates to be local
my $feature = Bio::SeqFeature::Generic->new ( -display_name => $tclg->{'accession'},
-start => $tclg->{'start'}-$start+1,
-end => $tclg->{'end'}-$start+1,
-strand => $tclg->{'strand'});
push @dnadnafeat, $feature;
}
my $col = new Bio::SeqFeature::Collection();
my $totaladded = $col->add_features(\@dnadnafeat);
foreach my $f (@features) {
$debug && print STDOUT "Feature: ".$f->display_name."\t".$f->start."\t".$f->end."\n";
my @subset = $col->features_in_range (-range=>$f, -contain=> 0);
foreach my $s (@subset) {
$debug && print STDOUT "In range: ".$s->display_name."\t".$s->start."\t".$s->end."\n";
my $posrelative = &relative_positioning ($s->display_name, $s->start, $s->end, $f->display_name, $f->start, $f->end);
my $inter = $f->intersection($s);
$debug && print STDOUT "Intersection ".$inter->length."\n";
my $coverage = ($inter->length/$s->length)*100;
$debug && print STDOUT "Coverage ".$coverage."\n";
if ($coverage > $tclg_coverage) {
my %tclg_ins;
$tclg_ins{'display_name'} = $s->display_name;
$tclg_ins{'dS_s'} = $posrelative->{'dS_s'};
$tclg_ins{'dE_e'} = $posrelative->{'dE_e'};
$tclg_ins{'label'} = $posrelative->{'label'};
$tclg_ins{'comment'} = $posrelative->{'comment'};
$tclg_ins{'coverage'} = ($inter->length/$s->length)*100;
$tclg_ins{'trapblock_id'} = $f->display_name;
$tclg_ins{'trap_id'} = $trap_id;
$tclg_ins{'strand'} = $s->strand;
my $st = &prepare_stmt ($trapdb, \%tclg_ins);
my $insid = &insert_set ($trapdb, $st, 'trapblock_tclg_annotation');
$debug && print STDOUT "Inserted trapblock_tclg_annotation with id $insid\n";
}
}
}
}
### Get overlap with repeats
if ($do_ensrepeats) {
$debug && print STDOUT "Checking for reapet features from $ensdbname\n";
my @repeatfeat;
#my $slice = $ensdb->get_SliceAdaptor->fetch_by_region ($region,$chr,$start,$end);
my $slice_adaptor = $registry->get_adaptor( 'Mouse', 'Core', 'Slice' );
my $slice = $slice_adaptor->fetch_by_region ($region,$chr,$start,$end);
foreach my $repeat (@{$slice->get_all_RepeatFeatures()}) {
$debug && print STDOUT "Repeat Name: ".$repeat->seqname." - Start : ".$repeat->start." - End:".$repeat->end."Strand:".$repeat->strand." - Score:".$repeat->score." - Hit start:".$repeat->hstart." - Hit end:".$repeat->hend." - Consensus:".$repeat->display_id."\n";
### Checking only for more_complexity repeats
if ($repeat->display_id ne 'Low_complexity' && $repeat->display_id ne 'Simple_repeat') {
my $feature = Bio::SeqFeature::Generic->new( -display_name => $repeat->display_id,
-start => $repeat->hstart,
-end => $repeat->hend,
-strand => $repeat->hstrand);
push @repeatfeat ,$feature;
}
}
if (@repeatfeat) {
my $col = new Bio::SeqFeature::Collection();
my $totaladded = $col->add_features(\@repeatfeat);
foreach my $f (@features) {
$debug && print STDOUT "Feature: ".$f->display_name."\t".$f->start."\t".$f->end."\n";
my @subset = $col->features_in_range(-range=>$f, -contain=> 0);
foreach my $s (@subset) {
$debug && print STDOUT "In range: " . $s->display_name . "\t". $s->start . "\t". $s->end . "\n";
my $posrelative = &relative_positioning($s->display_name, $s->start, $s->end, $f->display_name, $f->start, $f->end);
my $inter = $f->intersection($s);
$debug && print STDOUT "Intersection ". $inter->length . "\n";
my $coverage = ($inter->length/$s->length)*100;
my %rep_ins;
$rep_ins{'display_name'} = $s->display_name;
$rep_ins{'dS_s'} = $posrelative->{'dS_s'};
$rep_ins{'dE_e'} = $posrelative->{'dE_e'};
$rep_ins{'label'} = $posrelative->{'label'};
$rep_ins{'comment'} = $posrelative->{'comment'};
$rep_ins{'coverage'} = ($inter->length/$s->length)*100;;
$rep_ins{'trapblock_id'}= $f->display_name;
$rep_ins{'trap_id'} = $trap_id;
my $st = &prepare_stmt($trapdb, \%rep_ins);
my $insid= &insert_set($trapdb, $st, 'trapblock_repeat_annotation');
$debug && print STDOUT "Inserted trapblock_repeat_annotation with id $insid\n";
}
}
} else {
$debug && print STDOUT "No repeats\n";
}
}
}
sub annotate_with_ensembl_gene {
my ($f, $slice) = @_;
my $genecount = 0;
my $transcriptcount = 0;
my $hashref;
my $yes_exon;
my $yes_gene;
foreach my $gene (@{$slice->get_all_Genes}) {
my $gene_feature = Bio::SeqFeature::Generic->new ( -display_name => $gene->stable_id,
-start => $gene->start,
-end => $gene->end,
-strand => $gene->strand);
my $ig = $f->intersection($gene_feature);
if ($ig) {
my $iglength = $ig->length;
$yes_gene = 1;
my $rank = 0;
$genecount++;
$debug && print STDOUT "\tGot a intersection between gene ". $gene->stable_id . " and Trapblock ". $f->display_name . "\n";
foreach my $transcript (@{$gene->get_all_Transcripts}) {
my $transcript_feature = Bio::SeqFeature::Generic->new (-display_name => $transcript->stable_id,
-start => $transcript->start,
-end => $transcript->end,
-strand => $transcript->strand);
my $it = $f->intersection($transcript_feature);
if ($it) {
$transcriptcount++;
my $rank = 0;
my $exoncount = 0;
foreach my $exon (@{$transcript->get_all_Exons}) {
my $exon_feature = Bio::SeqFeature::Generic->new ( -display_name => $exon->stable_id,
-start => $exon->start,
-end => $exon->end,
-strand => $exon->strand);
my $i = $f->intersection($exon_feature);
if ($i) {
$yes_exon = 1;
$exoncount++;
#we should only have one exon, after all this is trapblocks, small things...
$debug && print STDOUT "\t\tGot a intersection between exon ". $exon->stable_id . " and Trapblock ". $f->display_name . "\n";
$debug && print STDOUT "\t\tExon start: ".$exon->start." end: ".$exon->end."\n";
$debug && print STDOUT "\t\tTrapblock start: ".$f->start." end: ".$f->end."\n";
$debug && print STDOUT "\t\tIntersection start: ".$i->start." end: ".$i->end." length " . $i->length . "\n";
$debug && print STDOUT "\t\tExon length: ".$exon->length." and TrapBlock length: ". $f->length. "\n";
my $ilength = $i->length;
my $ecoverage = ($ilength/$exon->length)*100;
$debug && print STDOUT "\t \tthe exon " . $exon->stable_id. " coverage by trapblock ". $f->display_name ." is $ecoverage\n";
$hashref->{$gene->stable_id}{$transcript->stable_id}{$exon->stable_id}{'coverage'} = $ecoverage;
my $posrelative = &relative_positioning ($exon->stable_id, $exon->start, $exon->end, $f->display_name, $f->start, $f->end);
$hashref->{$gene->stable_id}{$transcript->stable_id}{$exon->stable_id}{'relative'} = $posrelative;
#rank the exons, give points for coverage and sharing of splice sites
$rank += $ecoverage;
if ($hashref->{$gene->stable_id}{$transcript->stable_id}{$exon->stable_id}{'relative'}{'dE_e'} == 0) {
$rank += 100;
}
if ($hashref->{$gene->stable_id}{$transcript->stable_id}{$exon->stable_id}{'relative'}{'dS_s'} == 0) {
$rank += 100;
}
$hashref->{$gene->stable_id}{$transcript->stable_id}{'rank'} = $rank;
my $transcoverage = ($ilength/$transcript->length)*100;
$hashref->{$gene->stable_id}{$transcript->stable_id}{'coverage'} = $transcoverage * 100;
}
}
if ($exoncount > 1) {
$debug && print STDOUT "\t\tgot more then one exon from the same transcript... exit\n";
#should exit...
}
}
}
unless ($yes_exon) {
$iglength += 1000; #for the perl issue to solve!!!
$debug && print STDOUT "\t\tgot zero exons, can't say which transcript is correct, not storing\n";
my $genecoverage = ($iglength/$gene->length)*100;
$debug && print STDOUT "\t\tNOTE: the intersection with the gene is $iglength and the coverage is $genecoverage\n";
$hashref->{$gene->stable_id}{'no_transcript'} = $genecoverage;
}
}
}
unless ($yes_gene) {
$debug && print STDOUT "\tno genes in this slice!!\n";
$hashref->{'no_gene'}++;
return $hashref;
}
return $hashref;
}
sub resolve_ensembl_conflicts {
my $hashref = shift;
#resolve transcript conflicts by the exon rank
my (%seengene, %addedranks, %addedtrans_coverage, %genecoverage);
foreach my $f (keys %{$hashref}) {
foreach my $g (keys %{$hashref->{$f}}){
last if ($g eq 'no_gene');
$seengene{$g}++;
foreach my $t (keys %{$hashref->{$f}{$g}}) {
if ($t eq 'no_transcript') {
my $v = $hashref->{$f}{$g}{'no_transcript'};
$genecoverage{$g} = $v;
last;
} else {
my $r = $hashref->{$f}{$g}{$t}{'rank'};
$addedranks{$g} += $r;
$addedtrans_coverage{$t} += $hashref->{$f}{$g}{$t}{'coverage'};
}
}
}
}
if (scalar (keys %seengene) > 1) {
my $mostseen = &check_if_topexists (\%seengene);
my $highestrank = &check_if_topexists (\%addedranks);
if ($mostseen && $highestrank) {
if ($highestrank eq $mostseen) {
$debug && print STDERR "\t\tThe most seen IS the highest ranking\n";
foreach my $f (keys %{$hashref}) {
foreach my $g (keys %{$hashref->{$f}}) {
last if ($g eq 'no_gene');
if ($g ne $mostseen) {
$debug && print STDERR "\t\t deleting $g\n";
delete $hashref->{$f}{$g};
} else {
my $ct;
foreach my $t (keys %{$hashref->{$f}{$g}}) {
$ct++;
}
if ($ct > 1) {
my $toptrans_coverage = &check_if_topexists(\%addedtrans_coverage);
if ($toptrans_coverage) {
foreach my $t (keys %{$hashref->{$f}{$g}}) {
if ($t ne $toptrans_coverage) {
next if ($t eq 'no_transcript');
$debug && print STDOUT "\t\t deleting $t\n";
delete $hashref->{$f}{$g}{$t};
}
}
}
}
}
}
}
} else {
$debug && print STDOUT "\t\tThe most seen ($mostseen) is not the the highestranking ($highestrank) using the rank only\n";
foreach my $f (keys %{$hashref}) {
foreach my $g (keys %{$hashref->{$f}}) {
last if ($g eq 'no_gene');
if ($g ne $highestrank) {
$debug && print STDERR "\t\t deleting $g\n";
delete $hashref->{$f}{$g};
} else {
my $ct;
foreach my $t (keys %{$hashref->{$f}{$g}}) {
$ct++;
}
if ($ct > 1) {
my $toptrans_coverage = &check_if_topexists (\%addedtrans_coverage);
if ($toptrans_coverage) {
foreach my $t (keys %{$hashref->{$f}{$g}}) {
if ($t ne $toptrans_coverage) {
next if ($t eq 'no_transcript');
$debug && print STDOUT "\t\t deleting $t\n";
delete $hashref->{$f}{$g}{$t};
}
}
}
}
}
}
}
}
} elsif ($highestrank) {
$debug && print STDOUT "\t\tThere is no mostseen but the highest ranking is $highestrank\n";
foreach my $f (keys %{$hashref}) {
foreach my $g (keys %{$hashref->{$f}}) {
last if ($g eq 'no_gene');
if ($g ne $highestrank) {
$debug && print STDOUT "\t\t deleting $g\n";
delete $hashref->{$f}{$g};
} else {
my $ct;
foreach my $t (keys %{$hashref->{$f}{$g}}) {
$ct++;
}
if ($ct > 1) {
my $toptrans_coverage = &check_if_topexists (\%addedtrans_coverage);
if ($toptrans_coverage) {
foreach my $t (keys %{$hashref->{$f}{$g}}) {
if ($t ne $toptrans_coverage) {
next if ($t eq 'no_transcript');
$debug && print STDOUT "\t\t deleting $t\n";
delete $hashref->{$f}{$g}{$t};
}
}
}
}
}
}
}
} elsif ($mostseen) {
$debug && print STDOUT "\t\tI dont think this a good parameter... $mostseen... but using\n";
foreach my $f (keys %{$hashref}) {
foreach my $g (keys %{$hashref->{$f}}) {
last if ($g eq 'no_gene');
if ($g ne $mostseen) {
$debug && print STDOUT "\t\t deleting $g\n";
delete $hashref->{$f}{$g};
} else {
my $ct;
foreach my $t (keys %{$hashref->{$f}{$g}}) {
$ct++;
}
if ($ct > 1) {
my $toptrans_coverage = &check_if_topexists (\%addedtrans_coverage);
if ($toptrans_coverage) {
foreach my $t (keys %{$hashref->{$f}{$g}}) {
if ($t ne $toptrans_coverage) {
next if ($t eq 'no_transcript');
$debug && print STDOUT "\t\t deleting $t\n";
delete $hashref->{$f}{$g}{$t};
}
}
}
}
}
}
}
} else {
$debug && print STDOUT "\t\treached here because got more than one gene with no transcripts\n using gene coverage to choose\n";
my $topgene = &check_if_topexists (\%genecoverage);
if ($topgene) {
foreach my $f (keys %{$hashref}) {
foreach my $gt (keys %{$hashref->{$f}}) {