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extractRNAsFromGenBank.pl
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extractRNAsFromGenBank.pl
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#!/usr/bin/perl
#
# INGLÊS/ENGLISH
# This program is distributed in the hope that it will be useful,
# but WITHOUT ANY WARRANTY; without even the implied warranty of
# MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
# GNU General Public License for more details.
# http://www.gnu.org/copyleft/gpl.html
#
#
# PORTUGUÊS/PORTUGUESE
# Este programa é distribuído na expectativa de ser útil aos seus
# usuários, porém NÃO TEM NENHUMA GARANTIA, EXPLÍCITAS OU IMPLÍCITAS,
# COMERCIAIS OU DE ATENDIMENTO A UMA DETERMINADA FINALIDADE. Consulte
# a Licença Pública Geral GNU para maiores detalhes.
# http://www.gnu.org/copyleft/gpl.html
#
# Copyright (C) 2012 Universidade de São Paulo
#
# Universidade de São Paulo
# Laboratório de Biologia do Desenvolvimento de Abelhas
# Núcleo de Bioinformática (LBDA-BioInfo)
#
# Daniel Guariz Pinheiro
# http://zulu.fmrp.usp.br/bioinfo
#
# $Id$
=head1 NAME
extractRNAsFromGenBank.pl
=head1 SYNOPSIS
perl extractRNAsFromGenBank.pl -i AmRefSeqDNA.gbk -o ./output
=head1 ABSTRACT
Extract sequences of 5' UTR, 3' UTR and CDS of mRNA sequences and sequences of non-coding
RNAs from GenBank chromosome files. The global statistics are displayed in STDERR output.
=head1 DESCRIPTION
Arguments:
-h/--help Help
-l/--level Log level [Default: FATAL]
OFF
FATAL
ERROR
WARN
INFO
DEBUG
TRACE
ALL
-i/--infile Input file path and name
-o/--outbase Output path and base name [Default: ./out]
=head1 AUTHOR
Daniel Guariz Pinheiro E<lt>[email protected]<gt>
Copyright (c) 2012 Universidade de São Paulo
=head1 LICENSE
GNU General Public License
http://www.gnu.org/copyleft/gpl.html
=cut
use strict;
use warnings;
use Readonly;
use Getopt::Long;
use File::Temp qw/ tempfile tempdir /;
use Bio::SeqIO;
use vars qw/$LOGGER/;
INIT {
use Log::Log4perl qw/:easy/;
Log::Log4perl->easy_init($FATAL);
$LOGGER = Log::Log4perl->get_logger($0);
}
my ($infile, $level, $outbase, $ethreep, $statsfile);
Usage("Too few arguments") if $#ARGV < 0;
GetOptions( "h|?|help" => sub { &Usage(); },
"i|infile=s" => \$infile,
"o|outbase=s" => \$outbase,
"l|level=s" => \$level,
"e|extend3p=s" => \$ethreep,
"s|statsfile=s" => \$statsfile
) or &Usage();
if ($level) {
my %LEVEL = (
'OFF' =>$OFF,
'FATAL' =>$FATAL,
'ERROR' =>$ERROR,
'WARN' =>$WARN,
'INFO' =>$INFO,
'DEBUG' =>$DEBUG,
'TRACE' =>$TRACE,
'ALL' =>$ALL);
$LOGGER->logdie("Wrong log level ($level). Choose one of: ".join(', ', keys %LEVEL)) unless (exists $LEVEL{$level});
Log::Log4perl->easy_init($LEVEL{$level});
}
$LOGGER->logdie("Missing input file") unless ($infile);
$LOGGER->logdie("Wrong input file") unless (-e $infile);
$outbase||='./emfgout';
my $in = Bio::SeqIO->new(-file=>$infile);
my %out;
$out{'3p'} =
Bio::SeqIO->new( -file => '>' . $outbase . '3p.fa', -format => 'FASTA' );
$out{'5p'} =
Bio::SeqIO->new( -file => '>' . $outbase . '5p.fa', -format => 'FASTA' );
$out{'CDS'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'CDS.fa', -format => 'FASTA' );
$out{'missedCDS'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'missedCDS.fa', -format => 'FASTA' );
$out{'chromosome'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'chromosome.fa', -format => 'FASTA' );
$out{'misc_RNA'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'misc_RNA.fa', -format => 'FASTA' );
$out{'ncRNA'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'ncRNA.fa', -format => 'FASTA' );
$out{'rRNA'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'rRNA.fa', -format => 'FASTA' );
$out{'tRNA'} =
Bio::SeqIO->new( -file => '>' . $outbase . 'tRNA.fa', -format => 'FASTA' );
if ($ethreep) {
$out{'3p-'.$ethreep} =
Bio::SeqIO->new( -file => '>' . $outbase . '3p-'.$ethreep.'.fa', -format => 'FASTA' );
}
my $tmpdir = tempdir( 'emfgXXXX', DIR=>'./', CLEANUP => 1 );
my %count = (
'chromosome' => 0,
'gene' => 0,
'mRNA' => 0,
'5pCDS3pmRNA' => {'all'=>0, 'small'=>0},
'5pCDSmRNA' => {'all'=>0, 'small'=>0},
'CDS3pmRNA' => {'all'=>0, 'small'=>0},
'CDSmRNA' => {'all'=>0, 'small'=>0},
'ncRNA' => 0,
'byGroup' => {}
);
my %threepext;
while ( my $seqobj = $in->next_seq()) {
my ($group) = ($1||$2||$3) if ($seqobj->description()=~/linkage group ([^, ]+)|(mitochondrion)|(GroupUn\d+)/);
$LOGGER->logdie("Not found linkage group: ".$seqobj->description()) unless ($group);
@{ $count{'byGroup'}->{$group} }{ 'gene', 'mRNA', '5pCDS3pmRNA', '5pCDSmRNA', 'CDS3pmRNA', 'CDSmRNA', 'ncRNA' } = (0, 0, {'all'=>0, 'small'=>0}, {'all'=>0, 'small'=>0}, {'all'=>0, 'small'=>0}, {'all'=>0, 'small'=>0}, 0) unless (exists $count{'byGroup'}->{$group});
$count{'chromosome'}++;
$LOGGER->info("Extracting features from : ".$seqobj->display_id());
my @features = $seqobj->get_SeqFeatures();
my ($fh, $filename) = tempfile( 'emfgXXXX', DIR => $tmpdir);
foreach my $feature ( @features ) {
# Transcript
if ( $feature->primary_tag() =~ /RNA/) {
my ($transcript_id, @db_xref) = ('', 0);
if ($feature->has_tag('transcript_id')) {
($transcript_id) = $feature->each_tag_value('transcript_id');
}
if ($feature->has_tag('db_xref')) {
@db_xref = $feature->each_tag_value('db_xref');
}
my $geneid = '?';
my $beebase = '?';
foreach my $xref (@db_xref) {
if ($xref=~/GeneID:(\d+)/) {
$geneid = $1;
}
elsif ($xref=~/BEEBASE:(\S+)/) {
$beebase = $1;
}
}
if ($ethreep) {
my ($ts, $te) = ($feature->start(), $feature->end());
if ($feature->strand() == 1) {
if ( ($te+1+$ethreep) <= $seqobj->length() ) {
$threepext{$transcript_id} = $seqobj->trunc($te+1, $te+1+$ethreep);
}
else {
if ( $te < $seqobj->length() ) {
$threepext{$transcript_id} = $seqobj->trunc($te+1, $seqobj->length());
}
}
}
else {
if ($ts >= $ethreep) {
$threepext{$transcript_id} = $seqobj->trunc($ts-1-$ethreep, $ts-1)->revcom();
}
else {
if ($ts>1) {
$threepext{$transcript_id} = $seqobj->trunc(1, $ts-1)->revcom();
}
}
}
}
my $rna_seq = $feature->spliced_seq();
print $fh $geneid,"\t",$beebase,"\t",$feature->primary_tag(),"\t",$transcript_id,"\t",$rna_seq->seq(),"\t",$feature->strand(),"\t",join(';', @db_xref),"\n";
}
# CDS
if ( $feature->primary_tag() eq 'CDS') {
my ($protein_id, @db_xref) = ('', 0);
if ($feature->has_tag('protein_id')) {
($protein_id) = $feature->each_tag_value('protein_id');
}
if ($feature->has_tag('db_xref')) {
@db_xref = $feature->each_tag_value('db_xref');
}
my $geneid = '?';
my $beebase = '?';
foreach my $xref (@db_xref) {
if ($xref=~/GeneID:(\d+)/) {
$geneid = $1;
}
elsif ($xref=~/BEEBASE:(\S+)/) {
$beebase = $1;
}
}
my $cds_seq = $feature->spliced_seq();
print $fh $geneid,"\t",$beebase,"\t",$feature->primary_tag(),"\t",$protein_id,"\t",$cds_seq->seq(),"\t",$feature->strand(),"\t",join(';', @db_xref),"\n";
}
}
# Sorting features
$LOGGER->info("\tSorting features in $filename to $filename.sort");
`sort -k1,1g -k2,2 -k3,3r -n $filename > $filename.sort`;
# Parsing features
$LOGGER->info("\tParsing features in $filename.sort");
open(FT, "<", "$filename.sort") or $LOGGER->logdie($!);
my %RNA;
my $lastgeneid = '';
my @m;
my %gene;
my %mRNA;
my %ncRNA;
while (<FT>) {
chomp;
my ($geneid, $beebase, $type, $acc, $seq, $strand, $other) = split(/\t/, $_);
$gene{$geneid} = 0 unless (exists $gene{$geneid});;
$gene{$geneid}++;
if ($lastgeneid ne $geneid) {
@m = ();
$lastgeneid = $geneid;
}
if ($type =~ /RNA/) {
$RNA{$acc} = { 'seq' => $seq,
'geneid' => $geneid,
'beebase' => $beebase,
'cds' => undef,
'type' => $type
};
if ( $type eq 'mRNA' ) {
push( @m, $acc );
$mRNA{$acc} = [];
}
else {
$ncRNA{$acc} = undef;
}
}
else {
my $found;
foreach my $mRNA_acc (@m) {
if ($RNA{$mRNA_acc}->{'seq'}=~/$seq/) {
$LOGGER->debug($mRNA_acc."\t".$acc."\n");
$RNA{$mRNA_acc}->{'cds'}->{$acc} = { 'start' => $-[0]+1,
'end' => $+[0] };
unless ($found) {
$found = 1;
push(@{$mRNA{$mRNA_acc}}, $acc);
}
else {
$LOGGER->logwarn("CDS ($acc) matches more than one mRNA");
}
}
}
unless (defined $found) {
$LOGGER->logwarn("Not found mRNA for this CDS ($acc). Trying to match the chromosome (".$seqobj->display_id().")");
$RNA{$seqobj->display_id().'.'.$acc} = { 'seq' => (($strand==1) ? $seqobj->seq() : $seqobj->revcom()->seq()),
'geneid' => $geneid||'-',
'beebase' => $beebase||'-',
'cds' => undef,
'type' => 'chromosome'
};
if ($RNA{$seqobj->display_id().'.'.$acc}->{'seq'}=~/$seq/) {
$RNA{$seqobj->display_id().'.'.$acc}->{'seq'}=$seq;
$RNA{$seqobj->display_id().'.'.$acc}->{'cds'}->{$acc} = { 'start' => 0,
'end' => length($seq) };
}
else {
$LOGGER->logdie("Not found match for this CDS ($acc) in the chromosome (".$seqobj->display_id().")");
}
}
}
}
close(FT);
open(DBG, ">>", "/tmp/MRNA.txt");
foreach my $k (keys %mRNA) {
print DBG $k,"\t",$group,"\n";
}
close(DBG);
$count{'gene'}+=scalar(keys %gene);
$count{'byGroup'}->{$group}->{'gene'}+=scalar(keys %gene);
$count{'mRNA'}+=scalar(keys %mRNA);
$count{'byGroup'}->{$group}->{'mRNA'}+=scalar(keys %mRNA);
$count{'ncRNA'}+=scalar(keys %ncRNA);
$count{'byGroup'}->{$group}->{'ncRNA'}+=scalar(keys %ncRNA);
$LOGGER->info("\tWriting sequence objects");
foreach my $RNA_acc (keys %RNA) {
my $seqobj = Bio::Seq->new( -seq => $RNA{$RNA_acc}->{'seq'},
-display_id => $RNA_acc,
-description=> "GeneID:$RNA{$RNA_acc}->{'geneid'}|BEEBASE:$RNA{$RNA_acc}->{'beebase'}"
);
my %valid;
my %seqlength;
if ($RNA{$RNA_acc}->{'type'} eq 'mRNA') {
if (scalar( keys %{$RNA{$RNA_acc}->{'cds'}})) {
foreach my $CDS_acc (keys %{$RNA{$RNA_acc}->{'cds'}}) {
if ($RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'start'} > 1) {
my $cdsobj = $seqobj->trunc(1, $RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'start'}-1);
$cdsobj->display_id( $cdsobj->display_id().'|5p'.'|'.$CDS_acc );
$valid{'5p'}->{ $CDS_acc } = $cdsobj;
}
if ($RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'end'} < $seqobj->length()) {
my $cdsobj = $seqobj->trunc($RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'end'}+1, $seqobj->length());
$cdsobj->display_id( $cdsobj->display_id().'|3p'.'|'.$CDS_acc );
$valid{'3p'}->{ $CDS_acc } = $cdsobj;
}
else {
if ($ethreep) {
if ($RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'end'}==$seqobj->length()) {
if (exists $threepext{$RNA_acc}) {
$valid{'3p-'.$ethreep}->{ $CDS_acc } = $threepext{$RNA_acc};
$valid{'3p-'.$ethreep}->{ $CDS_acc }->display_id($RNA_acc.'|3p-'.$ethreep.'|'.$CDS_acc);
$valid{'3p-'.$ethreep}->{ $CDS_acc }->description( "GeneID:$RNA{$RNA_acc}->{'geneid'}|BEEBASE:$RNA{$RNA_acc}->{'beebase'}" );
}
else {
$LOGGER->logdie("Not found 3p extension for $RNA_acc");
}
}
else {
$LOGGER->logdie("You couldn't be here.");
}
}
}
my $cdsobj = $seqobj->trunc($RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'start'}, $RNA{$RNA_acc}->{'cds'}->{$CDS_acc}->{'end'});
$cdsobj->display_id( $cdsobj->display_id().'|CDS'.'|'.$CDS_acc );
$valid{'CDS'}->{ $CDS_acc } = $cdsobj;
if (!exists $seqlength{'CDS'}) {
$seqlength{'CDS'} = $cdsobj->length();
}
else {
$seqlength{'CDS'} = $cdsobj->length() if ($seqlength{'CDS'} > $cdsobj->length());
}
}
}
else {
my $cdsobj = $seqobj;
$cdsobj->display_id( $cdsobj->display_id().'|missedCDS'.'|'.'-' );
$valid{'missedCDS'}->{ $RNA_acc } = $cdsobj;
}
}
else {
my $cdsobj = $seqobj;
$cdsobj->display_id( $cdsobj->display_id().'|'.$RNA{$RNA_acc}->{'type'}.'|'.'-' );
$valid{$RNA{$RNA_acc}->{'type'}}->{ $RNA_acc } = $cdsobj;
}
if (exists $valid{'CDS'}) {
if ( exists $valid{'5p'} ) {
if ( exists $valid{'3p'} ) {
$count{'5pCDS3pmRNA'}->{'all'}++;
$count{'byGroup'}->{$group}->{'5pCDS3pmRNA'}->{'all'}++;
if ( $seqlength{'CDS'} < 60 ) {
$count{'5pCDS3pmRNA'}->{'small'}++;
$count{'byGroup'}->{$group}->{'5pCDS3pmRNA'}->{'small'}++;
}
}
else {
$count{'5pCDSmRNA'}->{'all'}++;
$count{'byGroup'}->{$group}->{'5pCDSmRNA'}->{'all'}++;
if ( $seqlength{'CDS'} < 60 ) {
$count{'5pCDSmRNA'}->{'small'}++;
$count{'byGroup'}->{$group}->{'5pCDSmRNA'}->{'small'}++;
}
}
}
else {
if ( exists $valid{'3p'} )
{
$count{'CDS3pmRNA'}->{'all'}++;
$count{'byGroup'}->{$group}->{'CDS3pmRNA'}->{'all'}++;
if ( $seqlength{'CDS'} < 60 ) {
$count{'CDS3pmRNA'}->{'small'}++;
$count{'byGroup'}->{$group}->{'CDS3pmRNA'}->{'small'}++;
}
}
else {
$count{'CDSmRNA'}->{'all'}++;
$count{'byGroup'}->{$group}->{'CDSmRNA'}->{'all'}++;
}
}
}
foreach my $type (keys %valid) {
foreach my $s (keys %{$valid{$type}}) {
$LOGGER->logdie("Not found file for this type: $type") unless ($out{$type});
$out{$type}->write_seq( $valid{$type}->{$s} );
}
}
}
}
open(STAT, ">", $statsfile) or $LOGGER->logwarn($!);
print STAT "
*** GENERAL REPORT ***
Nº of groups scanned....: $count{'chromosome'}
Nº of genes found.......: $count{'gene'}
Nº of mRNAs found.......: $count{'mRNA'}
5p CDS 3p...........: $count{'5pCDS3pmRNA'}->{'all'}
Small...........: $count{'5pCDS3pmRNA'}->{'small'}
5p CDS..............: $count{'5pCDSmRNA'}->{'all'}
Small...........: $count{'5pCDSmRNA'}->{'small'}
CDS 3p..............: $count{'CDS3pmRNA'}->{'all'}
Small...........: $count{'CDS3pmRNA'}->{'small'}
CDS.................: $count{'CDSmRNA'}->{'all'}
Small...........: $count{'CDSmRNA'}->{'small'}
ncRNA...............: $count{'ncRNA'}
";
print STAT "
*** REPORT BY GROUP ***
";
foreach my $group (sort {$a cmp $b} keys %{$count{'byGroup'}} ) {
print STAT "
* $group
Nº of genes found.......: $count{'byGroup'}->{$group}->{'gene'}
Nº of mRNAs found.......: $count{'byGroup'}->{$group}->{'mRNA'}
5p CDS 3p...........: $count{'byGroup'}->{$group}->{'5pCDS3pmRNA'}->{'all'}
Small...........: $count{'byGroup'}->{$group}->{'5pCDS3pmRNA'}->{'small'}
5p CDS..............: $count{'byGroup'}->{$group}->{'5pCDSmRNA'}->{'all'}
Small...........: $count{'byGroup'}->{$group}->{'5pCDSmRNA'}->{'small'}
CDS 3p..............: $count{'byGroup'}->{$group}->{'CDS3pmRNA'}->{'all'}
Small...........: $count{'byGroup'}->{$group}->{'CDS3pmRNA'}->{'small'}
CDS.................: $count{'byGroup'}->{$group}->{'CDSmRNA'}->{'all'}
Small...........: $count{'byGroup'}->{$group}->{'CDSmRNA'}->{'small'}
ncRNA...............: $count{'byGroup'}->{$group}->{'ncRNA'}
";
}
close(STAT);
# Subroutines
sub Usage {
my ($msg) = @_;
Readonly my $USAGE => <<"END_USAGE";
Daniel Guariz Pinheiro (dgpinheiro\@gmail.com)
(c)2012 Universidade de São Paulo
Usage
$0 [-h/--help] [-l/--level <LEVEL>] -i <INFILE> -o <OUTBASE>
Argument(s)
-h --help Help
-l --level Log level [Default: FATAL]
-i --infile Input file path and name
-o --outbase Output path and base name [Default: ./out]
-s --statsfile Statistics file
END_USAGE
print STDERR "\nERR: $msg\n\n" if $msg;
print STDERR qq[$0 ] . q[$Revision$] . qq[\n];
print STDERR $USAGE;
exit(1);
}