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#!/usr/bin/perl
# gffGetmRNA.pl
# Creates mRNA fasta sequence files from a GFF with gene annotation
#
# Mario Stanke, January, 2010
#
use strict;
use Getopt::Long;
my $usage = "cat genes.gff | gffGetmRNA.pl --genome=s --mrna=s\n";
$usage .= " Makes a fasta file with mRMA sequences\n";
$usage .= " --genome=s Input a fasta file with the genomic sequences.\n";
$usage .= " --mrna=s Output fasta file with mRNA sequences.\n";
my ($seqfile, $mrnafile);
my %sequence = ();
my %ignorechr = ();
GetOptions('genome=s'=>\$seqfile,
'mrnae=s'=>\$mrnafile);
if (!defined($seqfile) || !defined($mrnafile)) {
print $usage;
exit;
}
my @gfflines; # complete input file
# hash of transcripts
# keys: chromosome names
# values: hash reference
# keys: transcript ids
# values: gff array reference
my %txs = ();
readGenome();
parseAndStoreGFF();
sortAndCheck();
open MRNA, ">$mrnafile" or die ("Could not open mrnafile for writing.");
printmrnas();
close MRNA;
if (keys %ignorechr > 0){
print "Annotation from these chromosomes was ignored because they were not contained in $seqfile.:\n"
. join (", ", sort keys %ignorechr) . "\n";
}
# Read in the sequence file in one chunk.
# And sort it in the sequence hash.
# Yes, this requires a lot of memory for large genomes.
sub readGenome {
my $seqname;
if ($seqfile){
open (SEQ, "<$seqfile") or die ("Could not open sequence file $seqfile\n");
$/=">";
while (<SEQ>){
s/>$//;
next unless /\S+/;
/(.*)\n/;
$seqname = $1;
my $sequencepart = $'; #'
$seqname =~ s/\s.*//; # seqname only up to first white space
$sequencepart =~ s/\s//g;
$sequence{$seqname} = $sequencepart;
}
print "Read in " . (scalar keys %sequence) . " sequence(s) from $seqfile.\n";
$/="\n";
}
}
#
# Read in the GFF file and store the exons by their transcript id
#
sub parseAndStoreGFF{
my ($txid, $type, $chr);
while(<STDIN>){
my @f = split /\t/;
next if (@f<8);
if ($f[8] =~ /(transcript_id|Transcript)."([^"]+)"/){ #"
$txid = $2;
} elsif ($f[8] =~ /gene_id."([^"]+)"/){ #"
$txid = $1;
} elsif ($f[8] =~ /Parent=Transcript:([^;]+)/){
$txid = $1;
} elsif ($f[8] =~ m/Parent=([^;]+)/){
$txid = $1;
} else {
$txid = $f[8];
}
$txid =~ s/\n//;
my ($chr,$type) = ($f[0],$f[2]);
if ($type eq "exon"){
$txs{$chr}{$txid} = [] if (!exists($txs{$chr}{$txid}));
push @{$txs{$chr}{$txid}}, \@f;
}
}
}
sub sortAndCheck {
foreach my $chr (sort keys %txs){
foreach my $txid (sort keys %{$txs{$chr}}){
@{$txs{$chr}{$txid}} = sort {$a->[3] <=> $b->[3]} @{$txs{$chr}{$txid}}; # sort by start position
my $strand;
my $end;
foreach my $exon (@{$txs{$chr}{$txid}}){
if (!defined($strand)){
$strand = $exon->[6];
} else {
die ("Strand not consistent in transcript $txid.") if ($strand ne $exon->[6]);
}
if (defined($end) && $end >= $exon->[3]){
die ("Exons overlapping in $txid.");
}
}
}
}
}
sub printmrnas() {
foreach my $chr (sort keys %txs){
if ($sequence{$chr}){
foreach my $txid (sort keys %{$txs{$chr}}){
my $seq = "";
foreach my $exon (@{$txs{$chr}{$txid}}){
die ("Coordinate of $txid out of sequence range. $exon->[4] >= " . length($sequence{$chr}) . "\n")
if ($exon->[4] >= length($sequence{$chr}));
my $seqpart = lc(substr($sequence{$chr}, $exon->[3]-1, $exon->[4] - $exon->[3] + 1));
$seq .= $seqpart;
}
$seq = rc($seq) if ($txs{$chr}{$txid}->[0]->[6] eq "-");
print MRNA ">$txid\n" . getFa($seq, 100);
}
} else {
$ignorechr{$chr}++;
}
}
}
# reverse complement
sub rc{
my $s = shift;
$s = reverse $s;
$s =~ tr/acgtACGT/tgcaTGCA/;
return $s;
}
sub getFa{
my $seq = shift;
my $cols = 100;
$cols = shift if (@_);
my $start = 0;
my $ret = "";
while (length($seq)-$start >= $cols) {
my $shortline = substr($seq, $start, $cols);
$ret .= "$shortline\n";
$start += $cols;
}
$ret .= substr($seq, $start, $cols) . "\n";
}
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