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check_primers_Fall2012.pl~
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check_primers_Fall2012.pl~
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#!/usr/bin/perl -w
use strict;
use Data::Dumper;
my $file = shift;
my $db1 = shift; #TE plus padding
my $db2 = shift; #TE only
my %students;
my %primers;
if ( !defined $file ) {
die "please provide a fasta file of primers in the following format:
ClassSection Student_name TE_id primerDesc PRIMER_foward PRIMER_rev
ClassSection Student_name TE_id primerDesc PRIMER_foward PRIMER_rev
ClassSection Student_name TE_id primerDesc PRIMER_foward PRIMER_rev
";
}
if ( !defined $db1 ) {
die "please provide a fasta to use as a db containing TE plus padding
";
}
if ( !defined $db2 ) {
die "please provide a fasta to use as a db of just the TE
";
}
open IN, $file or die "Can't open $file $!";
open OUT, ">$file.flank.fa" or die "Can't open $file.flank.fa for writting $!";
open OUTFlanktINNER , ">$file.flank_inner.fa" or die "Can't open $file.flank.fa for writting $!";
open OUTINNER, ">$file.inner.fa"
or die "Can't open $file.inner.fa for writting $!";
while ( my $line = <IN> ) {
chomp $line;
my ( $timeStamp, $student_name, $id, $desc, $primer1, $primer2 , $section) = split /\t/, $line;
if ( $desc eq 'Flank' ) {
$primers{$id}{p1}{seq} = $primer1;
$primers{$id}{p2}{seq} = $primer2;
$primers{$id}{p1}{len} = length($primer1);
$primers{$id}{p2}{len} = length($primer2);
my $seq_for_blast = $primer1 . "NNNNNNNNNN" . $primer2;
$primers{$id}{student}=$student_name;
$students{$student_name}=0;
print OUT ">$id\t$desc\n$seq_for_blast\n";
}
elsif ( $desc eq 'Inner' ) {
$primers{$id}{p3}{seq} = $primer2;
$primers{$id}{p3}{len} = length($primer2);
my $seq_for_blast = $primer1 . "NNNNNNNNNN" . $primer2;
print OUTINNER ">$id\t$desc\n$primer2\n";
print OUTFlanktINNER ">$id\t$desc\n$seq_for_blast\n";
}
}
`blastall -F F -p blastn -i $file.flank.fa -d $db1 -o $file.flank.blastout -m8`;
`blastall -F F -p blastn -i $file.inner.fa -d $db2 -o $file.inner.blastout -m8`;
`blastall -F F -p blastn -i $file.flank_inner.fa -d $db1 -o $file.flank_inner.blastout -m8`;
open BLASTOUT, "$file.flank.blastout" or die "Can't open $file.flank.blastout";
#14|TE Flank 2838|ZM_hAT_noncoding_5|padding|400|te|chr9:136082011..136082378|pcr|chr9:136081611..136082778 100.00 20 0 0 1 20 472 491 9e-05 40.1
while ( my $line = <BLASTOUT> ) {
chomp $line;
my (
$primer_pair, $desc,$subject, $perId, $alignLen,
$mm, $gapOpenings, $qStart, $qEnd,
$sStart, $sEnd, $e, $score
) = split /\t/, $line;
$subject =~ s/^(\d+\|.+?)\|padding.+/$1/;
my $p1_len = $primers{$primer_pair}{p1}{len};
my $p2_len = $primers{$primer_pair}{p2}{len};
next if ( ( $alignLen != $p1_len ) and ( $alignLen != $p2_len ) );
#next if $mm > 0;
next if ( $perId != 100 );
if ( $sStart > $sEnd ) {
( $sStart, $sEnd ) = ( $sEnd, $sStart );
}
if ( exists $primers{$primer_pair}{blast}{$subject} ) {
my $stored_start = $primers{$primer_pair}{blast}{$subject}{sStart};
my $stored_end = $primers{$primer_pair}{blast}{$subject}{sEnd};
if ( $sStart < $stored_start ) {
$primers{$primer_pair}{blast}{$subject}{sStart} = $sStart;
}
if ( $sEnd > $stored_end ) {
$primers{$primer_pair}{blast}{$subject}{sEnd} = $sEnd;
}
}
else {
$primers{$primer_pair}{blast}{$subject}{sStart} = $sStart;
$primers{$primer_pair}{blast}{$subject}{sEnd} = $sEnd;
}
$primers{$primer_pair}{blast}{$subject}{count}++;
$primers{$primer_pair}{blast}{$subject}{alnLen} = $alignLen;
$primers{$primer_pair}{blast}{$subject}{mm} = $mm;
$primers{$primer_pair}{blast}{$subject}{perID} = $perId;
$primers{$primer_pair}{blast}{$subject}{e} = $e;
$primers{$primer_pair}{blast}{$subject}{line} = $line;
$primers{$primer_pair}{subject}{$subject} = 1;
$primers{$primer_pair}{inner}{$subject}{inner} = 0 ;
}
open BLASTOUT_flank_inner, "$file.flank_inner.blastout" or die "Can't open $file.flank_inner.blastout";
while ( my $line = <BLASTOUT_flank_inner> ) {
chomp $line;
my (
$primer_pair, $desc,$subject, $perId, $alignLen,
$mm, $gapOpenings, $qStart, $qEnd,
$sStart, $sEnd, $e, $score
) = split /\t/, $line;
$subject =~ s/^(\d+\|.+?)\|padding.+/$1/;
my $p1_len = $primers{$primer_pair}{p1}{len};
my $p2_len = $primers{$primer_pair}{p2}{len};
next if ( ( $alignLen != $p1_len ) and ( $alignLen != $p2_len ) );
next if ( $perId != 100 );
if ( $sStart > $sEnd ) {
( $sStart, $sEnd ) = ( $sEnd, $sStart );
}
if ( exists $primers{$primer_pair}{inner}{$subject}{sStart} ) {
my $stored_start = $primers{$primer_pair}{inner}{$subject}{sStart};
my $stored_end = $primers{$primer_pair}{inner}{$subject}{sEnd};
if ( $sStart < $stored_start ) {
$primers{$primer_pair}{inner}{$subject}{sStart} = $sStart;
}
if ( $sEnd > $stored_end ) {
$primers{$primer_pair}{inner}{$subject}{sEnd} = $sEnd;
}
}
else {
$primers{$primer_pair}{inner}{$subject}{sStart} = $sStart;
$primers{$primer_pair}{inner}{$subject}{sEnd} = $sEnd;
}
$primers{$primer_pair}{inner}{$subject}{count}++;
$primers{$primer_pair}{inner}{$subject}{alnLen} = $alignLen;
$primers{$primer_pair}{inner}{$subject}{mm} = $mm;
$primers{$primer_pair}{inner}{$subject}{perID} = $perId;
$primers{$primer_pair}{inner}{$subject}{e} = $e;
$primers{$primer_pair}{inner}{$subject}{line} = $line;
}
open BLASTOUT_inner, "$file.inner.blastout"
or die "Can't open $file.inner.blastout";
#14|TE Inner 2834|ZM_hAT_noncoding_5|methyl|chr8:29147579..29147766|complete|chr8:29147384..29147766 100.00 20 0 0 1 20 250 231 5e-06 40.1
while ( my $line = <BLASTOUT_inner> ) {
chomp $line;
my (
$primer_pair, $desc, $subject, $perId, $alignLen,
$mm, $gapOpenings, $qStart, $qEnd,
$sStart, $sEnd, $e, $score
) = split /\t/, $line;
$subject =~ s/^(\d+\|.+?)\|methy.+/$1/;
my $p3_len = $primers{$primer_pair}{p3}{len};
next if ( $alignLen != $p3_len );
next if ( $perId != 100 );
if (exists $primers{$primer_pair}{blast}{$subject}){
#does the 3rd primer fall match to the same TE
$primers{$primer_pair}{inner}{$subject}{inner} =1 ;
}
}
print
"name\tprimer_set\thit\tflank_product_size\tinner_product_size\tcorrect_hit\tcorrect_inner\tp1\tp2\tp3\n";
my $student_count = scalar keys %students;
my $to_print;
foreach my $primer_set ( keys %primers ) {
foreach my $sub ( keys %{ $primers{$primer_set}{blast} } ) {
my %hits;
my @values;
my @lines;
my ($loc) = $primer_set =~ /^(\d+)/;
my $correct_matches = 'yes';
my $correct_inner = 'yes';
my $count = $primers{$primer_set}{blast}{$sub}{count};
if ( $count != 2) { #2 primer hits
$correct_matches = 'no';
}
if ( $primers{$primer_set}{inner}{$sub} == 0 )
{ #1=correct position, 0=incorrect position
$correct_inner = 'no';
}
my $flank_start = $primers{$primer_set}{blast}{$sub}{sStart};
my $flank_end = $primers{$primer_set}{blast}{$sub}{sEnd};
my $inner_end = exists $primers{$primer_set}{inner}{$sub}{sEnd} ? $primers{$primer_set}{inner}{$sub}{sEnd} : 0 ;
my $p1 = uc $primers{$primer_set}{p1}{seq};
my $p2 = uc $primers{$primer_set}{p2}{seq};
my $p3 = uc $primers{$primer_set}{p3}{seq};
my $flank_product_size =
$correct_matches eq 'yes' ? $flank_end - $flank_start + 1 : 'N/A';
my $inner_product_size =
$correct_inner eq 'yes' ? $inner_end - $flank_start + 1 : 'N/A';
my $in_range = 'no';
my ($actual_loc) = $sub =~ /^(\d+)/;
#print Dumper \%primers;
if ( $loc == $actual_loc ) {
$in_range = 'yes';
}
my $name = $primers{$primer_set}{student};
if ($correct_matches eq 'yes'){
print
"$name\t$primer_set\t$sub\t$flank_product_size\t$inner_product_size\t$in_range\t$correct_inner\t$p1\t$p2\t$p3\n";
}elsif ($in_range eq 'yes' and $correct_inner eq 'yes'){
print
"$name\t$primer_set\t$sub\t$flank_product_size\t(incorrectMatchNumber=$count)\t$in_range\t$correct_inner\t$p1\t$p2\t$p3\n";
$students{$name}{err}{flank}="the 2 flanking primers have $count match(es) but should have 2, 1 for each";
}
if (($correct_matches eq 'yes') and ($in_range eq 'yes') and ($correct_inner eq 'yes')){
my $line = "$name\t$loc" . "_p1\t$p1\n$name\t$loc" . "_p2\t$p2\n$name\t$loc" . "_p3\t$p3\n";
$to_print .= $line;
$students{$name}=1;
}
}
}
print "\n\nstudent_count\t$student_count\n";
print "\n\n$to_print\n";
foreach my $name (keys %students){
my $complete = $students{$name};
my $err_flank;
if (exists $students{$name}{err}{flank}){
$err_flank = $students{$name}{err}{flank};
}
print "$name\t$err_flank\tp1\t-\n$name\t$err_flank\tp2\t-\n$name\t\tp3\t-\n" if !$complete;
}