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#!/usr/bin/perl
# Author: cckim@stanford.edu
# Description: This program designs primers for constructing knockouts
# of genes by transformation of PCR products (ref: Datsenko & Wanner,
# PNAS 2000). A tab-delimited file containing ORF START STOP is read,
# and primers flanking the start & stop coordinates are designed based
# on the user-designated sequence file. In addition, primers flanking
# the knockout regions are chosen for PCR screening purposes once the
# knockout is generated. The script uses Bioperl in order to
# determine the primer sequences, which requires getting subsequences
# and reverse complementing some of the objects.
# make_primers.pl
# Purpose: Design primers for the Wanner method of PCR product-based knockouts
# Input: FASTA sequence file, tab-delimited coordinates file
# Output: Primer output file
# July 4, 2001
# Charles C. Kim
###########
# MODULES #
###########
use Bio::Seq;
use Getopt::Std;
#############
# VARIABLES #
#############
$upgap = 0; # the number of nt upstream of the 5' end to include in the deletion
$downgap = 0; # the number of nucleotides downstream of the 3' end to include
# in the deletion
$oligolength = 40; # the length of the homologous region on each primer
$seqfile = ''; # don't specify these filenames unless you want to run
$coordfile = ''; # the program on these filenames exclusively
$outfile = ''; #
%fiveprime_primers = (
"P1" => "GTGTAGGCTGGAGCTGCTTC",
);
%threeprime_primers = (
"P2" => "CATATGAATATCCTCCTTAG",
"P4" => "ATTCCGGGGATCCGTCGACC",
);
#########
# FILES #
#########
getopts('s:c:o:'); # sequence file, coordinates file, output file
$seqfile = $opt_s if $opt_s;
$coordfile = $opt_c if $opt_c;
$outfile = $opt_o if $opt_o;
&open_readfile(*SEQFILE, 'sequence', $seqfile);
&open_readfile(*COORDFILE, 'coordinate', $coordfile);
&open_writefile(*PRIMERFILE, 'output', $outfile);
########
# MAIN #
########
$seq = '';
$count = 0;
while (<SEQFILE>) {
if (/>/) {
$count++;
if ($count > 1) {
die "More than one sequence present in the input file\n";
}
next;
}
chomp($_);
$_ =~ tr/gatc/GATC/;
$seq .= $_;
}
close SEQFILE;
$seq = Bio::Seq-> new('-seq'=>$seq );
while (<COORDFILE>) {
chomp($_);
next if !$_;
(my $name, my $start, my $stop) = split(/\t/, $_);
if ($start < $stop) {
$upprimer = $seq->subseq($start-$oligolength-$upgap, $start-1-$upgap);
$downprimer = $seq->subseq($stop+1+$downgap,$stop+$oligolength+$downgap);
$downprimer = Bio::Seq->new('-seq'=>$downprimer);
$downprimer = $downprimer->revcom();
$downprimer = $downprimer->seq();
$uppcr = $seq->subseq($start-$oligolength-$upgap-20,$start-1-$upgap-$oligolength);
$downpcr = $seq->subseq($stop+1+$downgap+$oligolength,$stop+$oligolength+$downgap+20);
$downpcr = Bio::Seq->new('-seq'=>$downpcr);
$downpcr = $downpcr->revcom();
$downpcr = $downpcr->seq();
}
elsif ($start > $stop) {
$upprimer = $seq->subseq($start+$upgap+1,$start+$oligolength+$upgap);
$downprimer = $seq->subseq($stop-$oligolength-$downgap, $stop-1-$downgap);
$upprimer = Bio::Seq->new('-seq'=>$upprimer);
$upprimer = $upprimer->revcom();
$upprimer = $upprimer->seq();
$uppcr = $seq->subseq($start+$oligolength+$upgap+1,$start+$oligolength+$upgap+20);
$downpcr = $seq->subseq($stop-$oligolength-$downgap-20,$stop-1-$downgap-$oligolength);
$uppcr = Bio::Seq->new('-seq'=>$uppcr);
$uppcr = $uppcr->revcom();
$uppcr = $uppcr->seq();
}
else { die "Problem with start and stop coordinates\n"; }
print PRIMERFILE "$name\n";
print PRIMERFILE "5'pcr\t$uppcr\n";
print PRIMERFILE "3'pcr\t$downpcr\n";
print PRIMERFILE "\tExpected wildtype product size: ",abs($start-$stop)+121," bp\n";
foreach $entry (sort keys %fiveprime_primers) {
print PRIMERFILE "5'+$entry\t$upprimer$fiveprime_primers{$entry}\n";
}
foreach $entry (sort keys %threeprime_primers) {
print PRIMERFILE "3'+$entry\t$downprimer$threeprime_primers{$entry}\n";
}
print PRIMERFILE "\n";
$upprimer = '';
$downprimer = '';
$uppcr = '';
$downpcr = '';
}
###############
# SUBROUTINES #
###############
sub open_readfile {
my $filehandle = $_[0];
my $filetype = $_[1] if $_[1];
my $filename = $_[2] if $_[2];
unless ($filename) {
print "Enter $filetype filename: ";
chomp ($filename=<STDIN>);
}
unless (-e $filename) { die "$filename not found\n"; }
open $filehandle,'<', $filename or die "Could not read file '$filename': $!\n";
$filehandle = '';
$filetype = '';
$filename = '';
}
sub open_writefile {
my $filehandle = $_[0];
my $filetype = $_[1] if $_[1];
my $filename = $_[2] if $_[2];
unless ($filename) {
print "Enter $filetype filename: ";
chomp ($filename=<STDIN>);
}
if (-e $filename) {
print "$filename already exists! Overwrite (Y/N)? ";
chomp ($_ = <STDIN>);
while (/[^yn]/i) {
print 'Y or N, please: ';
chomp ($_ = <STDIN>);
}
if (/n/i) { die "$filename not overwritten.\n"; }
else { open $filehandle, '>', $filename or die "Could nott write file '$filename': $!\n"; }
}
else { open $filehandle, '>', $filename or die "Could not write file '$filename': $!\n"; }
$filehandle = '';
$filetype = '';
$filename = '';
}
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