examples/make_primers.pl

   1 #!/usr/bin/perl -w
   2 # $Id$
   3 # Author: cckim@stanford.edu
   4
   5 # Description: This program designs primers for constructing knockouts
   6 # of genes by transformation of PCR products (ref: Datsenko & Wanner,
   7 # PNAS 2000).  A tab-delimtied file containing ORF START STOP is read,
   8 # and primers flanking the start & stop coordinates are designed based
   9 # on the user-designated sequence file.  In addition, primers flanking
  10 # the knockout regions are chosen for PCR screening purposes once the
  11 # knockout is generated.  The script uses Bioperl in order to
  12 # determine the primer sequences, which requires getting subsequences
  13 # and reverse complementing some of the objects.
  14
  15 # make_primers.pl
  16 # Purpose: Design primers for the Wanner method of PCR product-based knockouts
  17 # Input: FASTA sequence file, tab-delimited coordinates file
  18 # Output: Primer output file
  19 # July 4, 2001
  20 # Charles C. Kim
  21
  22 ###########
  23 # MODULES #
  24 ###########
  25 use Bio::Seq;
  26 use Getopt::Std;
  27
  28 #############
  29 # VARIABLES #
  30 #############
  31 $upgap = 0; # the number of nt upstream of the 5' end to include in the deletion
  32 $downgap = 0; # the number of nucleotides downstream of the 3' end to include
  33               # in the deletion
  34 $oligolength = 40; # the length of the homologous region on each primer
  35 $seqfile = '';   # don't specify these filenames unless you want to run
  36 $coordfile = ''; # the program on these filenames exclusively
  37 $outfile = '';   #
  38 %fiveprime_primers = (
  39                       "P1" => "GTGTAGGCTGGAGCTGCTTC",
  40                       );
  41 %threeprime_primers = (
  42                        "P2" => "CATATGAATATCCTCCTTAG",
  43                        "P4" => "ATTCCGGGGATCCGTCGACC",
  44                        );
  45
  46 #########
  47 # FILES #
  48 #########
  49 getopts('s:c:o:');  # sequence file, coordinates file, output file
  50
  51 $seqfile = $opt_s if $opt_s;
  52 $coordfile = $opt_c if $opt_c;
  53 $outfile = $opt_o if $opt_o;
  54
  55 &open_readfile(*SEQFILE, 'sequence', $seqfile);
  56 &open_readfile(*COORDFILE, 'coordinate', $coordfile);
  57 &open_writefile(*PRIMERFILE, 'output', $outfile);
  58
  59 ########
  60 # MAIN #
  61 ########
  62
  63 $seq = '';
  64 $count = 0;
  65 while (<SEQFILE>) {
  66     if (/>/) {
  67         $count++;
  68         if ($count > 1) {
  69             die "More than one sequence present in the input file\n";
  70         }
  71         next;
  72     }
  73     chomp($_);
  74     $_ =~ tr/gatc/GATC/;
  75     $seq .= $_;
  76 }
  77 close SEQFILE;
  78
  79 $seq = Bio::Seq-> new('-seq'=>$seq );
  80
  81 while (<COORDFILE>) {
  82     chomp($_);
  83     next if !$_;
  84     (my $name, my $start, my $stop) = split(/\t/, $_);
  85     if ($start < $stop) {
  86         $upprimer = $seq->subseq($start-$oligolength-$upgap, $start-1-$upgap);
  87         $downprimer = $seq->subseq($stop+1+$downgap,$stop+$oligolength+$downgap);
  88         $downprimer = Bio::Seq->new('-seq'=>$downprimer);
  89         $downprimer = $downprimer->revcom();
  90         $downprimer = $downprimer->seq();
  91         $uppcr = $seq->subseq($start-$oligolength-$upgap-20,$start-1-$upgap-$oligolength);
  92         $downpcr = $seq->subseq($stop+1+$downgap+$oligolength,$stop+$oligolength+$downgap+20);
  93         $downpcr = Bio::Seq->new('-seq'=>$downpcr);
  94         $downpcr = $downpcr->revcom();
  95         $downpcr = $downpcr->seq();
  96     }
  97     elsif ($start > $stop) {
  98         $upprimer = $seq->subseq($start+$upgap+1,$start+$oligolength+$upgap);
  99         $downprimer = $seq->subseq($stop-$oligolength-$downgap, $stop-1-$downgap);
 100         $upprimer = Bio::Seq->new('-seq'=>$upprimer);
 101         $upprimer = $upprimer->revcom();
 102         $upprimer = $upprimer->seq();
 103         $uppcr = $seq->subseq($start+$oligolength+$upgap+1,$start+$oligolength+$upgap+20);
 104         $downpcr = $seq->subseq($stop-$oligolength-$downgap-20,$stop-1-$downgap-$oligolength);
 105         $uppcr = Bio::Seq->new('-seq'=>$uppcr);
 106         $uppcr = $uppcr->revcom();
 107         $uppcr = $uppcr->seq();
 108     }
 109     else { die "Problem with start and stop coordinates\n"; }
 110     print PRIMERFILE "$name\n";
 111     print PRIMERFILE "5'pcr\t$uppcr\n";
 112     print PRIMERFILE "3'pcr\t$downpcr\n";
 113     print PRIMERFILE "\tExpected wildtype product size: ",abs($start-$stop)+121," bp\n";
 114     foreach $entry (sort keys %fiveprime_primers) {
 115         print PRIMERFILE "5'+$entry\t$upprimer$fiveprime_primers{$entry}\n";
 116     }
 117     foreach $entry (sort keys %threeprime_primers) {
 118         print PRIMERFILE "3'+$entry\t$downprimer$threeprime_primers{$entry}\n";
 119     }
 120     print PRIMERFILE "\n";
 121     $upprimer = '';
 122     $downprimer = '';
 123     $uppcr = '';
 124     $downpcr = '';
 125 }
 126
 127
 128 ###############
 129 # SUBROUTINES #
 130 ###############
 131
 132 sub open_readfile {
 133     my $filehandle = $_[0];
 134     my $filetype = $_[1] if $_[1];
 135     my $filename = $_[2] if $_[2];
 136     unless ($filename) {
 137         print "Enter $filetype filename: ";
 138         chomp ($filename=<STDIN>);
 139     }
 140     unless (-e $filename) { die "$filename not found\n"; }
 141     open($filehandle,$filename) or die "Couldn't open $filename\n";
 142     $filehandle = '';
 143     $filetype = '';
 144     $filename = '';
 145 }
 146
 147 sub open_writefile {
 148     my $filehandle = $_[0];
 149     my $filetype = $_[1] if $_[1];
 150     my $filename = $_[2] if $_[2];
 151     unless ($filename) {
 152         print "Enter $filetype filename: ";
 153         chomp ($filename=<STDIN>);
 154     }
 155     if (-e $filename) {
 156         print "$filename already exists!  Overwrite (Y/N)? ";
 157         chomp ($_ = <STDIN>);
 158         while (/[^yn]/i) {
 159             print 'Y or N, please: ';
 160             chomp ($_ = <STDIN>);
 161         }
 162         if (/n/i) { die "$filename not overwritten.\n"; }
 163         else { open($filehandle, ">$filename") or die "Couldn't open $filename\n"; }
 164     }
 165     else { open($filehandle, ">$filename") or die "Couldn't open $filename\n"; }
 166     $filehandle = '';
 167     $filetype = '';
 168     $filename = '';
 169 }