examples/make_primers.pl

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