# # This is the original copyright statement. I have relied on Chad's module # extensively for this module. # # Copyright (c) 1997-2001 bioperl, Chad Matsalla. All Rights Reserved. # This module is free software; you can redistribute it and/or # modify it under the same terms as Perl itself. # # Copyright Chad Matsalla # # You may distribute this module under the same terms as perl itself # POD documentation - main docs before the code # # But I have modified lots of it, so I guess I should add: # # Copyright (c) 2003 bioperl, Rob Edwards. All Rights Reserved. # This module is free software; you can redistribute it and/or # modify it under the same terms as Perl itself. # # Copyright Rob Edwards # # You may distribute this module under the same terms as perl itself # POD documentation - main docs before the code =head1 NAME Bio::Tools::Run::Primer3 - Create input for and work with the output from the program primer3 =head1 SYNOPSIS # design some primers. # the output will be put into temp.out use Bio::Tools::Run::Primer3; use Bio::SeqIO; my $seqio=Bio::SeqIO->new(-file=>'data/dna1.fa'); my $seq=$seqio->next_seq; my $primer3 = Bio::Tools::Run::Primer3->new(-seq=>$seq, -outfile=>"temp.out"); unless ($primer3->executable) {print STDERR "primer3 can not be found. Is it installed?\n"; exit(-1)} # what are the arguments, and what do they mean? my $args=$primer3->arguments; print "ARGUMENT\tMEANING\n"; foreach my $key (keys %{$args}) {print "$key\t", $$args{$key}, "\n"} # set the maximum and minimum Tm of the primer $primer3->add_targets('PRIMER_MIN_TM'=>56, 'PRIMER_MAX_TM'=>90); # design the primers. This runs primer3 and returns a # Bio::Tools::Run::Primer3 object with the results $results=$primer3->run; # see the Bio::Tools::Run::Primer3 pod for # things that you can get from this. For example: print "There were ", $results->number_of_results, " primers\n"; =head1 DESCRIPTION Bio::Tools::Primer3 creates the input files needed to design primers using primer3 and provides mechanisms to access data in the primer3 output files. This module provides a bioperl interface to the program primer3. See http://www-genome.wi.mit.edu/genome_software/other/primer3.html for details and to download the software. This module is based on one written by Chad Matsalla (bioinformatics1@dieselwurks.com) I have ripped some of his code, and added a lot of my own. I hope he's not mad at me! The original version was designed to work with PrimedSeq and Bio::SeqFeature::Primer. I will try and include those compatibilities too, but as of this writing neither are fully developed, so we'll see how far we get. =head1 FEEDBACK =head2 Mailing Lists User feedback is an integral part of the evolution of this and other Bioperl modules. Send your comments and suggestions preferably to one of the Bioperl mailing lists. Your participation is much appreciated. bioperl-l@bioperl.org - General discussion http://www.bioperl.org/MailList.html - About the mailing lists =head2 Reporting Bugs Report bugs to the Bioperl bug tracking system to help us keep track the bugs and their resolution. Bug reports can be submitted via email or the web: bioperl-bugs@bio.perl.org http://bugzilla.bioperl.org/ =head1 AUTHOR - Rob Edwards redwards@utmem.edu Based heavily on work of Chad Matsalla bioinformatics1@dieselwurks.com =head1 CONTRIBUTORS Shawn Hoon shawnh@fugu-sg.org =head1 APPENDIX The rest of the documentation details each of the object methods. Internal methods are usually preceded with a _ =cut # Let the code begin... package Bio::Tools::Run::Primer3; use vars qw(@ISA); use strict; use Bio::Root::Root; use Bio::Tools::Primer3; use Bio::Tools::Run::WrapperBase; use vars qw($AUTOLOAD @ISA @PRIMER3_PARAMS %OK_FIELD); @ISA = qw(Bio::Root::Root Bio::Tools::Run::WrapperBase); BEGIN { @PRIMER3_PARAMS=qw( PROGRAM EXCLUDED_REGION INCLUDED_REGION PRIMER_COMMENT PRIMER_DNA_CONC PRIMER_EXPLAIN_FLAG PRIMER_FILE_FLAG PRIMER_FIRST_BASE_INDEX PRIMER_GC_CLAMP PRIMER_INTERNAL_OLIGO_DNA_CONC PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION PRIMER_INTERNAL_OLIGO_INPUT PRIMER_INTERNAL_OLIGO_MAX_GC PRIMER_INTERNAL_OLIGO_MAX_MISHYB PRIMER_INTERNAL_OLIGO_MAX_POLY_X PRIMER_INTERNAL_OLIGO_MAX_SIZE PRIMER_INTERNAL_OLIGO_MAX_TM PRIMER_INTERNAL_OLIGO_MIN_GC PRIMER_INTERNAL_OLIGO_MIN_QUALITY PRIMER_INTERNAL_OLIGO_MIN_SIZE PRIMER_INTERNAL_OLIGO_MIN_TM PRIMER_INTERNAL_OLIGO_MISHYB_LIBRARY PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT PRIMER_INTERNAL_OLIGO_OPT_SIZE PRIMER_INTERNAL_OLIGO_OPT_TM PRIMER_INTERNAL_OLIGO_SALT_CONC PRIMER_INTERNAL_OLIGO_SELF_ANY PRIMER_INTERNAL_OLIGO_SELF_END PRIMER_IO_WT_COMPL_ANY PRIMER_IO_WT_COMPL_END PRIMER_IO_WT_END_QUAL PRIMER_IO_WT_GC_PERCENT_GT PRIMER_IO_WT_GC_PERCENT_LT PRIMER_IO_WT_NUM_NS PRIMER_IO_WT_REP_SIM PRIMER_IO_WT_SEQ_QUAL PRIMER_IO_WT_SIZE_GT PRIMER_IO_WT_SIZE_LT PRIMER_IO_WT_TM_GT PRIMER_IO_WT_TM_LT PRIMER_LEFT_INPUT PRIMER_LIBERAL_BASE PRIMER_MAX_DIFF_TM PRIMER_MAX_END_STABILITY PRIMER_MAX_GC PRIMER_MAX_MISPRIMING PRIMER_MAX_POLY_X PRIMER_MAX_SIZE PRIMER_MAX_TM PRIMER_MIN_END_QUALITY PRIMER_MIN_GC PRIMER_MIN_QUALITY PRIMER_MIN_SIZE PRIMER_MIN_TM PRIMER_MISPRIMING_LIBRARY PRIMER_NUM_NS_ACCEPTED PRIMER_NUM_RETURN PRIMER_OPT_GC_PERCENT PRIMER_OPT_SIZE PRIMER_OPT_TM PRIMER_PAIR_MAX_MISPRIMING PRIMER_PAIR_WT_COMPL_ANY PRIMER_PAIR_WT_COMPL_END PRIMER_PAIR_WT_DIFF_TM PRIMER_PAIR_WT_IO_PENALTY PRIMER_PAIR_WT_PRODUCT_SIZE_GT PRIMER_PAIR_WT_PRODUCT_SIZE_LT PRIMER_PAIR_WT_PRODUCT_TM_GT PRIMER_PAIR_WT_PRODUCT_TM_LT PRIMER_PAIR_WT_PR_PENALTY PRIMER_PAIR_WT_REP_SIM PRIMER_PICK_ANYWAY PRIMER_PICK_INTERNAL_OLIGO PRIMER_PRODUCT_MAX_TM PRIMER_PRODUCT_MIN_TM PRIMER_PRODUCT_OPT_SIZE PRIMER_PRODUCT_OPT_TM PRIMER_PRODUCT_SIZE_RANGE PRIMER_QUALITY_RANGE_MAX PRIMER_QUALITY_RANGE_MIN PRIMER_RIGHT_INPUT PRIMER_SALT_CONC PRIMER_SELF_ANY PRIMER_SELF_END PRIMER_SEQUENCE_ID PRIMER_SEQUENCE_QUALITY PRIMER_START_CODON_POSITION PRIMER_TASK PRIMER_WT_COMPL_ANY PRIMER_WT_COMPL_END PRIMER_WT_END_QUAL PRIMER_WT_END_STABILITY PRIMER_WT_GC_PERCENT_GT PRIMER_WT_GC_PERCENT_LT PRIMER_WT_NUM_NS PRIMER_WT_POS_PENALTY PRIMER_WT_REP_SIM PRIMER_WT_SEQ_QUAL PRIMER_WT_SIZE_GT PRIMER_WT_SIZE_LT PRIMER_WT_TM_GT PRIMER_WT_TM_LT SEQUENCE TARGET ); foreach my $attr (@PRIMER3_PARAMS) {$OK_FIELD{$attr}++} } sub AUTOLOAD { my $self = shift; my $attr = $AUTOLOAD; $attr =~ s/.*:://; $attr = uc $attr; $self->throw("Unallowed parameter: $attr !") unless $OK_FIELD{$attr}; $self->{$attr} = shift if @_; return $self->{$attr}; } =head2 new() Title : new() Usage : my $primer3 = Bio::Tools::Primer3->new(-file=>$file) to read a primer3 output file. my $primer3 = Bio::Tools::Primer3->new(-seq=>sequence object) design primers against sequence Function: Start primer3 working and adds a sequence. At the moment it will not clear out the old sequence, but I suppose it should. Returns : Doesn't return anything. If called with a filename will allow you to retrieve the results Args : -seq (optional) Bio::Seq object of sequence. This is required to run primer3 but can be added later with add_targets() -outfile file name to output results to (note can also be added with $primer3->outfile_name -verbose (optional) set verbose output Notes : =cut sub new { my($class,%args) = @_; my $self = $class->SUPER::new(%args); $self->io->_initialize_io(); if ($args{-program}) {$self->{program}=$args{-program}} unless (defined $self->{program}) { $self->{program}=$self->executable; } if ($args{'-verbose'}) {$self->{'verbose'}=1} if ($args{'-seq'}) { $self->{'seqobject'}=$args{'-seq'}; my @input; push (@input, ("PRIMER_SEQUENCE_ID=".$self->{'seqobject'}->id),("SEQUENCE=".$self->{'seqobject'}->seq)); $self->{'primer3_input'}=\@input; } if ($args{'-outfile'}) {$self->{_outfilename}=$args{'-outfile'}} return $self; } =head2 program_name Title : program_name Usage : $primer3->program_name() Function: holds the program name Returns: string Args : None =cut sub program_name { return 'primer3'; } =head2 program_dir Title : program_dir Usage : $factory->program_dir(@params) Function: returns the program directory, obtiained from ENV variable. Returns: string Args : =cut sub program_dir { return Bio::Root::IO->catfile($ENV{PRIMER3}) if $ENV{PRIMER3}; } =head2 add_targets() Title : add_targets() Usage : $primer3->add_targets(key=>value) Function: Add any legal value to the input command line. Returns : Returns the number of arguments added. Args : Use $primer3->arguments to find a list of all the values that are allowed, or see the primer3 docs. Notes : This will only do limited error checking at the moment, but it should work. =cut sub add_targets { my ($self, %args)=@_; my $added_args; # a count of what we have added. my $inputarray = $self->{'primer3_input'}; foreach my $key (keys %args) { # we will allow them to add a sequence before checking for arguments if ((uc($key) eq "-SEQ") || (uc($key) eq "-SEQUENCE")) { # adding a new sequence. We need to separate them with an = $self->{'seqobject'}=$args{$key}; if (defined $$inputarray[0]) {push (@$inputarray, "=")} push (@$inputarray, ("PRIMER_SEQUENCE_ID=".$self->{'seqobject'}->id), ("SEQUENCE=".$self->{'seqobject'}->seq)); next; } unless ($self->{'no_param_checks'}) { unless ($OK_FIELD{$key}) {$self->warn("Parameter $key is not a valid Primer3 parameter"); next} } if (uc($key) eq "INCLUDED_REGION") { # this must be a comma separated start, length. unless ($args{$key} =~ /\,/) { my $sequencelength; # we don't have a length, hence we need to add the length of the sequence less the start. foreach my $input (@$inputarray) { if ($input =~ /SEQUENCE=(.*)/) {$sequencelength=length($1)} } my $length_of_included = $sequencelength-$args{$key}; $args{$key} .= ",".$length_of_included; } } elsif (uc($key) eq "PRIMER_MIN_SIZE") { # minimum size must be less than MAX size and greater than zero if (exists $args{"PRIMER_MAX_SIZE"}) { unless ($args{"PRIMER_MAX_SIZE"} > $args{"PRIMER_MIN_SIZE"}) { $self->warn('Maximum primer size (PRIMER_MAX_SIZE) must be greater than minimum primer size (PRIMER_MIN_SIZE)'); } } if ($args{$key} < 0) { $self->warn('Minimum primer size (PRIMER_MIN_SIZE) must be greater than 0'); } } elsif ($key eq "PRIMER_MAX_SIZE") { if ($args{$key}>35) {$self->warn('Maximum primer size (PRIMER_MAX_SIZE) must be less than 35')} } # need a check to see whether this is already in the array # and finally add the argument to the list. my $toadd=uc($key)."=".$args{$key}; my $replaced; # don't add it if it is replacing something! my @new_array; foreach my $input (@$inputarray) { my ($array_key, $array_value) = split /=/, $input; if (uc($array_key) eq uc($key)) {push @new_array, $toadd; $replaced=1} else {push @new_array, $input} } unless ($replaced) {push @new_array, $toadd} @$inputarray=@new_array; if ($self->{'verbose'}) {print STDERR "Updated ", uc($key), " to $args{$key}\n"} $added_args++; } $self->{'primer3_input'}=$inputarray; return $added_args; } =head2 run() Title : run() Usage : $primer3->run(); Function: Run the primer3 program with the arguments that you have supplied. Returns : A Bio::Tools::Run::Primer3 object containing the results. Args : None. Note : See the Bio::Tools::Primer3 documentation for those functions. =cut sub run { my($self,%args) = @_; my $executable = $self->{program_dir}.$self->{program}; my $input = $self->{'primer3_input'}; unless (-e $executable) { $self->throw("$executable was not found. Do not know where primer3 is!"); exit(-1); } # note that I write this to a temp file because we need both read and write access to primer3, therefore, # we can't use a simple pipe. if ($self->{'verbose'}) {print STDERR "TRYING\n", join "\n", @{$self->{'primer3_input'}}, "=\n"} # make a temporary file and print the instructions to it. my ($temphandle, $tempfile)=$self->io->tempfile; print $temphandle join "\n", @{$self->{'primer3_input'}}, "=\n"; open (RESULTS, "$executable < $tempfile|") || $self->throw("Can't open RESULTS"); if ($self->{'_outfilename'}) { # I can't figure out how to use either of these to write the results out. # neither work, what am I doing wrong or missing in the docs? # $self->{output}=$self->_initialize_io(-file=>$self->{'outfile'}); # $self->{output}=$self->io; # OK, for now, I will just do it myself, because I need this to check the parser :) open (OUT, ">".$self->{'_outfilename'}) || $self->throw("Can't open ".$self->{'_outfilename'}." for writing"); } my @results; while () { if ($self->{'_outfilename'}) { # this should work, but isn't #$self->{output}->_print($_); print OUT $_; } chomp; my ($return, $value) = split /=/; $self->{'results'}->{$return} = $value; } close RESULTS; #clean up the output file if ($self->{'_outfilename'}) { # $self->{output}->close(); close OUT; } $self->cleanup; # convert the results to individual results $self->{results_obj}=new Bio::Tools::Primer3; $self->{results_obj}->_set_variable('results', $self->{results}); $self->{results_obj}->_set_variable('seqobject', $self->{seqobject}); $self->{results_separated}= $self->{results_obj}->_separate(); return $self->{results_obj}; } =head2 arguments() Title : arguments() Usage : $hashref = $primer3->arguments(); Function: Describes the options that you can set through Bio::Tools::Primer3, with a brief (one line) description of what they are and their default values Returns : A string (if an argument is supplied) or a reference to a hash. Args : If supplied with an argument will return a string of its description. If no arguments are supplied, will return all the arguments as a reference to a hash Notes : Much of this is taken from the primer3 README file, and you should read that file for a more detailed description. =cut sub arguments { my ($self, $required) = @_; unless ($self->{'input_options'}) {$self->_input_args} if ($required) {return ${$self->{'input_options'}}{'$required'}} else {return $self->{'input_options'}} } =head2 _input_args() Title : _input_args() Usage : an internal method to set the input arguments for Primer3 Function: Define a hash with keys for each of the input arguments and values as a short one line description Returns : A reference to a hash. Args : None. Notes : much of this is taken from the primer3 README file, and you should read that file for a more detailed description. =cut sub _input_args { my($self) = shift; # just return functions that we can set and what they are my %hash=( 'PRIMER_SEQUENCE_ID'=>'(string, optional) an id. Optional. Note must be present if PRIMER_FILE_FLAG is set', 'SEQUENCE'=>'(nucleotide sequence, REQUIRED) The sequence itself. Cannot contain newlines', 'INCLUDED_REGION'=>'(interval, optional) Where to pick primers from. In form ,. Based on zero indexing!', 'TARGET'=>'(interval list, default empty) Regions that must be included in the product. The value should be a space-separated list of ,', 'EXCLUDED_REGION'=>'(interval list, default empty) Regions that must NOT be included in the product. The value should be a space-separated list of ,', 'PRIMER_COMMENT'=>'(string) This is ignored, so we will just save, and return it', 'PRIMER_SEQUENCE_QUALITY'=>'(quality list, default empty) A list of space separated integers with one per base. Could adapt a Phred object to this.', 'PRIMER_LEFT_INPUT'=>'(nucleotide sequence, default empty) If you know the left primer sequence, put it here', 'PRIMER_RIGHT_INPUT'=>'(nucleotide sequence, default empty) If you know the right primer sequence, put it here', 'PRIMER_START_CODON_POSITION'=>'(int, default -1000000) Location of known start codons for designing in frame primers.', 'PRIMER_PICK_ANYWAY'=>'boolean, default 0) Pick a primer, even if we have violated some constraints.', 'PRIMER_MISPRIMING_LIBRARY'=>'(string, optional) A file containing sequences to avoid amplifying. Should be fasta format, but see primer3 docs for constraints.', 'PRIMER_MAX_MISPRIMING'=>'(decimal,9999.99, default 12.00) Weighting for PRIMER_MISPRIMING_LIBRARY', 'PRIMER_PAIR_MAX_MISPRIMING'=>'(decimal,9999.99, default 24.00 Weighting for PRIMER_MISPRIMING_LIBRARY', 'PRIMER_PRODUCT_MAX_TM'=>'(float, default 1000000.0) The maximum allowed Tm of the product.', 'PRIMER_PRODUCT_MIN_TM'=>'(float, default -1000000.0) The minimum allowed Tm of the product', 'PRIMER_EXPLAIN_FLAG'=>'(boolean, default 0) If set it will print a bunch of information out.', 'PRIMER_PRODUCT_SIZE_RANGE'=>'(size range list, default 100-300) space separated list of product sizes eg - -', 'PRIMER_PICK_INTERNAL_OLIGO'=>'(boolean, default 0) if set, a hybridization probe will be selected', 'PRIMER_GC_CLAMP'=>'(int, default 0) Number of Gs and Cs at the 3 prime end.', 'PRIMER_OPT_SIZE'=>'(int, default 20) Optimal primer size. Primers will be close to this value in length', 'PRIMER_MIN_SIZE'=>'(int, default 18) Minimum size. Must be 0 < PRIMER_MIN_SIZE < PRIMER_MAX_SIZE ', 'PRIMER_MAX_SIZE'=>'(int, default 27) Maximum size. Must be < 35.', 'PRIMER_OPT_TM'=>'(float, default 60.0C) Optimum Tm of a primer.', 'PRIMER_MIN_TM'=>'(float, default 57.0C) Minimum Tm of a primer', 'PRIMER_MAX_TM'=>'(float, default 63.0C) Maximum Tm of a primer', 'PRIMER_MAX_DIFF_TM'=>'(float, default 100.0C) acceptable difference in Tms', 'PRIMER_MIN_GC'=>'(float, default 20.0%) Minimum allowable GCs', 'PRIMER_OPT_GC_PERCENT'=>'(float, default 50.0%) Optimal GCs', 'PRIMER_MAX_GC'=>'(float, default 80.0%) Maximum allowable GCs', 'PRIMER_SALT_CONC'=>'(float, default 50.0 mM) Salt concentration required for Tm calcs.', 'PRIMER_DNA_CONC'=>'(float, default 50.0 nM) DNA concentration required for Tm calcs. ', 'PRIMER_NUM_NS_ACCEPTED'=>'(int, default 0) Maximum number of unknown bases (N) allowable in any primer.', 'PRIMER_SELF_ANY'=>'(decimal,9999.99, default 8.00) Maximum aligment score for within and between primers when checking for hairpin loops', 'PRIMER_SELF_END'=>'(decimal 9999.99, default 3.00) Maximum aligment score for within and between primers when checking for primer dimers', 'PRIMER_FILE_FLAG'=>'(boolean, default 0) Output .for and .rev with all acceptable forward and reverse primers', 'PRIMER_MAX_POLY_X'=>'(int, default 5) The maximum allowable length of a mononucleotide repeat.', 'PRIMER_LIBERAL_BASE'=>'(boolean, default 0) Use IUPAC codes (well, just change them to N). Note must also set PRIMER_NUM_NS_ACCEPTED', 'PRIMER_NUM_RETURN'=>'(int, default 5) Number of primers to return', 'PRIMER_FIRST_BASE_INDEX'=>'(int, default 0) Index of the first base. Do not change this or allow it to be changed, as we will have to mess with subseqs and whatnot.', 'PRIMER_MIN_QUALITY'=>'(int, default 0) Minimum sequence quality calculated from PRIMER_SEQUENCE_QUALITY', 'PRIMER_MIN_END_QUALITY'=>'(int, default 0) Minimum sequence quality calculated from PRIMER_SEQUENCE_QUALITY at 5 prime 5 bases', 'PRIMER_QUALITY_RANGE_MIN'=>'(int, default 0) Minimum sequence quality calculated from PRIMER_SEQUENCE_QUALITY', 'PRIMER_QUALITY_RANGE_MAX'=>'(int, default 100) Maximum sequence quality calculated from PRIMER_SEQUENCE_QUALITY', 'PRIMER_MAX_END_STABILITY'=>'(float 999.9999, default 100.0) Maximum stability for the five 3 prime bases of a primer. Bigger numbers mean more stable 3 prime ends.', 'PRIMER_PRODUCT_OPT_TM'=>'(float, default 0.0) Optimum melting temperature for the PCR product. 0 means no optimum.', 'PRIMER_PRODUCT_OPT_SIZE'=>'(int, default 0) Optimum size for the PCR product. 0 means no optimum.', 'PRIMER_TASK'=>'(string, default pick_pcr_primers) Choose from pick_pcr_primers, pick_pcr_primers_and_hyb_probe, pick_left_only, pick_right_only, pick_hyb_probe_only', 'PRIMER_WT_TM_GT'=>'(float, default 1.0) Penalty weight for primers with Tm over PRIMER_OPT_TM.', 'PRIMER_WT_TM_LT'=>'(float, default 1.0) Penalty weight for primers with Tm under PRIMER_OPT_TM.', 'PRIMER_WT_SIZE_LT'=>'(float, default 1.0) Penalty weight for primers shorter than PRIMER_OPT_SIZE.', 'PRIMER_WT_SIZE_GT'=>'(float, default 1.0) Penalty weight for primers longer than PRIMER_OPT_SIZE.', 'PRIMER_WT_GC_PERCENT_LT'=>'(float, default 1.0) Penalty weight for primers with GC percent greater than PRIMER_OPT_GC_PERCENT.', 'PRIMER_WT_GC_PERCENT_GT'=>'(float, default 1.0) Penalty weight for primers with GC percent greater than PRIMER_OPT_GC_PERCENT.', 'PRIMER_WT_COMPL_ANY'=>'(float, default 0.0)', 'PRIMER_WT_COMPL_END'=>'(float, default 0.0)', 'PRIMER_WT_NUM_NS'=>'(float, default 0.0)', 'PRIMER_WT_REP_SIM'=>'(float, default 0.0)', 'PRIMER_WT_SEQ_QUAL'=>'(float, default 0.0)', 'PRIMER_WT_END_QUAL'=>'(float, default 0.0)', 'PRIMER_WT_POS_PENALTY'=>'(float, default 0.0)', 'PRIMER_WT_END_STABILITY'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_PR_PENALTY'=>'(float, default 1.0)', 'PRIMER_PAIR_WT_IO_PENALTY'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_DIFF_TM'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_COMPL_ANY'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_COMPL_END'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_PRODUCT_TM_LT'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_PRODUCT_TM_GT'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_PRODUCT_SIZE_GT'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_PRODUCT_SIZE_LT'=>'(float, default 0.0)', 'PRIMER_PAIR_WT_REP_SIM'=>'(float, default 0.0)', 'PRIMER_INTERNAL_OLIGO_EXCLUDED_REGION'=>'(interval list, default empty) Internal oligos must ignore these regions', 'PRIMER_INTERNAL_OLIGO_INPUT'=>'(nucleotide sequence, default empty) Known sequence of an internal oligo', 'PRIMER_INTERNAL_OLIGO_OPT_SIZE'=>'(int, default 20)', 'PRIMER_INTERNAL_OLIGO_MIN_SIZE'=>'(int, default 18)', 'PRIMER_INTERNAL_OLIGO_MAX_SIZE'=>'(int, default 27)', 'PRIMER_INTERNAL_OLIGO_OPT_TM'=>'(float, default 60.0 degrees C)', 'PRIMER_INTERNAL_OLIGO_OPT_GC_PERCENT'=>'(float, default 50.0%)', 'PRIMER_INTERNAL_OLIGO_MIN_TM'=>'(float, default 57.0 degrees C)', 'PRIMER_INTERNAL_OLIGO_MAX_TM'=>'(float, default 63.0 degrees C)', 'PRIMER_INTERNAL_OLIGO_MIN_GC'=>'(float, default 20.0%)', 'PRIMER_INTERNAL_OLIGO_MAX_GC'=>'(float, default 80.0%)', 'PRIMER_INTERNAL_OLIGO_SALT_CONC'=>'(float, default 50.0 mM)', 'PRIMER_INTERNAL_OLIGO_DNA_CONC'=>'(float, default 50.0 nM)', 'PRIMER_INTERNAL_OLIGO_SELF_ANY'=>'(decimal 9999.99, default 12.00)', 'PRIMER_INTERNAL_OLIGO_MAX_POLY_X'=>'(int, default 5)', 'PRIMER_INTERNAL_OLIGO_SELF_END'=>'(decimal 9999.99, default 12.00)', 'PRIMER_INTERNAL_OLIGO_MISHYB_LIBRARY'=>'(string, optional)', 'PRIMER_INTERNAL_OLIGO_MAX_MISHYB'=>'(decimal,9999.99, default 12.00)', 'PRIMER_INTERNAL_OLIGO_MIN_QUALITY'=>'(int, default 0)', 'PRIMER_IO_WT_TM_GT'=>'(float, default 1.0)', 'PRIMER_IO_WT_TM_LT'=>'(float, default 1.0)', 'PRIMER_IO_WT_GC_PERCENT_GT'=>'(float, default 1.0)', 'PRIMER_IO_WT_GC_PERCENT_LT'=>'(float, default 1.0)', 'PRIMER_IO_WT_SIZE_LT'=>'(float, default 1.0)', 'PRIMER_IO_WT_SIZE_GT'=>'(float, default 1.0)', 'PRIMER_IO_WT_COMPL_ANY'=>'(float, default 0.0)', 'PRIMER_IO_WT_COMPL_END'=>'(float, default 0.0)', 'PRIMER_IO_WT_NUM_NS'=>'(float, default 0.0)', 'PRIMER_IO_WT_REP_SIM'=>'(float, default 0.0)', 'PRIMER_IO_WT_SEQ_QUAL'=>'(float, default 0.0)', 'PRIMER_IO_WT_END_QUAL'=>'(float, default 0.0)'); $self->{'input_options'}=\%hash; return \%hash; } ############################################################################################################# # COMMENTS # # # # These comments are taken from the primer3 README file. They have an abbreviated explanation of what's # # what. You should read the readme file for more information. # # # # I have used this list to generate defaults, and handle output from primer. # # # # Note that I have ignored some experimental and/or deprecated things. # # # # Rob Edwards 3/15/03 # # # # # ############################################################################################################# # OUTPUT TAGS #each tage has PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO,PAIR}__ # PRIMER_ERROR (string) describes user-correctible errors detected in the input. Absent if no errors. # PRIMER_LEFT (start, length) The selected left primer. Start is the 0-based index of the FIRST base, length its length. # PRIMER_RIGHT (start, length) The selected right primer. Start is the 0-based index of the LAST base, length its length. # PRIMER_INTERNAL_OLIGO (start, length) The selected internal oligo.Start is the 0-based index of start base of the primer. # PRIMER_PRODUCT_SIZE (integer) The product size of the PCR product. # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_EXPLAIN (string) String containing statistics on the possiblities that primer3 considered in selecting a single oligo. # PRIMER_PAIR_EXPLAIN (string) String containing statistics on picking a primer pair (plus internal oligo if requested). # PRIMER_PAIR_PENALTY (float) The score for this pair (lower is better). # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_PENALTY (float) The score for the primer selected (lower is better). # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_SEQUENCE (string) The sequence of the oligo. All seqs are 5'->3' # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_TM (float) The Tm for the selected oligo. # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_GC_PERCENT (float) The percent GC for the selected oligo # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_SELF_ANY (float) Score of complimentarity for the oligo # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_SELF_END (float) Score of complimentarity for the oligo # PRIMER_PAIR_COMPL_ANY (float) Score of complimentarity for the pair # PRIMER_PAIR_COMPL_END (float) Score of complimentarity for the pair # PRIMER_WARNING (string) Warnings generated by primer (separated by semicolons); # PRIMER_{LEFT,RIGHT,PAIR}_MISPRIMING_SCORE (float, string) Maximum mispriming score for the primer, string is the id of corresponding library sequence. # PRIMER_PRODUCT_TM (float) Tm of the product # PRIMER_PRODUCT_TM_OLIGO_TM_DIFF (float) Tm difference # PRIMER_PAIR_T_OPT_A (float) # PRIMER_INTERNAL_OLIGO_MISHYB_SCORE (float, string) Maximum mishybridization score and id of the sequence in MISHYB_LIBRARY # PRIMER_{LEFT,RIGHT,INTERNAL_OLIGO}_MIN_SEQ_QUALITY (int) Minimum SEQUENCE quality from (e.g. phred scores) # PRIMER_{LEFT,RIGHT}_END_STABILITY (float) Delta G of disruption of the five 3' bases of the oligo. # PRIMER_STOP_CODON_POSITION (int) Position of the first base of the stop codon, if Primer3 found one, or -1 if Primer3 did not. # PRIMER3 EXIT STATUS CODES # 0 on normal operation # -1 under the following conditions: # illegal command-line arguments. # unable to fflush stdout. # unable to open (for writing and creating) a .for, .rev # or .int file (probably due to a protection problem). # -2 on out-of-memory # -3 empty input # -4 error in a "Global" input tag (message in PRIMER_ERROR). 1;