lib/SGN/Controller/Primer3.pm

   1 package SGN::Controller::Primer3;
   2
   3 =head1 NAME
   4
   5 Primer3 picks primers for PCR reactions, considering as criteria: oligonucleotide melting temperature, size, GC content, primer-dimer possibilities, PCR product size, positional contraints within the source (template) sequence, possibilities for ectopic priming (amplifying the wrong sequence), and many other contraints.
   6
   7 =cut
   8
   9 use Moose;
  10 use namespace::autoclean;
  11 use YAML::Any qw/LoadFile/;
  12
  13 use URI::FromHash 'uri';
  14 use List::Compare;
  15 use File::Temp qw / tempfile /;
  16 use File::Slurp;
  17
  18 use DBI;
  19 use HTML::FormFu;
  20
  21 BEGIN { extends 'Catalyst::Controller' }
  22
  23 =head1 PUBLIC ACTIONS
  24
  25 =head2 index
  26
  27 Public path: /primer3/create
  28
  29 Display a Primer3 input form.
  30
  31 =cut
  32
  33 sub create :Path('/primer3/') :Args(0) {
  34     my ( $self, $c ) = @_;
  35         my $form = HTML::FormFu->new(LoadFile($c->path_to(qw{forms primer3 formfu_create.yaml})));
  36
  37     # Set the template
  38     $c->stash(template => 'primer3/primer3form.mas',
  39         form => $form);
  40 }
  41
  42
  43 =head2
  44
  45 Submit input and return results.
  46
  47 =cut
  48
  49 sub update :Path('/primer3/update') :Args(0) {
  50         my ($self, $c) = @_;
  51
  52         # Get all parameters from the form and store them in their respective variables.
  53         my $SEQUENCE_EXCLUDED_REGION = $c->request->params->{SEQUENCE_EXCLUDED_REGION};
  54         my $SEQUENCE_INCLUDED_REGION = $c->request->params->{SEQUENCE_INCLUDED_REGION};
  55         my $SEQUENCE_PRIMER_REVCOMP = $c->request->params->{SEQUENCE_PRIMER_REVCOMP};
  56         my $SEQUENCE_FORCE_LEFT_END = $c->request->params->{SEQUENCE_FORCE_LEFT_END};
  57         my $SEQUENCE_INTERNAL_EXCLUDED_REGION = $c->request->params->{SEQUENCE_INTERNAL_EXCLUDED_REGION};
  58         my $SEQUENCE_QUALITY = $c->request->params->{SEQUENCE_QUALITY};
  59         my $SEQUENCE_FORCE_LEFT_START = $c->request->params->{SEQUENCE_FORCE_LEFT_START};
  60         my $SEQUENCE_INTERNAL_OLIGO = $c->request->params->{SEQUENCE_INTERNAL_OLIGO};
  61         my $SEQUENCE_START_CODON_POSITION = $c->request->params->{SEQUENCE_START_CODON_POSITION};
  62         my $SEQUENCE_FORCE_RIGHT_END = $c->request->params->{SEQUENCE_FORCE_RIGHT_END};
  63         my $SEQUENCE_OVERLAP_JUNCTION_LIST = $c->request->params->{SEQUENCE_OVERLAP_JUNCTION_LIST};
  64         my $SEQUENCE_TARGET = $c->request->params->{SEQUENCE_TARGET};
  65         my $SEQUENCE_FORCE_RIGHT_START = $c->request->params->{SEQUENCE_FORCE_RIGHT_START};
  66         my $SEQUENCE_PRIMER = $c->request->params->{SEQUENCE_PRIMER};
  67         my $SEQUENCE_TEMPLATE = $c->request->params->{SEQUENCE_TEMPLATE};
  68         my $SEQUENCE_ID = $c->request->params->{SEQUENCE_ID};
  69         my $SEQUENCE_PRIMER_PAIR_OK_REGION_LIST = $c->request->params->{SEQUENCE_PRIMER_PAIR_OK_REGION_LIST};
  70         my $PRIMER_DNA_CONC = $c->request->params->{PRIMER_DNA_CONC};
  71         my $PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS = $c->request->params->{PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS};
  72         my $PRIMER_PAIR_WT_PRODUCT_SIZE_GT = $c->request->params->{PRIMER_PAIR_WT_PRODUCT_SIZE_GT};
  73         my $PRIMER_DNTP_CONC = $c->request->params->{PRIMER_DNTP_CONC};
  74         my $PRIMER_LOWERCASE_MASKING = $c->request->params->{PRIMER_LOWERCASE_MASKING};
  75         my $PRIMER_PAIR_WT_PRODUCT_SIZE_LT = $c->request->params->{PRIMER_PAIR_WT_PRODUCT_SIZE_LT};
  76         my $PRIMER_EXPLAIN_FLAG = $c->request->params->{PRIMER_EXPLAIN_FLAG};
  77         my $PRIMER_MAX_END_GC = $c->request->params->{PRIMER_MAX_END_GC};
  78         my $PRIMER_PAIR_WT_PRODUCT_TM_GT = $c->request->params->{PRIMER_PAIR_WT_PRODUCT_TM_GT};
  79         my $PRIMER_FIRST_BASE_INDEX = $c->request->params->{PRIMER_FIRST_BASE_INDEX};
  80         my $PRIMER_MAX_END_STABILITY = $c->request->params->{PRIMER_MAX_END_STABILITY};
  81         my $PRIMER_PAIR_WT_PRODUCT_TM_LT = $c->request->params->{PRIMER_PAIR_WT_PRODUCT_TM_LT};
  82         my $PRIMER_GC_CLAMP = $c->request->params->{PRIMER_GC_CLAMP};
  83         my $PRIMER_MAX_GC = $c->request->params->{PRIMER_MAX_GC};
  84         my $PRIMER_PAIR_WT_PR_PENALTY = $c->request->params->{PRIMER_PAIR_WT_PR_PENALTY};
  85         my $PRIMER_INSIDE_PENALTY = $c->request->params->{PRIMER_INSIDE_PENALTY};
  86         my $PRIMER_MAX_HAIRPIN_TH = $c->request->params->{PRIMER_MAX_HAIRPIN_TH};
  87         my $PRIMER_PAIR_WT_TEMPLATE_MISPRIMING = $c->request->params->{PRIMER_PAIR_WT_TEMPLATE_MISPRIMING};
  88         my $PRIMER_INTERNAL_DNA_CONC = $c->request->params->{PRIMER_INTERNAL_DNA_CONC};
  89         my $PRIMER_MAX_LIBRARY_MISPRIMING = $c->request->params->{PRIMER_MAX_LIBRARY_MISPRIMING};
  90         my $PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH = $c->request->params->{PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH};
  91         my $PRIMER_INTERNAL_DNTP_CONC = $c->request->params->{PRIMER_INTERNAL_DNTP_CONC};
  92         my $PRIMER_MAX_NS_ACCEPTED = $c->request->params->{PRIMER_MAX_NS_ACCEPTED};
  93         my $PRIMER_PICK_ANYWAY = $c->request->params->{PRIMER_PICK_ANYWAY};
  94         my $PRIMER_INTERNAL_MAX_GC = $c->request->params->{PRIMER_INTERNAL_MAX_GC};
  95         my $PRIMER_MAX_POLY_X = $c->request->params->{PRIMER_MAX_POLY_X};
  96         my $PRIMER_PICK_INTERNAL_OLIGO = $c->request->params->{PRIMER_PICK_INTERNAL_OLIGO};
  97         my $PRIMER_INTERNAL_MAX_HAIRPIN_TH = $c->request->params->{PRIMER_INTERNAL_MAX_HAIRPIN_TH};
  98         my $PRIMER_MAX_SELF_ANY = $c->request->params->{PRIMER_MAX_SELF_ANY};
  99         my $PRIMER_PICK_LEFT_PRIMER = $c->request->params->{PRIMER_PICK_LEFT_PRIMER};
 100         my $PRIMER_INTERNAL_MAX_LIBRARY_MISHYB = $c->request->params->{PRIMER_INTERNAL_MAX_LIBRARY_MISHYB};
 101         my $PRIMER_MAX_SELF_ANY_TH = $c->request->params->{PRIMER_MAX_SELF_ANY_TH};
 102         my $PRIMER_PICK_RIGHT_PRIMER = $c->request->params->{PRIMER_PICK_RIGHT_PRIMER};
 103         my $PRIMER_INTERNAL_MAX_NS_ACCEPTED = $c->request->params->{PRIMER_INTERNAL_MAX_NS_ACCEPTED};
 104         my $PRIMER_MAX_SELF_END = $c->request->params->{PRIMER_MAX_SELF_END};
 105         my $PRIMER_PRODUCT_MAX_TM = $c->request->params->{PRIMER_PRODUCT_MAX_TM};
 106         my $PRIMER_INTERNAL_MAX_POLY_X = $c->request->params->{PRIMER_INTERNAL_MAX_POLY_X};
 107         my $PRIMER_MAX_SELF_END_TH = $c->request->params->{PRIMER_MAX_SELF_END_TH};
 108         my $PRIMER_PRODUCT_MIN_TM = $c->request->params->{PRIMER_PRODUCT_MIN_TM};
 109         my $PRIMER_INTERNAL_MAX_SELF_ANY = $c->request->params->{PRIMER_INTERNAL_MAX_SELF_ANY};
 110         my $PRIMER_MAX_SIZE = $c->request->params->{PRIMER_MAX_SIZE};
 111         my $PRIMER_PRODUCT_OPT_SIZE = $c->request->params->{PRIMER_PRODUCT_OPT_SIZE};
 112         my $PRIMER_INTERNAL_MAX_SELF_ANY_TH = $c->request->params->{PRIMER_INTERNAL_MAX_SELF_ANY_TH};
 113         my $PRIMER_MAX_TEMPLATE_MISPRIMING = $c->request->params->{PRIMER_MAX_TEMPLATE_MISPRIMING};
 114         my $PRIMER_PRODUCT_OPT_TM = $c->request->params->{PRIMER_PRODUCT_OPT_TM};
 115         my $PRIMER_INTERNAL_MAX_SELF_END = $c->request->params->{PRIMER_INTERNAL_MAX_SELF_END};
 116         my $PRIMER_MAX_TEMPLATE_MISPRIMING_TH = $c->request->params->{PRIMER_MAX_TEMPLATE_MISPRIMING_TH};
 117         my $PRIMER_PRODUCT_SIZE_RANGE = $c->request->params->{PRIMER_PRODUCT_SIZE_RANGE};
 118         my $PRIMER_INTERNAL_MAX_SELF_END_TH = $c->request->params->{PRIMER_INTERNAL_MAX_SELF_END_TH};
 119         my $PRIMER_MAX_TM = $c->request->params->{PRIMER_MAX_TM};
 120         my $PRIMER_QUALITY_RANGE_MAX = $c->request->params->{PRIMER_QUALITY_RANGE_MAX};
 121         my $PRIMER_INTERNAL_MAX_SIZE = $c->request->params->{PRIMER_INTERNAL_MAX_SIZE};
 122         my $PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION = $c->request->params->{PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION};
 123         my $PRIMER_QUALITY_RANGE_MIN = $c->request->params->{PRIMER_QUALITY_RANGE_MIN};
 124         my $PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION = $c->request->params->{PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION};
 125         my $PRIMER_SALT_CORRECTIONS = $c->request->params->{PRIMER_SALT_CORRECTIONS};
 126         my $PRIMER_MIN_END_QUALITY = $c->request->params->{PRIMER_MIN_END_QUALITY};
 127         my $PRIMER_SALT_DIVALENT = $c->request->params->{PRIMER_SALT_DIVALENT};
 128         my $PRIMER_INTERNAL_MAX_TM = $c->request->params->{PRIMER_INTERNAL_MAX_TM};
 129         my $PRIMER_MIN_GC = $c->request->params->{PRIMER_MIN_GC};
 130         my $PRIMER_SALT_MONOVALENT = $c->request->params->{PRIMER_SALT_MONOVALENT};
 131         my $PRIMER_INTERNAL_MIN_GC = $c->request->params->{PRIMER_INTERNAL_MIN_GC};
 132         my $PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE = $c->request->params->{PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE};
 133         my $PRIMER_SEQUENCING_ACCURACY = $c->request->params->{PRIMER_SEQUENCING_ACCURACY};
 134         my $PRIMER_INTERNAL_MIN_QUALITY = $c->request->params->{PRIMER_INTERNAL_MIN_QUALITY};
 135         my $PRIMER_MIN_QUALITY = $c->request->params->{PRIMER_MIN_QUALITY};
 136         my $PRIMER_SEQUENCING_INTERVAL = $c->request->params->{PRIMER_SEQUENCING_INTERVAL};
 137         my $PRIMER_INTERNAL_MIN_SIZE = $c->request->params->{PRIMER_INTERNAL_MIN_SIZE};
 138         my $PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE = $c->request->params->{PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE};
 139         my $PRIMER_SEQUENCING_LEAD = $c->request->params->{PRIMER_SEQUENCING_LEAD};
 140         my $PRIMER_INTERNAL_MIN_TM = $c->request->params->{PRIMER_INTERNAL_MIN_TM};
 141         my $PRIMER_MIN_SIZE = $c->request->params->{PRIMER_MIN_SIZE};
 142         my $PRIMER_SEQUENCING_SPACING = $c->request->params->{PRIMER_SEQUENCING_SPACING};
 143         my $PRIMER_INTERNAL_MISHYB_LIBRARY = $c->request->params->{PRIMER_INTERNAL_MISHYB_LIBRARY};
 144         my $PRIMER_MIN_THREE_PRIME_DISTANCE = $c->request->params->{PRIMER_MIN_THREE_PRIME_DISTANCE};
 145         my $PRIMER_TASK = $c->request->params->{PRIMER_TASK};
 146         my $PRIMER_INTERNAL_OPT_GC_PERCENT = $c->request->params->{PRIMER_INTERNAL_OPT_GC_PERCENT};
 147         my $PRIMER_MIN_TM = $c->request->params->{PRIMER_MIN_TM};
 148         my $PRIMER_THERMODYNAMIC_ALIGNMENT = $c->request->params->{PRIMER_THERMODYNAMIC_ALIGNMENT};
 149         my $PRIMER_INTERNAL_OPT_SIZE = $c->request->params->{PRIMER_INTERNAL_OPT_SIZE};
 150         my $PRIMER_MISPRIMING_LIBRARY = $c->request->params->{PRIMER_MISPRIMING_LIBRARY};
 151         my $PRIMER_THERMODYNAMIC_PARAMETERS_PATH = $c->request->params->{PRIMER_THERMODYNAMIC_PARAMETERS_PATH};
 152         my $PRIMER_INTERNAL_OPT_TM = $c->request->params->{PRIMER_INTERNAL_OPT_TM};
 153         my $PRIMER_NUM_RETURN = $c->request->params->{PRIMER_NUM_RETURN};
 154         my $PRIMER_TM_FORMULA = $c->request->params->{PRIMER_TM_FORMULA};
 155         my $PRIMER_INTERNAL_SALT_DIVALENT = $c->request->params->{PRIMER_INTERNAL_SALT_DIVALENT};
 156         my $PRIMER_OPT_GC_PERCENT = $c->request->params->{PRIMER_OPT_GC_PERCENT};
 157         my $PRIMER_WT_END_QUAL = $c->request->params->{PRIMER_WT_END_QUAL};
 158         my $PRIMER_INTERNAL_SALT_MONOVALENT = $c->request->params->{PRIMER_INTERNAL_SALT_MONOVALENT};
 159         my $PRIMER_OPT_SIZE = $c->request->params->{PRIMER_OPT_SIZE};
 160         my $PRIMER_WT_END_STABILITY = $c->request->params->{PRIMER_WT_END_STABILITY};
 161         my $PRIMER_INTERNAL_WT_END_QUAL = $c->request->params->{PRIMER_INTERNAL_WT_END_QUAL};
 162         my $PRIMER_OPT_TM = $c->request->params->{PRIMER_OPT_TM};
 163         my $PRIMER_WT_GC_PERCENT_GT = $c->request->params->{PRIMER_WT_GC_PERCENT_GT};
 164         my $PRIMER_INTERNAL_WT_GC_PERCENT_GT = $c->request->params->{PRIMER_INTERNAL_WT_GC_PERCENT_GT};
 165         my $PRIMER_OUTSIDE_PENALTY = $c->request->params->{PRIMER_OUTSIDE_PENALTY};
 166         my $PRIMER_WT_GC_PERCENT_LT = $c->request->params->{PRIMER_WT_GC_PERCENT_LT};
 167         my $PRIMER_INTERNAL_WT_GC_PERCENT_LT = $c->request->params->{PRIMER_INTERNAL_WT_GC_PERCENT_LT};
 168         my $PRIMER_PAIR_MAX_COMPL_ANY = $c->request->params->{PRIMER_PAIR_MAX_COMPL_ANY};
 169         my $PRIMER_WT_HAIRPIN_TH = $c->request->params->{PRIMER_WT_HAIRPIN_TH};
 170         my $PRIMER_INTERNAL_WT_HAIRPIN_TH = $c->request->params->{PRIMER_INTERNAL_WT_HAIRPIN_TH};
 171         my $PRIMER_PAIR_MAX_COMPL_ANY_TH = $c->request->params->{PRIMER_PAIR_MAX_COMPL_ANY_TH};
 172         my $PRIMER_WT_LIBRARY_MISPRIMING = $c->request->params->{PRIMER_WT_LIBRARY_MISPRIMING};
 173         my $PRIMER_INTERNAL_WT_LIBRARY_MISHYB = $c->request->params->{PRIMER_INTERNAL_WT_LIBRARY_MISHYB};
 174         my $PRIMER_PAIR_MAX_COMPL_END = $c->request->params->{PRIMER_PAIR_MAX_COMPL_END};
 175         my $PRIMER_WT_NUM_NS = $c->request->params->{PRIMER_WT_NUM_NS};
 176         my $PRIMER_INTERNAL_WT_NUM_NS = $c->request->params->{PRIMER_INTERNAL_WT_NUM_NS};
 177         my $PRIMER_PAIR_MAX_COMPL_END_TH = $c->request->params->{PRIMER_PAIR_MAX_COMPL_END_TH};
 178         my $PRIMER_WT_POS_PENALTY = $c->request->params->{PRIMER_WT_POS_PENALTY};
 179         my $PRIMER_INTERNAL_WT_SELF_ANY = $c->request->params->{PRIMER_INTERNAL_WT_SELF_ANY};
 180         my $PRIMER_PAIR_MAX_DIFF_TM = $c->request->params->{PRIMER_PAIR_MAX_DIFF_TM};
 181         my $PRIMER_WT_SELF_ANY = $c->request->params->{PRIMER_WT_SELF_ANY};
 182         my $PRIMER_INTERNAL_WT_SELF_ANY_TH = $c->request->params->{PRIMER_INTERNAL_WT_SELF_ANY_TH};
 183         my $PRIMER_WT_SELF_ANY_TH = $c->request->params->{PRIMER_WT_SELF_ANY_TH};
 184         my $PRIMER_INTERNAL_WT_SELF_END = $c->request->params->{PRIMER_INTERNAL_WT_SELF_END};
 185         my $PRIMER_PAIR_MAX_LIBRARY_MISPRIMING = $c->request->params->{PRIMER_PAIR_MAX_LIBRARY_MISPRIMING};
 186         my $PRIMER_WT_SELF_END = $c->request->params->{PRIMER_WT_SELF_END};
 187         my $PRIMER_INTERNAL_WT_SELF_END_TH = $c->request->params->{PRIMER_INTERNAL_WT_SELF_END_TH};
 188         my $PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING = $c->request->params->{PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING};
 189         my $PRIMER_WT_SELF_END_TH = $c->request->params->{PRIMER_WT_SELF_END_TH};
 190         my $PRIMER_INTERNAL_WT_SEQ_QUAL = $c->request->params->{PRIMER_INTERNAL_WT_SEQ_QUAL};
 191         my $PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH = $c->request->params->{PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH};
 192         my $PRIMER_WT_SEQ_QUAL = $c->request->params->{PRIMER_WT_SEQ_QUAL};
 193         my $PRIMER_INTERNAL_WT_SIZE_GT = $c->request->params->{PRIMER_INTERNAL_WT_SIZE_GT};
 194         my $PRIMER_PAIR_WT_COMPL_ANY = $c->request->params->{PRIMER_PAIR_WT_COMPL_ANY};
 195         my $PRIMER_WT_SIZE_GT = $c->request->params->{PRIMER_WT_SIZE_GT};
 196         my $PRIMER_INTERNAL_WT_SIZE_LT = $c->request->params->{PRIMER_INTERNAL_WT_SIZE_LT};
 197         my $PRIMER_PAIR_WT_COMPL_ANY_TH = $c->request->params->{PRIMER_PAIR_WT_COMPL_ANY_TH};
 198         my $PRIMER_WT_SIZE_LT = $c->request->params->{PRIMER_WT_SIZE_LT};
 199         my $PRIMER_PAIR_WT_COMPL_END = $c->request->params->{PRIMER_PAIR_WT_COMPL_END};
 200         my $PRIMER_WT_TEMPLATE_MISPRIMING = $c->request->params->{PRIMER_WT_TEMPLATE_MISPRIMING};
 201         my $PRIMER_PAIR_WT_COMPL_END_TH = $c->request->params->{PRIMER_PAIR_WT_COMPL_END_TH};
 202         my $PRIMER_WT_TEMPLATE_MISPRIMING_TH = $c->request->params->{PRIMER_WT_TEMPLATE_MISPRIMING_TH};
 203         my $PRIMER_INTERNAL_WT_TM_GT = $c->request->params->{PRIMER_INTERNAL_WT_TM_GT};
 204         my $PRIMER_PAIR_WT_DIFF_TM = $c->request->params->{PRIMER_PAIR_WT_DIFF_TM};
 205         my $PRIMER_WT_TM_GT = $c->request->params->{PRIMER_WT_TM_GT};
 206         my $PRIMER_INTERNAL_WT_TM_LT = $c->request->params->{PRIMER_INTERNAL_WT_TM_LT};
 207         my $PRIMER_PAIR_WT_IO_PENALTY = $c->request->params->{PRIMER_PAIR_WT_IO_PENALTY};
 208         my $PRIMER_WT_TM_LT = $c->request->params->{PRIMER_WT_TM_LT};
 209         my $PRIMER_LIBERAL_BASE = $c->request->params->{PRIMER_LIBERAL_BASE};
 210         my $PRIMER_PAIR_WT_LIBRARY_MISPRIMING = $c->request->params->{PRIMER_PAIR_WT_LIBRARY_MISPRIMING};
 211
 212
 213         # Remove all non-alphabetic characters (makes FASTA input ok)
 214         $SEQUENCE_TEMPLATE =~ s/[^a-zA-Z]*//g;
 215
 216         # Store Boulder-IO formatted input in text variable.
 217
 218         my $text =  qq(SEQUENCE_EXCLUDED_REGION=$SEQUENCE_EXCLUDED_REGION
 219 SEQUENCE_INCLUDED_REGION=$SEQUENCE_INCLUDED_REGION
 220 SEQUENCE_PRIMER_REVCOMP=$SEQUENCE_PRIMER_REVCOMP
 221 SEQUENCE_FORCE_LEFT_END=$SEQUENCE_FORCE_LEFT_END
 222 SEQUENCE_INTERNAL_EXCLUDED_REGION=$SEQUENCE_INTERNAL_EXCLUDED_REGION
 223 SEQUENCE_QUALITY=$SEQUENCE_QUALITY
 224 SEQUENCE_FORCE_LEFT_START=$SEQUENCE_FORCE_LEFT_START
 225 SEQUENCE_INTERNAL_OLIGO=$SEQUENCE_INTERNAL_OLIGO
 226 SEQUENCE_START_CODON_POSITION=$SEQUENCE_START_CODON_POSITION
 227 SEQUENCE_FORCE_RIGHT_END=$SEQUENCE_FORCE_RIGHT_END
 228 SEQUENCE_OVERLAP_JUNCTION_LIST=$SEQUENCE_OVERLAP_JUNCTION_LIST
 229 SEQUENCE_TARGET=$SEQUENCE_TARGET
 230 SEQUENCE_FORCE_RIGHT_START=$SEQUENCE_FORCE_RIGHT_START
 231 SEQUENCE_PRIMER=$SEQUENCE_PRIMER
 232 SEQUENCE_TEMPLATE=$SEQUENCE_TEMPLATE
 233 SEQUENCE_ID=$SEQUENCE_ID
 234 SEQUENCE_PRIMER_PAIR_OK_REGION_LIST=$SEQUENCE_PRIMER_PAIR_OK_REGION_LIST
 235 PRIMER_DNA_CONC=$PRIMER_DNA_CONC
 236 PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS=$PRIMER_LIB_AMBIGUITY_CODES_CONSENSUS
 237 PRIMER_PAIR_WT_PRODUCT_SIZE_GT=$PRIMER_PAIR_WT_PRODUCT_SIZE_GT
 238 PRIMER_DNTP_CONC=$PRIMER_DNTP_CONC
 239 PRIMER_LOWERCASE_MASKING=$PRIMER_LOWERCASE_MASKING
 240 PRIMER_PAIR_WT_PRODUCT_SIZE_LT=$PRIMER_PAIR_WT_PRODUCT_SIZE_LT
 241 PRIMER_EXPLAIN_FLAG=$PRIMER_EXPLAIN_FLAG
 242 PRIMER_MAX_END_GC=$PRIMER_MAX_END_GC
 243 PRIMER_PAIR_WT_PRODUCT_TM_GT=$PRIMER_PAIR_WT_PRODUCT_TM_GT
 244 PRIMER_FIRST_BASE_INDEX=$PRIMER_FIRST_BASE_INDEX
 245 PRIMER_MAX_END_STABILITY=$PRIMER_MAX_END_STABILITY
 246 PRIMER_PAIR_WT_PRODUCT_TM_LT=$PRIMER_PAIR_WT_PRODUCT_TM_LT
 247 PRIMER_GC_CLAMP=$PRIMER_GC_CLAMP
 248 PRIMER_MAX_GC=$PRIMER_MAX_GC
 249 PRIMER_PAIR_WT_PR_PENALTY=$PRIMER_PAIR_WT_PR_PENALTY
 250 PRIMER_INSIDE_PENALTY=$PRIMER_INSIDE_PENALTY
 251 PRIMER_MAX_HAIRPIN_TH=$PRIMER_MAX_HAIRPIN_TH
 252 PRIMER_PAIR_WT_TEMPLATE_MISPRIMING=$PRIMER_PAIR_WT_TEMPLATE_MISPRIMING
 253 PRIMER_INTERNAL_DNA_CONC=$PRIMER_INTERNAL_DNA_CONC
 254 PRIMER_MAX_LIBRARY_MISPRIMING=$PRIMER_MAX_LIBRARY_MISPRIMING
 255 PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH=$PRIMER_PAIR_WT_TEMPLATE_MISPRIMING_TH
 256 PRIMER_INTERNAL_DNTP_CONC=$PRIMER_INTERNAL_DNTP_CONC
 257 PRIMER_MAX_NS_ACCEPTED=$PRIMER_MAX_NS_ACCEPTED
 258 PRIMER_PICK_ANYWAY=$PRIMER_PICK_ANYWAY
 259 PRIMER_INTERNAL_MAX_GC=$PRIMER_INTERNAL_MAX_GC
 260 PRIMER_MAX_POLY_X=$PRIMER_MAX_POLY_X
 261 PRIMER_PICK_INTERNAL_OLIGO=$PRIMER_PICK_INTERNAL_OLIGO
 262 PRIMER_INTERNAL_MAX_HAIRPIN_TH=$PRIMER_INTERNAL_MAX_HAIRPIN_TH
 263 PRIMER_MAX_SELF_ANY=$PRIMER_MAX_SELF_ANY
 264 PRIMER_PICK_LEFT_PRIMER=$PRIMER_PICK_LEFT_PRIMER
 265 PRIMER_INTERNAL_MAX_LIBRARY_MISHYB=$PRIMER_INTERNAL_MAX_LIBRARY_MISHYB
 266 PRIMER_MAX_SELF_ANY_TH=$PRIMER_MAX_SELF_ANY_TH
 267 PRIMER_PICK_RIGHT_PRIMER=$PRIMER_PICK_RIGHT_PRIMER
 268 PRIMER_INTERNAL_MAX_NS_ACCEPTED=$PRIMER_INTERNAL_MAX_NS_ACCEPTED
 269 PRIMER_MAX_SELF_END=$PRIMER_MAX_SELF_END
 270 PRIMER_PRODUCT_MAX_TM=$PRIMER_PRODUCT_MAX_TM
 271 PRIMER_INTERNAL_MAX_POLY_X=$PRIMER_INTERNAL_MAX_POLY_X
 272 PRIMER_MAX_SELF_END_TH=$PRIMER_MAX_SELF_END_TH
 273 PRIMER_PRODUCT_MIN_TM=$PRIMER_PRODUCT_MIN_TM
 274 PRIMER_INTERNAL_MAX_SELF_ANY=$PRIMER_INTERNAL_MAX_SELF_ANY
 275 PRIMER_MAX_SIZE=$PRIMER_MAX_SIZE
 276 PRIMER_PRODUCT_OPT_SIZE=$PRIMER_PRODUCT_OPT_SIZE
 277 PRIMER_INTERNAL_MAX_SELF_ANY_TH=$PRIMER_INTERNAL_MAX_SELF_ANY_TH
 278 PRIMER_MAX_TEMPLATE_MISPRIMING=$PRIMER_MAX_TEMPLATE_MISPRIMING
 279 PRIMER_PRODUCT_OPT_TM=$PRIMER_PRODUCT_OPT_TM
 280 PRIMER_INTERNAL_MAX_SELF_END=$PRIMER_INTERNAL_MAX_SELF_END
 281 PRIMER_MAX_TEMPLATE_MISPRIMING_TH=$PRIMER_MAX_TEMPLATE_MISPRIMING_TH
 282 PRIMER_PRODUCT_SIZE_RANGE=$PRIMER_PRODUCT_SIZE_RANGE
 283 PRIMER_INTERNAL_MAX_SELF_END_TH=$PRIMER_INTERNAL_MAX_SELF_END_TH
 284 PRIMER_MAX_TM=$PRIMER_MAX_TM
 285 PRIMER_QUALITY_RANGE_MAX=$PRIMER_QUALITY_RANGE_MAX
 286 PRIMER_INTERNAL_MAX_SIZE=$PRIMER_INTERNAL_MAX_SIZE
 287 PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION=$PRIMER_MIN_3_PRIME_OVERLAP_OF_JUNCTION
 288 PRIMER_QUALITY_RANGE_MIN=$PRIMER_QUALITY_RANGE_MIN
 289 PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION=$PRIMER_MIN_5_PRIME_OVERLAP_OF_JUNCTION
 290 PRIMER_SALT_CORRECTIONS=$PRIMER_SALT_CORRECTIONS
 291 PRIMER_MIN_END_QUALITY=$PRIMER_MIN_END_QUALITY
 292 PRIMER_SALT_DIVALENT=$PRIMER_SALT_DIVALENT
 293 PRIMER_INTERNAL_MAX_TM=$PRIMER_INTERNAL_MAX_TM
 294 PRIMER_MIN_GC=$PRIMER_MIN_GC
 295 PRIMER_SALT_MONOVALENT=$PRIMER_SALT_MONOVALENT
 296 PRIMER_INTERNAL_MIN_GC=$PRIMER_INTERNAL_MIN_GC
 297 PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE=$PRIMER_MIN_LEFT_THREE_PRIME_DISTANCE
 298 PRIMER_SEQUENCING_ACCURACY=$PRIMER_SEQUENCING_ACCURACY
 299 PRIMER_INTERNAL_MIN_QUALITY=$PRIMER_INTERNAL_MIN_QUALITY
 300 PRIMER_MIN_QUALITY=$PRIMER_MIN_QUALITY
 301 PRIMER_SEQUENCING_INTERVAL=$PRIMER_SEQUENCING_INTERVAL
 302 PRIMER_INTERNAL_MIN_SIZE=$PRIMER_INTERNAL_MIN_SIZE
 303 PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE=$PRIMER_MIN_RIGHT_THREE_PRIME_DISTANCE
 304 PRIMER_SEQUENCING_LEAD=$PRIMER_SEQUENCING_LEAD
 305 PRIMER_INTERNAL_MIN_TM=$PRIMER_INTERNAL_MIN_TM
 306 PRIMER_MIN_SIZE=$PRIMER_MIN_SIZE
 307 PRIMER_SEQUENCING_SPACING=$PRIMER_SEQUENCING_SPACING
 308 PRIMER_INTERNAL_MISHYB_LIBRARY=$PRIMER_INTERNAL_MISHYB_LIBRARY
 309 PRIMER_MIN_THREE_PRIME_DISTANCE=$PRIMER_MIN_THREE_PRIME_DISTANCE
 310 PRIMER_TASK=$PRIMER_TASK
 311 PRIMER_INTERNAL_OPT_GC_PERCENT=$PRIMER_INTERNAL_OPT_GC_PERCENT
 312 PRIMER_MIN_TM=$PRIMER_MIN_TM
 313 PRIMER_THERMODYNAMIC_ALIGNMENT=$PRIMER_THERMODYNAMIC_ALIGNMENT
 314 PRIMER_INTERNAL_OPT_SIZE=$PRIMER_INTERNAL_OPT_SIZE
 315 PRIMER_MISPRIMING_LIBRARY=$PRIMER_MISPRIMING_LIBRARY
 316 PRIMER_THERMODYNAMIC_PARAMETERS_PATH=$PRIMER_THERMODYNAMIC_PARAMETERS_PATH
 317 PRIMER_INTERNAL_OPT_TM=$PRIMER_INTERNAL_OPT_TM
 318 PRIMER_NUM_RETURN=$PRIMER_NUM_RETURN
 319 PRIMER_TM_FORMULA=$PRIMER_TM_FORMULA
 320 PRIMER_INTERNAL_SALT_DIVALENT=$PRIMER_INTERNAL_SALT_DIVALENT
 321 PRIMER_OPT_GC_PERCENT=$PRIMER_OPT_GC_PERCENT
 322 PRIMER_WT_END_QUAL=$PRIMER_WT_END_QUAL
 323 PRIMER_INTERNAL_SALT_MONOVALENT=$PRIMER_INTERNAL_SALT_MONOVALENT
 324 PRIMER_OPT_SIZE=$PRIMER_OPT_SIZE
 325 PRIMER_WT_END_STABILITY=$PRIMER_WT_END_STABILITY
 326 PRIMER_INTERNAL_WT_END_QUAL=$PRIMER_INTERNAL_WT_END_QUAL
 327 PRIMER_OPT_TM=$PRIMER_OPT_TM
 328 PRIMER_WT_GC_PERCENT_GT=$PRIMER_WT_GC_PERCENT_GT
 329 PRIMER_INTERNAL_WT_GC_PERCENT_GT=$PRIMER_INTERNAL_WT_GC_PERCENT_GT
 330 PRIMER_OUTSIDE_PENALTY=$PRIMER_OUTSIDE_PENALTY
 331 PRIMER_WT_GC_PERCENT_LT=$PRIMER_WT_GC_PERCENT_LT
 332 PRIMER_INTERNAL_WT_GC_PERCENT_LT=$PRIMER_INTERNAL_WT_GC_PERCENT_LT
 333 PRIMER_PAIR_MAX_COMPL_ANY=$PRIMER_PAIR_MAX_COMPL_ANY
 334 PRIMER_WT_HAIRPIN_TH=$PRIMER_WT_HAIRPIN_TH
 335 PRIMER_INTERNAL_WT_HAIRPIN_TH=$PRIMER_INTERNAL_WT_HAIRPIN_TH
 336 PRIMER_PAIR_MAX_COMPL_ANY_TH=$PRIMER_PAIR_MAX_COMPL_ANY_TH
 337 PRIMER_WT_LIBRARY_MISPRIMING=$PRIMER_WT_LIBRARY_MISPRIMING
 338 PRIMER_INTERNAL_WT_LIBRARY_MISHYB=$PRIMER_INTERNAL_WT_LIBRARY_MISHYB
 339 PRIMER_PAIR_MAX_COMPL_END=$PRIMER_PAIR_MAX_COMPL_END
 340 PRIMER_WT_NUM_NS=$PRIMER_WT_NUM_NS
 341 PRIMER_INTERNAL_WT_NUM_NS=$PRIMER_INTERNAL_WT_NUM_NS
 342 PRIMER_PAIR_MAX_COMPL_END_TH=$PRIMER_PAIR_MAX_COMPL_END_TH
 343 PRIMER_WT_POS_PENALTY=$PRIMER_WT_POS_PENALTY
 344 PRIMER_INTERNAL_WT_SELF_ANY=$PRIMER_INTERNAL_WT_SELF_ANY
 345 PRIMER_PAIR_MAX_DIFF_TM=$PRIMER_PAIR_MAX_DIFF_TM
 346 PRIMER_WT_SELF_ANY=$PRIMER_WT_SELF_ANY
 347 PRIMER_INTERNAL_WT_SELF_ANY_TH=$PRIMER_INTERNAL_WT_SELF_ANY_TH
 348 PRIMER_WT_SELF_ANY_TH=$PRIMER_WT_SELF_ANY_TH
 349 PRIMER_INTERNAL_WT_SELF_END=$PRIMER_INTERNAL_WT_SELF_END
 350 PRIMER_PAIR_MAX_LIBRARY_MISPRIMING=$PRIMER_PAIR_MAX_LIBRARY_MISPRIMING
 351 PRIMER_WT_SELF_END=$PRIMER_WT_SELF_END
 352 PRIMER_INTERNAL_WT_SELF_END_TH=$PRIMER_INTERNAL_WT_SELF_END_TH
 353 PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING=$PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING
 354 PRIMER_WT_SELF_END_TH=$PRIMER_WT_SELF_END_TH
 355 PRIMER_INTERNAL_WT_SEQ_QUAL=$PRIMER_INTERNAL_WT_SEQ_QUAL
 356 PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH=$PRIMER_PAIR_MAX_TEMPLATE_MISPRIMING_TH
 357 PRIMER_WT_SEQ_QUAL=$PRIMER_WT_SEQ_QUAL
 358 PRIMER_INTERNAL_WT_SIZE_GT=$PRIMER_INTERNAL_WT_SIZE_GT
 359 PRIMER_PAIR_WT_COMPL_ANY=$PRIMER_PAIR_WT_COMPL_ANY
 360 PRIMER_WT_SIZE_GT=$PRIMER_WT_SIZE_GT
 361 PRIMER_INTERNAL_WT_SIZE_LT=$PRIMER_INTERNAL_WT_SIZE_LT
 362 PRIMER_PAIR_WT_COMPL_ANY_TH=$PRIMER_PAIR_WT_COMPL_ANY_TH
 363 PRIMER_WT_SIZE_LT=$PRIMER_WT_SIZE_LT
 364 PRIMER_PAIR_WT_COMPL_END=$PRIMER_PAIR_WT_COMPL_END
 365 PRIMER_WT_TEMPLATE_MISPRIMING=$PRIMER_WT_TEMPLATE_MISPRIMING
 366 PRIMER_PAIR_WT_COMPL_END_TH=$PRIMER_PAIR_WT_COMPL_END_TH
 367 PRIMER_WT_TEMPLATE_MISPRIMING_TH=$PRIMER_WT_TEMPLATE_MISPRIMING_TH
 368 PRIMER_INTERNAL_WT_TM_GT=$PRIMER_INTERNAL_WT_TM_GT
 369 PRIMER_PAIR_WT_DIFF_TM=$PRIMER_PAIR_WT_DIFF_TM
 370 PRIMER_WT_TM_GT=$PRIMER_WT_TM_GT
 371 PRIMER_INTERNAL_WT_TM_LT=$PRIMER_INTERNAL_WT_TM_LT
 372 PRIMER_PAIR_WT_IO_PENALTY=$PRIMER_PAIR_WT_IO_PENALTY
 373 PRIMER_WT_TM_LT=$PRIMER_WT_TM_LT
 374 PRIMER_LIBERAL_BASE=$PRIMER_LIBERAL_BASE
 375 PRIMER_PAIR_WT_LIBRARY_MISPRIMING=$PRIMER_PAIR_WT_LIBRARY_MISPRIMING
 376 =
 377 );
 378
 379         my $filename = "primer3.txt";
 380         my $tempdir = File::Spec->catfile($c->config->{basepath}, $c->tempfiles_subdir('primer3'), $filename);
 381
 382
 383         my @text = split("\n", $text);
 384         my $filteredtext;
 385         foreach my $item (@text) {
 386                 if ($item !~ m/=$/) {
 387                         $filteredtext .= $item."\n";
 388                 }
 389         }
 390         $filteredtext .= "=";
 391
 392         write_file($tempdir, $filteredtext);
 393
 394         system("primer3_core < $tempdir > $tempdir.results");
 395
 396         open (RESULTS, "$tempdir.results");
 397         my %results;
 398         while (<RESULTS>) {
 399                 chomp;
 400                 my @stuff = split(/=/, $_);
 401                 $results{$stuff[0]} = $stuff[1];
 402         }
 403         close (RESULTS);
 404
 405         $c->stash(template => 'primer3/primer3results.mas',
 406                   resultshash => \%results
 407         );
 408     }
 409
 410
 411
 412 __PACKAGE__->meta->make_immutable;