bio-polyploid-tools 0.5.2 → 0.6.0

checksums.yaml

@@ -1,7 +1,7 @@
 ---
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-  data.tar.gz: a093ad10be19c610df004bc2b0ad8834dcd7ca00
+  metadata.gz: 76d57ca94d667e3aff5324977e57d8b7a5f0d91e
+  data.tar.gz: ccd2fad348fbc8a68927b944adc8e13b7d776edf
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+  data.tar.gz: c387e7b9a9b8d5c6bbb8e286a2fa71e4ecd92093578e58d04d260f5d8f7ba66f47d03856486c030f91488f39078129b27f600329596faec2183e0c521eaf6785

data/VERSION

@@ -1 +1 @@
-0.5.2
+0.6.0

data/bin/polymarker.rb

@@ -36,6 +36,8 @@ options[:bucket] = 1
 options[:model] = "est2genome"
 options[:arm_selection] = arm_selection_functions[:arm_selection_embl] ;
 options[:flanking_size] = 150;
+options[:variation_free_region] = 0 
+options[:extract_found_contigs] = false
 options[:primer_3_preferences] = {
       :primer_product_size_range => "50-150" ,
       :primer_max_size => 25 , 
@@ -60,6 +62,11 @@ OptionParser.new do |opts|
   opts.on("-s", "--snp_list FILE", "File with the list of snps to search from, requires --reference to get the sequence using a position") do |o|
     options[:snp_list] = o
   end
+
+  opts.on("-t", "--mutant_list FILE", "File with the list of positions with mutation and the mutation line.\n\
+    requires --reference to get the sequence using a position") do |o|
+    options[:mutant_list] = o
+  end
   
   opts.on("-r", "--reference FILE", "Fasta file with the sequence for the markers (to complement --snp_list)") do |o|
     options[:reference] = o
@@ -71,9 +78,9 @@ OptionParser.new do |opts|
   
   opts.on("-e", "--exonerate_model MODEL", "Model to be used in exonerate to search for the contigs") do |o|
      options[:model] = o
-   end
+  end
 
-   opts.on("-a", "--arm_selection arm_selection_embl|arm_selection_morex|arm_selection_first_two", "Function to decide the chromome arm") do |o|
+  opts.on("-a", "--arm_selection arm_selection_embl|arm_selection_morex|arm_selection_first_two", "Function to decide the chromome arm") do |o|
     options[:arm_selection] = arm_selection_functions[o.to_sym];
    end
   
@@ -81,8 +88,20 @@ OptionParser.new do |opts|
     options[:primer_3_preferences] = Bio::DB::Primer3.read_primer_preferences(o, options[:primer_3_preferences] )
   end
 
+  opts.on("-v", "--variation_free_region INT", "If present, avoid generating the common primer if there are homoeologous SNPs within the specified distance") do |o|
+    options[:variation_free_region] = o.to_i
+  end
+
+  opts.on("-x", "--extract_found_contigs", "If present, save in a separate file the contigs with matches. Useful to debug.") do |o|
+    options[:extract_found_contigs] = true
+  end
+
+  opts.on("-P", "--primers_to_order")do
+    #TODO: have a string with the tails, optional. 
+    options[:primers_to_order] = true
+  end
+
   
-    
 end.parse!
 
 if options[:primer_3_preferences][:primer_product_size_range]
@@ -108,8 +127,9 @@ snp_in="B"
 
 fasta_reference = nil
 #test_file="/Users/ramirezr/Dropbox/JIC/PrimersToTest/test_primers_nick_and_james_1.csv"
-test_file=options[:marker_list]
+test_file=options[:marker_list]  if options[:marker_list]
 test_file=options[:snp_list] if options[:snp_list]
+test_file=options[:mutant_list] if options[:mutant_list]
 fasta_reference = options[:reference]
 output_folder="#{test_file}_primer_design_#{Time.now.strftime('%Y%m%d-%H%M%S')}" 
 output_folder= options[:output_folder] if  options[:output_folder]
@@ -122,12 +142,12 @@ primer_3_input="#{output_folder}/primer_3_input_temp"
 primer_3_output="#{output_folder}/primer_3_output_temp"
 exons_filename="#{output_folder}/exons_genes_and_contigs.fa"
 output_primers="#{output_folder}/primers.csv"
+output_to_order="#{output_folder}/primers_to_order.csv"
 @status_file="#{output_folder}/status.txt"
 
 primer_3_config=File.expand_path(File.dirname(__FILE__) + '/../conf/primer3_config')
 model=options[:model] 
 
-
 def write_status(status)
   f=File.open(@status_file, "a")
   f.puts "#{Time.now.to_s},#{status}"
@@ -148,9 +168,6 @@ if fasta_reference
   p "Fasta reference: #{fasta_reference}"
 end
 
-
-
-
 #1. Read all the SNP files 
 #chromosome = nil
 write_status "Reading SNPs"
@@ -167,13 +184,22 @@ File.open(test_file) do | f |
        region = fasta_reference_db.index.region_for_entry(snp.gene).get_full_region
        snp.template_sequence = fasta_reference_db.fetch_sequence(region)
      else
-        $stderr.puts "ERROR: Unable to find entry for #{snp.gene}"
+        write_status "WARN: Unable to find entry for #{snp.gene}"
+      end
+    elsif options[:mutant_list] and options[:reference] #List and fasta file
+      snp = Bio::PolyploidTools::SNPMutant.parse(line)
+      entry = fasta_reference_db.index.region_for_entry(snp.contig)
+      if entry
+       region = fasta_reference_db.index.region_for_entry(snp.contig).get_full_region
+       snp.full_sequence = fasta_reference_db.fetch_sequence(region)
+     else
+        write_status "WARN: Unable to find entry for #{snp.gene}"
       end
-
     else
       rise Bio::DB::Exonerate::ExonerateException.new "Wrong number of arguments. " 
     end
     rise Bio::DB::Exonerate::ExonerateException.new "No SNP for line '#{line}'" if snp == nil
+  
     snp.snp_in = snp_in
     snp.original_name = original_name
     if snp.position 
@@ -206,7 +232,7 @@ file.close
 #chr_group = chromosome[0]
 write_status "Searching markers in genome"
 exo_f = File.open(exonerate_file, "w")
-contigs_f = File.open(temp_contigs, "w")
+contigs_f = File.open(temp_contigs, "w") if options[:extract_found_contigs]
 filename=path_to_contigs 
 puts filename
 target=filename
@@ -224,13 +250,13 @@ Bio::DB::Exonerate.align({:query=>temp_fasta_query, :target=>target, :model=>mod
       raise ExonerateException.new,  "Entry not found! #{aln.target_id}. Make sure that the #{target_id}.fai was generated properly." if entry == nil
       region = entry.get_full_region
       seq = fasta_file.fetch_sequence(region)
-      contigs_f.puts(">#{aln.target_id}\n#{seq}")
+      contigs_f.puts(">#{aln.target_id}\n#{seq}") if options[:extract_found_contigs]
     end
   end  
 end
  
-exo_f.close()
-contigs_f.close()
+exo_f.close() 
+contigs_f.close() if options[:extract_found_contigs]
 
 #4. Load all the results from exonerate and get the input filename for primer3
 #Custom arm selection function that only uses the first two characters. Maybe
@@ -241,12 +267,13 @@ write_status "Reading best alignment on each chromosome"
 container= Bio::PolyploidTools::ExonContainer.new
 container.flanking_size=options[:flanking_size] 
 container.gene_models(temp_fasta_query)
-container.chromosomes(temp_contigs)
+container.chromosomes(fasta_reference)
 container.add_parental({:name=>snp_in})
 container.add_parental({:name=>original_name})
 snps.each do |snp|
   snp.container = container
   snp.flanking_size = container.flanking_size
+  snp.variation_free_region = options[:variation_free_region]
   container.add_snp(snp)
 end
 container.add_alignments({:exonerate_file=>exonerate_file, :arm_selection=>options[:arm_selection] , :min_identity=>min_identity})
@@ -281,6 +308,8 @@ kasp_container.add_primers_file(primer_3_output) if added_exons > 0
 header = "Marker,SNP,RegionSize,chromosome,total_contigs,contig_regions,SNP_type,#{original_name},#{snp_in},common,primer_type,orientation,#{original_name}_TM,#{snp_in}_TM,common_TM,selected_from,product_size,errors"
 File.open(output_primers, 'w') { |f| f.write("#{header}\n#{kasp_container.print_primers}") }
 
+File.open(output_to_order, "w") { |io|  io.write(kasp_container.print_primers_with_tails()) }
+
 write_status "DONE"
 rescue StandardError => e
   write_status "ERROR\t#{e.message}"

data/bin/snp_position_to_polymarker.rb

@@ -28,7 +28,7 @@ OptionParser.new do |opts|
   
   opts.banner = "Usage: snp_postion_to_polymarker.rb [options]"
 
-  opts.on("-s", "--sno_file CSV", "CSV file with the following columnns:\nID,Allele_1,position,Allele_1,target_chromosome") do |o|
+  opts.on("-s", "--snp_file CSV", "CSV file with the following columnns:\nID,Allele_1,position,Allele_1,target_chromosome") do |o|
     options[:snp_file] = o
   end
   opts.on("-r", "--reference FASTA", "reference with the genes/contings/marker seuqnece") do |o|

data/bio-polyploid-tools.gemspec

@@ -2,16 +2,16 @@
 # DO NOT EDIT THIS FILE DIRECTLY
 # Instead, edit Jeweler::Tasks in Rakefile, and run 'rake gemspec'
 # -*- encoding: utf-8 -*-
-# stub: bio-polyploid-tools 0.5.2 ruby lib
+# stub: bio-polyploid-tools 0.6.0 ruby lib
 
 Gem::Specification.new do |s|
   s.name = "bio-polyploid-tools"
-  s.version = "0.5.2"
+  s.version = "0.6.0"
 
   s.required_rubygems_version = Gem::Requirement.new(">= 0") if s.respond_to? :required_rubygems_version=
   s.require_paths = ["lib"]
   s.authors = ["Ricardo H.  Ramirez-Gonzalez"]
-  s.date = "2014-10-28"
+  s.date = "2015-02-15"
   s.description = "Repository of tools developed in TGAC and Crop Genetics in JIC to work with polyploid wheat"
   s.email = "ricardo.ramirez-gonzalez@tgac.ac.uk"
   s.executables = ["bfr.rb", "count_variations.rb", "filter_blat_by_target_coverage.rb", "find_best_blat_hit.rb", "find_best_exonerate.rb", "hexaploid_primers.rb", "homokaryot_primers.rb", "map_markers_to_contigs.rb", "markers_in_region.rb", "polymarker.rb", "snp_position_to_polymarker.rb", "snps_between_bams.rb"]
@@ -81,6 +81,7 @@ Gem::Specification.new do |s|
     "lib/bio/PolyploidTools/Marker.rb",
     "lib/bio/PolyploidTools/PrimerRegion.rb",
     "lib/bio/PolyploidTools/SNP.rb",
+    "lib/bio/PolyploidTools/SNPMutant.rb",
     "lib/bio/PolyploidTools/SNPSequence.rb",
     "lib/bio/db/exonerate.rb",
     "lib/bio/db/primer3.rb",
@@ -91,6 +92,8 @@ Gem::Specification.new do |s|
     "test/data/BS00068396_51_contigs.fa",
     "test/data/BS00068396_51_exonerate.tab",
     "test/data/BS00068396_51_genes.txt",
+    "test/data/IWGSC_CSS_1AL_scaff_1455974.fa",
+    "test/data/IWGSC_CSS_1AL_scaff_1455974_aln_contigs.fa",
     "test/data/LIB1716.bam",
     "test/data/LIB1716.bam.bai",
     "test/data/LIB1719.bam",
@@ -111,6 +114,7 @@ Gem::Specification.new do |s|
     "test/data/patological_cases5D.csv",
     "test/data/primer_3_input_header_test",
     "test/data/short_primer_design_test.csv",
+    "test/data/test_from_mutant.csv",
     "test/data/test_iselect.csv",
     "test/data/test_iselect_reference.fa",
     "test/data/test_iselect_reference.fa.fai",

data/lib/bio/PolyploidTools/SNP.rb

@@ -14,6 +14,7 @@ module Bio::PolyploidTools
     attr_accessor :genomes_count
     attr_accessor :primer_3_min_seq_length 
     attr_accessor :chromosome
+    attr_accessor :variation_free_region
 
     #Format: 
     #Gene_name,Original,SNP_Pos,pos,chromosome
@@ -37,6 +38,7 @@ module Bio::PolyploidTools
     def initialize
       @genomes_count = 3 #TODO: if we want to use this with other polyploids, me need to set this as a variable in the main script. 
       @primer_3_min_seq_length = 50
+      @variation_free_region = 0
     end
 
     def to_polymarker_sequence(flanking_size)
@@ -260,8 +262,14 @@ module Bio::PolyploidTools
           sequence = reverse_complement_string(sequence)
           orientation = "reverse"
         end
+        if @variation_free_region > 0
+          check_str = sequence[right+1, @variation_free_region]
+          return nil if check_str != check_str.downcase
+        end
+
       end
 
+
       str = "SEQUENCE_ID=#{opts[:name]} #{orientation}\n"
       str << "SEQUENCE_FORCE_LEFT_END=#{left}\n"
       str << "SEQUENCE_FORCE_RIGHT_END=#{right}\n" if opts[:right_pos]
@@ -279,8 +287,6 @@ module Bio::PolyploidTools
         str << "SEQUENCE_FORCE_LEFT_END=#{left}\n"
         str << "SEQUENCE_TEMPLATE=#{sequence}\n"
         str << "=\n"
-      else
-
       end
 
       str

data/lib/bio/PolyploidTools/SNPMutant.rb

@@ -0,0 +1,85 @@
+
+require_relative "SNPSequence"
+require 'bio-samtools'
+module Bio::PolyploidTools
+  class SNPSequenceException < RuntimeError 
+  end
+
+  class SNPMutant < SNPSequence
+
+    attr_accessor :library, :contig, :chr
+    #Format: 
+    #seqid,library,position,wt_base,mut_base
+    #IWGSC_CSS_1AL_scaff_1455974,Kronos2281,127,C,T
+    def self.parse(reg_str)
+      reg_str.chomp!
+      snp = SNPMutant.new
+
+      arr = reg_str.split(",")
+      
+      throw SNPSequenceException.new "Need five fields to parse, and got #{arr.size} in #{reg_str}" unless arr.size == 5
+      
+      snp.contig, snp.library, snp.position, snp.original, snp.snp = reg_str.split(",")
+      snp.position = snp.position.to_i
+      snp.gene = "EMPTY"
+      begin
+        toks = snp.contig.split('_')
+        #1AL_1455974_Kronos2281_127C
+        #snp.chr = contig.split('_')[2][0,2] #This parses the default from the IWGSC. We may want to make this a lambda  
+        #snp.chr = toks[2][0,2]
+        name = toks[2] + "_" + toks[4] + "_" + snp.library + "_" + snp.position.to_s 
+        snp.gene = name
+        snp.chromosome = toks[2][0,2]
+        snp.chr = snp.chromosome
+        
+      rescue Exception => e
+        $stderr.puts "WARN: snp.chr couldnt be set, the sequence id to parse was #{snp.contig}. We expect something like: IWGSC_CSS_1AL_scaff_1455974"
+        snp.gene = "Error"
+        $stderr.puts e
+      end
+      
+      snp.exon_list = Hash.new()
+      snp.flanking_size=100
+      snp
+    end
+    
+    def full_sequence=(seq)
+      self.template_sequence = seq
+      self.sequence_original = self.to_polymarker_sequence(self.flanking_size)
+      self.parse_sequence_snp
+    end
+
+    def full_sequence()
+      self.template_sequence
+    end
+
+    def chromosome_group
+      chr[0]
+    end
+    
+    def chromosome_genome
+      chr[1]
+    end
+    
+    def chromosome_genome
+      return chr[3] if chr[3]
+      return nil
+    end
+      
+    def parse_sequence_snp
+      pos = 0
+      match_data = /(?<pre>\w*)\[(?<org>[ACGT])\/(?<snp>[ACGT])\](?<pos>\w*)/.match(sequence_original.strip)
+      if match_data
+        @position = Regexp.last_match(:pre).size + 1
+        @original = Regexp.last_match(:org)
+        @snp = Regexp.last_match(:snp)
+        amb_base = Bio::NucleicAcid.to_IUAPC("#{@original}#{@snp}")
+        @template_sequence = "#{Regexp.last_match(:pre)}#{amb_base}#{Regexp.last_match(:pos)}"
+        
+     end 
+    end
+    
+   
+
+  end
+end

data/lib/bio/PolyploidTools/SNPSequence.rb

@@ -41,14 +41,14 @@ module Bio::PolyploidTools
       pos = 0
       match_data = /(?<pre>\w*)\[(?<org>[ACGT])\/(?<snp>[ACGT])\](?<pos>\w*)/.match(sequence_original.strip)
       if match_data
-      @position = Regexp.last_match(:pre).size + 1
-      @original = Regexp.last_match(:org)
-      @snp = Regexp.last_match(:snp)
-      
-      amb_base = Bio::NucleicAcid.to_IUAPC("#{@original}#{@snp}")
-      
-      @template_sequence = "#{Regexp.last_match(:pre)}#{amb_base}#{Regexp.last_match(:pos)}"
-      
+        @position = Regexp.last_match(:pre).size + 1
+        @original = Regexp.last_match(:org)
+        @snp = Regexp.last_match(:snp)
+        
+        amb_base = Bio::NucleicAcid.to_IUAPC("#{@original}#{@snp}")
+        
+        @template_sequence = "#{Regexp.last_match(:pre)}#{amb_base}#{Regexp.last_match(:pos)}"
+        
      end 
     end
     

data/lib/bio/db/primer3.rb

@@ -17,18 +17,18 @@ module Bio::DB::Primer3
   end
 
   def self.prepare_input_file(file, opts2={})
-     opts = {
-      :primer_product_size_range => "50-150" ,
-      :primer_max_size => 25 , 
-      :primer_lib_ambiguity_codes_consensus => 1,
-      :primer_liberal_base => 1, 
-      :primer_num_return => 5,
-      :primer_explain_flag => 1,
-      :primer_thermodynamic_parameters_path => File.expand_path(File.dirname(__FILE__) + '../../../../conf/primer3_config/') + '/'
+   opts = {
+    :primer_product_size_range => "50-150" ,
+    :primer_max_size => 25 , 
+    :primer_lib_ambiguity_codes_consensus => 1,
+    :primer_liberal_base => 1, 
+    :primer_num_return => 5,
+    :primer_explain_flag => 1,
+    :primer_thermodynamic_parameters_path => File.expand_path(File.dirname(__FILE__) + '../../../../conf/primer3_config/') + '/'
     }.merge(opts2)
     
     opts.each do |key,value|
-        file.puts "#{key.to_s.upcase}=#{value}\n"
+      file.puts "#{key.to_s.upcase}=#{value}\n"
     end
     # file.puts "="
   end
@@ -68,7 +68,7 @@ module Bio::DB::Primer3
       @primers_line_1 = SortedSet.new
       @primers_line_2 = SortedSet.new
       @regions = SortedSet.new
-       @primer3_errors = Set.new
+      @primer3_errors = Set.new
     end
 
     def line_2_name
@@ -106,24 +106,23 @@ module Bio::DB::Primer3
       nil
     end
 
-
-    def print_primers
-#TODO: Retrieve error messages
+    def values
+      return @values if @values
       left_start = 0
       left_end = 0
       right_start = 0
       right_end = 0
       total_columns_before_messages=17
-      values = Array.new
-      #values << "#{gene},,#{template_length},"
-      values << gene
-      values << "#{original}#{position}#{snp}"
-      values << template_length
-      values << snp_from.chromosome
-      values << regions.size
-      values << regions.join("|")
+      @values = Array.new
+      #@values << "#{gene},,#{template_length},"
+      @values << gene
+      @values << "#{original}#{position}#{snp}"
+      @values << template_length
+      @values << snp_from.chromosome
+      @values << regions.size
+      @values << regions.join("|")
       if primer3_line_1 and primer3_line_2
-        values <<  primer3_line_1.polymorphism
+        @values <<  primer3_line_1.polymorphism
 
         #Block that searches both if both pairs have a TM
         primer_2 = primer3_line_2.left_primer_with_coordinates(primer3_line_1.left_coordinates, primer3_line_1.orientation)
@@ -133,27 +132,27 @@ module Bio::DB::Primer3
         #  $stderr.puts primer_1
         #  $stderr.puts primer_2
         if primer3_line_1 < primer3_line_2 and primer_2_tm != "NA"
-          values << primer3_line_1.left_primer
-          values << primer_2
-          values << primer3_line_1.right_primer 
-          values << primer3_line_1.type.to_s 
-          values << primer3_line_1.orientation.to_s 
-          values << primer3_line_1.best_pair.left.tm 
-          values << primer_2_tm
-          values << primer3_line_1.best_pair.right.tm
-          values << "first" 
-          values << primer3_line_1.best_pair.product_size
+          @values << primer3_line_1.left_primer
+          @values << primer_2
+          @values << primer3_line_1.right_primer 
+          @values << primer3_line_1.type.to_s 
+          @values << primer3_line_1.orientation.to_s 
+          @values << primer3_line_1.best_pair.left.tm 
+          @values << primer_2_tm
+          @values << primer3_line_1.best_pair.right.tm
+          @values << "first" 
+          @values << primer3_line_1.best_pair.product_size
         elsif  primer_1_tm != "NA"
-          values << primer_1
-          values << primer3_line_2.left_primer
-          values << primer3_line_2.right_primer
-          values << primer3_line_2.type.to_s
-          values << primer3_line_2.orientation.to_s
-          values << primer_1_tm
-          values << primer3_line_2.best_pair.left.tm
-          values << primer3_line_2.best_pair.right.tm
-          values << "second"
-          values << primer3_line_2.best_pair.product_size
+          @values << primer_1
+          @values << primer3_line_2.left_primer
+          @values << primer3_line_2.right_primer
+          @values << primer3_line_2.type.to_s
+          @values << primer3_line_2.orientation.to_s
+          @values << primer_1_tm
+          @values << primer3_line_2.best_pair.left.tm
+          @values << primer3_line_2.best_pair.right.tm
+          @values << "second"
+          @values << primer3_line_2.best_pair.product_size
         else
 
           first_candidate = find_primer_pair_first
@@ -172,80 +171,99 @@ module Bio::DB::Primer3
 
           if first_candidate and second_candidate and first_candidate < second_candidate 
             #puts "A"
-            values << first_candidate.left_primer
-            values << primer_2
-            values << first_candidate.right_primer 
-            values << first_candidate.type.to_s 
-            values << first_candidate.orientation.to_s 
-            values << first_candidate.best_pair.left.tm 
-            values << primer_2_tm
-            values << first_candidate.best_pair.right.tm
-            values << "first" 
-            values << first_candidate.best_pair.product_size
+            @values << first_candidate.left_primer
+            @values << primer_2
+            @values << first_candidate.right_primer 
+            @values << first_candidate.type.to_s 
+            @values << first_candidate.orientation.to_s 
+            @values << first_candidate.best_pair.left.tm 
+            @values << primer_2_tm
+            @values << first_candidate.best_pair.right.tm
+            @values << "first" 
+            @values << first_candidate.best_pair.product_size
           elsif  second_candidate 
             #puts "B"
-            values << primer_1
-            values << second_candidate.left_primer
-            values << second_candidate.right_primer
-            values << second_candidate.type.to_s
-            values << second_candidate.orientation.to_s
-            values << primer_1_tm
-            values << second_candidate.best_pair.left.tm
-            values << second_candidate.best_pair.right.tm
-            values << "second"
-            values << second_candidate.best_pair.product_size
+            @values << primer_1
+            @values << second_candidate.left_primer
+            @values << second_candidate.right_primer
+            @values << second_candidate.type.to_s
+            @values << second_candidate.orientation.to_s
+            @values << primer_1_tm
+            @values << second_candidate.best_pair.left.tm
+            @values << second_candidate.best_pair.right.tm
+            @values << "second"
+            @values << second_candidate.best_pair.product_size
           elsif  first_candidate 
             #puts "C"
-            values << first_candidate.left_primer
-            values << primer_2
-            values << first_candidate.right_primer
-            values << first_candidate.type.to_s
-            values << first_candidate.orientation.to_s
-            values << primer_2_tm
-            values << first_candidate.best_pair.left.tm
-            values << first_candidate.best_pair.right.tm
-            values << "first"
-            values << first_candidate.best_pair.product_size
-#          else
-#            values << "" 
+            @values << first_candidate.left_primer
+            @values << primer_2
+            @values << first_candidate.right_primer
+            @values << first_candidate.type.to_s
+            @values << first_candidate.orientation.to_s
+            @values << primer_2_tm
+            @values << first_candidate.best_pair.left.tm
+            @values << first_candidate.best_pair.right.tm
+            @values << "first"
+            @values << first_candidate.best_pair.product_size
           end
-
         end
 
       elsif primer3_line_1 
-        values << primer3_line_1.polymorphism
-        values << primer3_line_1.left_primer
-        values << primer3_line_1.left_primer_snp(self) 
-        values << primer3_line_1.right_primer 
-        values << primer3_line_1.type.to_s 
-        values << primer3_line_1.orientation.to_s      
-        values << primer3_line_1.best_pair.left.tm 
-        values << "NA"
-        values << primer3_line_1.best_pair.right.tm
-
-        values << "first+"
-        values << primer3_line_1.best_pair.product_size
+        @values << primer3_line_1.polymorphism
+        @values << primer3_line_1.left_primer
+        @values << primer3_line_1.left_primer_snp(self) 
+        @values << primer3_line_1.right_primer 
+        @values << primer3_line_1.type.to_s 
+        @values << primer3_line_1.orientation.to_s      
+        @values << primer3_line_1.best_pair.left.tm 
+        @values << "NA"
+        @values << primer3_line_1.best_pair.right.tm
+
+        @values << "first+"
+        @values << primer3_line_1.best_pair.product_size
       elsif primer3_line_2 
-        values << primer3_line_2.polymorphism
-        values << primer3_line_2.left_primer_snp(self) 
-        values << primer3_line_2.left_primer
-        values << primer3_line_2.right_primer
-        values << primer3_line_2.type.to_s
-        values << primer3_line_2.orientation.to_s
-        values << "NA"
-        values << primer3_line_2.best_pair.left.tm
-        values << primer3_line_2.best_pair.right.tm
-        values << "second+"
-        values << primer3_line_2.best_pair.product_size
+        @values << primer3_line_2.polymorphism
+        @values << primer3_line_2.left_primer_snp(self) 
+        @values << primer3_line_2.left_primer
+        @values << primer3_line_2.right_primer
+        @values << primer3_line_2.type.to_s
+        @values << primer3_line_2.orientation.to_s
+        @values << "NA"
+        @values << primer3_line_2.best_pair.left.tm
+        @values << primer3_line_2.best_pair.right.tm
+        @values << "second+"
+        @values << primer3_line_2.best_pair.product_size
 
       end 
-      if values.size < total_columns_before_messages
-        values[total_columns_before_messages] = primer3_errors.to_a.join("|")
+      if @values.size < total_columns_before_messages
+        @values[total_columns_before_messages] = primer3_errors.to_a.join("|")
       else
-        values << nil
+        @values << nil
       end
+      return @values
+    end
+
+    def print_primers
+      self.values.join(",")
+    end
+
+    def found_primers?
+      return self.values[7] && self.values[7] != nil
+    end
+
+    def first_primer
+      return self.values[7] if self.values[7] && self.values[7] != nil
+      return ""
+    end
+
+    def second_primer
+      return self.values[8] if self.values[8] && self.values[8] != nil
+      return ""
+    end
 
-      values.join(",")
+    def common_primer
+      return self.values[9] if self.values[9]&& self.values[9] != nil
+      return ""
     end
 
     def self.parse(reg_str)
@@ -273,10 +291,10 @@ module Bio::DB::Primer3
     def add_record(primer3record)
       @primer3_errors = Set.new unless @primer3_errors
       @template_length = primer3record.sequence_template.size
-       if primer3record.primer_error != nil 
-          primer3_errors << primer3record.primer_error
-          return
-        end
+      if primer3record.primer_error != nil 
+        primer3_errors << primer3record.primer_error
+        return
+      end
       case
 
       when primer3record.line == @line_1
@@ -451,7 +469,7 @@ module Bio::DB::Primer3
       @scores[:chromosome_semispecific] = 100
       @scores[:chromosome_nonspecific] = 0
       @scores[:exon] = 50
-    
+
     end
 
     def snp
@@ -654,7 +672,7 @@ module Bio::DB::Primer3
 
     attr_accessor :line_1, :line_2
     attr_accessor :snp_hash
-   
+
 
     def add_snp_file(filename)
       @snp_hash=Hash.new unless @snp_hash
@@ -696,6 +714,19 @@ module Bio::DB::Primer3
       end
       return str
     end
+
+    def print_primers_with_tails(tail_a: "GAAGGTCGGAGTCAACGGATT", tail_b: "GAAGGTGACCAAGTTCATGCT")
+      str = ""
+      snp_hash.each do |k, snp|  
+        if snp.found_primers?
+          str << snp.gene << snp.original << "\t" << tail_a << snp.first_primer << "\n"
+          str << snp.gene << snp.snp      << "\t" << tail_b << snp.second_primer << "\n"
+          str << snp.gene                 << "\t"           << snp.common_primer << "\n"
+        end
+      end
+      return str
+    end
+
   end
 end