bio-polyploid-tools 0.7.3 → 0.8.0

data/bin/snp_position_to_polymarker.rb

@@ -42,7 +42,8 @@ OptionParser.new do |opts|
   opts.on("-f", "--flanking_size INT", "Flanking size around the SNP") do |o|
     options[:flanking_size] = o.to_i
   end
-  opts.on("-t", "--mutant_list FILE", "File with the list of positions with mutation and the mutation line.\n\
+
+  opts.on("-t", "--mutant_list FILE", "File with the list of positions with mutation and the mutation line. Example: IWGSC_CSS_1AL_scaff_1455974,Kronos2281,127,C,T\n\
     requires --reference to get the sequence using a position") do |o|
     options[:mutant_list] = o
      test_file = o 
@@ -76,9 +77,10 @@ File.open(test_file) do | f |
        		if region != lastRegion
              lastTemplate = fasta_reference_db.fetch_sequence(region)
           end
-          snp.template_sequence = lastTemplate
+          snp.full_sequence = lastTemplate
           lastRegion = region
-       		out.puts "#{snp.gene}_#{snp.snp_id_in_seq},#{snp.chromosome},#{snp.to_polymarker_sequence(options[:flanking_size])}"
+
+       		out.puts "#{snp.gene}_#{snp.snp_id_in_seq},#{snp.chromosome},#{snp.sequence_original}"
     	else
     	   $stderr.puts "ERROR: Unable to find entry for #{snp.gene}"
     	end

data/bin/snps_between_bams.rb

@@ -54,7 +54,6 @@ fasta_db.index.entries.each do | r |
 
 
   begin
-<<<<<<< HEAD
     reg_a = bam1.fetch_region({:region=>region,  :min_cov=>min_cov, :A=>1})
     reg_b = bam2.fetch_region({:region=>region,  :min_cov=>min_cov, :A=>1})
     cons_1 = reg_a.consensus
@@ -85,34 +84,6 @@ fasta_db.index.entries.each do | r |
     fasta_file.puts ">#{r.id}_2"
     fasta_file.puts "#{cons_2}"
     
-=======
-
-    cons_1 = bam1.consensus_with_ambiguities({:region=>region, :case=>true, :min_cov=>min_cov})
-    cons_2 = bam2.consensus_with_ambiguities({:region=>region, :case=>true, :min_cov=>min_cov})
-    if cons_1 != cons_2
-
-      snps_1 = cons_1.count_ambiguities
-      snps_2 = cons_2.count_ambiguities
-
-      snps_tot = Bio::Sequence.snps_between(cons_1, cons_2)
-
-      snps_per_1k_1   = (block_size * snps_1.to_f   ) / region.size
-      snps_per_1k_2   = (block_size * snps_2.to_f   ) / region.size
-      snps_per_1k_tot = (block_size * snps_tot.to_f ) / region.size
-
-      hist_1[snps_per_1k_1.to_i] += 1
-      hist_2[snps_per_1k_2.to_i] += 1
-
-      table_file.print "#{r.id}\t#{region.size}\t"
-      table_file.print "#{snps_1}\t#{called_1}\t#{snps_per_1k_1}\t"
-      table_file.print "#{snps_2}\t#{called_2}\t#{snps_per_1k_2}\t"
-      table_file.print "#{snps_tot}\t#{snps_per_1k_tot}\n"
-      fasta_file.puts ">#{r.id}_1"
-      fasta_file.puts "#{cons_1}"
-      fasta_file.puts ">#{r.id}_2"
-      fasta_file.puts "#{cons_2}"
-    end
->>>>>>> 1b60bd09fdb1b087d6cb53c643ff36e536efe4a3
   rescue Exception => e
     $stderr.puts "Unable to process #{region}: #{e.to_s}"
   end

data/bin/vcfLineToTable.rb

@@ -0,0 +1,56 @@
+require 'bio-samtools'
+require 'optparse'
+
+$: << File.expand_path(File.dirname(__FILE__) + '/../lib')
+$: << File.expand_path('.')
+path=File.expand_path(File.dirname(__FILE__) + '/../lib/bioruby-polyploid-tools.rb')
+
+
+
+
+def parseVCFheader(head_line="")
+	##INFO=<ID=NS,Number=1,Type=Integer,Description="Number of samples with data">
+
+	m=/##INFO=<ID=(.+),Number=(.+),Type=(.+),Description="(.+)">/.match(head_line)
+	{:id=>m[1],:number=>m[2],:type=>m[3],:desc=>m[4]}
+
+end
+
+
+header_info = Hash.new
+ARGF.each_line do |line|  
+	h = nil
+	h = parseVCFheader(line) if line.start_with? "##INFO"
+
+	header_info[h[:id]] = h[:desc] if h
+	#puts header_info.inspect
+	next if line.start_with? "##"
+	if line.start_with? "#CHROM"
+		arr = line.split
+		arr = arr.drop(9)
+		arr2 = arr.map { |s| [s.clone().prepend('Cov'), s.clone().prepend('Hap') ]}
+		#header += arr2.join("\t")
+		#puts header
+		next
+	end
+
+	line.chomp!
+		
+	vcf = Bio::DB::Vcf.new(line, arr)
+#	puts arr.join("\t") if vcf.info["TYPE"] == "snp"
+#	puts vcf.inspect
+	#pus vcf.pos.inspect
+	#next if vcf.info["AO"].to_i != 1
+	vcf.info.each_pair { |name, val| puts "#{name}\t#{val}\t#{header_info[name]}" }
+
+    arr2 = Array.new
+    puts "____"
+    i = 0
+	vcf.samples.each do |sample|
+		#puts sample.inspect
+		puts sample[1].keys.join("\t") if i == 0
+        puts sample[1].values.join("\t")
+        i+=1
+    end
+
+end

data/bio-polyploid-tools.gemspec

@@ -1,44 +1,52 @@
-# Generated by jeweler
+# Generated by juwelier
 # DO NOT EDIT THIS FILE DIRECTLY
-# Instead, edit Jeweler::Tasks in Rakefile, and run 'rake gemspec'
+# Instead, edit Juwelier::Tasks in Rakefile, and run 'rake gemspec'
 # -*- encoding: utf-8 -*-
-# stub: bio-polyploid-tools 0.7.3 ruby lib
+# stub: bio-polyploid-tools 0.8.0 ruby lib
 
 Gem::Specification.new do |s|
-  s.name = "bio-polyploid-tools"
-  s.version = "0.7.3"
+  s.name = "bio-polyploid-tools".freeze
+  s.version = "0.8.0"
 
-  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 = "2015-08-10"
-  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", "filter_exonerate_by_identity.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"]
+  s.required_rubygems_version = Gem::Requirement.new(">= 0".freeze) if s.respond_to? :required_rubygems_version=
+  s.require_paths = ["lib".freeze]
+  s.authors = ["Ricardo H.  Ramirez-Gonzalez".freeze]
+  s.date = "2018-01-18"
+  s.description = "Repository of tools developed at Crop Genetics in JIC to work with polyploid wheat".freeze
+  s.email = "ricardo.ramirez-gonzalez@jic.ac.uk".freeze
+  s.executables = ["bfr.rb".freeze, "blast_triads.rb".freeze, "blast_triads_promoters.rb".freeze, "count_variations.rb".freeze, "filter_blat_by_target_coverage.rb".freeze, "filter_exonerate_by_identity.rb".freeze, "find_best_blat_hit.rb".freeze, "find_best_exonerate.rb".freeze, "find_homoeologue_variations.rb".freeze, "get_longest_hsp_blastx_triads.rb".freeze, "hexaploid_primers.rb".freeze, "homokaryot_primers.rb".freeze, "mafft_triads.rb".freeze, "mafft_triads_promoters.rb".freeze, "map_markers_to_contigs.rb".freeze, "markers_in_region.rb".freeze, "polymarker.rb".freeze, "polymarker_capillary.rb".freeze, "snp_position_to_polymarker.rb".freeze, "snps_between_bams.rb".freeze, "vcfLineToTable.rb".freeze]
   s.extra_rdoc_files = [
     "README",
     "README.md"
   ]
   s.files = [
+    ".travis.yml",
     "Gemfile",
-    "Gemfile.lock",
     "README",
     "README.md",
     "Rakefile",
     "VERSION",
     "bin/bfr.rb",
+    "bin/blast_triads.rb",
+    "bin/blast_triads_promoters.rb",
     "bin/count_variations.rb",
     "bin/filter_blat_by_target_coverage.rb",
     "bin/filter_exonerate_by_identity.rb",
     "bin/find_best_blat_hit.rb",
     "bin/find_best_exonerate.rb",
+    "bin/find_homoeologue_variations.rb",
+    "bin/get_longest_hsp_blastx_triads.rb",
     "bin/hexaploid_primers.rb",
     "bin/homokaryot_primers.rb",
+    "bin/mafft_triads.rb",
+    "bin/mafft_triads_promoters.rb",
     "bin/map_markers_to_contigs.rb",
     "bin/markers_in_region.rb",
     "bin/polymarker.rb",
+    "bin/polymarker_capillary.rb",
     "bin/snp_position_to_polymarker.rb",
     "bin/snps_between_bams.rb",
+    "bin/vcfLineToTable.rb",
     "bio-polyploid-tools.gemspec",
     "conf/defaults.rb",
     "conf/primer3_config/dangle.dh",
@@ -80,21 +88,29 @@ Gem::Specification.new do |s|
     "lib/bio/PolyploidTools/ChromosomeArm.rb",
     "lib/bio/PolyploidTools/ExonContainer.rb",
     "lib/bio/PolyploidTools/Marker.rb",
+    "lib/bio/PolyploidTools/NoSNPSequence.rb",
     "lib/bio/PolyploidTools/PrimerRegion.rb",
     "lib/bio/PolyploidTools/SNP.rb",
     "lib/bio/PolyploidTools/SNPMutant.rb",
     "lib/bio/PolyploidTools/SNPSequence.rb",
+    "lib/bio/db/blast.rb",
     "lib/bio/db/exonerate.rb",
     "lib/bio/db/primer3.rb",
     "lib/bioruby-polyploid-tools.rb",
     "test/data/BS00068396_51.fa",
+    "test/data/BS00068396_51_blast.tab",
     "test/data/BS00068396_51_contigs.aln",
     "test/data/BS00068396_51_contigs.dnd",
     "test/data/BS00068396_51_contigs.fa",
+    "test/data/BS00068396_51_contigs.nhr",
+    "test/data/BS00068396_51_contigs.nin",
+    "test/data/BS00068396_51_contigs.nsq",
     "test/data/BS00068396_51_exonerate.tab",
+    "test/data/BS00068396_51_for_polymarker.fa",
     "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/IWGSC_CSS_1AL_scaff_1455974_aln_contigs.fa.fai",
     "test/data/LIB1716.bam",
     "test/data/LIB1716.bam.bai",
     "test/data/LIB1719.bam",
@@ -109,9 +125,31 @@ Gem::Specification.new do |s|
     "test/data/PST130_reverse_primer.csv",
     "test/data/S22380157.fa",
     "test/data/S22380157.fa.fai",
+    "test/data/S22380157.vcf",
+    "test/data/S58861868/LIB1716.bam",
+    "test/data/S58861868/LIB1716.sam",
+    "test/data/S58861868/LIB1719.bam",
+    "test/data/S58861868/LIB1719.sam",
+    "test/data/S58861868/LIB1721.bam",
+    "test/data/S58861868/LIB1721.sam",
+    "test/data/S58861868/LIB1722.bam",
+    "test/data/S58861868/LIB1722.sam",
+    "test/data/S58861868/S58861868.fa",
+    "test/data/S58861868/S58861868.fa.fai",
+    "test/data/S58861868/S58861868.vcf",
+    "test/data/S58861868/header.txt",
+    "test/data/S58861868/merged.bam",
+    "test/data/S58861868/merged_reheader.bam",
+    "test/data/S58861868/merged_reheader.bam.bai",
     "test/data/Test3Aspecific.csv",
     "test/data/Test3Aspecific_contigs.fa",
     "test/data/bfr_out_test.csv",
+    "test/data/headerMergeed.txt",
+    "test/data/headerS2238015",
+    "test/data/mergedLibs.bam",
+    "test/data/mergedLibsReheader.bam",
+    "test/data/mergedLibsSorted.bam",
+    "test/data/mergedLibsSorted.bam.bai",
     "test/data/patological_cases5D.csv",
     "test/data/primer_3_input_header_test",
     "test/data/short_primer_design_test.csv",
@@ -122,38 +160,42 @@ Gem::Specification.new do |s|
     "test/data/test_primer3_error.csv",
     "test/data/test_primer3_error_contigs.fa",
     "test/test_bfr.rb",
+    "test/test_blast.rb",
     "test/test_exon_container.rb",
     "test/test_exonearate.rb",
     "test/test_snp_parsing.rb",
     "test/test_wrong_selection.sh"
   ]
-  s.homepage = "http://github.com/tgac/bioruby-polyploid-tools"
-  s.licenses = ["MIT"]
-  s.rubygems_version = "2.4.7"
-  s.summary = "Tool to work with polyploids, NGS and molecular biology"
+  s.homepage = "http://github.com/tgac/bioruby-polyploid-tools".freeze
+  s.licenses = ["MIT".freeze]
+  s.rubygems_version = "2.7.4".freeze
+  s.summary = "Tool to work with polyploids, NGS and molecular biology".freeze
 
   if s.respond_to? :specification_version then
     s.specification_version = 4
 
     if Gem::Version.new(Gem::VERSION) >= Gem::Version.new('1.2.0') then
-      s.add_runtime_dependency(%q<bio>, [">= 1.4.3"])
-      s.add_runtime_dependency(%q<bio-samtools>, [">= 2.0.4"])
-      s.add_runtime_dependency(%q<rake>, [">= 0"])
-      s.add_runtime_dependency(%q<jeweler>, [">= 0"])
-      s.add_runtime_dependency(%q<systemu>, [">= 2.5.2"])
+      s.add_runtime_dependency(%q<bio>.freeze, [">= 1.5.1"])
+      s.add_runtime_dependency(%q<bio-samtools>.freeze, [">= 2.6.2"])
+      s.add_runtime_dependency(%q<systemu>.freeze, [">= 2.5.2"])
+      s.add_development_dependency(%q<shoulda>.freeze, [">= 2.10"])
+      s.add_development_dependency(%q<test-unit>.freeze, [">= 0"])
+      s.add_development_dependency(%q<juwelier>.freeze, [">= 0"])
     else
-      s.add_dependency(%q<bio>, [">= 1.4.3"])
-      s.add_dependency(%q<bio-samtools>, [">= 2.0.4"])
-      s.add_dependency(%q<rake>, [">= 0"])
-      s.add_dependency(%q<jeweler>, [">= 0"])
-      s.add_dependency(%q<systemu>, [">= 2.5.2"])
+      s.add_dependency(%q<bio>.freeze, [">= 1.5.1"])
+      s.add_dependency(%q<bio-samtools>.freeze, [">= 2.6.2"])
+      s.add_dependency(%q<systemu>.freeze, [">= 2.5.2"])
+      s.add_dependency(%q<shoulda>.freeze, [">= 2.10"])
+      s.add_dependency(%q<test-unit>.freeze, [">= 0"])
+      s.add_dependency(%q<juwelier>.freeze, [">= 0"])
     end
   else
-    s.add_dependency(%q<bio>, [">= 1.4.3"])
-    s.add_dependency(%q<bio-samtools>, [">= 2.0.4"])
-    s.add_dependency(%q<rake>, [">= 0"])
-    s.add_dependency(%q<jeweler>, [">= 0"])
-    s.add_dependency(%q<systemu>, [">= 2.5.2"])
+    s.add_dependency(%q<bio>.freeze, [">= 1.5.1"])
+    s.add_dependency(%q<bio-samtools>.freeze, [">= 2.6.2"])
+    s.add_dependency(%q<systemu>.freeze, [">= 2.5.2"])
+    s.add_dependency(%q<shoulda>.freeze, [">= 2.10"])
+    s.add_dependency(%q<test-unit>.freeze, [">= 0"])
+    s.add_dependency(%q<juwelier>.freeze, [">= 0"])
   end
 end
 

data/lib/bio/BFRTools.rb

@@ -114,6 +114,7 @@ module Bio::BFRTools
       self.entry = reg.entry
       self.start = reg.start
       self.end   = reg.end
+      @BFRs = nil
       opts[:region] = reg
       @container = opts[:container]
 

data/lib/bio/PolyploidTools/ChromosomeArm.rb

@@ -22,6 +22,7 @@ module Bio::PolyploidTools
      # puts entry
       @fasta_db.fetch_sequence(entry.get_full_region)
     end
+
     #Loads all the chromosome arms in a folder. 
     #The current version requires that all the references end with .fa, and start with XXX_*.fa
     #Where XXX is the chromosome name
@@ -29,16 +30,11 @@ module Bio::PolyploidTools
       chromosomeArms = Hash.new
       
       Dir.foreach(path_to_contigs) do |filename |
-        
         if  File.fnmatch("*.fa", filename)
           
           parsed = /^(?<arm>\d\w+)/.match(filename)
-         
           target="#{path_to_contigs}/#{filename}"
-          
-         
-         
-         # fasta_file = Bio::DB::Fasta::FastaFile.new(target)
+          #fasta_file = Bio::DB::Fasta::FastaFile.new(target)
           #fasta_file.load_fai_entries
           arm = ChromosomeArm.new(parsed[:arm], target)
           chromosomeArms[arm.name] = arm

data/lib/bio/PolyploidTools/ExonContainer.rb

@@ -19,15 +19,31 @@ module Bio::PolyploidTools
 
     def gene_models(path)
       @gene_models_db = Bio::DB::Fasta::FastaFile.new({:fasta=>path})
+      @gene_models_db.index
       @gene_models_path = path
     end
 
     #Returns the sequence for a region in the gene models (exon)
     def gene_model_sequence(region)
-      #puts region
-      seq=@gene_models_db.fetch_sequence(region)
-
+      #puts "Region: "
+      #puts region.inspect
+      target_reg = @gene_models_db.index.region_for_entry(region.entry)
+      #puts target_reg.inspect
+      region.end = target_reg.length if region.end > target_reg.length
+      #entries[region.entry]
 
+      seq=@gene_models_db.fetch_sequence(region)
+      #puts "sequence: "
+      #This is a patch that we need to fix in biosamtools: 
+      #puts seq
+      index = seq.index('>')
+      if(index )
+        index -= 1
+        #puts "Index: #{index}"
+        seq = seq.slice(0..index) 
+      end
+      #puts seq
+      seq
     end
 
     #Sets the reference file for the gene models
@@ -40,10 +56,10 @@ module Bio::PolyploidTools
     def chromosome_sequence(region)
       left_pad = 0
       #TODO: Padd if it goes to the right
-      if(region.start < 0)
+      if(region.start < 1)
         left_pad = region.start * -1
         left_pad += 1
-        region.start = 0
+        region.start = 1
       end
       str = "-" * left_pad << @chromosomes_db.fetch_sequence(region)
       #str << "n" * (region.size - str.size + 1) if region.size > str.size
@@ -116,12 +132,17 @@ module Bio::PolyploidTools
       @snp_map.each do | gene, snp_array|
         snp_array.each do |snp|
           #file.puts snp.primer_fasta_string 
-       
+          #puts "In print_fast_np_exones"
+          #puts snp.inspect
+
           begin 
             file.puts snp.aligned_sequences_fasta
           rescue Exception=>e
             @missing_exons << snp.to_s
-            $stderr.puts e.to_s
+            $stderr.puts "print_fasta_snp_exones:" + snp.to_s + ":" + e.to_s
+            $stderr.puts "Local position: #{snp.local_position}"
+            $stderr.puts "Local position: #{snp.parental_sequences.to_s}"
+            $stderr.puts e.backtrace
           end
         end
       end
@@ -143,8 +164,10 @@ module Bio::PolyploidTools
             end 
            rescue Exception=>e
               @missing_exons << snp.to_s
+             # $stderr.puts ""
 
-              $stderr.puts e.to_s
+              $stderr.puts "print_primer_3_exons: #{e.to_s} : snp.to_s"
+              $stderr.puts e.backtrace
             end
         end 
       end

data/lib/bio/PolyploidTools/NoSNPSequence.rb

@@ -0,0 +1,286 @@
+
+require_relative "SNP"
+require 'bio-samtools'
+module Bio::PolyploidTools
+  class SNPSequenceException < RuntimeError 
+  end
+
+  class NoSNPSequence < SNP
+
+    attr_accessor :sequence_original
+    #Format: 
+    #snp name,chromsome from contig,microarray sequence
+    #BS00068396_51,2AS,CGAAGCGATCCTACTACATTGCGTTCCTTTCCCACTCCCAGGTCCCCCTA[T/C]ATGCAGGATCTTGATTAGTCGTGTGAACAACTGAAATTTGAGCGCCACAA
+    def self.parse(reg_str)
+      reg_str.chomp!
+      snp = NoSNPSequence.new
+
+      arr = reg_str.split(",")
+      
+      if arr.size == 3
+        snp.gene, snp.chromosome, snp.sequence_original = reg_str.split(",")
+      elsif arr.size == 2
+       snp.gene, snp.sequence_original = arr
+     else
+       throw SNPSequenceException.new "Need two or three fields to parse, and got #{arr.size} in #{reg_str}"
+      end
+      #snp.position = snp.position.to_i
+      #snp.original.upcase!
+      #snp.snp.upcase!  
+      snp.chromosome. strip!
+      snp.snp_in = snp.chromosome
+      snp.parse_sequence_snp
+      snp.exon_list = Hash.new()
+      snp
+    end
+    
+    def parse_snp
+       
+    end
+
+    def parse_sequence_snp
+       @position = (sequence_original.length / 2).to_i 
+       @original = sequence_original[@position]
+       @snp = @original
+    end
+
+    def to_s
+      "#{gene}:#{chromosome}"
+    end
+
+    def sequences_to_align
+      @sequences_to_align = surrounding_exon_sequences unless @sequences_to_align
+      @sequences_to_align
+    end
+
+     def mask_aligned_chromosomal_snp(chromosome)
+      return nil if  aligned_sequences.values.size == 0
+      names = exon_sequences.keys
+
+      masked_snps = aligned_sequences[chromosome].downcase if aligned_sequences[chromosome]
+   
+      masked_snps = "-" * aligned_sequences.values[0].size  unless aligned_sequences[chromosome]
+      #TODO: Make this chromosome specific, even when we have more than one alignment going to the region we want.
+      i = 0
+      while i < masked_snps.size
+        different = 0
+        cov = 0
+        from_group = 0
+        names.each do | chr |
+          if aligned_sequences[chr] and aligned_sequences[chr][i]  != "-"
+            cov += 1 
+
+            from_group += 1 if chr[0] == chromosome_group
+            #puts "Comparing #{chromosome_group} and #{chr[0]} as chromosomes"
+            if chr != chromosome 
+              $stderr.puts "WARN: No base for #{masked_snps} : ##{i}" unless masked_snps[i].upcase
+              $stderr.puts "WARN: No base for #{aligned_sequences[chr]} : ##{i}" unless masked_snps[i].upcase
+              different += 1  if masked_snps[i].upcase != aligned_sequences[chr][i].upcase 
+            end
+          end
+        end
+        masked_snps[i] = "-" if different == 0
+        masked_snps[i] = "-" if cov == 1
+        masked_snps[i] = "*" if cov == 0
+        expected_snps = names.size - 1
+       #puts "Diferences: #{different} to expected: #{ expected_snps } [#{i}] Genome count (#{from_group} == #{genomes_count})"
+        
+        masked_snps[i] = masked_snps[i].upcase if different == expected_snps and from_group == genomes_count
+
+        i += 1
+      end
+      masked_snps
+    end
+
+    def count_deletions_around(position,target_chromosome)
+      first_aligned = aligned_sequences[target_chromosome]
+
+      pos_start = position - flanking_size
+      pos_end = position + flanking_size
+      pos_start = 0 if pos_start < 0
+      pos_end = first_aligned.size - 1 if pos_end >= first_aligned.size
+      count = 0
+      for i in pos_start..pos_end
+        has_del = false
+
+        aligned_sequences.each_pair do |name, val|  
+          has_del = true if val[i] == '-'
+          print "#{val[i]}\t"
+        end
+        count += 1 if has_del
+        print "#{count}\n"
+      end
+      return count
+    end
+
+    def primer_region(target_chromosome, parental_chr )
+      chromosome_seq = aligned_sequences[target_chromosome]
+      #chromosome_seq = "-" * parental.size unless chromosome_seq
+      if aligned_sequences.size == 0
+        #puts aligned_sequences.inspect
+        #puts surrounding_exon_sequences.inspect
+        #puts self.inspect
+        chromosome_seq = surrounding_exon_sequences[target_chromosome]
+
+      end
+      chromosome_seq = chromosome_seq.downcase
+
+      mask = mask_aligned_chromosomal_snp(target_chromosome)
+    
+      pr = PrimerRegion.new
+      pr.homoeologous = false
+      position_in_region = 0
+      parental = chromosome_seq.clone
+      (0..chromosome_seq.size-1).each do |i|
+
+        if chromosome_seq[i] != '-'
+          case   
+          when mask[i] == '-'
+            #When the mask doesnt detect a SNP, so we take the parental
+            parental[i] = chromosome_seq[i] unless Bio::NucleicAcid::is_unambiguous(parental[i])
+          when /[[:upper:]]/.match(mask[i])
+            #This is a good candidate for marking a SNP
+            #We validate that the consensus from the sam file accepts the variation from the chromosomal sequence
+            if parental[i] == '-'
+              parental[i] = mask[i]
+              pr.crhomosome_specific_intron << position_in_region
+            elsif Bio::NucleicAcid.is_valid(parental[i], mask[i])
+              parental[i] = mask[i]
+              pr.chromosome_specific << position_in_region if count_deletions_around(1,target_chromosome) < 3
+              pr.chromosome_specific_in_mask << i
+            end
+
+          when /[[:lower:]]/.match(mask[i])
+            #this is not that good candidate, but sitll gives specificity
+            if parental[i] == '-'
+              parental[i] = mask[i]
+              pr.almost_crhomosome_specific_intron << position_in_region
+            elsif Bio::NucleicAcid.is_valid(parental[i], mask[i])
+              parental[i] = mask[i].upcase
+              pr.almost_chromosome_specific << position_in_region
+              pr.almost_chromosome_specific_in_mask << i
+            end
+          end #Case closes
+          pr.position_in_mask_from_template[position_in_region] = i
+          position_in_region += 1
+        end #Closes region with bases 
+      end         
+
+      pr.sequence=parental.gsub('-','')
+      pr
+    end
+
+    def return_primer_3_string_test(opts={})
+
+      left = opts[:right_pos]
+      right = opts[:right_pos]
+      sequence =  opts[:sequence]
+      orientation = "forward"
+      if opts[:right_pos]
+        orientation = "forward"
+        if left > right
+          left = sequence.size - left - 1
+          right = sequence.size - right - 1
+          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]
+      str << "SEQUENCE_TEMPLATE=#{sequence}\n"
+      str << "=\n"
+
+
+      #In case that we don't have a right primer, we do both orientations
+      unless opts[:right_pos]
+        sequence =  opts[:sequence]    
+        left = sequence.size - left - 1
+        orientation = "reverse"
+        sequence = reverse_complement_string(sequence)
+        str << "SEQUENCE_ID=#{opts[:name]} #{orientation}\n"
+        str << "SEQUENCE_FORCE_LEFT_END=#{left}\n"
+        str << "SEQUENCE_TEMPLATE=#{sequence}\n"
+        str << "=\n"
+      end
+
+      str
+    end
+
+    def get_base_in_different_chromosome(position, target_chromosome)
+
+        aligned_sequences.each_pair do |name, val|  
+          next if target_chromosome == name
+          return val[position]
+        end
+    end
+
+    def primer_3_all_strings(target_chromosome, parental) 
+      pr = primer_region(target_chromosome, parental )
+      primer_3_propertes = Array.new
+
+      seq_original = String.new(pr.sequence)
+      #puts seq_original.size.to_s << "-" << primer_3_min_seq_length.to_s
+      return primer_3_propertes if seq_original.size < primer_3_min_seq_length
+
+      if pr.homoeologous
+        snp_type = "homoeologous"
+      else
+        snp_type = "non-homoeologous"
+      end
+
+      pr.chromosome_specific.each do |pos|
+        
+        seq_snp =  String.new(pr.sequence)
+        orgiginal_base = seq_snp[pos]
+        other_chromosome_base = get_base_in_different_chromosome(pos, target_chromosome)
+
+        args = {
+          :name =>"#{gene} A chromosome_specific exon #{snp_type} #{chromosome}", 
+          :left_pos => pos,  
+          :sequence=>seq_original
+        }
+        
+        
+        primer_3_propertes << return_primer_3_string(args)
+        args[:name] = "#{gene} B chromosome_specific exon #{snp_type} #{chromosome}"
+        args[:sequence] = seq_snp
+        #TODO: Find base from another chromosome
+        seq_snp[pos] =  other_chromosome_base.upcase
+        
+        primer_3_propertes << return_primer_3_string(args)
+      end
+
+  
+      primer_3_propertes
+    end
+
+    def aligned_sequences
+     
+      return @aligned_sequences if @aligned_sequences
+      if sequences_to_align.size == 1
+        @aligned_sequences = sequences_to_align
+        return @aligned_sequences
+      end
+      options = ['--maxiterate', '1000', '--localpair', '--quiet']
+      mafft = Bio::MAFFT.new( "mafft" , options)
+    #  puts "Before MAFT:#{sequences_to_align.inspect}"
+      report = mafft.query_align(sequences_to_align)
+      @aligned_sequences = report.alignment
+   #   puts "MAFFT: #{report.alignment.inspect}" 
+      @aligned_sequences
+    end
+    
+
+
+   
+
+  end
+end