bio-polyploid-tools 0.8.0 → 0.8.1

checksums.yaml

@@ -1,7 +1,7 @@
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data/README.md

@@ -1,10 +1,12 @@
-#bio-polyploid-tools
+# bio-polyploid-tools
+
+## Introduction
 
-##Introduction
 This tools are designed to deal with polyploid wheat. The first tool is to design KASP primers, making them as specific as possible. 
 
 
-##Installation
+## Installation
+
 ```sh
 gem install bio-polyploid-tools
 ```
@@ -13,13 +15,19 @@ You need to have in your ```$PATH``` the following programs:
 * [MAFFT](http://mafft.cbrc.jp/alignment/software/)
 * [primer3](http://primer3.sourceforge.net/releases.php)
 * [exonerate](http://www.ebi.ac.uk/~guy/exonerate/) 
+* [blast](https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE%3DBlastDocs&DOC_TYPE%3DDownload)
 
-The code has been developed on ruby 2.1.0, but it should work on 1.9.3 and above. 
+The code was originally developed on ruby 2.1.0, but it should work on 1.9.3 and above. However, it is only actively tested in currently supported ruby versions: 
+  
+  * 2.1.10
+  * 2.2.5
+  * 2.3.5
+  * 2.4.2
 
-#PolyMarker
 
-To run poolymerker with the CSS wheat contigs, you need to unzip the reference file from  [ensembl](http://ftp.ensemblgenomes.org/pub/release-25/plants/fasta/triticum_aestivum/dna/Triticum_aestivum.IWGSC2.25.dna.genome.fa.gz).
+# PolyMarker
 
+To run PolyMarker with the CSS wheat contigs, you need to unzip the reference file from  [ensembl](http://ftp.ensemblgenomes.org/pub/release-25/plants/fasta/triticum_aestivum/dna/Triticum_aestivum.IWGSC2.25.dna.genome.fa.gz).
 
 
 ```sh
@@ -80,7 +88,7 @@ If the flanking sequence is unknow, but the position on a reference is available
 * **alternative allele** The base in the alternative allele. 
 * **target chromosome** for the specific primers. Must be in line with the chromosome selection critieria. 
 
-####Example
+#### Example
 
 ```
 IWGSC_CSS_1AL_scaff_110,C,519,A,2A
@@ -89,7 +97,8 @@ IWGSC_CSS_1AL_scaff_110,C,519,A,2A
 This file format can be used with ```snp_positions_to_polymarker.rb``` to produce the input for the option```--marker_list```.
 
 
-###Custom reference sequences. 
+### Custom reference sequences. 
+
 By default, the contigs and pseudomolecules from [ensembl](ftp://ftp.ensemblgenomes.org/pub/release-25/plants/fasta/triticum_aestivum/dna/Triticum_aestivum.IWGSC2.25.dna.genome.fa.gz
 ) are used. However, it is possible to use a custom reference. To define the chromosome where each contig belongs the argument ```arm_selection``` is used.  The defailt uses ids like: ```IWGSC_CSS_1AL_scaff_110```, where the third field, separated by underscores is used. A simple way to add costum references is to rename the fasta file to follow that convention. Another way is to use the option ```--arm_selection arm_selection_first_two```, where only the first two characters in each contig is used as identifier, useful when pseudomolecules are named after the chromosomes (ie: ">1A" in the fasta file). 
 
@@ -117,33 +126,38 @@ To use blast instead of exonerate, use the following command:
 ```
 
 
-##Release Notes
+## Release Notes
+
+### 0.8
+
+* FEATURE: ```polymarker.rb``` added the flag ```--aligner blast|exonerate ``` which lets you pick between ```blast``` or ```exonerate``` as the aligner. For blast the default is to have the database with the same name as the ```--contigs``` file. However, it is possible to use a different name vua the option ```--database```.
+
+### 0.7.3
 
-###0.7.3
 * FEATURE: ```polymarker.rb``` Added to the flag ```--arm_selection``` the option ```scaffold```, which now supports a scaffold specific primer.
 * FEATURE: ```snp_position_to_polymarker``` Added the option ```--mutant_list```  to prepare files for PolyMarker from files with the following columns ```ID,Allele_1,position,Allele_1,target_chromosome```. 
 
-###0.7.2
+### 0.7.2
+
 * FEATURE: Added a flag ```min_identity``` to set the minimum identity to consider a hit. The default is 90
 
-###0.7.1
+### 0.7.1
 * BUGFIX: Now the parser for ```arm_selection_embl``` works with the mixture of contigs and pseudomolecules
 *  DOC: Added documentation on how to use custom references.  
 
-###0.7.0
+### 0.7.0
 * Added flag ```genomes_count``` for number of genomes, to be used on tetraploids, etc. 
 
-###0.6.1
+### 0.6.1
 
 
 * polymarker.rb now validates that all the files exist. 
 * BUGFIX: A reference was required even when it was not used to generate contigs. 
 
-#Notes
-
+# Notes
 
-* If the SNP is in a gap in the alignment to the chromosomes, it is ignored. 
 
+* BUG: If the SNP is in a gap in the alignment to the chromosomes, it is ignored. 
 * BUG: Blocks with NNNs are picked and treated as semi-specific. 
 * BUG: If the name of the reference have space, the ID is not chopped. ">gene_1 (G12A)" shouls be treated as ">gene_1". 
 * TODO: Add a parameter file to configure the alignments. 

data/VERSION

@@ -1 +1 @@
-0.8.0
+0.8.1

data/bin/homokaryot_primers.rb

@@ -180,4 +180,4 @@ end
 
 kasp_container.add_primers_file(primer_3_output)
 header = "Marker,SNP,RegionSize,SNP_type,#{snp_in},#{original_name},common,primer_type,orientation,#{snp_in}_TM,#{original_name}_TM,common_TM,selected_from,product_size"
-File.open(output_primers, 'w') { |f| f.write("#{header}\n#{kasp_container.print_primers}") }
+File.open(output_primers, 'w') { |f| f.write("#{header}\n#{kasp_container.print_primers}") }

data/bin/polymarker.rb

@@ -12,6 +12,11 @@ require path
 
 arm_selection_functions = Hash.new;
 
+arm_selection_functions[:arm_selection_nrgenes] = lambda do | contig_name |
+#example format: chr2A
+  ret = contig_name[3,2]
+  return ret
+end
 
 arm_selection_functions[:arm_selection_first_two] = lambda do | contig_name |
   contig_name.gsub!(/chr/,"")
@@ -417,4 +422,4 @@ rescue StandardError => e
 rescue Exception => e
   write_status "ERROR\t#{e.message}"
   raise e  
-end
+end

data/bin/snp_position_to_polymarker.rb

@@ -71,16 +71,26 @@ File.open(test_file) do | f |
          snp = Bio::PolyploidTools::SNPMutant.parse(line)
          entry = fasta_reference_db.index.region_for_entry(snp.contig)
       end
-    	
+    	#puts line
     	if entry
        		region = entry.get_full_region
-       		if region != lastRegion
-             lastTemplate = fasta_reference_db.fetch_sequence(region)
-          end
-          snp.full_sequence = lastTemplate
+          snp_name = snp.snp_id_in_seq
+
+       		#if region != lastRegion
+          #  lastTemplate = fasta_reference_db.fetch_sequence(region)
+          #end
+          start, total, new_position = snp.to_polymarker_coordinates(options[:flanking_size])
+          region.start = start 
+          region.end = start + total
+          #puts region
+          local_template = fasta_reference_db.fetch_sequence(region)
+
+          snp.position = new_position
+
+          snp.template_sequence = local_template
           lastRegion = region
 
-       		out.puts "#{snp.gene}_#{snp.snp_id_in_seq},#{snp.chromosome},#{snp.sequence_original}"
+       		out.puts "#{snp.gene}_#{snp_name},#{snp.chromosome},#{snp.to_polymarker_sequence(options[:flanking_size])}"
     	else
     	   $stderr.puts "ERROR: Unable to find entry for #{snp.gene}"
     	end

data/bio-polyploid-tools.gemspec

@@ -2,16 +2,16 @@
 # DO NOT EDIT THIS FILE DIRECTLY
 # Instead, edit Juwelier::Tasks in Rakefile, and run 'rake gemspec'
 # -*- encoding: utf-8 -*-
-# stub: bio-polyploid-tools 0.8.0 ruby lib
+# stub: bio-polyploid-tools 0.8.1 ruby lib
 
 Gem::Specification.new do |s|
   s.name = "bio-polyploid-tools".freeze
-  s.version = "0.8.0"
+  s.version = "0.8.1"
 
   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.date = "2018-01-19"
   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]
@@ -102,6 +102,10 @@ Gem::Specification.new do |s|
     "test/data/BS00068396_51_contigs.aln",
     "test/data/BS00068396_51_contigs.dnd",
     "test/data/BS00068396_51_contigs.fa",
+    "test/data/BS00068396_51_contigs.fa.fai",
+    "test/data/BS00068396_51_contigs.fa.nhr",
+    "test/data/BS00068396_51_contigs.fa.nin",
+    "test/data/BS00068396_51_contigs.fa.nsq",
     "test/data/BS00068396_51_contigs.nhr",
     "test/data/BS00068396_51_contigs.nin",
     "test/data/BS00068396_51_contigs.nsq",

data/lib/bio/PolyploidTools/ExonContainer.rb

@@ -116,13 +116,13 @@ module Bio::PolyploidTools
         target_region = exon.target_region
         exon_start_offset = exon.query_region.start - gene_region.start
         chr_local_pos=local_pos_in_gene + target_region.start + 1
-        ret_str << ">#{chromosome}_SNP-#{chr_local_pos} #{exon.to_s} #{target_region.orientation}\n"
-        to_print = "-" * exon_start_offset 
-        chr_seq = chromosome_sequence(exon.target_region).to_s
-        l_pos = exon_start_offset + local_pos_in_gene
-        to_print <<  chr_seq
+        ret_str  << ">#{chromosome}_SNP-#{chr_local_pos} #{exon.to_s} #{target_region.orientation}\n"
+        to_print =  "-" * exon_start_offset 
+        chr_seq  =  chromosome_sequence(exon.target_region).to_s
+        l_pos    =  exon_start_offset + local_pos_in_gene
+        to_print << chr_seq
         to_print[local_pos_in_gene] = to_print[local_pos_in_gene].upcase
-        ret_str << to_print
+        ret_str  << to_print
       end
       ret_str
     end

data/lib/bio/PolyploidTools/SNP.rb

@@ -16,6 +16,8 @@ module Bio::PolyploidTools
     attr_accessor :chromosome
     attr_accessor :variation_free_region
 
+
+
     #Format: 
     #Gene_name,Original,SNP_Pos,pos,chromosome
     #A_comp0_c0_seq1,C,519,A,2A
@@ -30,7 +32,7 @@ module Bio::PolyploidTools
       snp.snp.upcase!
       snp.snp.strip!  
       snp.chromosome.strip!
-      snp.exon_list = Hash.new()
+   
       snp.use_reference = false
       snp
     end
@@ -60,6 +62,16 @@ module Bio::PolyploidTools
       @primer_3_min_seq_length = 50
       @variation_free_region = 0
       @contig = false
+      @exon_list = Hash.new {|hsh, key| hsh[key] = [] }
+    end
+
+    def to_polymarker_coordinates(flanking_size, total:nil)
+      start = position - flanking_size + 1
+      start = 0 if start < 0
+      total = flanking_size * 2 unless total
+      total += 1
+      new_position = position - start + 2
+      [start , total, new_position ]
     end
 
     def to_polymarker_sequence(flanking_size, total:nil)
@@ -103,8 +115,7 @@ module Bio::PolyploidTools
       end
 
     def add_exon(exon, arm)
-      @exon_list[arm] = exon unless @exon_list[arm]
-      @exon_list[arm] = exon if exon.record.score > @exon_list[arm].record.score
+      exon_list[arm] << exon    
     end
 
     def covered_region
@@ -115,28 +126,28 @@ module Bio::PolyploidTools
          reg.orientation = :forward
          reg.start = self.position - self.flanking_size
          reg.end = self.position + self.flanking_size
-         
          reg.start = 1 if reg.start < 1
-         
          return reg
       end
       
       min = @position
       max = @position
-     # puts "Calculating covered region for #{self.inspect}"
-    #  puts "#{@exon_list.inspect}"
-      #raise SNPException.new "Exons haven't been loaded for #{self.to_s}" if @exon_list.size == 0
+      # puts "Calculating covered region for #{self.inspect}"
+      # puts "#{@exon_list.inspect}"
+      # raise SNPException.new "Exons haven't been loaded for #{self.to_s}" if @exon_list.size == 0
       if @exon_list.size == 0
         min = self.position - self.flanking_size
         min = 1 if min < 1
         max =  self.position + self.flanking_size
       end
-      @exon_list.each do | chromosome, exon |
-       # puts exon.inspect
-        reg = exon.query_region
-        min = reg.start if reg.start < min
-        max = reg.end if reg.end > max
+      @exon_list.each do | chromosome, exon_arr |
+        exon_arr.each do | exon |
+          reg = exon.query_region
+          min = reg.start if reg.start < min
+          max = reg.end if reg.end > max
+        end
       end
+
       reg = Bio::DB::Fasta::Region.new()
       reg.entry = gene
       reg.orientation = :forward
@@ -168,24 +179,6 @@ module Bio::PolyploidTools
       pos + left_padding
     end
 
-    def exon_fasta_string
-      gene_region = self.covered_region
-      local_pos_in_gene = self.local_position
-      ret_str = ""
-      container.parents.each  do |name, bam|
-        ret_str << ">#{gene_region.entry}-#{self.position}_#{name} Overlapping_exons:#{gene_region.to_s} localSNPpo:#{local_pos_in_gene+1}\n" 
-        to_print = parental_sequences[name]
-        ret_str << to_print << "\n"
-      end
-      self.exon_sequences.each do | chromosome, exon_seq | 
-        ret_str << ">#{chromosome}\n#{exon_seq}\n"
-      end
-      mask = masked_chromosomal_snps("1BS", flanking_size)
-      ret_str << ">Mask\n#{mask}\n"
-      ret_str
-    end
-
-
     def primer_fasta_string
       gene_region = self.covered_region
       local_pos_in_gene = self.local_position
@@ -209,12 +202,15 @@ module Bio::PolyploidTools
     end
 
     def primer_region(target_chromosome, parental )
+  
       parental = aligned_sequences[parental].downcase
+      names = aligned_sequences.keys
+      target_chromosome = get_target_sequence(names, target_chromosome)
+  
       chromosome_seq = aligned_sequences[target_chromosome]
       chromosome_seq = "-" * parental.size unless chromosome_seq
       chromosome_seq = chromosome_seq.downcase
       mask = mask_aligned_chromosomal_snp(target_chromosome)
-      #puts "'#{mask}'"
 
       pr = PrimerRegion.new
       position_in_region = 0
@@ -291,8 +287,9 @@ module Bio::PolyploidTools
 
       end
 
-
-      str = "SEQUENCE_ID=#{opts[:name]} #{orientation}\n"
+      #puts "__"
+      #puts self.inspect
+      str = "SEQUENCE_ID=#{opts[:name]} #{orientation} \n"
       str << "SEQUENCE_FORCE_LEFT_END=#{left}\n" unless  opts[:extra_f]
       str << "SEQUENCE_FORCE_RIGHT_END=#{right}\n" if opts[:right_pos]
       str << extra if extra
@@ -326,10 +323,10 @@ module Bio::PolyploidTools
       primer_3_propertes = Array.new
 
       seq_original = String.new(pr.sequence)
-      puts seq_original.size.to_s << "-" << primer_3_min_seq_length.to_s 
+      #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
       #puts self.inspect
-      puts pr.snp_pos.to_s << "(" << seq_original.length.to_s << ")"
+      #puts pr.snp_pos.to_s << "(" << seq_original.length.to_s << ")"
       
       seq_original[pr.snp_pos] = self.original
       seq_original_reverse = reverse_complement_string(seq_original)
@@ -432,12 +429,13 @@ module Bio::PolyploidTools
         
         seq[local_pos_in_gene] = self.snp if name == self.snp_in    
         @parental_sequences [name] = seq
-        puts name
-        puts seq
       end
       @parental_sequences
     end
 
+
+
+
     def surrounding_parental_sequences
       return @surrounding_parental_sequences if @surrounding_parental_sequences
       gene_region = self.covered_region
@@ -450,11 +448,15 @@ module Bio::PolyploidTools
           seq =  bam.consensus_with_ambiguities({:region=>gene_region}).to_s
         else
           seq = container.gene_model_sequence(gene_region)
-           
-           unless name == self.snp_in
-              #puts "Modifing original: #{name} #{seq}"  
-              seq[local_pos_in_gene] = self.original 
-            end
+          #puts "#{name} #{self.snp_in}"
+          #puts "Modifing original: #{name}\n#{seq}"  
+          unless name == self.snp_in
+
+            seq[local_pos_in_gene] = self.original 
+          else
+            seq[local_pos_in_gene] = self.snp 
+          end
+          #puts "#{seq}"  
         end
         seq[local_pos_in_gene] = seq[local_pos_in_gene].upcase
         seq[local_pos_in_gene] = self.snp if name == self.snp_in  
@@ -522,71 +524,101 @@ module Bio::PolyploidTools
       ret_str 
     end
 
+
+    def get_snp_position_after_trim
+      local_pos_in_gene = self.local_position
+      ideal_min = self.local_position - flanking_size 
+      ideal_max = self.local_position + flanking_size
+      left_pad = 0
+      if ideal_min < 0
+        left_pad = ideal_min * -1
+        ideal_min = 0 
+      end
+      local_pos_in_gene - ideal_min
+    end
+
     def aligned_snp_position
       return @aligned_snp_position if @aligned_snp_position
+      #puts self.inspect
       pos = -1
       parental_strings = Array.new
       parental_sequences.keys.each do | par |
-        
         parental_strings << aligned_sequences[par]
       end
-      template_sequence = nil
-      aligned_sequences.keys.each do |temp |
-        template_sequence = aligned_sequences[ temp ] if  aligned_sequences[ temp ][0] != "-"
-      end
       $stderr.puts "WARN: #{self.to_s} #{parental_sequences.keys} is not of size 2 (#{parental_strings.size})" if parental_strings.size != 2
 
+      local_pos_in_parental = get_snp_position_after_trim
       i = 0
-      differences = 0
-      local_pos_in_gene = flanking_size
-      local_pos = 0
-      started = false
-#TODO: Validate the cases when the alignment has padding on the left on all the chromosomes
-      #unless parental_strings[0] 
-        #puts "parental hash: #{parental_sequences}"
-        #puts "Aligned sequences: #{aligned_sequences.to_fasta}"
-       # puts "parental_strings: #{parental_strings.to_s}"
-      #end
       while i < parental_strings[0].size  do
-        if local_pos_in_gene == local_pos
+        if local_pos_in_parental == 0 and parental_strings[0][i] != "-"
           pos = i
           if parental_strings[0][i] == parental_strings[1][i]
             $stderr.puts "WARN: #{self.to_s} doesn't have a SNP in the marked place (#{i})! \n#{parental_strings[0]}\n#{parental_strings[1]}"
           end 
-    
-        end
-
-        started = true if template_sequence[i] != "-" 
-        if started == false or template_sequence[i] != "-" 
-          local_pos += 1
         end
+      
+        local_pos_in_parental -= 1 if parental_strings[0][i] != "-"
         i += 1
       end
       @aligned_snp_position = pos
       return pos
     end
 
+    def get_target_sequence(names, chromosome)
+      
+      best = chromosome
+      best_score = 0
+      names.each do |e|  
+        arr = e.split("_")
+        if arr.length == 3
+          score = arr[2].to_f
+          if score >best_score
+            best_score = score 
+            best = e
+          end
+        end
+      end
+      best
+    end
+
+    
+
     def mask_aligned_chromosomal_snp(chromosome)
-      names = exon_sequences.keys
+      names = aligned_sequences.keys
       parentals =  parental_sequences.keys
 
+      position_after_trim = get_snp_position_after_trim
+
+      names = names - parentals
       local_pos_in_gene = aligned_snp_position
-      masked_snps = aligned_sequences[chromosome].downcase if aligned_sequences[chromosome]
-      masked_snps = "-" * aligned_sequences.values[0].size  unless aligned_sequences[chromosome]
+
+      best_target = get_target_sequence(names, chromosome)
+      masked_snps = aligned_sequences[best_target].downcase if aligned_sequences[best_target]
+      masked_snps = "-" * aligned_sequences.values[0].size  unless aligned_sequences[best_target]
       #TODO: Make this chromosome specific, even when we have more than one alignment going to the region we want.
+      #puts "mask_aligned_chromosomal_snp(#{chromosome})"
+      #puts names
       i = 0
-      while i < masked_snps.size
+      for  i in 0..masked_snps.size-1
+        #puts i
         different = 0
         cov = 0
         from_group = 0
         nCount = 0
+        seen = []
         names.each do | chr |
           if aligned_sequences[chr] and aligned_sequences[chr][i]  != "-"
+            #puts aligned_sequences[chr][i]
             cov += 1 
             nCount += 1 if aligned_sequences[chr][i] == 'N' or  aligned_sequences[chr][i] == 'n' # maybe fix this to use ambiguity codes instead. 
-            from_group += 1 if chr[0] == chromosome_group
+            
+            if chr[0] == chromosome_group and  not seen.include? chr[1]
+              seen << chr[1]
+              from_group += 1 
+
+            end
             #puts "Comparing #{chromosome_group} and #{chr[0]} as chromosomes"
-            if chr != chromosome 
+            if chr != best_target 
               $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 
@@ -598,12 +630,15 @@ module Bio::PolyploidTools
         masked_snps[i] = "-" if nCount > 0
         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})"
+
+       #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
+        #puts "#{i}:#{masked_snps[i]}"
 
         if i == local_pos_in_gene
           masked_snps[i] = "&"
+          #puts "#{i}:#{masked_snps[i]}___"
           bases = ""
           names.each do | chr |
             bases << aligned_sequences[chr][i]  if aligned_sequences[chr] and aligned_sequences[chr][i]  != "-"
@@ -617,62 +652,22 @@ module Bio::PolyploidTools
           end 
 
         end
-        i += 1
-      end
-      masked_snps
-    end
-
-    def masked_chromosomal_snps(chromosome)
-      chromosomes = exon_sequences
-      names = chromosomes.keys
-      masked_snps = chromosomes[chromosome].tr("-","+") if chromosomes[chromosome]
-      masked_snps = "-" * covered_region.size unless chromosomes[chromosome]
-      local_pos_in_gene = self.local_position
-      ideal_min = local_pos_in_gene - flanking_size
-      ideal_max = local_pos_in_gene + flanking_size
-      i = 0
-      while i < masked_snps.size  do
-        if i > ideal_min and i <= ideal_max
-
-          different = 0
-          cov = 0
-          names.each do | chr |
-            if chromosomes[chr][i]  != "-"
-              cov += 1 
-              if chr != chromosome and masked_snps[i] != "+"
-                different += 1  if masked_snps[i] != chromosomes[chr][i] 
-              end
-            end
-
-          end
-          masked_snps[i] = "-" if different == 0 and  masked_snps[i] != "+"
-          masked_snps[i] = "-" if cov < 2
-          masked_snps[i] = masked_snps[i].upcase if different > 1   
-
-        else
-          masked_snps[i] = "*"
-        end   
-        if i == local_pos_in_gene
-          masked_snps[i] = "&"
-        end
-        i += 1
+        #i += 1
       end
       masked_snps
     end
 
+    
     def surrounding_masked_chromosomal_snps(chromosome)
 
       chromosomes = surrounding_exon_sequences
       names = chromosomes.keys
+      get_target_sequence(names)
       masked_snps = chromosomes[chromosome].tr("-","+") if chromosomes[chromosome]
       masked_snps = "-" * (flanking_size * 2 ) unless chromosomes[chromosome]
       local_pos_in_gene = flanking_size 
-      # ideal_min = local_pos_in_gene - flanking_size
-      #ideal_max = local_pos_in_gene + flanking_size
       i = 0
       while i < masked_snps.size  do
-
-
         different = 0
         cov = 0
         names.each do | chr |
@@ -682,13 +677,11 @@ module Bio::PolyploidTools
               different += 1  if masked_snps[i] != chromosomes[chr][i] 
             end
           end
-
         end
         masked_snps[i] = "-" if different == 0 and  masked_snps[i] != "+"
         masked_snps[i] = "-" if cov < 2
         masked_snps[i] = masked_snps[i].upcase if different > 1   
 
-
         if i == local_pos_in_gene
           masked_snps[i] = "&"
         end
@@ -699,18 +692,19 @@ module Bio::PolyploidTools
 
     def surrounding_exon_sequences
       return @surrounding_exon_sequences if @surrounding_exon_sequences
+      gene_region = self.covered_region
       @surrounding_exon_sequences =  Bio::Alignment::SequenceHash.new
-      self.exon_list.each do |chromosome, exon| 
-        #puts "surrounding_exon_sequences #{flanking_size}"
-        #puts chromosome
-        #puts exon
-        flanquing_region  = exon.target_flanking_region_from_position(position,flanking_size)
-        #TODO: Padd when the exon goes over the regions... 
-        
-        #Ignoring when the exon is in a gap
-        unless exon.snp_in_gap 
-          exon_seq = container.chromosome_sequence(flanquing_region)
-          @surrounding_exon_sequences[chromosome] = exon_seq
+      self.exon_list.each do |chromosome, exon_arr| 
+        exon_arr.each do |exon|
+          exon_start_offset = exon.query_region.start - gene_region.start
+          flanquing_region  = exon.target_flanking_region_from_position(position,flanking_size)
+          #TODO: Padd when the exon goes over the regions... 
+          #puts flanquing_region.inspect
+          #Ignoring when the exon is in a gap
+          unless exon.snp_in_gap 
+            exon_seq = container.chromosome_sequence(flanquing_region)
+            @surrounding_exon_sequences["#{chromosome}_#{flanquing_region.start}_#{exon.record.score}"] = exon_seq
+          end
         end
       end
       @surrounding_exon_sequences
@@ -722,18 +716,21 @@ module Bio::PolyploidTools
       gene_region = self.covered_region
       local_pos_in_gene = self.local_position
       @exon_sequences = Bio::Alignment::SequenceHash.new
-      self.exon_list.each do |chromosome, exon| 
-        exon_start_offset = exon.query_region.start - gene_region.start
-        exon_seq  = "-" * exon_start_offset 
-        exon_seq << container.chromosome_sequence(exon.target_region).to_s
-        #puts exon_seq
-       # l_pos = exon_start_offset + local_pos_in_gene
-        unless exon.snp_in_gap
-          #puts "local position: #{local_pos_in_gene}"
-          #puts "Exon_seq: #{exon_seq}"
-          exon_seq[local_pos_in_gene] = exon_seq[local_pos_in_gene].upcase
-          exon_seq << "-" * (gene_region.size - exon_seq.size + 1)
-          @exon_sequences[chromosome] = exon_seq
+      self.exon_list.each do |chromosome, exon_arr| 
+        exon_arr.each do |exon|
+          exon_start_offset = exon.query_region.start - gene_region.start
+          exon_seq  = "-" * exon_start_offset 
+          exon_seq << container.chromosome_sequence(exon.target_region).to_s
+          #puts exon_seq
+          #l_pos = exon_start_offset + local_pos_in_gene
+          unless exon.snp_in_gap
+            #puts "local position: #{local_pos_in_gene}"
+            #puts "Exon_seq: #{exon_seq}"
+            exon_seq[local_pos_in_gene] = exon_seq[local_pos_in_gene].upcase
+            exon_seq << "-" * (gene_region.size - exon_seq.size + 1)
+            #puts exon.inspect
+            @exon_sequences["#{chromosome}_#{exon.query_region.start}_#{exon.record.score}"] = exon_seq
+          end
         end
       end
       @exon_sequences[@chromosome] = "-" * gene_region.size unless @exon_sequences[@chromosome]

data/lib/bio/PolyploidTools/SNPMutant.rb

@@ -38,7 +38,6 @@ module Bio::PolyploidTools
         $stderr.puts e
       end
       
-      snp.exon_list = Hash.new()
       snp.flanking_size=100
       snp.region_size = region_size.to_i if region_size
       snp.flanking_size = parsed_flanking.to_i if parsed_flanking

data/lib/bio/PolyploidTools/SNPSequence.rb

@@ -29,7 +29,7 @@ module Bio::PolyploidTools
       #snp.snp.upcase!  
       snp.chromosome. strip!
       snp.parse_sequence_snp
-      snp.exon_list = Hash.new()
+  
       snp
     end
     

data/lib/bio/db/primer3.rb

@@ -113,6 +113,8 @@ module Bio::DB::Primer3
       right_start = 0
       right_end = 0
       total_columns_before_messages=17
+      #puts "Values in primer3"
+      #puts snp_from.inspect
       @values = Array.new
       #@values << "#{gene},,#{template_length},"
       @values << gene
@@ -763,7 +765,7 @@ module Bio::DB::Primer3
       snp.line_1 = @line_1
       snp.line_2 = @line_2 
       snp.snp_from = snp_in
-      snp.regions = snp_in.exon_list.values.collect { |x| x.target_region.to_s }
+      snp.regions = snp_in.exon_list.values.collect { |x|  x.collect {|y| y.target_region.to_s }}
       @snp_hash[snp.to_s] = snp
       snp
     end

data/test/data/BS00068396_51_contigs.fa.fai

@@ -0,0 +1,4 @@
+2AS_5222932	6364	46	6364	6365
+2BS_5245544	8836	6457	8836	8837
+2BS_5163353	11974	15341	11974	11975
+2DS_5334799	7226	27363	7226	7227

metadata

@@ -1,14 +1,14 @@
 --- !ruby/object:Gem::Specification
 name: bio-polyploid-tools
 version: !ruby/object:Gem::Version
-  version: 0.8.0
+  version: 0.8.1
 platform: ruby
 authors:
 - Ricardo H.  Ramirez-Gonzalez
 autorequire: 
 bindir: bin
 cert_chain: []
-date: 2018-01-18 00:00:00.000000000 Z
+date: 2018-01-19 00:00:00.000000000 Z
 dependencies:
 - !ruby/object:Gem::Dependency
   name: bio
@@ -206,6 +206,10 @@ files:
 - test/data/BS00068396_51_contigs.aln
 - test/data/BS00068396_51_contigs.dnd
 - test/data/BS00068396_51_contigs.fa
+- test/data/BS00068396_51_contigs.fa.fai
+- test/data/BS00068396_51_contigs.fa.nhr
+- test/data/BS00068396_51_contigs.fa.nin
+- test/data/BS00068396_51_contigs.fa.nsq
 - test/data/BS00068396_51_contigs.nhr
 - test/data/BS00068396_51_contigs.nin
 - test/data/BS00068396_51_contigs.nsq