bio-polymarker 1.3.2

data/bin/polymarker_capillary.rb

@@ -0,0 +1,443 @@
+#!/usr/bin/env ruby
+require 'bio'
+require 'bio-samtools-wrapper'
+require 'pathname'
+require 'optparse'
+
+$: << File.expand_path(File.dirname(__FILE__) + '/../lib')
+$: << File.expand_path('.')
+path= File.expand_path(File.dirname(__FILE__) + '/../lib/bio-polymarker.rb')
+require path
+
+def log(msg)
+  time=Time.now.strftime("%Y-%m-%d %H:%M:%S.%L")
+  puts "#{time}: #{msg}"
+end
+
+
+
+#reference='wheat_6x_ty_mm_mutations_10mutants_for_validations/scaffolds_with_mm.fa'
+#markers='wheat_6x_ty_mm_mutations_10mutants_for_validations/CadMulitMap.fa'
+#output_folder='wheat_6x_ty_mm_mutations_10mutants_for_validations/PolyMarker'
+
+options = Hash.new
+
+options[:primer_3_preferences] = {
+  :primer_product_size_range => "100-900" ,
+  :primer_max_size => 25 , 
+  :primer_lib_ambiguity_codes_consensus => 1,
+  :primer_liberal_base => 1, 
+  :primer_min_left_three_prime_distance => 5,
+  :primer_min_right_three_prime_distance => 5,
+  :primer_num_return =>1,
+  :primer_explain_flag => 1,
+  :primer_thermodynamic_parameters_path=>File.expand_path(File.dirname(__FILE__) + '../../conf/primer3_config/') + '/'
+}
+options[:genomes_count] = 3
+options[:allow_non_specific] = false
+options[:aligner] = :blast
+options[:arm_selection]
+model="ungapped" 
+options[:arm_selection] = Bio::PolyploidTools::ChromosomeArm.getArmSelection("nrgene")
+options[:database]  = false 
+
+OptionParser.new do |opts|
+  opts.banner = "Usage: polymarker_deletions.rb [options]"
+
+  opts.on("-r", "--reference FILE", "Fasta file with the assembly") do |o|
+    options[:reference] = o
+  end
+
+  opts.on("-m", "--sequences FILE", "Fasta file with the sequences to amplify. the format must be Chromosome:start-end. Chromosome 
+    should match the names to the entries in the fasta files as it is used as main target") do |o|
+    options[:markers] = o
+  end
+
+  opts.on("-o", "--output FOLDER", "Path to a folder where the outputs are going to be stored") do |o|
+    options[:output_folder] = o
+  end
+  opts.on("-g", "--genomes_count INT", "Number of genomes (default 3, for hexaploid)") do |o|
+    options[:genomes_count] = o.to_i
+  end
+  opts.on("-A", "--allow_non_specific", "If used, semi-specific and non-specific primers will be produced") do |o|
+    options[:allow_non_specific] = true
+  end
+
+  opts.on("-d", "--database PREFIX", "Path to the blast database. Only used if the aligner is blast. The default is the name of the contigs file without extension.") do |o|
+    options[:database] = o
+  end
+
+
+  opts.on("-a", "--arm_selection #{Bio::PolyploidTools::ChromosomeArm.getValidFunctions.join('|')}", "Function to decide the chromome arm") do |o|
+    options[:arm_selection] = Bio::PolyploidTools::ChromosomeArm.getArmSelection(o)
+  end
+
+end.parse!
+
+
+#puts options.inspect
+reference     = options[:reference]
+markers       = options[:markers]
+output_folder = options[:output_folder]
+allow_non_specific = options[:allow_non_specific]
+
+options[:database] = options[:reference] unless  options[:database] 
+temp_fasta_query="#{output_folder}/to_align.fa"
+log "Output folder: #{output_folder}"
+exonerate_file="#{output_folder}/exonerate_tmp.tab"
+Dir.mkdir(output_folder)
+arm_selection = options[:arm_selection]
+
+module Bio::PolyploidTools
+  
+  class SequenceToAmplify < SNP
+
+    def self.select_chromosome(gene_name, arm_selection)
+      #m=/##INFO=<ID=(.+),Number=(.+),Type=(.+),Description="(.+)">/.match(gene_name)
+      #m=/TraesCS(\d{1})(\w{1})(\d{2})G(\d+)/.match(gene_name)
+      #ret = {:group : m[1],
+      #       :genome : m[2],:version=>m[3],:chr_id=>m[4]}
+    
+      
+      #arr = contig_name.split('_')
+      #ret = "U"
+      #ret = arr[2][0,2] if arr.size >= 3
+      #ret = "3B" if arr.size == 2 and arr[0] == "v443"
+      #ret = arr[0][0,2] if arr.size == 1   
+      #ret = "#{m[1]}#{m[2]}"
+      #puts ret
+      ret = arm_selection.call(gene_name)
+      return ret
+    end
+
+    attr_accessor :sequence_original
+    attr_accessor :rstart
+    attr_accessor :rend
+    attr_accessor :includeNoSpecific
+    #Format: 
+    #A fasta entry with the id: contig:start-end
+    #The sequence can be prodcued with samtools faidx
+    def self.parse(fasta_entry, arm_selection)
+      #puts fasta_entry.definition
+      snp = SequenceToAmplify.new
+      match_data = /(?<rname>\w*):(?<rstart>\w*)-(?<rend>\w*)/.match(fasta_entry.definition)
+      #puts match_data.inspect
+      rName = Regexp.last_match(:rname)
+      rStart =  Regexp.last_match(:rstart).to_i
+      rEnd =  Regexp.last_match(:rend).to_i
+      snp.gene = fasta_entry.definition
+      #snp.chromosome=rName
+      #puts "Gene: #{snp.gene}"
+      snp.chromosome=select_chromosome(fasta_entry.definition, arm_selection)
+      #puts "#{rName}: #{snp.chromosome}"
+      snp.sequence_original = fasta_entry.seq
+      snp.template_sequence = fasta_entry.seq.upcase
+      snp.snp_in = "B"
+      snp.rstart = rStart
+      snp.rend = rEnd
+
+      snp.position   = snp.sequence_original.size / 2
+      snp.original   = snp.sequence_original[snp.position]
+      
+      tmp =  Bio::Sequence::NA.new(snp.original)
+      rev = tmp.complement
+      snp.snp = rev
+      snp.exon_list = Hash.new()
+      snp
+    end
+
+    def primer_3_all_strings(target_chromosome, parental, max_specific_primers: 20, flanking_size:500) 
+      #puts target_chromosome 
+      #puts parental
+      #puts aligned_sequences.to_fasta
+      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
+      #puts "___"
+      #puts pr.inspect
+      return primer_3_propertes if seq_original.size < primer_3_min_seq_length
+      #puts "((("
+      return primer_3_propertes unless pr.snp_pos == flanking_size
+      #puts "Sequence origina: #{ self.original}" 
+      #puts pr.to_fasta
+      #puts "Postion: #{pr.snp_pos}"
+      seq_original[pr.snp_pos] = self.original
+      seq_original_reverse = reverse_complement_string(seq_original)
+
+      seq_snp =  String.new(pr.sequence)
+      seq_snp[pr.snp_pos] =  self.snp
+      seq_snp_reverse = reverse_complement_string(seq_snp)
+
+      rev_pos = seq_snp.size - position
+
+      if pr.homoeologous
+        snp_type = "homoeologous"
+      else
+        snp_type = "non-homoeologous"
+      end
+      left_pos = Array.new
+      right_pos = Array.new
+      l_pos = pr.snp_pos
+      pr.chromosome_specific.shuffle.each {|pos| left_pos  << pos if pos < l_pos - 50 }
+      pr.chromosome_specific.shuffle.each {|pos| right_pos << pos if pos > l_pos + 50}
+      
+      pr.crhomosome_specific_intron.shuffle.each {|pos| left_pos  << pos if pos < l_pos - 50}
+      pr.crhomosome_specific_intron.shuffle.each {|pos| right_pos << pos if pos > l_pos + 50}
+
+      prepareLRPrimers(left_pos, right_pos, "chromosome_specific" , snp_type,seq_original, primer_3_propertes)
+      if includeNoSpecific and (right_pos.size == 0 or right_pos.size == 0)
+        left_pos = Array.new
+        right_pos = Array.new
+        l_pos = pr.snp_pos
+        pr.almost_chromosome_specific.each {|pos| left_pos  << pos if pos < l_pos - 50 }
+        pr.almost_chromosome_specific.each {|pos| right_pos << pos if pos > l_pos + 50}
+        
+        pr.almost_crhomosome_specific_intron.each {|pos| left_pos  << pos if pos < l_pos - 50}
+        pr.almost_crhomosome_specific_intron.each {|pos| right_pos << pos if pos > l_pos + 50}
+
+        prepareLRPrimers(left_pos, right_pos, "chromosome_semispecific" ,snp_type, seq_original, primer_3_propertes)
+        args = {
+          :name =>"#{gene}:#{original}#{position}#{snp} #{original_name} chromosome_nonspecific exon #{snp_type} #{chromosome}", 
+            :left_pos => 350,
+            :extra_f=>"SEQUENCE_TARGET=350,400\n",
+            :extra_r=>"SEQUENCE_TARGET=350,400\n",
+            :sequence=>seq_original}
+            str =  return_primer_3_string(args)
+
+        primer_3_propertes << str
+      end
+      primer_3_propertes
+    end
+
+    def prepareLRPrimers(left_pos, right_pos, type , snp_type, seq_original,primer_3_propertes)
+      count = 0
+      left_pos.each do |l|
+        right_pos.each do |r|
+          args = {:name =>"#{gene}:#{original}#{position}#{snp} #{original_name} #{type} exon #{snp_type} #{chromosome}", 
+          :left_pos => l, 
+          :right_pos => r, 
+          :sequence=>seq_original}
+
+          primer_3_propertes << return_primer_3_string(args)
+          count += 1
+#          return if count > 25
+        end
+      end
+    end
+
+    def parental_sequences
+      return @parental_sequences if @parental_sequences
+      gene_region = self.covered_region
+      local_pos_in_gene = self.position
+      
+      @parental_sequences = Bio::Alignment::SequenceHash.new
+      container.parents.each  do |name, bam|
+        seq = self.sequence_original.clone.downcase
+        
+        if name == self.snp_in
+          #puts self.snp
+          seq[local_pos_in_gene] = self.snp
+        else
+          #puts self.original
+          seq[local_pos_in_gene] = self.original
+        end
+        seq[local_pos_in_gene] = seq[local_pos_in_gene].upcase    
+        @parental_sequences [name] = seq
+        #puts name 
+        #puts self.snp_in
+        #puts seq
+      end
+      @parental_sequences
+    end
+  end
+end
+
+
+snps = Array.new
+file = Bio::FastaFormat.open(markers)
+file.each do |entry|
+
+  begin
+    #puts entry.inspect
+    tmp = Bio::PolyploidTools::SequenceToAmplify.parse(entry, arm_selection)
+    snps << tmp if tmp
+  rescue Exception => e
+    log "ERROR\t#{e.message}"
+    $stderr.puts "Unable to generate the marker for: #{entry.definition}"
+    $stderr.puts e.backtrace
+  end
+
+end
+file.close
+
+
+
+exo_f = File.open(exonerate_file, "w")
+target=reference
+
+fasta_file = Bio::DB::Fasta::FastaFile.new(fasta: target)
+fasta_file.load_fai_entries
+min_identity = 90
+found_contigs = Set.new
+
+
+def do_align(aln, exo_f, found_contigs, min_identity,fasta_file,options)
+  if aln.identity > min_identity
+    exo_f.puts aln.line
+    unless found_contigs.include?(aln.target_id) #We only add once each contig. Should reduce the size of the output file. 
+      found_contigs.add(aln.target_id)
+      entry = fasta_file.index.region_for_entry(aln.target_id)
+      raise ExonerateException.new,  "Entry not found! #{aln.target_id}. Make sure that the #{target_id}.fai was generated properly." if entry == nil
+      if options[:extract_found_contigs]
+        region = entry.get_full_region
+        seq = fasta_file.fetch_sequence(region)
+        contigs_f.puts(">#{aln.target_id}\n#{seq}") 
+      end
+    end
+  end  
+
+end
+
+Bio::DB::Blast.align({:query=>markers, :target=>options[:database], :model=>model, :max_hits=>options[:max_hits]}) do |aln|
+  do_align(aln, exo_f, found_contigs,min_identity, fasta_file,options)
+end if options[:aligner] == :blast
+
+Bio::DB::Exonerate.align({:query=>markers, :target=>target, :model=>model}) do |aln|
+  do_align(aln, exo_f, found_contigs, min_identity,fasta_file,options)
+end if options[:aligner] == :exonerate
+
+exo_f.close
+
+container= Bio::PolyploidTools::ExonContainer.new
+container.flanking_size=500 
+container.gene_models(markers)
+container.chromosomes(target)
+container.add_parental({:name=>"A"})
+container.add_parental({:name=>"B"})
+#puts "SNPs size: #{snps.size}"
+snps.each do |snp|
+  snp.snp_in = "B"
+  snp.container = container
+  snp.flanking_size = container.flanking_size
+  snp.genomes_count = options[:genomes_count]
+  snp.includeNoSpecific = allow_non_specific
+  container.add_snp(snp)
+end
+
+container.add_alignments({:exonerate_file=>exonerate_file, 
+  :arm_selection=> arm_selection,
+  :min_identity=>min_identity})
+
+
+exons_filename="#{output_folder}/localAlignment.fa"
+file = File.open(exons_filename, "w")
+container.print_fasta_snp_exones(file)
+file.close
+
+
+
+primer_3_input  ="#{output_folder}/primer3_input.txt"
+primer_3_output ="#{output_folder}/primer3_output.txt"
+
+
+
+file = File.open(primer_3_input, "w")
+snp_in="B"
+Bio::DB::Primer3.prepare_input_file(file, options[:primer_3_preferences])
+added_exons = container.print_primer_3_exons(file, nil, snp_in)
+file.close
+
+Bio::DB::Primer3.run({:in=>primer_3_input, :out=>primer_3_output}) if added_exons > 0
+
+masks_output = "#{output_folder}/masks_designed.fa"
+output_file  = "#{output_folder}/primers.csv"
+file = File.open(masks_output, "w")
+out  = File.open(output_file,  "w")
+
+out.puts ["Id","specificity","inside","type","target","orientation","product_size",
+  "left_position","left_tm","left_sequence",
+"right_position","right_tm","right_sequence"].join ","
+class Bio::DB::Primer3::Primer3Record
+  attr_accessor :primerPairs
+end
+
+printed_counts = Hash.new(0)
+Bio::DB::Primer3::Primer3Record.parse_file(primer_3_output ) do | primer3record |
+  #puts primer3record.inspect
+  next if primer3record.primer_left_num_returned.to_i == 0
+  
+  seq_id = primer3record.sequence_id
+  printed_counts[seq_id] += 1
+  next if printed_counts[seq_id] > 10
+  excluded = "-"
+  exArr = excluded.split(",")
+  st = exArr[0].to_i
+  ed = exArr[1].to_i
+  tot = ed + st
+  
+  excluded="#{st}-#{tot}"
+  seq_len = primer3record.sequence_template.length
+  printed = 0
+
+  sequence_template = primer3record.sequence_template
+  sequence_mask = "-" * st 
+  sequence_mask << "*" * ed 
+  sequence_mask << "-" * (seq_len - sequence_mask.length)
+
+  file.puts ">#{seq_id}\n#{sequence_template}"
+  file.puts ">#{seq_id}:mask\n#{sequence_mask}"
+  
+   primer3record.primerPairs.each do |p| 
+    #puts p.inspect
+    printed += 1   
+    lArr =  p.left.coordinates
+    lArr[1] = lArr[0] + lArr[1]
+    rArr =  p.right.coordinates
+    rArr[1] = rArr[0] - rArr[1]
+    toPrint = Array.new
+    toPrint <<  seq_id.split(" ")
+    #toPrint <<  seq_len
+    toPrint <<  p.product_size
+    toPrint <<  lArr.join("-")
+    toPrint <<  p.left.tm
+    toPrint <<  p.left.sequence
+    toPrint <<  rArr.join("-")
+    toPrint <<  p.right.tm
+    toPrint <<  p.right.sequence
+
+    middle = 501 
+    #toPrint << lArr[0]
+    #toPrint << rArr[0]
+    #toPrint << middle - lArr[0]
+    #toPrint << rArr[0] - middle
+#Start End LeftDistance  RightDistance
+
+    out.puts toPrint.join(",")
+
+    sequence_primers = sequence_mask.clone
+    a = lArr[0] 
+    b = lArr[1] - 1
+    #puts   sequence_template[a..b]
+    sequence_primers[a..b] = sequence_template[a..b]
+    b = rArr[0] 
+    a = rArr[1] + 1
+
+    sequence_primers[a..b] = sequence_template[a..b]
+
+    file.puts ">#{seq_id}:primerPair:#{printed}\n#{sequence_primers}"
+  end 
+
+  if printed == 0
+    toPrint = Array.new
+    toPrint << seq_id.split(" ")
+    toPrint << excluded
+    toPrint << seq_len
+    out.puts toPrint.join(",")
+  end
+
+end
+out.close
+file.close
+