Class: Cheripic::Variants

Inherits:
Object
  • Object
show all
Extended by:
Forwardable
Includes:
Enumerable
Defined in:
lib/cheripic/variants.rb

Overview

A Variants object for each analysis pipeline that stores assembly details and extracts pileups for each contig assembly and pileup details are stored as hashes of Contig and ContigPileups objects

Instance Attribute Summary collapse

Instance Method Summary collapse

Constructor Details

#initialize(options) ⇒ Variants

creates a Variants object using user input files

Parameters:

  • options (OpenStruct) —

    a hash of required input files as keys and file paths as values



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# File 'lib/cheripic/variants.rb', line 64

def initialize(options)
  @params = options
  @assembly = {}
  @pileups = {}
  Bio::FastaFormat.open(@params.assembly).each do |entry|
    if entry.seq.length == 0
      logger.error "No sequence found for entry #{entry.entry_id}"
      raise VariantsError
    end
    contig = Contig.new(entry)
    if @assembly.key?(contig.id)
      logger.error "fasta id already found in the file for #{contig.id}"
      logger.error 'make sure there are no duplicate entries in the fasta file'
      raise VariantsError
    end
    @assembly[contig.id] = contig
    @pileups[contig.id] = ContigPileups.new(contig.id)
  end
  @pileups_analyzed = false
  unless @params.repeats_file == ''
    store_repeat_regions
  end
end

Instance Attribute Details

#assembly ⇒ Hash (readonly)

Returns a hash of contig ids from assembly as keys and respective Contig objects as values.

Returns:

  • (Hash) —

    a hash of contig ids from assembly as keys and respective Contig objects as values



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# File 'lib/cheripic/variants.rb', line 55

class Variants

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_key, :each_value, :size, :length, :[]
  attr_reader :assembly, :pileups, :pileups_analyzed

  # creates a Variants object using user input files
  # @param options [OpenStruct] a hash of required input files as keys and file paths as values
  def initialize(options)
    @params = options
    @assembly = {}
    @pileups = {}
    Bio::FastaFormat.open(@params.assembly).each do |entry|
      if entry.seq.length == 0
        logger.error "No sequence found for entry #{entry.entry_id}"
        raise VariantsError
      end
      contig = Contig.new(entry)
      if @assembly.key?(contig.id)
        logger.error "fasta id already found in the file for #{contig.id}"
        logger.error 'make sure there are no duplicate entries in the fasta file'
        raise VariantsError
      end
      @assembly[contig.id] = contig
      @pileups[contig.id] = ContigPileups.new(contig.id)
    end
    @pileups_analyzed = false
    unless @params.repeats_file == ''
      store_repeat_regions
    end
  end

  # reads repeat masker output file and stores masked regions to ignore variants in thos regions
  def store_repeat_regions
    File.foreach(@params.repeats_file) do |line|
      line.strip!
      next if line =~ /^SW/ or line =~ /^score/ or line == ''
      info = line.split("\s")
      pileups_obj = @pileups[info[4]]
      index = pileups_obj.masked_regions.length
      pileups_obj.masked_regions[index + 1][:begin] = info[5].to_i
      pileups_obj.masked_regions[index + 1][:end] = info[6].to_i
    end
  end

  # Reads and store pileup data for each of input bulk and parents pileup files
  # And sets pileups_analyzed to true that pileups files are processed
  def analyse_pileups
    if @params.input_format == 'bam'
      @vcf_hash = Vcf.filtering(@params.mut_bulk_vcf, @params.bg_bulk_vcf)
    end
    i{mut_bulk bg_bulk mut_parent bg_parent}.each do | input |
      infile = @params[input]
      if infile != ''
        logger.info "processing #{input} file"
        if @params.input_format == 'pileup'
          extract_pileup(infile, input)
        else
          extract_bam_pileup(infile, input)
        end
      end
    end

    @pileups_analyzed = true
  end

  # Bam object is read and each contig mean and std deviation of depth calculated
  # @param bamobject [Bio::DB::Sam]
  # Open3 capture returns string output, so careful not to give whole genome or big contigs for depth analysis
  def set_max_depth(bamobject, bamfile)
    logger.info "processing #{bamfile} file for depth"
    all_depths = []
    bq = Options.base_quality
    mq = Options.mapping_quality
    @assembly.each_key do | id |
      contig_obj = @assembly[id]
      len = contig_obj.length
      data = bamobject.depth(:r => "#{id}", :Q => bq, :q => mq)
      depths = []
      data.split("\n").each do |line|
        info = line.split("\t")
        depths << info[2].to_i
      end
      variance = 0
      mean_depth = depths.reduce(0, :+) / len.to_f
      depths.each do |value|
        variance += (value.to_f - mean_depth)**2
      end
      all_depths << mean_depth
      contig_obj.sd_depth = Math.sqrt(variance)
      contig_obj.mean_depth = mean_depth
    end
    # setting max depth as 3 times the average depth
    mean_coverage = all_depths.reduce(0, :+) / @assembly.length.to_f
    Options.maxdepth = Options.max_d_multiple * mean_coverage
  end

  # Input pileup file is read and positions are selected that pass the thresholds
  # @param pileupfile [String] path to the pileup file to read
  # @param sym [Symbol] Symbol of the pileup file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_pileup(pileupfile, sym)
    # read mpileup file and process each variant
    File.foreach(pileupfile) do |line|
      pileup = Pileup.new(line)
      if pileup.is_var
        contig_obj = @pileups[pileup.ref_name]
        contig_obj.send(sym).store(pileup.pos, pileup)
      end
    end
  end

  # Input bamfile is read and selected positions pileups are stored
  # @param bamfile [String] path to the bam file to read
  # @param sym [Symbol] Symbol of the bam file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_bam_pileup(bamfile, sym)
    bq = Options.base_quality
    mq = Options.mapping_quality
    bamobject = Bio::DB::Sam.new(:bam=>bamfile, :fasta=>@params.assembly)
    bamobject.index unless bamobject.indexed?

    # check if user has set max depth or set to zero to ignore
    max_d = Options.maxdepth
    # or calculate from bamfile
    if Options.max_d_multiple > 0
      set_max_depth(bamobject, bamfile)
      max_d = Options.maxdepth
      logger.info "max depth used for #{sym} file\t#{max_d}"
    end

    @vcf_hash.each_key do | id |
      positions = @vcf_hash[id][:het].keys
      positions << @vcf_hash[id][:hom].keys
      positions.flatten!
      next if positions.empty?
      contig_obj = @pileups[id]
      positions.each do | pos |
        command = "#{bamobject.samtools} mpileup -r #{id}:#{pos}-#{pos} -Q #{bq} -q #{mq} -B -f #{@params.assembly} #{bamfile}"
        stdout, stderr, status = Open3.capture3(command)
        unless status.success?
          logger.error "resulted in exit code #{status.exitstatus} using #{command}"
          logger.error "stderr output is: #{stderr}"
          raise CheripicError
        end
        stdout.chomp!
        if stdout == '' or stdout.split("\t")[3].to_i == 0 or stdout =~ /^\t0/
          logger.info "pileup data empty for\t#{id}\t#{pos}"
        else
          pileup = Pileup.new(stdout)
          unless max_d == 0 or pileup.coverage <= max_d
            logger.info "pileup coverage is higher than max\t#{pileup.to_s}"
            next
          end
          contig_obj.send(sym).store(pos, pileup)
        end
      end
    end
  end

  # Once pileup files are analysed and variants are extracted from each bulk;
  # bulks are compared to identify and isolate variants for downstream analysis.
  # If polyploidy set to trye and mut_parent and bg_parent bulks are provided
  # hemisnps in parents are extracted for bulk frequency ratio analysis
  def compare_pileups
    unless @pileups_analyzed
      self.analyse_pileups
    end
    @assembly.each_key do | id |
      contig = @assembly[id]
      # extract parental hemi snps for polyploids before bulks are compared
      if Options.polyploidy
        if @params.mut_parent != '' or @params.bg_parent != ''
          @pileups[id].hemisnps_in_parent
        end
      end
      contig.hm_pos, contig.ht_pos, contig.hemi_pos = @pileups[id].bulks_compared
    end
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for homozygosity enrichment score (hme_score)
  def hmes_frags
    # calculate every time method gets called
    @hmes_frags = select_contigs(:hme_score)
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for bulk frequency ratio (bfr_score)
  def bfr_frags
    unless defined?(@bfr_frags)
      @bfr_frags = select_contigs(:bfr_score)
    end
    @bfr_frags
  end

  # Applies selection procedure on assembly contigs based on the ratio_type provided.
  # If use_all_contigs is set to false then contigs without any variant are discarded for :hme_score
  # while contigs without any hemisnps are discarded for :bfr_score
  # If include_low_hmes is set to false then contigs are further filtered based on a cut off value of the score
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  def select_contigs(ratio_type)
    selected_contigs ={}
    use_all_contigs = Options.use_all_contigs
    @assembly.each_key do | frag |
      if use_all_contigs
        selected_contigs[frag] = @assembly[frag]
      else
        if ratio_type == :hme_score
          # selecting fragments which have a variant
          if @assembly[frag].hm_num + @assembly[frag].ht_num > 2 * Options.hmes_adjust
            selected_contigs[frag] = @assembly[frag]
          end
        else # ratio_type == :bfr_score
          # in polyploidy scenario selecting fragments with at least one bfr position
          if @assembly[frag].hemi_num > 0
            selected_contigs[frag] = @assembly[frag]
          end
        end
      end
    end
    selected_contigs = filter_contigs(selected_contigs, ratio_type)
    if use_all_contigs
      logger.info "No filtering was applied to fragments\n"
    else
      logger.info "Selected #{selected_contigs.length} out of #{@assembly.length} fragments with #{ratio_type} score\n"
    end
    selected_contigs
  end

  # Filters out contigs below a cutoff for selected ratio_type
  # a cutoff value is calculated based on ratio_type provided
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def filter_contigs(selected_contigs, ratio_type)
    cutoff = get_cutoff(selected_contigs, ratio_type)
    selected_contigs.each_key do | frag |
      if selected_contigs[frag].send(ratio_type) < cutoff
        selected_contigs.delete(frag)
      end
    end
    selected_contigs
  end

  # Cut off value calculation used to filter out low scored contigs.
  #
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def get_cutoff(selected_contigs, ratio_type)
    include_low_hmes = Options.include_low_hmes
    # set minimum cut off hme_score or bfr_score to pick fragments with variants
    # calculate min hme score for back or out crossed data or bfr_score for polypoidy data
    # if no filtering applied set cutoff to 1.1
    if include_low_hmes
      cutoff = 0.0
    else
      if ratio_type == :hme_score
        adjust = Options.hmes_adjust
        if Options.cross_type == 'back'
          cutoff = (1.0/adjust) + 1.0
        else # outcross
          cutoff = (2.0/adjust) + 1.0
        end
      else # ratio_type is bfr_score
        cutoff = bfr_cutoff(selected_contigs)
      end
    end
    cutoff
  end

  # Cut off value calculation for bfr contigs.
  # ratio value at index 0.1% length of an array or at index zero of an array that contains decreasing order of bfr ratios
  # @param selected_contigs [Hash] a hash of contigs with selected bfr score, a subset of assembly hash
  def bfr_cutoff(selected_contigs, prop=0.1)
    ratios = []
    selected_contigs.each_key do | frag |
      ratios << selected_contigs[frag].bfr_score
    end
    ratios.sort!.reverse!
    index = (ratios.length * prop)/100
    # set a minmum index to get at least one contig
    if index < 1
      index = 1
    end
    ratios[index - 1]
  end

  # Method is to discard homozygous variant positions for which background bulk
  # pileup shows a fraction value higher than 0.35 for variant allele/non-reference allele
  # a recessive variant is expected to have 1/3rd frequency in background bulk
  def verify_bg_bulk_pileup
    unless defined?(@hmes_frags)
      self.hmes_frags
    end
    @hmes_frags.each_key do | frag |
      positions = @assembly[frag].hm_pos.keys
      contig_pileup_obj = @pileups[frag]
      positions.each do | pos |
        if contig_pileup_obj.mut_bulk.key?(pos)
          mut_pileup = contig_pileup_obj.mut_bulk[pos]
          if mut_pileup.is_var
            if contig_pileup_obj.bg_bulk.key?(pos)
              bg_pileup = contig_pileup_obj.bg_bulk[pos]
              if bg_pileup.non_ref_ratio > 0.35
                @assembly[frag].hm_pos.delete(pos)
              end
            end
          else
            # this should not happen, may be catch as as an error
            @assembly[frag].hm_pos.delete(pos)
          end
        else
          # this should not happen, may be catch as as an error
          @assembly[frag].hm_pos.delete(pos)
        end
      end
    end
    # recalculate hmes_frags once pileups are verified
    self.hmes_frags
  end

end

#bfr_frags ⇒ Object (readonly)

From Assembly contig objects, contigs are selected based on user selected options for bulk frequency ratio (bfr_score)



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# File 'lib/cheripic/variants.rb', line 55

class Variants

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_key, :each_value, :size, :length, :[]
  attr_reader :assembly, :pileups, :pileups_analyzed

  # creates a Variants object using user input files
  # @param options [OpenStruct] a hash of required input files as keys and file paths as values
  def initialize(options)
    @params = options
    @assembly = {}
    @pileups = {}
    Bio::FastaFormat.open(@params.assembly).each do |entry|
      if entry.seq.length == 0
        logger.error "No sequence found for entry #{entry.entry_id}"
        raise VariantsError
      end
      contig = Contig.new(entry)
      if @assembly.key?(contig.id)
        logger.error "fasta id already found in the file for #{contig.id}"
        logger.error 'make sure there are no duplicate entries in the fasta file'
        raise VariantsError
      end
      @assembly[contig.id] = contig
      @pileups[contig.id] = ContigPileups.new(contig.id)
    end
    @pileups_analyzed = false
    unless @params.repeats_file == ''
      store_repeat_regions
    end
  end

  # reads repeat masker output file and stores masked regions to ignore variants in thos regions
  def store_repeat_regions
    File.foreach(@params.repeats_file) do |line|
      line.strip!
      next if line =~ /^SW/ or line =~ /^score/ or line == ''
      info = line.split("\s")
      pileups_obj = @pileups[info[4]]
      index = pileups_obj.masked_regions.length
      pileups_obj.masked_regions[index + 1][:begin] = info[5].to_i
      pileups_obj.masked_regions[index + 1][:end] = info[6].to_i
    end
  end

  # Reads and store pileup data for each of input bulk and parents pileup files
  # And sets pileups_analyzed to true that pileups files are processed
  def analyse_pileups
    if @params.input_format == 'bam'
      @vcf_hash = Vcf.filtering(@params.mut_bulk_vcf, @params.bg_bulk_vcf)
    end
    i{mut_bulk bg_bulk mut_parent bg_parent}.each do | input |
      infile = @params[input]
      if infile != ''
        logger.info "processing #{input} file"
        if @params.input_format == 'pileup'
          extract_pileup(infile, input)
        else
          extract_bam_pileup(infile, input)
        end
      end
    end

    @pileups_analyzed = true
  end

  # Bam object is read and each contig mean and std deviation of depth calculated
  # @param bamobject [Bio::DB::Sam]
  # Open3 capture returns string output, so careful not to give whole genome or big contigs for depth analysis
  def set_max_depth(bamobject, bamfile)
    logger.info "processing #{bamfile} file for depth"
    all_depths = []
    bq = Options.base_quality
    mq = Options.mapping_quality
    @assembly.each_key do | id |
      contig_obj = @assembly[id]
      len = contig_obj.length
      data = bamobject.depth(:r => "#{id}", :Q => bq, :q => mq)
      depths = []
      data.split("\n").each do |line|
        info = line.split("\t")
        depths << info[2].to_i
      end
      variance = 0
      mean_depth = depths.reduce(0, :+) / len.to_f
      depths.each do |value|
        variance += (value.to_f - mean_depth)**2
      end
      all_depths << mean_depth
      contig_obj.sd_depth = Math.sqrt(variance)
      contig_obj.mean_depth = mean_depth
    end
    # setting max depth as 3 times the average depth
    mean_coverage = all_depths.reduce(0, :+) / @assembly.length.to_f
    Options.maxdepth = Options.max_d_multiple * mean_coverage
  end

  # Input pileup file is read and positions are selected that pass the thresholds
  # @param pileupfile [String] path to the pileup file to read
  # @param sym [Symbol] Symbol of the pileup file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_pileup(pileupfile, sym)
    # read mpileup file and process each variant
    File.foreach(pileupfile) do |line|
      pileup = Pileup.new(line)
      if pileup.is_var
        contig_obj = @pileups[pileup.ref_name]
        contig_obj.send(sym).store(pileup.pos, pileup)
      end
    end
  end

  # Input bamfile is read and selected positions pileups are stored
  # @param bamfile [String] path to the bam file to read
  # @param sym [Symbol] Symbol of the bam file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_bam_pileup(bamfile, sym)
    bq = Options.base_quality
    mq = Options.mapping_quality
    bamobject = Bio::DB::Sam.new(:bam=>bamfile, :fasta=>@params.assembly)
    bamobject.index unless bamobject.indexed?

    # check if user has set max depth or set to zero to ignore
    max_d = Options.maxdepth
    # or calculate from bamfile
    if Options.max_d_multiple > 0
      set_max_depth(bamobject, bamfile)
      max_d = Options.maxdepth
      logger.info "max depth used for #{sym} file\t#{max_d}"
    end

    @vcf_hash.each_key do | id |
      positions = @vcf_hash[id][:het].keys
      positions << @vcf_hash[id][:hom].keys
      positions.flatten!
      next if positions.empty?
      contig_obj = @pileups[id]
      positions.each do | pos |
        command = "#{bamobject.samtools} mpileup -r #{id}:#{pos}-#{pos} -Q #{bq} -q #{mq} -B -f #{@params.assembly} #{bamfile}"
        stdout, stderr, status = Open3.capture3(command)
        unless status.success?
          logger.error "resulted in exit code #{status.exitstatus} using #{command}"
          logger.error "stderr output is: #{stderr}"
          raise CheripicError
        end
        stdout.chomp!
        if stdout == '' or stdout.split("\t")[3].to_i == 0 or stdout =~ /^\t0/
          logger.info "pileup data empty for\t#{id}\t#{pos}"
        else
          pileup = Pileup.new(stdout)
          unless max_d == 0 or pileup.coverage <= max_d
            logger.info "pileup coverage is higher than max\t#{pileup.to_s}"
            next
          end
          contig_obj.send(sym).store(pos, pileup)
        end
      end
    end
  end

  # Once pileup files are analysed and variants are extracted from each bulk;
  # bulks are compared to identify and isolate variants for downstream analysis.
  # If polyploidy set to trye and mut_parent and bg_parent bulks are provided
  # hemisnps in parents are extracted for bulk frequency ratio analysis
  def compare_pileups
    unless @pileups_analyzed
      self.analyse_pileups
    end
    @assembly.each_key do | id |
      contig = @assembly[id]
      # extract parental hemi snps for polyploids before bulks are compared
      if Options.polyploidy
        if @params.mut_parent != '' or @params.bg_parent != ''
          @pileups[id].hemisnps_in_parent
        end
      end
      contig.hm_pos, contig.ht_pos, contig.hemi_pos = @pileups[id].bulks_compared
    end
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for homozygosity enrichment score (hme_score)
  def hmes_frags
    # calculate every time method gets called
    @hmes_frags = select_contigs(:hme_score)
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for bulk frequency ratio (bfr_score)
  def bfr_frags
    unless defined?(@bfr_frags)
      @bfr_frags = select_contigs(:bfr_score)
    end
    @bfr_frags
  end

  # Applies selection procedure on assembly contigs based on the ratio_type provided.
  # If use_all_contigs is set to false then contigs without any variant are discarded for :hme_score
  # while contigs without any hemisnps are discarded for :bfr_score
  # If include_low_hmes is set to false then contigs are further filtered based on a cut off value of the score
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  def select_contigs(ratio_type)
    selected_contigs ={}
    use_all_contigs = Options.use_all_contigs
    @assembly.each_key do | frag |
      if use_all_contigs
        selected_contigs[frag] = @assembly[frag]
      else
        if ratio_type == :hme_score
          # selecting fragments which have a variant
          if @assembly[frag].hm_num + @assembly[frag].ht_num > 2 * Options.hmes_adjust
            selected_contigs[frag] = @assembly[frag]
          end
        else # ratio_type == :bfr_score
          # in polyploidy scenario selecting fragments with at least one bfr position
          if @assembly[frag].hemi_num > 0
            selected_contigs[frag] = @assembly[frag]
          end
        end
      end
    end
    selected_contigs = filter_contigs(selected_contigs, ratio_type)
    if use_all_contigs
      logger.info "No filtering was applied to fragments\n"
    else
      logger.info "Selected #{selected_contigs.length} out of #{@assembly.length} fragments with #{ratio_type} score\n"
    end
    selected_contigs
  end

  # Filters out contigs below a cutoff for selected ratio_type
  # a cutoff value is calculated based on ratio_type provided
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def filter_contigs(selected_contigs, ratio_type)
    cutoff = get_cutoff(selected_contigs, ratio_type)
    selected_contigs.each_key do | frag |
      if selected_contigs[frag].send(ratio_type) < cutoff
        selected_contigs.delete(frag)
      end
    end
    selected_contigs
  end

  # Cut off value calculation used to filter out low scored contigs.
  #
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def get_cutoff(selected_contigs, ratio_type)
    include_low_hmes = Options.include_low_hmes
    # set minimum cut off hme_score or bfr_score to pick fragments with variants
    # calculate min hme score for back or out crossed data or bfr_score for polypoidy data
    # if no filtering applied set cutoff to 1.1
    if include_low_hmes
      cutoff = 0.0
    else
      if ratio_type == :hme_score
        adjust = Options.hmes_adjust
        if Options.cross_type == 'back'
          cutoff = (1.0/adjust) + 1.0
        else # outcross
          cutoff = (2.0/adjust) + 1.0
        end
      else # ratio_type is bfr_score
        cutoff = bfr_cutoff(selected_contigs)
      end
    end
    cutoff
  end

  # Cut off value calculation for bfr contigs.
  # ratio value at index 0.1% length of an array or at index zero of an array that contains decreasing order of bfr ratios
  # @param selected_contigs [Hash] a hash of contigs with selected bfr score, a subset of assembly hash
  def bfr_cutoff(selected_contigs, prop=0.1)
    ratios = []
    selected_contigs.each_key do | frag |
      ratios << selected_contigs[frag].bfr_score
    end
    ratios.sort!.reverse!
    index = (ratios.length * prop)/100
    # set a minmum index to get at least one contig
    if index < 1
      index = 1
    end
    ratios[index - 1]
  end

  # Method is to discard homozygous variant positions for which background bulk
  # pileup shows a fraction value higher than 0.35 for variant allele/non-reference allele
  # a recessive variant is expected to have 1/3rd frequency in background bulk
  def verify_bg_bulk_pileup
    unless defined?(@hmes_frags)
      self.hmes_frags
    end
    @hmes_frags.each_key do | frag |
      positions = @assembly[frag].hm_pos.keys
      contig_pileup_obj = @pileups[frag]
      positions.each do | pos |
        if contig_pileup_obj.mut_bulk.key?(pos)
          mut_pileup = contig_pileup_obj.mut_bulk[pos]
          if mut_pileup.is_var
            if contig_pileup_obj.bg_bulk.key?(pos)
              bg_pileup = contig_pileup_obj.bg_bulk[pos]
              if bg_pileup.non_ref_ratio > 0.35
                @assembly[frag].hm_pos.delete(pos)
              end
            end
          else
            # this should not happen, may be catch as as an error
            @assembly[frag].hm_pos.delete(pos)
          end
        else
          # this should not happen, may be catch as as an error
          @assembly[frag].hm_pos.delete(pos)
        end
      end
    end
    # recalculate hmes_frags once pileups are verified
    self.hmes_frags
  end

end

#hmes_frags ⇒ Object (readonly)

From Assembly contig objects, contigs are selected based on user selected options for homozygosity enrichment score (hme_score)



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# File 'lib/cheripic/variants.rb', line 55

class Variants

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_key, :each_value, :size, :length, :[]
  attr_reader :assembly, :pileups, :pileups_analyzed

  # creates a Variants object using user input files
  # @param options [OpenStruct] a hash of required input files as keys and file paths as values
  def initialize(options)
    @params = options
    @assembly = {}
    @pileups = {}
    Bio::FastaFormat.open(@params.assembly).each do |entry|
      if entry.seq.length == 0
        logger.error "No sequence found for entry #{entry.entry_id}"
        raise VariantsError
      end
      contig = Contig.new(entry)
      if @assembly.key?(contig.id)
        logger.error "fasta id already found in the file for #{contig.id}"
        logger.error 'make sure there are no duplicate entries in the fasta file'
        raise VariantsError
      end
      @assembly[contig.id] = contig
      @pileups[contig.id] = ContigPileups.new(contig.id)
    end
    @pileups_analyzed = false
    unless @params.repeats_file == ''
      store_repeat_regions
    end
  end

  # reads repeat masker output file and stores masked regions to ignore variants in thos regions
  def store_repeat_regions
    File.foreach(@params.repeats_file) do |line|
      line.strip!
      next if line =~ /^SW/ or line =~ /^score/ or line == ''
      info = line.split("\s")
      pileups_obj = @pileups[info[4]]
      index = pileups_obj.masked_regions.length
      pileups_obj.masked_regions[index + 1][:begin] = info[5].to_i
      pileups_obj.masked_regions[index + 1][:end] = info[6].to_i
    end
  end

  # Reads and store pileup data for each of input bulk and parents pileup files
  # And sets pileups_analyzed to true that pileups files are processed
  def analyse_pileups
    if @params.input_format == 'bam'
      @vcf_hash = Vcf.filtering(@params.mut_bulk_vcf, @params.bg_bulk_vcf)
    end
    i{mut_bulk bg_bulk mut_parent bg_parent}.each do | input |
      infile = @params[input]
      if infile != ''
        logger.info "processing #{input} file"
        if @params.input_format == 'pileup'
          extract_pileup(infile, input)
        else
          extract_bam_pileup(infile, input)
        end
      end
    end

    @pileups_analyzed = true
  end

  # Bam object is read and each contig mean and std deviation of depth calculated
  # @param bamobject [Bio::DB::Sam]
  # Open3 capture returns string output, so careful not to give whole genome or big contigs for depth analysis
  def set_max_depth(bamobject, bamfile)
    logger.info "processing #{bamfile} file for depth"
    all_depths = []
    bq = Options.base_quality
    mq = Options.mapping_quality
    @assembly.each_key do | id |
      contig_obj = @assembly[id]
      len = contig_obj.length
      data = bamobject.depth(:r => "#{id}", :Q => bq, :q => mq)
      depths = []
      data.split("\n").each do |line|
        info = line.split("\t")
        depths << info[2].to_i
      end
      variance = 0
      mean_depth = depths.reduce(0, :+) / len.to_f
      depths.each do |value|
        variance += (value.to_f - mean_depth)**2
      end
      all_depths << mean_depth
      contig_obj.sd_depth = Math.sqrt(variance)
      contig_obj.mean_depth = mean_depth
    end
    # setting max depth as 3 times the average depth
    mean_coverage = all_depths.reduce(0, :+) / @assembly.length.to_f
    Options.maxdepth = Options.max_d_multiple * mean_coverage
  end

  # Input pileup file is read and positions are selected that pass the thresholds
  # @param pileupfile [String] path to the pileup file to read
  # @param sym [Symbol] Symbol of the pileup file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_pileup(pileupfile, sym)
    # read mpileup file and process each variant
    File.foreach(pileupfile) do |line|
      pileup = Pileup.new(line)
      if pileup.is_var
        contig_obj = @pileups[pileup.ref_name]
        contig_obj.send(sym).store(pileup.pos, pileup)
      end
    end
  end

  # Input bamfile is read and selected positions pileups are stored
  # @param bamfile [String] path to the bam file to read
  # @param sym [Symbol] Symbol of the bam file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_bam_pileup(bamfile, sym)
    bq = Options.base_quality
    mq = Options.mapping_quality
    bamobject = Bio::DB::Sam.new(:bam=>bamfile, :fasta=>@params.assembly)
    bamobject.index unless bamobject.indexed?

    # check if user has set max depth or set to zero to ignore
    max_d = Options.maxdepth
    # or calculate from bamfile
    if Options.max_d_multiple > 0
      set_max_depth(bamobject, bamfile)
      max_d = Options.maxdepth
      logger.info "max depth used for #{sym} file\t#{max_d}"
    end

    @vcf_hash.each_key do | id |
      positions = @vcf_hash[id][:het].keys
      positions << @vcf_hash[id][:hom].keys
      positions.flatten!
      next if positions.empty?
      contig_obj = @pileups[id]
      positions.each do | pos |
        command = "#{bamobject.samtools} mpileup -r #{id}:#{pos}-#{pos} -Q #{bq} -q #{mq} -B -f #{@params.assembly} #{bamfile}"
        stdout, stderr, status = Open3.capture3(command)
        unless status.success?
          logger.error "resulted in exit code #{status.exitstatus} using #{command}"
          logger.error "stderr output is: #{stderr}"
          raise CheripicError
        end
        stdout.chomp!
        if stdout == '' or stdout.split("\t")[3].to_i == 0 or stdout =~ /^\t0/
          logger.info "pileup data empty for\t#{id}\t#{pos}"
        else
          pileup = Pileup.new(stdout)
          unless max_d == 0 or pileup.coverage <= max_d
            logger.info "pileup coverage is higher than max\t#{pileup.to_s}"
            next
          end
          contig_obj.send(sym).store(pos, pileup)
        end
      end
    end
  end

  # Once pileup files are analysed and variants are extracted from each bulk;
  # bulks are compared to identify and isolate variants for downstream analysis.
  # If polyploidy set to trye and mut_parent and bg_parent bulks are provided
  # hemisnps in parents are extracted for bulk frequency ratio analysis
  def compare_pileups
    unless @pileups_analyzed
      self.analyse_pileups
    end
    @assembly.each_key do | id |
      contig = @assembly[id]
      # extract parental hemi snps for polyploids before bulks are compared
      if Options.polyploidy
        if @params.mut_parent != '' or @params.bg_parent != ''
          @pileups[id].hemisnps_in_parent
        end
      end
      contig.hm_pos, contig.ht_pos, contig.hemi_pos = @pileups[id].bulks_compared
    end
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for homozygosity enrichment score (hme_score)
  def hmes_frags
    # calculate every time method gets called
    @hmes_frags = select_contigs(:hme_score)
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for bulk frequency ratio (bfr_score)
  def bfr_frags
    unless defined?(@bfr_frags)
      @bfr_frags = select_contigs(:bfr_score)
    end
    @bfr_frags
  end

  # Applies selection procedure on assembly contigs based on the ratio_type provided.
  # If use_all_contigs is set to false then contigs without any variant are discarded for :hme_score
  # while contigs without any hemisnps are discarded for :bfr_score
  # If include_low_hmes is set to false then contigs are further filtered based on a cut off value of the score
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  def select_contigs(ratio_type)
    selected_contigs ={}
    use_all_contigs = Options.use_all_contigs
    @assembly.each_key do | frag |
      if use_all_contigs
        selected_contigs[frag] = @assembly[frag]
      else
        if ratio_type == :hme_score
          # selecting fragments which have a variant
          if @assembly[frag].hm_num + @assembly[frag].ht_num > 2 * Options.hmes_adjust
            selected_contigs[frag] = @assembly[frag]
          end
        else # ratio_type == :bfr_score
          # in polyploidy scenario selecting fragments with at least one bfr position
          if @assembly[frag].hemi_num > 0
            selected_contigs[frag] = @assembly[frag]
          end
        end
      end
    end
    selected_contigs = filter_contigs(selected_contigs, ratio_type)
    if use_all_contigs
      logger.info "No filtering was applied to fragments\n"
    else
      logger.info "Selected #{selected_contigs.length} out of #{@assembly.length} fragments with #{ratio_type} score\n"
    end
    selected_contigs
  end

  # Filters out contigs below a cutoff for selected ratio_type
  # a cutoff value is calculated based on ratio_type provided
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def filter_contigs(selected_contigs, ratio_type)
    cutoff = get_cutoff(selected_contigs, ratio_type)
    selected_contigs.each_key do | frag |
      if selected_contigs[frag].send(ratio_type) < cutoff
        selected_contigs.delete(frag)
      end
    end
    selected_contigs
  end

  # Cut off value calculation used to filter out low scored contigs.
  #
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def get_cutoff(selected_contigs, ratio_type)
    include_low_hmes = Options.include_low_hmes
    # set minimum cut off hme_score or bfr_score to pick fragments with variants
    # calculate min hme score for back or out crossed data or bfr_score for polypoidy data
    # if no filtering applied set cutoff to 1.1
    if include_low_hmes
      cutoff = 0.0
    else
      if ratio_type == :hme_score
        adjust = Options.hmes_adjust
        if Options.cross_type == 'back'
          cutoff = (1.0/adjust) + 1.0
        else # outcross
          cutoff = (2.0/adjust) + 1.0
        end
      else # ratio_type is bfr_score
        cutoff = bfr_cutoff(selected_contigs)
      end
    end
    cutoff
  end

  # Cut off value calculation for bfr contigs.
  # ratio value at index 0.1% length of an array or at index zero of an array that contains decreasing order of bfr ratios
  # @param selected_contigs [Hash] a hash of contigs with selected bfr score, a subset of assembly hash
  def bfr_cutoff(selected_contigs, prop=0.1)
    ratios = []
    selected_contigs.each_key do | frag |
      ratios << selected_contigs[frag].bfr_score
    end
    ratios.sort!.reverse!
    index = (ratios.length * prop)/100
    # set a minmum index to get at least one contig
    if index < 1
      index = 1
    end
    ratios[index - 1]
  end

  # Method is to discard homozygous variant positions for which background bulk
  # pileup shows a fraction value higher than 0.35 for variant allele/non-reference allele
  # a recessive variant is expected to have 1/3rd frequency in background bulk
  def verify_bg_bulk_pileup
    unless defined?(@hmes_frags)
      self.hmes_frags
    end
    @hmes_frags.each_key do | frag |
      positions = @assembly[frag].hm_pos.keys
      contig_pileup_obj = @pileups[frag]
      positions.each do | pos |
        if contig_pileup_obj.mut_bulk.key?(pos)
          mut_pileup = contig_pileup_obj.mut_bulk[pos]
          if mut_pileup.is_var
            if contig_pileup_obj.bg_bulk.key?(pos)
              bg_pileup = contig_pileup_obj.bg_bulk[pos]
              if bg_pileup.non_ref_ratio > 0.35
                @assembly[frag].hm_pos.delete(pos)
              end
            end
          else
            # this should not happen, may be catch as as an error
            @assembly[frag].hm_pos.delete(pos)
          end
        else
          # this should not happen, may be catch as as an error
          @assembly[frag].hm_pos.delete(pos)
        end
      end
    end
    # recalculate hmes_frags once pileups are verified
    self.hmes_frags
  end

end

#pileups ⇒ Hash (readonly)

Returns a hash of contig ids from assembly as keys and respective ContigPileups objects as values.

Returns:

  • (Hash) —

    a hash of contig ids from assembly as keys and respective ContigPileups objects as values



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# File 'lib/cheripic/variants.rb', line 55

class Variants

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_key, :each_value, :size, :length, :[]
  attr_reader :assembly, :pileups, :pileups_analyzed

  # creates a Variants object using user input files
  # @param options [OpenStruct] a hash of required input files as keys and file paths as values
  def initialize(options)
    @params = options
    @assembly = {}
    @pileups = {}
    Bio::FastaFormat.open(@params.assembly).each do |entry|
      if entry.seq.length == 0
        logger.error "No sequence found for entry #{entry.entry_id}"
        raise VariantsError
      end
      contig = Contig.new(entry)
      if @assembly.key?(contig.id)
        logger.error "fasta id already found in the file for #{contig.id}"
        logger.error 'make sure there are no duplicate entries in the fasta file'
        raise VariantsError
      end
      @assembly[contig.id] = contig
      @pileups[contig.id] = ContigPileups.new(contig.id)
    end
    @pileups_analyzed = false
    unless @params.repeats_file == ''
      store_repeat_regions
    end
  end

  # reads repeat masker output file and stores masked regions to ignore variants in thos regions
  def store_repeat_regions
    File.foreach(@params.repeats_file) do |line|
      line.strip!
      next if line =~ /^SW/ or line =~ /^score/ or line == ''
      info = line.split("\s")
      pileups_obj = @pileups[info[4]]
      index = pileups_obj.masked_regions.length
      pileups_obj.masked_regions[index + 1][:begin] = info[5].to_i
      pileups_obj.masked_regions[index + 1][:end] = info[6].to_i
    end
  end

  # Reads and store pileup data for each of input bulk and parents pileup files
  # And sets pileups_analyzed to true that pileups files are processed
  def analyse_pileups
    if @params.input_format == 'bam'
      @vcf_hash = Vcf.filtering(@params.mut_bulk_vcf, @params.bg_bulk_vcf)
    end
    i{mut_bulk bg_bulk mut_parent bg_parent}.each do | input |
      infile = @params[input]
      if infile != ''
        logger.info "processing #{input} file"
        if @params.input_format == 'pileup'
          extract_pileup(infile, input)
        else
          extract_bam_pileup(infile, input)
        end
      end
    end

    @pileups_analyzed = true
  end

  # Bam object is read and each contig mean and std deviation of depth calculated
  # @param bamobject [Bio::DB::Sam]
  # Open3 capture returns string output, so careful not to give whole genome or big contigs for depth analysis
  def set_max_depth(bamobject, bamfile)
    logger.info "processing #{bamfile} file for depth"
    all_depths = []
    bq = Options.base_quality
    mq = Options.mapping_quality
    @assembly.each_key do | id |
      contig_obj = @assembly[id]
      len = contig_obj.length
      data = bamobject.depth(:r => "#{id}", :Q => bq, :q => mq)
      depths = []
      data.split("\n").each do |line|
        info = line.split("\t")
        depths << info[2].to_i
      end
      variance = 0
      mean_depth = depths.reduce(0, :+) / len.to_f
      depths.each do |value|
        variance += (value.to_f - mean_depth)**2
      end
      all_depths << mean_depth
      contig_obj.sd_depth = Math.sqrt(variance)
      contig_obj.mean_depth = mean_depth
    end
    # setting max depth as 3 times the average depth
    mean_coverage = all_depths.reduce(0, :+) / @assembly.length.to_f
    Options.maxdepth = Options.max_d_multiple * mean_coverage
  end

  # Input pileup file is read and positions are selected that pass the thresholds
  # @param pileupfile [String] path to the pileup file to read
  # @param sym [Symbol] Symbol of the pileup file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_pileup(pileupfile, sym)
    # read mpileup file and process each variant
    File.foreach(pileupfile) do |line|
      pileup = Pileup.new(line)
      if pileup.is_var
        contig_obj = @pileups[pileup.ref_name]
        contig_obj.send(sym).store(pileup.pos, pileup)
      end
    end
  end

  # Input bamfile is read and selected positions pileups are stored
  # @param bamfile [String] path to the bam file to read
  # @param sym [Symbol] Symbol of the bam file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_bam_pileup(bamfile, sym)
    bq = Options.base_quality
    mq = Options.mapping_quality
    bamobject = Bio::DB::Sam.new(:bam=>bamfile, :fasta=>@params.assembly)
    bamobject.index unless bamobject.indexed?

    # check if user has set max depth or set to zero to ignore
    max_d = Options.maxdepth
    # or calculate from bamfile
    if Options.max_d_multiple > 0
      set_max_depth(bamobject, bamfile)
      max_d = Options.maxdepth
      logger.info "max depth used for #{sym} file\t#{max_d}"
    end

    @vcf_hash.each_key do | id |
      positions = @vcf_hash[id][:het].keys
      positions << @vcf_hash[id][:hom].keys
      positions.flatten!
      next if positions.empty?
      contig_obj = @pileups[id]
      positions.each do | pos |
        command = "#{bamobject.samtools} mpileup -r #{id}:#{pos}-#{pos} -Q #{bq} -q #{mq} -B -f #{@params.assembly} #{bamfile}"
        stdout, stderr, status = Open3.capture3(command)
        unless status.success?
          logger.error "resulted in exit code #{status.exitstatus} using #{command}"
          logger.error "stderr output is: #{stderr}"
          raise CheripicError
        end
        stdout.chomp!
        if stdout == '' or stdout.split("\t")[3].to_i == 0 or stdout =~ /^\t0/
          logger.info "pileup data empty for\t#{id}\t#{pos}"
        else
          pileup = Pileup.new(stdout)
          unless max_d == 0 or pileup.coverage <= max_d
            logger.info "pileup coverage is higher than max\t#{pileup.to_s}"
            next
          end
          contig_obj.send(sym).store(pos, pileup)
        end
      end
    end
  end

  # Once pileup files are analysed and variants are extracted from each bulk;
  # bulks are compared to identify and isolate variants for downstream analysis.
  # If polyploidy set to trye and mut_parent and bg_parent bulks are provided
  # hemisnps in parents are extracted for bulk frequency ratio analysis
  def compare_pileups
    unless @pileups_analyzed
      self.analyse_pileups
    end
    @assembly.each_key do | id |
      contig = @assembly[id]
      # extract parental hemi snps for polyploids before bulks are compared
      if Options.polyploidy
        if @params.mut_parent != '' or @params.bg_parent != ''
          @pileups[id].hemisnps_in_parent
        end
      end
      contig.hm_pos, contig.ht_pos, contig.hemi_pos = @pileups[id].bulks_compared
    end
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for homozygosity enrichment score (hme_score)
  def hmes_frags
    # calculate every time method gets called
    @hmes_frags = select_contigs(:hme_score)
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for bulk frequency ratio (bfr_score)
  def bfr_frags
    unless defined?(@bfr_frags)
      @bfr_frags = select_contigs(:bfr_score)
    end
    @bfr_frags
  end

  # Applies selection procedure on assembly contigs based on the ratio_type provided.
  # If use_all_contigs is set to false then contigs without any variant are discarded for :hme_score
  # while contigs without any hemisnps are discarded for :bfr_score
  # If include_low_hmes is set to false then contigs are further filtered based on a cut off value of the score
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  def select_contigs(ratio_type)
    selected_contigs ={}
    use_all_contigs = Options.use_all_contigs
    @assembly.each_key do | frag |
      if use_all_contigs
        selected_contigs[frag] = @assembly[frag]
      else
        if ratio_type == :hme_score
          # selecting fragments which have a variant
          if @assembly[frag].hm_num + @assembly[frag].ht_num > 2 * Options.hmes_adjust
            selected_contigs[frag] = @assembly[frag]
          end
        else # ratio_type == :bfr_score
          # in polyploidy scenario selecting fragments with at least one bfr position
          if @assembly[frag].hemi_num > 0
            selected_contigs[frag] = @assembly[frag]
          end
        end
      end
    end
    selected_contigs = filter_contigs(selected_contigs, ratio_type)
    if use_all_contigs
      logger.info "No filtering was applied to fragments\n"
    else
      logger.info "Selected #{selected_contigs.length} out of #{@assembly.length} fragments with #{ratio_type} score\n"
    end
    selected_contigs
  end

  # Filters out contigs below a cutoff for selected ratio_type
  # a cutoff value is calculated based on ratio_type provided
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def filter_contigs(selected_contigs, ratio_type)
    cutoff = get_cutoff(selected_contigs, ratio_type)
    selected_contigs.each_key do | frag |
      if selected_contigs[frag].send(ratio_type) < cutoff
        selected_contigs.delete(frag)
      end
    end
    selected_contigs
  end

  # Cut off value calculation used to filter out low scored contigs.
  #
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def get_cutoff(selected_contigs, ratio_type)
    include_low_hmes = Options.include_low_hmes
    # set minimum cut off hme_score or bfr_score to pick fragments with variants
    # calculate min hme score for back or out crossed data or bfr_score for polypoidy data
    # if no filtering applied set cutoff to 1.1
    if include_low_hmes
      cutoff = 0.0
    else
      if ratio_type == :hme_score
        adjust = Options.hmes_adjust
        if Options.cross_type == 'back'
          cutoff = (1.0/adjust) + 1.0
        else # outcross
          cutoff = (2.0/adjust) + 1.0
        end
      else # ratio_type is bfr_score
        cutoff = bfr_cutoff(selected_contigs)
      end
    end
    cutoff
  end

  # Cut off value calculation for bfr contigs.
  # ratio value at index 0.1% length of an array or at index zero of an array that contains decreasing order of bfr ratios
  # @param selected_contigs [Hash] a hash of contigs with selected bfr score, a subset of assembly hash
  def bfr_cutoff(selected_contigs, prop=0.1)
    ratios = []
    selected_contigs.each_key do | frag |
      ratios << selected_contigs[frag].bfr_score
    end
    ratios.sort!.reverse!
    index = (ratios.length * prop)/100
    # set a minmum index to get at least one contig
    if index < 1
      index = 1
    end
    ratios[index - 1]
  end

  # Method is to discard homozygous variant positions for which background bulk
  # pileup shows a fraction value higher than 0.35 for variant allele/non-reference allele
  # a recessive variant is expected to have 1/3rd frequency in background bulk
  def verify_bg_bulk_pileup
    unless defined?(@hmes_frags)
      self.hmes_frags
    end
    @hmes_frags.each_key do | frag |
      positions = @assembly[frag].hm_pos.keys
      contig_pileup_obj = @pileups[frag]
      positions.each do | pos |
        if contig_pileup_obj.mut_bulk.key?(pos)
          mut_pileup = contig_pileup_obj.mut_bulk[pos]
          if mut_pileup.is_var
            if contig_pileup_obj.bg_bulk.key?(pos)
              bg_pileup = contig_pileup_obj.bg_bulk[pos]
              if bg_pileup.non_ref_ratio > 0.35
                @assembly[frag].hm_pos.delete(pos)
              end
            end
          else
            # this should not happen, may be catch as as an error
            @assembly[frag].hm_pos.delete(pos)
          end
        else
          # this should not happen, may be catch as as an error
          @assembly[frag].hm_pos.delete(pos)
        end
      end
    end
    # recalculate hmes_frags once pileups are verified
    self.hmes_frags
  end

end

#pileups_analyzed ⇒ Boolean (readonly)

Returns a Boolean option to check if pileups for the assembly are analyzed or not.

Returns:

  • (Boolean) —

    a Boolean option to check if pileups for the assembly are analyzed or not



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# File 'lib/cheripic/variants.rb', line 55

class Variants

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_key, :each_value, :size, :length, :[]
  attr_reader :assembly, :pileups, :pileups_analyzed

  # creates a Variants object using user input files
  # @param options [OpenStruct] a hash of required input files as keys and file paths as values
  def initialize(options)
    @params = options
    @assembly = {}
    @pileups = {}
    Bio::FastaFormat.open(@params.assembly).each do |entry|
      if entry.seq.length == 0
        logger.error "No sequence found for entry #{entry.entry_id}"
        raise VariantsError
      end
      contig = Contig.new(entry)
      if @assembly.key?(contig.id)
        logger.error "fasta id already found in the file for #{contig.id}"
        logger.error 'make sure there are no duplicate entries in the fasta file'
        raise VariantsError
      end
      @assembly[contig.id] = contig
      @pileups[contig.id] = ContigPileups.new(contig.id)
    end
    @pileups_analyzed = false
    unless @params.repeats_file == ''
      store_repeat_regions
    end
  end

  # reads repeat masker output file and stores masked regions to ignore variants in thos regions
  def store_repeat_regions
    File.foreach(@params.repeats_file) do |line|
      line.strip!
      next if line =~ /^SW/ or line =~ /^score/ or line == ''
      info = line.split("\s")
      pileups_obj = @pileups[info[4]]
      index = pileups_obj.masked_regions.length
      pileups_obj.masked_regions[index + 1][:begin] = info[5].to_i
      pileups_obj.masked_regions[index + 1][:end] = info[6].to_i
    end
  end

  # Reads and store pileup data for each of input bulk and parents pileup files
  # And sets pileups_analyzed to true that pileups files are processed
  def analyse_pileups
    if @params.input_format == 'bam'
      @vcf_hash = Vcf.filtering(@params.mut_bulk_vcf, @params.bg_bulk_vcf)
    end
    i{mut_bulk bg_bulk mut_parent bg_parent}.each do | input |
      infile = @params[input]
      if infile != ''
        logger.info "processing #{input} file"
        if @params.input_format == 'pileup'
          extract_pileup(infile, input)
        else
          extract_bam_pileup(infile, input)
        end
      end
    end

    @pileups_analyzed = true
  end

  # Bam object is read and each contig mean and std deviation of depth calculated
  # @param bamobject [Bio::DB::Sam]
  # Open3 capture returns string output, so careful not to give whole genome or big contigs for depth analysis
  def set_max_depth(bamobject, bamfile)
    logger.info "processing #{bamfile} file for depth"
    all_depths = []
    bq = Options.base_quality
    mq = Options.mapping_quality
    @assembly.each_key do | id |
      contig_obj = @assembly[id]
      len = contig_obj.length
      data = bamobject.depth(:r => "#{id}", :Q => bq, :q => mq)
      depths = []
      data.split("\n").each do |line|
        info = line.split("\t")
        depths << info[2].to_i
      end
      variance = 0
      mean_depth = depths.reduce(0, :+) / len.to_f
      depths.each do |value|
        variance += (value.to_f - mean_depth)**2
      end
      all_depths << mean_depth
      contig_obj.sd_depth = Math.sqrt(variance)
      contig_obj.mean_depth = mean_depth
    end
    # setting max depth as 3 times the average depth
    mean_coverage = all_depths.reduce(0, :+) / @assembly.length.to_f
    Options.maxdepth = Options.max_d_multiple * mean_coverage
  end

  # Input pileup file is read and positions are selected that pass the thresholds
  # @param pileupfile [String] path to the pileup file to read
  # @param sym [Symbol] Symbol of the pileup file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_pileup(pileupfile, sym)
    # read mpileup file and process each variant
    File.foreach(pileupfile) do |line|
      pileup = Pileup.new(line)
      if pileup.is_var
        contig_obj = @pileups[pileup.ref_name]
        contig_obj.send(sym).store(pileup.pos, pileup)
      end
    end
  end

  # Input bamfile is read and selected positions pileups are stored
  # @param bamfile [String] path to the bam file to read
  # @param sym [Symbol] Symbol of the bam file used to write selected variants
  # pileup information to respective ContigPileups object
  def extract_bam_pileup(bamfile, sym)
    bq = Options.base_quality
    mq = Options.mapping_quality
    bamobject = Bio::DB::Sam.new(:bam=>bamfile, :fasta=>@params.assembly)
    bamobject.index unless bamobject.indexed?

    # check if user has set max depth or set to zero to ignore
    max_d = Options.maxdepth
    # or calculate from bamfile
    if Options.max_d_multiple > 0
      set_max_depth(bamobject, bamfile)
      max_d = Options.maxdepth
      logger.info "max depth used for #{sym} file\t#{max_d}"
    end

    @vcf_hash.each_key do | id |
      positions = @vcf_hash[id][:het].keys
      positions << @vcf_hash[id][:hom].keys
      positions.flatten!
      next if positions.empty?
      contig_obj = @pileups[id]
      positions.each do | pos |
        command = "#{bamobject.samtools} mpileup -r #{id}:#{pos}-#{pos} -Q #{bq} -q #{mq} -B -f #{@params.assembly} #{bamfile}"
        stdout, stderr, status = Open3.capture3(command)
        unless status.success?
          logger.error "resulted in exit code #{status.exitstatus} using #{command}"
          logger.error "stderr output is: #{stderr}"
          raise CheripicError
        end
        stdout.chomp!
        if stdout == '' or stdout.split("\t")[3].to_i == 0 or stdout =~ /^\t0/
          logger.info "pileup data empty for\t#{id}\t#{pos}"
        else
          pileup = Pileup.new(stdout)
          unless max_d == 0 or pileup.coverage <= max_d
            logger.info "pileup coverage is higher than max\t#{pileup.to_s}"
            next
          end
          contig_obj.send(sym).store(pos, pileup)
        end
      end
    end
  end

  # Once pileup files are analysed and variants are extracted from each bulk;
  # bulks are compared to identify and isolate variants for downstream analysis.
  # If polyploidy set to trye and mut_parent and bg_parent bulks are provided
  # hemisnps in parents are extracted for bulk frequency ratio analysis
  def compare_pileups
    unless @pileups_analyzed
      self.analyse_pileups
    end
    @assembly.each_key do | id |
      contig = @assembly[id]
      # extract parental hemi snps for polyploids before bulks are compared
      if Options.polyploidy
        if @params.mut_parent != '' or @params.bg_parent != ''
          @pileups[id].hemisnps_in_parent
        end
      end
      contig.hm_pos, contig.ht_pos, contig.hemi_pos = @pileups[id].bulks_compared
    end
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for homozygosity enrichment score (hme_score)
  def hmes_frags
    # calculate every time method gets called
    @hmes_frags = select_contigs(:hme_score)
  end

  # From Assembly contig objects, contigs are selected based on user selected options
  # for bulk frequency ratio (bfr_score)
  def bfr_frags
    unless defined?(@bfr_frags)
      @bfr_frags = select_contigs(:bfr_score)
    end
    @bfr_frags
  end

  # Applies selection procedure on assembly contigs based on the ratio_type provided.
  # If use_all_contigs is set to false then contigs without any variant are discarded for :hme_score
  # while contigs without any hemisnps are discarded for :bfr_score
  # If include_low_hmes is set to false then contigs are further filtered based on a cut off value of the score
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  def select_contigs(ratio_type)
    selected_contigs ={}
    use_all_contigs = Options.use_all_contigs
    @assembly.each_key do | frag |
      if use_all_contigs
        selected_contigs[frag] = @assembly[frag]
      else
        if ratio_type == :hme_score
          # selecting fragments which have a variant
          if @assembly[frag].hm_num + @assembly[frag].ht_num > 2 * Options.hmes_adjust
            selected_contigs[frag] = @assembly[frag]
          end
        else # ratio_type == :bfr_score
          # in polyploidy scenario selecting fragments with at least one bfr position
          if @assembly[frag].hemi_num > 0
            selected_contigs[frag] = @assembly[frag]
          end
        end
      end
    end
    selected_contigs = filter_contigs(selected_contigs, ratio_type)
    if use_all_contigs
      logger.info "No filtering was applied to fragments\n"
    else
      logger.info "Selected #{selected_contigs.length} out of #{@assembly.length} fragments with #{ratio_type} score\n"
    end
    selected_contigs
  end

  # Filters out contigs below a cutoff for selected ratio_type
  # a cutoff value is calculated based on ratio_type provided
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def filter_contigs(selected_contigs, ratio_type)
    cutoff = get_cutoff(selected_contigs, ratio_type)
    selected_contigs.each_key do | frag |
      if selected_contigs[frag].send(ratio_type) < cutoff
        selected_contigs.delete(frag)
      end
    end
    selected_contigs
  end

  # Cut off value calculation used to filter out low scored contigs.
  #
  # @param ratio_type [Symbol] ratio_type is either :hme_score or :bfr_score
  # @param selected_contigs [Hash] a hash of contigs with selected ratio_type, a subset of assembly hash
  def get_cutoff(selected_contigs, ratio_type)
    include_low_hmes = Options.include_low_hmes
    # set minimum cut off hme_score or bfr_score to pick fragments with variants
    # calculate min hme score for back or out crossed data or bfr_score for polypoidy data
    # if no filtering applied set cutoff to 1.1
    if include_low_hmes
      cutoff = 0.0
    else
      if ratio_type == :hme_score
        adjust = Options.hmes_adjust
        if Options.cross_type == 'back'
          cutoff = (1.0/adjust) + 1.0
        else # outcross
          cutoff = (2.0/adjust) + 1.0
        end
      else # ratio_type is bfr_score
        cutoff = bfr_cutoff(selected_contigs)
      end
    end
    cutoff
  end

  # Cut off value calculation for bfr contigs.
  # ratio value at index 0.1% length of an array or at index zero of an array that contains decreasing order of bfr ratios
  # @param selected_contigs [Hash] a hash of contigs with selected bfr score, a subset of assembly hash
  def bfr_cutoff(selected_contigs, prop=0.1)
    ratios = []
    selected_contigs.each_key do | frag |
      ratios << selected_contigs[frag].bfr_score
    end
    ratios.sort!.reverse!
    index = (ratios.length * prop)/100
    # set a minmum index to get at least one contig
    if index < 1
      index = 1
    end
    ratios[index - 1]
  end

  # Method is to discard homozygous variant positions for which background bulk
  # pileup shows a fraction value higher than 0.35 for variant allele/non-reference allele
  # a recessive variant is expected to have 1/3rd frequency in background bulk
  def verify_bg_bulk_pileup
    unless defined?(@hmes_frags)
      self.hmes_frags
    end
    @hmes_frags.each_key do | frag |
      positions = @assembly[frag].hm_pos.keys
      contig_pileup_obj = @pileups[frag]
      positions.each do | pos |
        if contig_pileup_obj.mut_bulk.key?(pos)
          mut_pileup = contig_pileup_obj.mut_bulk[pos]
          if mut_pileup.is_var
            if contig_pileup_obj.bg_bulk.key?(pos)
              bg_pileup = contig_pileup_obj.bg_bulk[pos]
              if bg_pileup.non_ref_ratio > 0.35
                @assembly[frag].hm_pos.delete(pos)
              end
            end
          else
            # this should not happen, may be catch as as an error
            @assembly[frag].hm_pos.delete(pos)
          end
        else
          # this should not happen, may be catch as as an error
          @assembly[frag].hm_pos.delete(pos)
        end
      end
    end
    # recalculate hmes_frags once pileups are verified
    self.hmes_frags
  end

end

Instance Method Details

#analyse_pileups ⇒ Object

Reads and store pileup data for each of input bulk and parents pileup files And sets pileups_analyzed to true that pileups files are processed



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# File 'lib/cheripic/variants.rb', line 103

def analyse_pileups
  if @params.input_format == 'bam'
    @vcf_hash = Vcf.filtering(@params.mut_bulk_vcf, @params.bg_bulk_vcf)
  end
  i{mut_bulk bg_bulk mut_parent bg_parent}.each do | input |
    infile = @params[input]
    if infile != ''
      logger.info "processing #{input} file"
      if @params.input_format == 'pileup'
        extract_pileup(infile, input)
      else
        extract_bam_pileup(infile, input)
      end
    end
  end

  @pileups_analyzed = true
end

#bfr_cutoff(selected_contigs, prop = 0.1) ⇒ Object

Cut off value calculation for bfr contigs. ratio value at index 0.1% length of an array or at index zero of an array that contains decreasing order of bfr ratios

Parameters:

  • selected_contigs (Hash) —

    a hash of contigs with selected bfr score, a subset of assembly hash



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# File 'lib/cheripic/variants.rb', line 329

def bfr_cutoff(selected_contigs, prop=0.1)
  ratios = []
  selected_contigs.each_key do | frag |
    ratios << selected_contigs[frag].bfr_score
  end
  ratios.sort!.reverse!
  index = (ratios.length * prop)/100
  # set a minmum index to get at least one contig
  if index < 1
    index = 1
  end
  ratios[index - 1]
end

#compare_pileups ⇒ Object

Once pileup files are analysed and variants are extracted from each bulk; bulks are compared to identify and isolate variants for downstream analysis. If polyploidy set to trye and mut_parent and bg_parent bulks are provided hemisnps in parents are extracted for bulk frequency ratio analysis



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# File 'lib/cheripic/variants.rb', line 220

def compare_pileups
  unless @pileups_analyzed
    self.analyse_pileups
  end
  @assembly.each_key do | id |
    contig = @assembly[id]
    # extract parental hemi snps for polyploids before bulks are compared
    if Options.polyploidy
      if @params.mut_parent != '' or @params.bg_parent != ''
        @pileups[id].hemisnps_in_parent
      end
    end
    contig.hm_pos, contig.ht_pos, contig.hemi_pos = @pileups[id].bulks_compared
  end
end

#extract_bam_pileup(bamfile, sym) ⇒ Object

Input bamfile is read and selected positions pileups are stored pileup information to respective ContigPileups object

Parameters:

  • bamfile (String) —

    path to the bam file to read

  • sym (Symbol) —

    Symbol of the bam file used to write selected variants



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# File 'lib/cheripic/variants.rb', line 172

def extract_bam_pileup(bamfile, sym)
  bq = Options.base_quality
  mq = Options.mapping_quality
  bamobject = Bio::DB::Sam.new(:bam=>bamfile, :fasta=>@params.assembly)
  bamobject.index unless bamobject.indexed?

  # check if user has set max depth or set to zero to ignore
  max_d = Options.maxdepth
  # or calculate from bamfile
  if Options.max_d_multiple > 0
    set_max_depth(bamobject, bamfile)
    max_d = Options.maxdepth
    logger.info "max depth used for #{sym} file\t#{max_d}"
  end

  @vcf_hash.each_key do | id |
    positions = @vcf_hash[id][:het].keys
    positions << @vcf_hash[id][:hom].keys
    positions.flatten!
    next if positions.empty?
    contig_obj = @pileups[id]
    positions.each do | pos |
      command = "#{bamobject.samtools} mpileup -r #{id}:#{pos}-#{pos} -Q #{bq} -q #{mq} -B -f #{@params.assembly} #{bamfile}"
      stdout, stderr, status = Open3.capture3(command)
      unless status.success?
        logger.error "resulted in exit code #{status.exitstatus} using #{command}"
        logger.error "stderr output is: #{stderr}"
        raise CheripicError
      end
      stdout.chomp!
      if stdout == '' or stdout.split("\t")[3].to_i == 0 or stdout =~ /^\t0/
        logger.info "pileup data empty for\t#{id}\t#{pos}"
      else
        pileup = Pileup.new(stdout)
        unless max_d == 0 or pileup.coverage <= max_d
          logger.info "pileup coverage is higher than max\t#{pileup.to_s}"
          next
        end
        contig_obj.send(sym).store(pos, pileup)
      end
    end
  end
end

#extract_pileup(pileupfile, sym) ⇒ Object

Input pileup file is read and positions are selected that pass the thresholds pileup information to respective ContigPileups object

Parameters:

  • pileupfile (String) —

    path to the pileup file to read

  • sym (Symbol) —

    Symbol of the pileup file used to write selected variants



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# File 'lib/cheripic/variants.rb', line 157

def extract_pileup(pileupfile, sym)
  # read mpileup file and process each variant
  File.foreach(pileupfile) do |line|
    pileup = Pileup.new(line)
    if pileup.is_var
      contig_obj = @pileups[pileup.ref_name]
      contig_obj.send(sym).store(pileup.pos, pileup)
    end
  end
end

#filter_contigs(selected_contigs, ratio_type) ⇒ Object

Filters out contigs below a cutoff for selected ratio_type a cutoff value is calculated based on ratio_type provided

Parameters:

  • ratio_type (Symbol) —

    ratio_type is either :hme_score or :bfr_score

  • selected_contigs (Hash) —

    a hash of contigs with selected ratio_type, a subset of assembly hash



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# File 'lib/cheripic/variants.rb', line 290

def filter_contigs(selected_contigs, ratio_type)
  cutoff = get_cutoff(selected_contigs, ratio_type)
  selected_contigs.each_key do | frag |
    if selected_contigs[frag].send(ratio_type) < cutoff
      selected_contigs.delete(frag)
    end
  end
  selected_contigs
end

#get_cutoff(selected_contigs, ratio_type) ⇒ Object

Cut off value calculation used to filter out low scored contigs.

Parameters:

  • ratio_type (Symbol) —

    ratio_type is either :hme_score or :bfr_score

  • selected_contigs (Hash) —

    a hash of contigs with selected ratio_type, a subset of assembly hash



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# File 'lib/cheripic/variants.rb', line 304

def get_cutoff(selected_contigs, ratio_type)
  include_low_hmes = Options.include_low_hmes
  # set minimum cut off hme_score or bfr_score to pick fragments with variants
  # calculate min hme score for back or out crossed data or bfr_score for polypoidy data
  # if no filtering applied set cutoff to 1.1
  if include_low_hmes
    cutoff = 0.0
  else
    if ratio_type == :hme_score
      adjust = Options.hmes_adjust
      if Options.cross_type == 'back'
        cutoff = (1.0/adjust) + 1.0
      else # outcross
        cutoff = (2.0/adjust) + 1.0
      end
    else # ratio_type is bfr_score
      cutoff = bfr_cutoff(selected_contigs)
    end
  end
  cutoff
end

#select_contigs(ratio_type) ⇒ Object

Applies selection procedure on assembly contigs based on the ratio_type provided. If use_all_contigs is set to false then contigs without any variant are discarded for :hme_score while contigs without any hemisnps are discarded for :bfr_score If include_low_hmes is set to false then contigs are further filtered based on a cut off value of the score

Parameters:

  • ratio_type (Symbol) —

    ratio_type is either :hme_score or :bfr_score



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# File 'lib/cheripic/variants.rb', line 257

def select_contigs(ratio_type)
  selected_contigs ={}
  use_all_contigs = Options.use_all_contigs
  @assembly.each_key do | frag |
    if use_all_contigs
      selected_contigs[frag] = @assembly[frag]
    else
      if ratio_type == :hme_score
        # selecting fragments which have a variant
        if @assembly[frag].hm_num + @assembly[frag].ht_num > 2 * Options.hmes_adjust
          selected_contigs[frag] = @assembly[frag]
        end
      else # ratio_type == :bfr_score
        # in polyploidy scenario selecting fragments with at least one bfr position
        if @assembly[frag].hemi_num > 0
          selected_contigs[frag] = @assembly[frag]
        end
      end
    end
  end
  selected_contigs = filter_contigs(selected_contigs, ratio_type)
  if use_all_contigs
    logger.info "No filtering was applied to fragments\n"
  else
    logger.info "Selected #{selected_contigs.length} out of #{@assembly.length} fragments with #{ratio_type} score\n"
  end
  selected_contigs
end

#set_max_depth(bamobject, bamfile) ⇒ Object

Bam object is read and each contig mean and std deviation of depth calculated Open3 capture returns string output, so careful not to give whole genome or big contigs for depth analysis

Parameters:



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# File 'lib/cheripic/variants.rb', line 125

def set_max_depth(bamobject, bamfile)
  logger.info "processing #{bamfile} file for depth"
  all_depths = []
  bq = Options.base_quality
  mq = Options.mapping_quality
  @assembly.each_key do | id |
    contig_obj = @assembly[id]
    len = contig_obj.length
    data = bamobject.depth(:r => "#{id}", :Q => bq, :q => mq)
    depths = []
    data.split("\n").each do |line|
      info = line.split("\t")
      depths << info[2].to_i
    end
    variance = 0
    mean_depth = depths.reduce(0, :+) / len.to_f
    depths.each do |value|
      variance += (value.to_f - mean_depth)**2
    end
    all_depths << mean_depth
    contig_obj.sd_depth = Math.sqrt(variance)
    contig_obj.mean_depth = mean_depth
  end
  # setting max depth as 3 times the average depth
  mean_coverage = all_depths.reduce(0, :+) / @assembly.length.to_f
  Options.maxdepth = Options.max_d_multiple * mean_coverage
end

#store_repeat_regions ⇒ Object

reads repeat masker output file and stores masked regions to ignore variants in thos regions



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# File 'lib/cheripic/variants.rb', line 89

def store_repeat_regions
  File.foreach(@params.repeats_file) do |line|
    line.strip!
    next if line =~ /^SW/ or line =~ /^score/ or line == ''
    info = line.split("\s")
    pileups_obj = @pileups[info[4]]
    index = pileups_obj.masked_regions.length
    pileups_obj.masked_regions[index + 1][:begin] = info[5].to_i
    pileups_obj.masked_regions[index + 1][:end] = info[6].to_i
  end
end

#verify_bg_bulk_pileup ⇒ Object

Method is to discard homozygous variant positions for which background bulk pileup shows a fraction value higher than 0.35 for variant allele/non-reference allele a recessive variant is expected to have 1/3rd frequency in background bulk



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# File 'lib/cheripic/variants.rb', line 346

def verify_bg_bulk_pileup
  unless defined?(@hmes_frags)
    self.hmes_frags
  end
  @hmes_frags.each_key do | frag |
    positions = @assembly[frag].hm_pos.keys
    contig_pileup_obj = @pileups[frag]
    positions.each do | pos |
      if contig_pileup_obj.mut_bulk.key?(pos)
        mut_pileup = contig_pileup_obj.mut_bulk[pos]
        if mut_pileup.is_var
          if contig_pileup_obj.bg_bulk.key?(pos)
            bg_pileup = contig_pileup_obj.bg_bulk[pos]
            if bg_pileup.non_ref_ratio > 0.35
              @assembly[frag].hm_pos.delete(pos)
            end
          end
        else
          # this should not happen, may be catch as as an error
          @assembly[frag].hm_pos.delete(pos)
        end
      else
        # this should not happen, may be catch as as an error
        @assembly[frag].hm_pos.delete(pos)
      end
    end
  end
  # recalculate hmes_frags once pileups are verified
  self.hmes_frags
end