Class: Transrate::Assembly

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

Overview

Container for a transcriptome assembly and its associated metadata.

Instance Attribute Summary collapse

Instance Method Summary collapse

Constructor Details

#initialize(file) ⇒ Assembly

Create a new Assembly.



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# File 'lib/transrate/assembly.rb', line 41

def initialize file
  @file = File.expand_path file
  unless File.exist? @file
    raise IOError.new "Assembly file doesn't exist: #{@file}"
  end
  @assembly = {}
  @n_bases = 0
  Bio::FastaFormat.open(file).each do |entry|
    @n_bases += entry.length
    contig = Contig.new(entry)
    @assembly[contig.name] = contig
  end
  @contig_metrics = ContigMetrics.new self
end

Instance Attribute Details

#assembly ⇒ Array<Bio::FastaFormat> (readonly) 



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# File 'lib/transrate/assembly.rb', line 25

class Assembly

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_value, :<<, :size, :length, :[]

  attr_accessor :file
  attr_reader :assembly
  attr_reader :has_run
  attr_accessor :n_bases
  attr_reader :n50
  attr_accessor :contig_metrics

  # Create a new Assembly.
  #
  # @param file [String] path to the assembly FASTA file
  def initialize file
    @file = File.expand_path file
    unless File.exist? @file
      raise IOError.new "Assembly file doesn't exist: #{@file}"
    end
    @assembly = {}
    @n_bases = 0
    Bio::FastaFormat.open(file).each do |entry|
      @n_bases += entry.length
      contig = Contig.new(entry)
      @assembly[contig.name] = contig
    end
    @contig_metrics = ContigMetrics.new self
  end

  # Generate and store the basic statistics for this assembly
  #
  # @param threads [Integer] number of threads to use
  def run threads=8
    stats = self.basic_stats threads
    stats.each_pair do |key, value|
      ivar = "@#{key.gsub(/\ /, '_')}".to_sym
      attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
      # creates accessors for the variables in stats
      singleton_class.class_eval { attr_accessor attr_ivar }
      self.instance_variable_set(ivar, value)
    end
    @contig_metrics.run
    @has_run = true
  end

  # Return a hash of statistics about this assembly. Stats are
  # calculated in parallel by splitting the assembly into
  # equal-sized bins and calling Assembly#basic_bin_stat on each
  # bin in a separate thread.
  #
  # @param threads [Integer] number of threads to use
  #
  # @return [Hash] basic statistics about the assembly
  def basic_stats threads=1
    return @basic_stats if @basic_stats
    bin = @assembly.values
    @basic_stats = basic_bin_stats bin
    @basic_stats
  end # basic_stats


  # Calculate basic statistics in an single thread for a bin
  # of contigs.
  #
  # Basic statistics are:
  #
  # - N10, N30, N50, N70, N90
  # - number of contigs >= 1,000 base pairs long
  # - number of contigs >= 10,000 base pairs long
  # - length of the shortest contig
  # - length of the longest contig
  # - number of contigs in the bin
  # - mean contig length
  # - total number of nucleotides in the bin
  # - mean % of contig length covered by the longest ORF
  #
  # @param [Array] bin An array of Bio::Sequence objects
  # representing contigs in the assembly

  def basic_bin_stats bin

    # cumulative length is a float so we can divide it
    # accurately later to get the mean length
    cumulative_length = 0.0

    # we'll calculate Nx for x in [10, 30, 50, 70, 90]
    # to do this we create a stack of the x values and
    # pop the first one to set the first cutoff. when
    # the cutoff is reached we store the nucleotide length and pop
    # the next value to set the next cutoff. we take a copy
    # of the Array so we can use the intact original to collect
    # the results later
    x = [90, 70, 50, 30, 10]
    x2 = x.clone
    cutoff = x2.pop / 100.0
    res = []
    n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
    # sort the contigs in ascending length order
    # and iterate over them
    bin.sort_by! { |c| c.seq.length }
    bin.each do |contig|

      # increment our long contig counters if this
      # contig is above the thresholds
      if contig.length < 200
        # ignore contigs less than 200 bases,
        # but record how many there are
        n_under_200 += 1
        next
      end
      n_over_1k += 1 if contig.length > 1_000
      n_over_10k += 1 if contig.length > 10_000

      # add the length of the longest orf to the
      # running total
      orf_length = contig.orf_length
      orf_length_sum += orf_length
      # only consider orfs that are realistic length
      # (here we set minimum amino acid length as 50)
      n_with_orf += 1 if orf_length > 149

      # increment the cumulative length and check whether the Nx
      # cutoff has been reached. if it has, store the Nx value and
      # get the next cutoff
      cumulative_length += contig.length
      if cumulative_length >= @n_bases * cutoff
        res << contig.length
        if x2.empty?
          cutoff = 1
        else
          cutoff = x2.pop / 100.0
        end
      end

    end

    # if there aren't enough sequences we might have no value for some
    # of the Nx. Fill the empty ones in with the longest contig length.
    while res.length < x.length do
      res << bin.last.length
    end

    # calculate and return the statistics as a hash
    mean = cumulative_length / @assembly.size
    ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
    {
      'n_seqs' => bin.size,
      'smallest' => bin.first.length,
      'largest' => bin.last.length,
      'n_bases' => n_bases,
      'mean_len' => mean,
      'n_under_200' => n_under_200,
      'n_over_1k' => n_over_1k,
      'n_over_10k' => n_over_10k,
      'n_with_orf' => n_with_orf,
      'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
    }.merge ns

  end # basic_bin_stats

  # Calls *block* with two arguments, the contig and an array
  # of integer per-base coverage counts.
  #
  # @param bam [Bio::Db::Sam] a bam alignment of reads against this assembly
  # @param block [Block] the block to call
  def each_with_coverage(bam, &block)
    logger.debug 'enumerating assembly with coverage'
    # generate coverage with samtools
    covfile = Samtools.coverage bam
    # get an assembly enumerator
    assembly_enum = @assembly.to_enum
    contig_name, contig = assembly_enum.next
    # precreate an array of the correct size to contain
    # coverage. this is necessary because samtools mpileup
    # doesn't print a result line for bases with 0 coverage
    contig.coverage = Array.new(contig.length, 0)
    # the columns we need
    name_i, pos_i, cov_i = 0, 1, 3
    # parse the coverage file
    File.open(covfile).each_line do |line|
      cols = line.chomp.split("\t")
      unless (cols && cols.length > 4)
        # last line
        break
      end
      # extract the columns
      name = Bio::FastaDefline.new(cols[name_i]).entry_id
      pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
      unless contig_name == name
        while contig_name != name
          begin
            block.call(contig, contig.coverage)
            contig_name, contig = assembly_enum.next
            contig.coverage = Array.new(contig.length, 0)
          rescue StopIteration => stop_error
            logger.error 'reached the end of assembly enumerator while ' +
                      'there were contigs left in the coverage results'
            logger.error "final assembly contig: #{@assembly.last.name}"
            logger.error "coverage contig: #{name}"
            raise stop_error
          end
        end
      end
      contig.coverage[pos - 1] = cov
    end
    # yield the final contig
    block.call(contig, contig.coverage)
  end

end

#contig_metrics ⇒ Object

Returns the value of attribute contig_metrics.



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# File 'lib/transrate/assembly.rb', line 36

def contig_metrics
  @contig_metrics
end

#file ⇒ String 



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# File 'lib/transrate/assembly.rb', line 25

class Assembly

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_value, :<<, :size, :length, :[]

  attr_accessor :file
  attr_reader :assembly
  attr_reader :has_run
  attr_accessor :n_bases
  attr_reader :n50
  attr_accessor :contig_metrics

  # Create a new Assembly.
  #
  # @param file [String] path to the assembly FASTA file
  def initialize file
    @file = File.expand_path file
    unless File.exist? @file
      raise IOError.new "Assembly file doesn't exist: #{@file}"
    end
    @assembly = {}
    @n_bases = 0
    Bio::FastaFormat.open(file).each do |entry|
      @n_bases += entry.length
      contig = Contig.new(entry)
      @assembly[contig.name] = contig
    end
    @contig_metrics = ContigMetrics.new self
  end

  # Generate and store the basic statistics for this assembly
  #
  # @param threads [Integer] number of threads to use
  def run threads=8
    stats = self.basic_stats threads
    stats.each_pair do |key, value|
      ivar = "@#{key.gsub(/\ /, '_')}".to_sym
      attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
      # creates accessors for the variables in stats
      singleton_class.class_eval { attr_accessor attr_ivar }
      self.instance_variable_set(ivar, value)
    end
    @contig_metrics.run
    @has_run = true
  end

  # Return a hash of statistics about this assembly. Stats are
  # calculated in parallel by splitting the assembly into
  # equal-sized bins and calling Assembly#basic_bin_stat on each
  # bin in a separate thread.
  #
  # @param threads [Integer] number of threads to use
  #
  # @return [Hash] basic statistics about the assembly
  def basic_stats threads=1
    return @basic_stats if @basic_stats
    bin = @assembly.values
    @basic_stats = basic_bin_stats bin
    @basic_stats
  end # basic_stats


  # Calculate basic statistics in an single thread for a bin
  # of contigs.
  #
  # Basic statistics are:
  #
  # - N10, N30, N50, N70, N90
  # - number of contigs >= 1,000 base pairs long
  # - number of contigs >= 10,000 base pairs long
  # - length of the shortest contig
  # - length of the longest contig
  # - number of contigs in the bin
  # - mean contig length
  # - total number of nucleotides in the bin
  # - mean % of contig length covered by the longest ORF
  #
  # @param [Array] bin An array of Bio::Sequence objects
  # representing contigs in the assembly

  def basic_bin_stats bin

    # cumulative length is a float so we can divide it
    # accurately later to get the mean length
    cumulative_length = 0.0

    # we'll calculate Nx for x in [10, 30, 50, 70, 90]
    # to do this we create a stack of the x values and
    # pop the first one to set the first cutoff. when
    # the cutoff is reached we store the nucleotide length and pop
    # the next value to set the next cutoff. we take a copy
    # of the Array so we can use the intact original to collect
    # the results later
    x = [90, 70, 50, 30, 10]
    x2 = x.clone
    cutoff = x2.pop / 100.0
    res = []
    n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
    # sort the contigs in ascending length order
    # and iterate over them
    bin.sort_by! { |c| c.seq.length }
    bin.each do |contig|

      # increment our long contig counters if this
      # contig is above the thresholds
      if contig.length < 200
        # ignore contigs less than 200 bases,
        # but record how many there are
        n_under_200 += 1
        next
      end
      n_over_1k += 1 if contig.length > 1_000
      n_over_10k += 1 if contig.length > 10_000

      # add the length of the longest orf to the
      # running total
      orf_length = contig.orf_length
      orf_length_sum += orf_length
      # only consider orfs that are realistic length
      # (here we set minimum amino acid length as 50)
      n_with_orf += 1 if orf_length > 149

      # increment the cumulative length and check whether the Nx
      # cutoff has been reached. if it has, store the Nx value and
      # get the next cutoff
      cumulative_length += contig.length
      if cumulative_length >= @n_bases * cutoff
        res << contig.length
        if x2.empty?
          cutoff = 1
        else
          cutoff = x2.pop / 100.0
        end
      end

    end

    # if there aren't enough sequences we might have no value for some
    # of the Nx. Fill the empty ones in with the longest contig length.
    while res.length < x.length do
      res << bin.last.length
    end

    # calculate and return the statistics as a hash
    mean = cumulative_length / @assembly.size
    ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
    {
      'n_seqs' => bin.size,
      'smallest' => bin.first.length,
      'largest' => bin.last.length,
      'n_bases' => n_bases,
      'mean_len' => mean,
      'n_under_200' => n_under_200,
      'n_over_1k' => n_over_1k,
      'n_over_10k' => n_over_10k,
      'n_with_orf' => n_with_orf,
      'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
    }.merge ns

  end # basic_bin_stats

  # Calls *block* with two arguments, the contig and an array
  # of integer per-base coverage counts.
  #
  # @param bam [Bio::Db::Sam] a bam alignment of reads against this assembly
  # @param block [Block] the block to call
  def each_with_coverage(bam, &block)
    logger.debug 'enumerating assembly with coverage'
    # generate coverage with samtools
    covfile = Samtools.coverage bam
    # get an assembly enumerator
    assembly_enum = @assembly.to_enum
    contig_name, contig = assembly_enum.next
    # precreate an array of the correct size to contain
    # coverage. this is necessary because samtools mpileup
    # doesn't print a result line for bases with 0 coverage
    contig.coverage = Array.new(contig.length, 0)
    # the columns we need
    name_i, pos_i, cov_i = 0, 1, 3
    # parse the coverage file
    File.open(covfile).each_line do |line|
      cols = line.chomp.split("\t")
      unless (cols && cols.length > 4)
        # last line
        break
      end
      # extract the columns
      name = Bio::FastaDefline.new(cols[name_i]).entry_id
      pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
      unless contig_name == name
        while contig_name != name
          begin
            block.call(contig, contig.coverage)
            contig_name, contig = assembly_enum.next
            contig.coverage = Array.new(contig.length, 0)
          rescue StopIteration => stop_error
            logger.error 'reached the end of assembly enumerator while ' +
                      'there were contigs left in the coverage results'
            logger.error "final assembly contig: #{@assembly.last.name}"
            logger.error "coverage contig: #{name}"
            raise stop_error
          end
        end
      end
      contig.coverage[pos - 1] = cov
    end
    # yield the final contig
    block.call(contig, contig.coverage)
  end

end

#has_run ⇒ BOOL (readonly) 



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# File 'lib/transrate/assembly.rb', line 25

class Assembly

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_value, :<<, :size, :length, :[]

  attr_accessor :file
  attr_reader :assembly
  attr_reader :has_run
  attr_accessor :n_bases
  attr_reader :n50
  attr_accessor :contig_metrics

  # Create a new Assembly.
  #
  # @param file [String] path to the assembly FASTA file
  def initialize file
    @file = File.expand_path file
    unless File.exist? @file
      raise IOError.new "Assembly file doesn't exist: #{@file}"
    end
    @assembly = {}
    @n_bases = 0
    Bio::FastaFormat.open(file).each do |entry|
      @n_bases += entry.length
      contig = Contig.new(entry)
      @assembly[contig.name] = contig
    end
    @contig_metrics = ContigMetrics.new self
  end

  # Generate and store the basic statistics for this assembly
  #
  # @param threads [Integer] number of threads to use
  def run threads=8
    stats = self.basic_stats threads
    stats.each_pair do |key, value|
      ivar = "@#{key.gsub(/\ /, '_')}".to_sym
      attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
      # creates accessors for the variables in stats
      singleton_class.class_eval { attr_accessor attr_ivar }
      self.instance_variable_set(ivar, value)
    end
    @contig_metrics.run
    @has_run = true
  end

  # Return a hash of statistics about this assembly. Stats are
  # calculated in parallel by splitting the assembly into
  # equal-sized bins and calling Assembly#basic_bin_stat on each
  # bin in a separate thread.
  #
  # @param threads [Integer] number of threads to use
  #
  # @return [Hash] basic statistics about the assembly
  def basic_stats threads=1
    return @basic_stats if @basic_stats
    bin = @assembly.values
    @basic_stats = basic_bin_stats bin
    @basic_stats
  end # basic_stats


  # Calculate basic statistics in an single thread for a bin
  # of contigs.
  #
  # Basic statistics are:
  #
  # - N10, N30, N50, N70, N90
  # - number of contigs >= 1,000 base pairs long
  # - number of contigs >= 10,000 base pairs long
  # - length of the shortest contig
  # - length of the longest contig
  # - number of contigs in the bin
  # - mean contig length
  # - total number of nucleotides in the bin
  # - mean % of contig length covered by the longest ORF
  #
  # @param [Array] bin An array of Bio::Sequence objects
  # representing contigs in the assembly

  def basic_bin_stats bin

    # cumulative length is a float so we can divide it
    # accurately later to get the mean length
    cumulative_length = 0.0

    # we'll calculate Nx for x in [10, 30, 50, 70, 90]
    # to do this we create a stack of the x values and
    # pop the first one to set the first cutoff. when
    # the cutoff is reached we store the nucleotide length and pop
    # the next value to set the next cutoff. we take a copy
    # of the Array so we can use the intact original to collect
    # the results later
    x = [90, 70, 50, 30, 10]
    x2 = x.clone
    cutoff = x2.pop / 100.0
    res = []
    n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
    # sort the contigs in ascending length order
    # and iterate over them
    bin.sort_by! { |c| c.seq.length }
    bin.each do |contig|

      # increment our long contig counters if this
      # contig is above the thresholds
      if contig.length < 200
        # ignore contigs less than 200 bases,
        # but record how many there are
        n_under_200 += 1
        next
      end
      n_over_1k += 1 if contig.length > 1_000
      n_over_10k += 1 if contig.length > 10_000

      # add the length of the longest orf to the
      # running total
      orf_length = contig.orf_length
      orf_length_sum += orf_length
      # only consider orfs that are realistic length
      # (here we set minimum amino acid length as 50)
      n_with_orf += 1 if orf_length > 149

      # increment the cumulative length and check whether the Nx
      # cutoff has been reached. if it has, store the Nx value and
      # get the next cutoff
      cumulative_length += contig.length
      if cumulative_length >= @n_bases * cutoff
        res << contig.length
        if x2.empty?
          cutoff = 1
        else
          cutoff = x2.pop / 100.0
        end
      end

    end

    # if there aren't enough sequences we might have no value for some
    # of the Nx. Fill the empty ones in with the longest contig length.
    while res.length < x.length do
      res << bin.last.length
    end

    # calculate and return the statistics as a hash
    mean = cumulative_length / @assembly.size
    ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
    {
      'n_seqs' => bin.size,
      'smallest' => bin.first.length,
      'largest' => bin.last.length,
      'n_bases' => n_bases,
      'mean_len' => mean,
      'n_under_200' => n_under_200,
      'n_over_1k' => n_over_1k,
      'n_over_10k' => n_over_10k,
      'n_with_orf' => n_with_orf,
      'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
    }.merge ns

  end # basic_bin_stats

  # Calls *block* with two arguments, the contig and an array
  # of integer per-base coverage counts.
  #
  # @param bam [Bio::Db::Sam] a bam alignment of reads against this assembly
  # @param block [Block] the block to call
  def each_with_coverage(bam, &block)
    logger.debug 'enumerating assembly with coverage'
    # generate coverage with samtools
    covfile = Samtools.coverage bam
    # get an assembly enumerator
    assembly_enum = @assembly.to_enum
    contig_name, contig = assembly_enum.next
    # precreate an array of the correct size to contain
    # coverage. this is necessary because samtools mpileup
    # doesn't print a result line for bases with 0 coverage
    contig.coverage = Array.new(contig.length, 0)
    # the columns we need
    name_i, pos_i, cov_i = 0, 1, 3
    # parse the coverage file
    File.open(covfile).each_line do |line|
      cols = line.chomp.split("\t")
      unless (cols && cols.length > 4)
        # last line
        break
      end
      # extract the columns
      name = Bio::FastaDefline.new(cols[name_i]).entry_id
      pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
      unless contig_name == name
        while contig_name != name
          begin
            block.call(contig, contig.coverage)
            contig_name, contig = assembly_enum.next
            contig.coverage = Array.new(contig.length, 0)
          rescue StopIteration => stop_error
            logger.error 'reached the end of assembly enumerator while ' +
                      'there were contigs left in the coverage results'
            logger.error "final assembly contig: #{@assembly.last.name}"
            logger.error "coverage contig: #{name}"
            raise stop_error
          end
        end
      end
      contig.coverage[pos - 1] = cov
    end
    # yield the final contig
    block.call(contig, contig.coverage)
  end

end

#n50 ⇒ Integer (readonly) 



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# File 'lib/transrate/assembly.rb', line 25

class Assembly

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_value, :<<, :size, :length, :[]

  attr_accessor :file
  attr_reader :assembly
  attr_reader :has_run
  attr_accessor :n_bases
  attr_reader :n50
  attr_accessor :contig_metrics

  # Create a new Assembly.
  #
  # @param file [String] path to the assembly FASTA file
  def initialize file
    @file = File.expand_path file
    unless File.exist? @file
      raise IOError.new "Assembly file doesn't exist: #{@file}"
    end
    @assembly = {}
    @n_bases = 0
    Bio::FastaFormat.open(file).each do |entry|
      @n_bases += entry.length
      contig = Contig.new(entry)
      @assembly[contig.name] = contig
    end
    @contig_metrics = ContigMetrics.new self
  end

  # Generate and store the basic statistics for this assembly
  #
  # @param threads [Integer] number of threads to use
  def run threads=8
    stats = self.basic_stats threads
    stats.each_pair do |key, value|
      ivar = "@#{key.gsub(/\ /, '_')}".to_sym
      attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
      # creates accessors for the variables in stats
      singleton_class.class_eval { attr_accessor attr_ivar }
      self.instance_variable_set(ivar, value)
    end
    @contig_metrics.run
    @has_run = true
  end

  # Return a hash of statistics about this assembly. Stats are
  # calculated in parallel by splitting the assembly into
  # equal-sized bins and calling Assembly#basic_bin_stat on each
  # bin in a separate thread.
  #
  # @param threads [Integer] number of threads to use
  #
  # @return [Hash] basic statistics about the assembly
  def basic_stats threads=1
    return @basic_stats if @basic_stats
    bin = @assembly.values
    @basic_stats = basic_bin_stats bin
    @basic_stats
  end # basic_stats


  # Calculate basic statistics in an single thread for a bin
  # of contigs.
  #
  # Basic statistics are:
  #
  # - N10, N30, N50, N70, N90
  # - number of contigs >= 1,000 base pairs long
  # - number of contigs >= 10,000 base pairs long
  # - length of the shortest contig
  # - length of the longest contig
  # - number of contigs in the bin
  # - mean contig length
  # - total number of nucleotides in the bin
  # - mean % of contig length covered by the longest ORF
  #
  # @param [Array] bin An array of Bio::Sequence objects
  # representing contigs in the assembly

  def basic_bin_stats bin

    # cumulative length is a float so we can divide it
    # accurately later to get the mean length
    cumulative_length = 0.0

    # we'll calculate Nx for x in [10, 30, 50, 70, 90]
    # to do this we create a stack of the x values and
    # pop the first one to set the first cutoff. when
    # the cutoff is reached we store the nucleotide length and pop
    # the next value to set the next cutoff. we take a copy
    # of the Array so we can use the intact original to collect
    # the results later
    x = [90, 70, 50, 30, 10]
    x2 = x.clone
    cutoff = x2.pop / 100.0
    res = []
    n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
    # sort the contigs in ascending length order
    # and iterate over them
    bin.sort_by! { |c| c.seq.length }
    bin.each do |contig|

      # increment our long contig counters if this
      # contig is above the thresholds
      if contig.length < 200
        # ignore contigs less than 200 bases,
        # but record how many there are
        n_under_200 += 1
        next
      end
      n_over_1k += 1 if contig.length > 1_000
      n_over_10k += 1 if contig.length > 10_000

      # add the length of the longest orf to the
      # running total
      orf_length = contig.orf_length
      orf_length_sum += orf_length
      # only consider orfs that are realistic length
      # (here we set minimum amino acid length as 50)
      n_with_orf += 1 if orf_length > 149

      # increment the cumulative length and check whether the Nx
      # cutoff has been reached. if it has, store the Nx value and
      # get the next cutoff
      cumulative_length += contig.length
      if cumulative_length >= @n_bases * cutoff
        res << contig.length
        if x2.empty?
          cutoff = 1
        else
          cutoff = x2.pop / 100.0
        end
      end

    end

    # if there aren't enough sequences we might have no value for some
    # of the Nx. Fill the empty ones in with the longest contig length.
    while res.length < x.length do
      res << bin.last.length
    end

    # calculate and return the statistics as a hash
    mean = cumulative_length / @assembly.size
    ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
    {
      'n_seqs' => bin.size,
      'smallest' => bin.first.length,
      'largest' => bin.last.length,
      'n_bases' => n_bases,
      'mean_len' => mean,
      'n_under_200' => n_under_200,
      'n_over_1k' => n_over_1k,
      'n_over_10k' => n_over_10k,
      'n_with_orf' => n_with_orf,
      'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
    }.merge ns

  end # basic_bin_stats

  # Calls *block* with two arguments, the contig and an array
  # of integer per-base coverage counts.
  #
  # @param bam [Bio::Db::Sam] a bam alignment of reads against this assembly
  # @param block [Block] the block to call
  def each_with_coverage(bam, &block)
    logger.debug 'enumerating assembly with coverage'
    # generate coverage with samtools
    covfile = Samtools.coverage bam
    # get an assembly enumerator
    assembly_enum = @assembly.to_enum
    contig_name, contig = assembly_enum.next
    # precreate an array of the correct size to contain
    # coverage. this is necessary because samtools mpileup
    # doesn't print a result line for bases with 0 coverage
    contig.coverage = Array.new(contig.length, 0)
    # the columns we need
    name_i, pos_i, cov_i = 0, 1, 3
    # parse the coverage file
    File.open(covfile).each_line do |line|
      cols = line.chomp.split("\t")
      unless (cols && cols.length > 4)
        # last line
        break
      end
      # extract the columns
      name = Bio::FastaDefline.new(cols[name_i]).entry_id
      pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
      unless contig_name == name
        while contig_name != name
          begin
            block.call(contig, contig.coverage)
            contig_name, contig = assembly_enum.next
            contig.coverage = Array.new(contig.length, 0)
          rescue StopIteration => stop_error
            logger.error 'reached the end of assembly enumerator while ' +
                      'there were contigs left in the coverage results'
            logger.error "final assembly contig: #{@assembly.last.name}"
            logger.error "coverage contig: #{name}"
            raise stop_error
          end
        end
      end
      contig.coverage[pos - 1] = cov
    end
    # yield the final contig
    block.call(contig, contig.coverage)
  end

end

#n_bases ⇒ Object

Returns the value of attribute n_bases.



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# File 'lib/transrate/assembly.rb', line 34

def n_bases
  @n_bases
end

#orss_ublast_db ⇒ String 



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# File 'lib/transrate/assembly.rb', line 25

class Assembly

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_value, :<<, :size, :length, :[]

  attr_accessor :file
  attr_reader :assembly
  attr_reader :has_run
  attr_accessor :n_bases
  attr_reader :n50
  attr_accessor :contig_metrics

  # Create a new Assembly.
  #
  # @param file [String] path to the assembly FASTA file
  def initialize file
    @file = File.expand_path file
    unless File.exist? @file
      raise IOError.new "Assembly file doesn't exist: #{@file}"
    end
    @assembly = {}
    @n_bases = 0
    Bio::FastaFormat.open(file).each do |entry|
      @n_bases += entry.length
      contig = Contig.new(entry)
      @assembly[contig.name] = contig
    end
    @contig_metrics = ContigMetrics.new self
  end

  # Generate and store the basic statistics for this assembly
  #
  # @param threads [Integer] number of threads to use
  def run threads=8
    stats = self.basic_stats threads
    stats.each_pair do |key, value|
      ivar = "@#{key.gsub(/\ /, '_')}".to_sym
      attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
      # creates accessors for the variables in stats
      singleton_class.class_eval { attr_accessor attr_ivar }
      self.instance_variable_set(ivar, value)
    end
    @contig_metrics.run
    @has_run = true
  end

  # Return a hash of statistics about this assembly. Stats are
  # calculated in parallel by splitting the assembly into
  # equal-sized bins and calling Assembly#basic_bin_stat on each
  # bin in a separate thread.
  #
  # @param threads [Integer] number of threads to use
  #
  # @return [Hash] basic statistics about the assembly
  def basic_stats threads=1
    return @basic_stats if @basic_stats
    bin = @assembly.values
    @basic_stats = basic_bin_stats bin
    @basic_stats
  end # basic_stats


  # Calculate basic statistics in an single thread for a bin
  # of contigs.
  #
  # Basic statistics are:
  #
  # - N10, N30, N50, N70, N90
  # - number of contigs >= 1,000 base pairs long
  # - number of contigs >= 10,000 base pairs long
  # - length of the shortest contig
  # - length of the longest contig
  # - number of contigs in the bin
  # - mean contig length
  # - total number of nucleotides in the bin
  # - mean % of contig length covered by the longest ORF
  #
  # @param [Array] bin An array of Bio::Sequence objects
  # representing contigs in the assembly

  def basic_bin_stats bin

    # cumulative length is a float so we can divide it
    # accurately later to get the mean length
    cumulative_length = 0.0

    # we'll calculate Nx for x in [10, 30, 50, 70, 90]
    # to do this we create a stack of the x values and
    # pop the first one to set the first cutoff. when
    # the cutoff is reached we store the nucleotide length and pop
    # the next value to set the next cutoff. we take a copy
    # of the Array so we can use the intact original to collect
    # the results later
    x = [90, 70, 50, 30, 10]
    x2 = x.clone
    cutoff = x2.pop / 100.0
    res = []
    n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
    # sort the contigs in ascending length order
    # and iterate over them
    bin.sort_by! { |c| c.seq.length }
    bin.each do |contig|

      # increment our long contig counters if this
      # contig is above the thresholds
      if contig.length < 200
        # ignore contigs less than 200 bases,
        # but record how many there are
        n_under_200 += 1
        next
      end
      n_over_1k += 1 if contig.length > 1_000
      n_over_10k += 1 if contig.length > 10_000

      # add the length of the longest orf to the
      # running total
      orf_length = contig.orf_length
      orf_length_sum += orf_length
      # only consider orfs that are realistic length
      # (here we set minimum amino acid length as 50)
      n_with_orf += 1 if orf_length > 149

      # increment the cumulative length and check whether the Nx
      # cutoff has been reached. if it has, store the Nx value and
      # get the next cutoff
      cumulative_length += contig.length
      if cumulative_length >= @n_bases * cutoff
        res << contig.length
        if x2.empty?
          cutoff = 1
        else
          cutoff = x2.pop / 100.0
        end
      end

    end

    # if there aren't enough sequences we might have no value for some
    # of the Nx. Fill the empty ones in with the longest contig length.
    while res.length < x.length do
      res << bin.last.length
    end

    # calculate and return the statistics as a hash
    mean = cumulative_length / @assembly.size
    ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
    {
      'n_seqs' => bin.size,
      'smallest' => bin.first.length,
      'largest' => bin.last.length,
      'n_bases' => n_bases,
      'mean_len' => mean,
      'n_under_200' => n_under_200,
      'n_over_1k' => n_over_1k,
      'n_over_10k' => n_over_10k,
      'n_with_orf' => n_with_orf,
      'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
    }.merge ns

  end # basic_bin_stats

  # Calls *block* with two arguments, the contig and an array
  # of integer per-base coverage counts.
  #
  # @param bam [Bio::Db::Sam] a bam alignment of reads against this assembly
  # @param block [Block] the block to call
  def each_with_coverage(bam, &block)
    logger.debug 'enumerating assembly with coverage'
    # generate coverage with samtools
    covfile = Samtools.coverage bam
    # get an assembly enumerator
    assembly_enum = @assembly.to_enum
    contig_name, contig = assembly_enum.next
    # precreate an array of the correct size to contain
    # coverage. this is necessary because samtools mpileup
    # doesn't print a result line for bases with 0 coverage
    contig.coverage = Array.new(contig.length, 0)
    # the columns we need
    name_i, pos_i, cov_i = 0, 1, 3
    # parse the coverage file
    File.open(covfile).each_line do |line|
      cols = line.chomp.split("\t")
      unless (cols && cols.length > 4)
        # last line
        break
      end
      # extract the columns
      name = Bio::FastaDefline.new(cols[name_i]).entry_id
      pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
      unless contig_name == name
        while contig_name != name
          begin
            block.call(contig, contig.coverage)
            contig_name, contig = assembly_enum.next
            contig.coverage = Array.new(contig.length, 0)
          rescue StopIteration => stop_error
            logger.error 'reached the end of assembly enumerator while ' +
                      'there were contigs left in the coverage results'
            logger.error "final assembly contig: #{@assembly.last.name}"
            logger.error "coverage contig: #{name}"
            raise stop_error
          end
        end
      end
      contig.coverage[pos - 1] = cov
    end
    # yield the final contig
    block.call(contig, contig.coverage)
  end

end

#ublast_db ⇒ String 



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# File 'lib/transrate/assembly.rb', line 25

class Assembly

  include Enumerable
  extend Forwardable
  def_delegators :@assembly, :each, :each_value, :<<, :size, :length, :[]

  attr_accessor :file
  attr_reader :assembly
  attr_reader :has_run
  attr_accessor :n_bases
  attr_reader :n50
  attr_accessor :contig_metrics

  # Create a new Assembly.
  #
  # @param file [String] path to the assembly FASTA file
  def initialize file
    @file = File.expand_path file
    unless File.exist? @file
      raise IOError.new "Assembly file doesn't exist: #{@file}"
    end
    @assembly = {}
    @n_bases = 0
    Bio::FastaFormat.open(file).each do |entry|
      @n_bases += entry.length
      contig = Contig.new(entry)
      @assembly[contig.name] = contig
    end
    @contig_metrics = ContigMetrics.new self
  end

  # Generate and store the basic statistics for this assembly
  #
  # @param threads [Integer] number of threads to use
  def run threads=8
    stats = self.basic_stats threads
    stats.each_pair do |key, value|
      ivar = "@#{key.gsub(/\ /, '_')}".to_sym
      attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
      # creates accessors for the variables in stats
      singleton_class.class_eval { attr_accessor attr_ivar }
      self.instance_variable_set(ivar, value)
    end
    @contig_metrics.run
    @has_run = true
  end

  # Return a hash of statistics about this assembly. Stats are
  # calculated in parallel by splitting the assembly into
  # equal-sized bins and calling Assembly#basic_bin_stat on each
  # bin in a separate thread.
  #
  # @param threads [Integer] number of threads to use
  #
  # @return [Hash] basic statistics about the assembly
  def basic_stats threads=1
    return @basic_stats if @basic_stats
    bin = @assembly.values
    @basic_stats = basic_bin_stats bin
    @basic_stats
  end # basic_stats


  # Calculate basic statistics in an single thread for a bin
  # of contigs.
  #
  # Basic statistics are:
  #
  # - N10, N30, N50, N70, N90
  # - number of contigs >= 1,000 base pairs long
  # - number of contigs >= 10,000 base pairs long
  # - length of the shortest contig
  # - length of the longest contig
  # - number of contigs in the bin
  # - mean contig length
  # - total number of nucleotides in the bin
  # - mean % of contig length covered by the longest ORF
  #
  # @param [Array] bin An array of Bio::Sequence objects
  # representing contigs in the assembly

  def basic_bin_stats bin

    # cumulative length is a float so we can divide it
    # accurately later to get the mean length
    cumulative_length = 0.0

    # we'll calculate Nx for x in [10, 30, 50, 70, 90]
    # to do this we create a stack of the x values and
    # pop the first one to set the first cutoff. when
    # the cutoff is reached we store the nucleotide length and pop
    # the next value to set the next cutoff. we take a copy
    # of the Array so we can use the intact original to collect
    # the results later
    x = [90, 70, 50, 30, 10]
    x2 = x.clone
    cutoff = x2.pop / 100.0
    res = []
    n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
    # sort the contigs in ascending length order
    # and iterate over them
    bin.sort_by! { |c| c.seq.length }
    bin.each do |contig|

      # increment our long contig counters if this
      # contig is above the thresholds
      if contig.length < 200
        # ignore contigs less than 200 bases,
        # but record how many there are
        n_under_200 += 1
        next
      end
      n_over_1k += 1 if contig.length > 1_000
      n_over_10k += 1 if contig.length > 10_000

      # add the length of the longest orf to the
      # running total
      orf_length = contig.orf_length
      orf_length_sum += orf_length
      # only consider orfs that are realistic length
      # (here we set minimum amino acid length as 50)
      n_with_orf += 1 if orf_length > 149

      # increment the cumulative length and check whether the Nx
      # cutoff has been reached. if it has, store the Nx value and
      # get the next cutoff
      cumulative_length += contig.length
      if cumulative_length >= @n_bases * cutoff
        res << contig.length
        if x2.empty?
          cutoff = 1
        else
          cutoff = x2.pop / 100.0
        end
      end

    end

    # if there aren't enough sequences we might have no value for some
    # of the Nx. Fill the empty ones in with the longest contig length.
    while res.length < x.length do
      res << bin.last.length
    end

    # calculate and return the statistics as a hash
    mean = cumulative_length / @assembly.size
    ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
    {
      'n_seqs' => bin.size,
      'smallest' => bin.first.length,
      'largest' => bin.last.length,
      'n_bases' => n_bases,
      'mean_len' => mean,
      'n_under_200' => n_under_200,
      'n_over_1k' => n_over_1k,
      'n_over_10k' => n_over_10k,
      'n_with_orf' => n_with_orf,
      'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
    }.merge ns

  end # basic_bin_stats

  # Calls *block* with two arguments, the contig and an array
  # of integer per-base coverage counts.
  #
  # @param bam [Bio::Db::Sam] a bam alignment of reads against this assembly
  # @param block [Block] the block to call
  def each_with_coverage(bam, &block)
    logger.debug 'enumerating assembly with coverage'
    # generate coverage with samtools
    covfile = Samtools.coverage bam
    # get an assembly enumerator
    assembly_enum = @assembly.to_enum
    contig_name, contig = assembly_enum.next
    # precreate an array of the correct size to contain
    # coverage. this is necessary because samtools mpileup
    # doesn't print a result line for bases with 0 coverage
    contig.coverage = Array.new(contig.length, 0)
    # the columns we need
    name_i, pos_i, cov_i = 0, 1, 3
    # parse the coverage file
    File.open(covfile).each_line do |line|
      cols = line.chomp.split("\t")
      unless (cols && cols.length > 4)
        # last line
        break
      end
      # extract the columns
      name = Bio::FastaDefline.new(cols[name_i]).entry_id
      pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
      unless contig_name == name
        while contig_name != name
          begin
            block.call(contig, contig.coverage)
            contig_name, contig = assembly_enum.next
            contig.coverage = Array.new(contig.length, 0)
          rescue StopIteration => stop_error
            logger.error 'reached the end of assembly enumerator while ' +
                      'there were contigs left in the coverage results'
            logger.error "final assembly contig: #{@assembly.last.name}"
            logger.error "coverage contig: #{name}"
            raise stop_error
          end
        end
      end
      contig.coverage[pos - 1] = cov
    end
    # yield the final contig
    block.call(contig, contig.coverage)
  end

end

Instance Method Details

#basic_bin_stats(bin) ⇒ Object

Calculate basic statistics in an single thread for a bin of contigs.

Basic statistics are:

  • N10, N30, N50, N70, N90

  • number of contigs >= 1,000 base pairs long

  • number of contigs >= 10,000 base pairs long

  • length of the shortest contig

  • length of the longest contig

  • number of contigs in the bin

  • mean contig length

  • total number of nucleotides in the bin

  • mean % of contig length covered by the longest ORF

representing contigs in the assembly



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# File 'lib/transrate/assembly.rb', line 106

def basic_bin_stats bin

  # cumulative length is a float so we can divide it
  # accurately later to get the mean length
  cumulative_length = 0.0

  # we'll calculate Nx for x in [10, 30, 50, 70, 90]
  # to do this we create a stack of the x values and
  # pop the first one to set the first cutoff. when
  # the cutoff is reached we store the nucleotide length and pop
  # the next value to set the next cutoff. we take a copy
  # of the Array so we can use the intact original to collect
  # the results later
  x = [90, 70, 50, 30, 10]
  x2 = x.clone
  cutoff = x2.pop / 100.0
  res = []
  n_under_200, n_over_1k, n_over_10k, n_with_orf, orf_length_sum = 0,0,0,0,0
  # sort the contigs in ascending length order
  # and iterate over them
  bin.sort_by! { |c| c.seq.length }
  bin.each do |contig|

    # increment our long contig counters if this
    # contig is above the thresholds
    if contig.length < 200
      # ignore contigs less than 200 bases,
      # but record how many there are
      n_under_200 += 1
      next
    end
    n_over_1k += 1 if contig.length > 1_000
    n_over_10k += 1 if contig.length > 10_000

    # add the length of the longest orf to the
    # running total
    orf_length = contig.orf_length
    orf_length_sum += orf_length
    # only consider orfs that are realistic length
    # (here we set minimum amino acid length as 50)
    n_with_orf += 1 if orf_length > 149

    # increment the cumulative length and check whether the Nx
    # cutoff has been reached. if it has, store the Nx value and
    # get the next cutoff
    cumulative_length += contig.length
    if cumulative_length >= @n_bases * cutoff
      res << contig.length
      if x2.empty?
        cutoff = 1
      else
        cutoff = x2.pop / 100.0
      end
    end

  end

  # if there aren't enough sequences we might have no value for some
  # of the Nx. Fill the empty ones in with the longest contig length.
  while res.length < x.length do
    res << bin.last.length
  end

  # calculate and return the statistics as a hash
  mean = cumulative_length / @assembly.size
  ns = Hash[x.map { |n| "n#{n}" }.zip(res)]
  {
    'n_seqs' => bin.size,
    'smallest' => bin.first.length,
    'largest' => bin.last.length,
    'n_bases' => n_bases,
    'mean_len' => mean,
    'n_under_200' => n_under_200,
    'n_over_1k' => n_over_1k,
    'n_over_10k' => n_over_10k,
    'n_with_orf' => n_with_orf,
    'mean_orf_percent' => 300 * orf_length_sum / (@assembly.size * mean)
  }.merge ns

end

#basic_stats(threads = 1) ⇒ Hash

Return a hash of statistics about this assembly. Stats are calculated in parallel by splitting the assembly into equal-sized bins and calling Assembly#basic_bin_stat on each bin in a separate thread.



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# File 'lib/transrate/assembly.rb', line 80

def basic_stats threads=1
  return @basic_stats if @basic_stats
  bin = @assembly.values
  @basic_stats = basic_bin_stats bin
  @basic_stats
end

#each_with_coverage(bam, &block) ⇒ Object

Calls block with two arguments, the contig and an array of integer per-base coverage counts.



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# File 'lib/transrate/assembly.rb', line 192

def each_with_coverage(bam, &block)
  logger.debug 'enumerating assembly with coverage'
  # generate coverage with samtools
  covfile = Samtools.coverage bam
  # get an assembly enumerator
  assembly_enum = @assembly.to_enum
  contig_name, contig = assembly_enum.next
  # precreate an array of the correct size to contain
  # coverage. this is necessary because samtools mpileup
  # doesn't print a result line for bases with 0 coverage
  contig.coverage = Array.new(contig.length, 0)
  # the columns we need
  name_i, pos_i, cov_i = 0, 1, 3
  # parse the coverage file
  File.open(covfile).each_line do |line|
    cols = line.chomp.split("\t")
    unless (cols && cols.length > 4)
      # last line
      break
    end
    # extract the columns
    name = Bio::FastaDefline.new(cols[name_i]).entry_id
    pos, cov =  cols[pos_i].to_i, cols[cov_i].to_i
    unless contig_name == name
      while contig_name != name
        begin
          block.call(contig, contig.coverage)
          contig_name, contig = assembly_enum.next
          contig.coverage = Array.new(contig.length, 0)
        rescue StopIteration => stop_error
          logger.error 'reached the end of assembly enumerator while ' +
                    'there were contigs left in the coverage results'
          logger.error "final assembly contig: #{@assembly.last.name}"
          logger.error "coverage contig: #{name}"
          raise stop_error
        end
      end
    end
    contig.coverage[pos - 1] = cov
  end
  # yield the final contig
  block.call(contig, contig.coverage)
end

#run(threads = 8) ⇒ Object

Generate and store the basic statistics for this assembly



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# File 'lib/transrate/assembly.rb', line 59

def run threads=8
  stats = self.basic_stats threads
  stats.each_pair do |key, value|
    ivar = "@#{key.gsub(/\ /, '_')}".to_sym
    attr_ivar = "#{key.gsub(/\ /, '_')}".to_sym
    # creates accessors for the variables in stats
    singleton_class.class_eval { attr_accessor attr_ivar }
    self.instance_variable_set(ivar, value)
  end
  @contig_metrics.run
  @has_run = true
end