Class: Bio::FinishM::RoundUp

Inherits:
Object
  • Object
show all
Includes:
Logging
Defined in:
lib/finishm/roundup.rb

Constant Summary collapse

DEFAULT_OPTIONS = 
{
:contig_end_length => 200,
:graph_search_leash_length => 20000,
:unscaffold_first => false,
:recoherence_kmer => 1,
:debug => false,
:gapfill_only => false,
:max_explore_nodes => 10000,
:max_gapfill_paths => 10,
:gapfill_with_max_coverage => false,
}

Instance Method Summary collapse

Methods included from Logging

#log

Instance Method Details

#add_options(optparse_object, options) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 16

def add_options(optparse_object, options)
  optparse_object.banner = "\nUsage: finishm roundup --genomes <genome1.fasta>[,<genome2.fasta>,...] --fastq-gz <reads..> --output-directory <directory>

Takes one or more genomes and tries to improve their quality by reducing the number of
scaffolds and N characters they contain.

Example:

finishm roundup --genomes genome1.fasta,genome2.fasta --fastq-gz reads.1.fq.gz,reads.2.fq.gz --output-directory finishm_roundup_results

That will create a collapsed de-Bruijn graph from reads.1.fq.gz and reads.2.fq.gz, then try to find connections between
the starts and the ends of the contigs in genome1.fasta. If any connections between contigs are mutually exclusive,
then they are incorporated into scaffolds together, and gapfilling is attempted. The final sequences are output in
the finishm_roundup_results directory in FASTA format. The procedure is then repeated for genome2.fasta.

\n\n"

  options.merge!(DEFAULT_OPTIONS)

  optparse_object.separator "\nRequired arguments:\n\n"
  optparse_object.on("--genomes FASTA_1[,FASTA_2...]", Array, "fasta files of genomes to be improved [required]") do |arg|
    options[:assembly_files] = arg
  end
  optparse_object.on("--output-directory PATH", "Output results to this directory [required]") do |arg|
    options[:output_directory] = arg
  end

  optparse_object.separator "\nThere must be some definition of reads too:\n\n" #TODO improve this help
  Bio::FinishM::ReadInput.new.add_options(optparse_object, options)

  optparse_object.separator "\nOptional arguments:\n\n"
  optparse_object.on("--overhang NUM", Integer, "Start assembling this far from the ends of the contigs [default: #{options[:contig_end_length] }]") do |arg|
    options[:contig_end_length] = arg.to_i
  end
  optparse_object.on("--recoherence-kmer NUM", Integer, "Use a kmer longer than the original velvet one, to help remove bubbles and circular paths [default: none]") do |arg|
    options[:recoherence_kmer] = arg
  end
  optparse_object.on("--leash-length NUM", Integer, "Don't explore too far in the graph, only this far and not much more [default: #{options[:graph_search_leash_length] }]") do |arg|
    options[:graph_search_leash_length] = arg
  end
  optparse_object.on("--unscaffold-first", "Break the scaffolds in the contigs file apart, and then wander between the resultant contigs. This option is only relevant to the wander step; gapfilling is attempted on all gaps by default. [default: #{options[:unscaffold_first] }]") do
    options[:unscaffold_first] = true
  end
  optparse_object.on("--gapfill-only", "Don't wander, just gapfill [default: #{options[:gapfill_only] }]") do
    options[:gapfill_only] = true
  end
  optparse_object.on("--max-gapfill-paths NUM", Integer, "When this number of paths is exceeded, don't gapfill, print as Ns [default: #{options[:max_gapfill_paths] }]") do |arg|
    options[:max_gapfill_paths] = arg
  end
  optparse_object.on("--max-explore-nodes NUM", Integer, "Only explore this many nodes. If max is reached, do not make connections. [default: #{options[:max_explore_nodes] }]") do |arg|
    options[:max_explore_nodes] = arg
  end
  optparse_object.on("--gapfill-with-max-coverage", "When gapfilling, take the path with maximal coverage and do not print variants [default: #{options[:gapfill_with_max_coverage] }]") do
    options[:gapfill_with_max_coverage] = true
  end
  optparse_object.on("--debug", "Build the graph, then drop to a pry console. [default: #{options[:debug] }]") do
    options[:debug] = true
  end
  optparse_object.on("--probe NUM",Integer,"debug mode: explore from this probe number (1-based index), gapfill only, no wander. [default: explore from all probes}]") do |arg|
    options[:interesting_probes] = [arg-1] #convert to 0-based index
    options[:gapfill_only] = true
  end
  #optparse_object.on("--proceed-on-short-contigs", "By default, when overly short contigs are encountered, finishm croaks. This option stops the croaking [default: #{options[:proceed_on_short_contigs] }]") do
  #  options[:proceed_on_short_contigs] = true
  #end

  Bio::FinishM::GraphGenerator.new.add_options optparse_object, options
end

#gapfill_a_scaffold(gapfiller, printer, master_graph, genome, scaffold_index, scaffold_direction, superscaffold_name, report, variants_file, options) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 295

def gapfill_a_scaffold(gapfiller, printer, master_graph, genome, scaffold_index, scaffold_direction, superscaffold_name, report, variants_file, options)
  connections = []
  genome.each_gap_probe_pair(scaffold_index) do |probe1, probe2|
    log.info "Gapfilling between probes #{probe1.number+1} and #{probe2.number+1}.."
    next unless options[:interesting_probes].nil? or
    options[:interesting_probes].include?(probe1.number) or
    options[:interesting_probes].include?(probe2.number)
    connections.push gapfiller.gapfill(master_graph, probe1.index, probe2.index, options)
  end
  log.debug "Found #{connections.length} connections gapfilling in scaffold #{scaffold_index}" if log.debug?

  all_variants = []
  num_gapfills = 0
  scaffold = genome.scaffolds[scaffold_index]
  gapfilled_sequence = genome.scaffolds[scaffold_index].contigs[0].sequence
  connections.each_with_index do |aconn, i|
    rhs_sequence = scaffold.contigs[i+1].sequence
    gapfilled_sequence, variants, gapfilled = piece_together_gapfill(
      printer, master_graph, gapfilled_sequence, aconn, rhs_sequence, genome.gap_length(scaffold_index, i),
      options[:max_gapfill_paths]
      )
    if gapfilled
      num_gapfills += 1
      variants.each{|v| all_variants << v}
      to_log = "Filled a gap on genome #{genome.filename}: scaffold #{scaffold.name}: #{scaffold.contigs[i].scaffold_position_end+1}-#{scaffold.contigs[i+1].scaffold_position_start-1}"
      report.puts to_log
      log.info to_log
    end
  end
  if scaffold_direction == false
    gapfilled_sequence = revcom(gapfilled_sequence)
    all_variants.each do |variant|
      variant.position = gapfilled_sequence.length - variant.position
      variant.reverse!
    end
  end
  all_variants.each do |variant|
    variant.reference_name = superscaffold_name
    variants_file.puts variant.vcf(gapfilled_sequence)
  end
  return gapfilled_sequence, num_gapfills, all_variants
end

#piece_together_gapfill(printer, master_graph, first_sequence, aconn, second_sequence, gap_length, max_gapfill_paths, options = {}) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 338

def piece_together_gapfill(printer, master_graph, first_sequence, aconn, second_sequence, gap_length, max_gapfill_paths, options={})
  scaffold_sequence = nil
  gapfilled = -1
  if aconn.paths.length != 0 and aconn.paths.length <= max_gapfill_paths
    aconn.collapse_paths_to_maximal_coverage_path! if options[:gapfill_with_max_coverage]
    scaffold_sequence, variants = printer.ready_two_contigs_and_connections(
      master_graph.graph, first_sequence, aconn, second_sequence, master_graph.velvet_sequences
      )
    if !scaffold_sequence.nil? #sometimes it is just impossible even if there is paths
      scaffold_sequence.gsub!('-','') #remove gaps i.e. where the consensus is a gap
      gapfilled = true
    end
  end
  if gapfilled != true
    # No paths found, or gapfilling failed at the final step Just fill with Ns like it was before
    scaffold_sequence = first_sequence + 'N'*gap_length + second_sequence
    gapfilled = false
  end

  return scaffold_sequence, variants, gapfilled
end

#revcom(seq) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 360

def revcom(seq)
  Bio::Sequence::NA.new(seq).reverse_complement.to_s.upcase
end

#run(options, argv = []) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 105

def run(options, argv=[])
  # Make sure output directory is writeable to avoid late croaking
  output_directory = setup_output_directory options[:output_directory]

  # Gather the probes from each genome supplied
  genomes = Bio::FinishM::InputGenome.parse_genome_fasta_files(
    options[:assembly_files],
    options[:contig_end_length],
    options
    )

  # Generate one velvet assembly to rule them all (forging the assembly is hard work..)
  probe_sequences = genomes.collect{|genome| genome.probe_sequences}.flatten
  # Generate the graph with the probe sequences in it.
  read_input = Bio::FinishM::ReadInput.new
  read_input.parse_options options
  master_graph = Bio::FinishM::GraphGenerator.new.generate_graph(probe_sequences, read_input, options)

  binding.pry if options[:debug]

  # For each genome, wander, gapfill, then output
  wanderer = Bio::FinishM::Wanderer.new
  gapfiller = Bio::FinishM::GapFiller.new
  printer = Bio::AssemblyGraphAlgorithms::ContigPrinter.new
  genomes.each do |genome|
    # wander using just the probes on the ends of the scaffolds
    connected_scaffolds = nil
    all_connections = []
    gaps_filled_in_genome = 0
    wandered_probe_indices = nil

    File.open(File.join(output_directory, File.basename(genome.filename)+".report.txt"),'w') do |report|
      report.puts "#{Time.now} FinishM report for roundup run with: #{options.inspect}"

      if options[:gapfill_only]
        log.info "Skipping wander, gapfilling only"
        connected_scaffolds = Bio::FinishM::ConnectionInterpreter.new([], (0...genome.scaffolds.length)).scaffolds([])
      else
        log.debug "Wandering.."
        connected_scaffolds, all_connections, wandered_probe_indices = wander_a_genome(wanderer, genome, master_graph, options, report)
      end

      # Write out all the connections
      File.open(File.join(output_directory, File.basename(genome.filename)+".connections.csv"),'w') do |con_file|
        all_connections.each do |connection|
          con_file.puts connection
        end
      end

      output_path = File.join(output_directory, File.basename(genome.filename)+".scaffolds.fasta")
      variants_path = File.join(output_directory, File.basename(genome.filename)+".at_least_half_completely_wrong.vcf")
      num_circular_scaffolds = 0

      File.open(output_path, 'w') do |output_file|
        File.open(variants_path,'w') do |variants_file|
          variants_file.puts %w(#CHROM POS ID REF ALT QUAL FILTER INFO).join("\t")
          # gapfill between
          # (1) interpreted_connections
          # (2) gaps that were present before above wander
          connected_scaffolds.each_with_index do |cross_scaffold_connection, connected_scaffold_index|
            superscaffold_name = "scaffold#{connected_scaffold_index+1}"

            pretend_contig = cross_scaffold_connection.contigs[0]
            first_scaffold_index = pretend_contig.sequence_index

            # Gapfill contigs within the scaffold on the extreme LHS
            scaffold_sequence, num_gaps, variants = gapfill_a_scaffold(gapfiller, printer, master_graph, genome, first_scaffold_index, pretend_contig.direction, superscaffold_name, report, variants_file, options)
            gaps_filled_in_genome += num_gaps

            last_contig = nil
            cross_scaffold_connection.contigs.each_with_index do |contig, superscaffold_index|
              unless last_contig.nil? #skip the first contig - it be done
                last_name = genome.scaffolds[last_contig.sequence_index].name
                current_name = genome.scaffolds[contig.sequence_index].name
                log.debug "Connecting #{last_name} and #{current_name}" if log.debug?

                # Ready the contig on the RHS of this join
                # Gapfill within the scaffold on the RHS of the new gap
                rhs_sequence, num_gaps, variants = gapfill_a_scaffold(gapfiller, printer, master_graph, genome, contig.sequence_index, contig.direction, superscaffold_name, report, variants_file, options)
                gaps_filled_in_genome += num_gaps

                # Gapfill across the new gap between scaffolds
                aconn = gapfiller.gapfill(master_graph,
                  last_contig.direction == true ? genome.last_probe(last_contig.sequence_index).index : genome.first_probe(last_contig.sequence_index).index,
                  contig.direction == true ? genome.first_probe(contig.sequence_index).index : genome.last_probe(contig.sequence_index).index,
                  options
                  )
                second_sequence = genome.scaffolds[contig.sequence_index].contigs[0].sequence
                log.debug "Found #{aconn.paths.length} connections between #{last_name} and #{current_name}" if log.debug?
                connected = false
                if aconn.paths.length > 0
                  aconn.collapse_paths_to_maximal_coverage_path! if options[:gapfill_with_max_coverage]
                  scaffold_sequence2, variants = printer.ready_two_contigs_and_connections(
                    master_graph.graph,
                    scaffold_sequence,
                    aconn,
                    rhs_sequence,
                    master_graph.velvet_sequences
                    )
                  if !scaffold_sequence2.nil?
                    scaffold_sequence = scaffold_sequence2
                    connected = true
                    # Print variants
                    # TODO: need to change coordinates of variants, particularly when >2 contigs are joined?
                    variants.each do |variant|
                      variant.reference_name = superscaffold_name
                      variants_file.puts variant.vcf(scaffold_sequence)
                    end
                  end
                end
                if !connected
                  # when this occurs, it is due to there being a circuit in the path, so no paths are printed.
                  # (at least for now) TODO: this could be improved.
                  # Just arbitrarily put in 100 N characters, to denote a join, but no gapfill

                  # (or, it could be impossible to join because of low coverage resulting in inability to get sequence from the node trail)
                  scaffold_sequence = scaffold_sequence+('N'*100)+rhs_sequence
                end
              end
              last_contig = contig
            end

            #Output the scaffold to the output directory
            descriptor = nil
            if cross_scaffold_connection.circular?
              descriptor = 'circular'
              num_circular_scaffolds += 1
            else
              descriptor = 'scaffold'
            end
            scaffold_names = cross_scaffold_connection.contigs.collect do |contig|
              genome.scaffolds[contig.sequence_index].name
            end
            output_file.puts ">#{superscaffold_name} #{descriptor} #{scaffold_names.join(':') }"
            scaffold_sequence.gsub! '-', '' #remove dashes since these make things fail downstream
            output_file.puts scaffold_sequence
          end

          num_connected_scaffolds = genome.scaffolds.length - connected_scaffolds.length
          log.info "Wrote #{connected_scaffolds.length} scaffolds to #{output_path}, after scaffolding #{num_connected_scaffolds} scaffolds together (#{num_circular_scaffolds} were circular) and filling #{gaps_filled_in_genome} gaps."
        end
      end
    end
  end
end

#setup_output_directory(given_directory) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 251

def setup_output_directory(given_directory)
  output_directory = File.absolute_path(given_directory)
  log.debug "Using output directory: #{output_directory}" if log.debug?

  if File.exist?(output_directory)
    if !File.directory?(output_directory)
      log.error "Specified --output-directory #{output_directory} exists but is a file and not a directory. Cannot continue."
      exit 1
    elsif !File.writable?(output_directory)
      log.error "Specified --output-directory #{output_directory} is not writeable. Cannot continue."
      exit 1
    else
      log.debug "Already existing output directory #{output_directory} seems usable"
    end
  else
    # Creating a new output directory
    Dir.mkdir(output_directory)
  end

  return output_directory
end

#validate_options(options, argv) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 85

def validate_options(options, argv)
  #TODO: give a better description of the error that has occurred
  #TODO: require reads options
  if argv.length != 0
    return "Dangling argument(s) found e.g. #{argv[0] }"
  else
    [
      :assembly_files,
      :output_directory,
      ].each do |sym|
        if options[sym].nil?
          return "No option found to specify #{sym}."
        end
      end

    #if return nil from here, options all were parsed successfully
    return Bio::FinishM::ReadInput.new.validate_options(options, [])
  end
end

#wander_a_genome(wanderer, genome, master_probed_graph, options, report) ⇒ Object 



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# File 'lib/finishm/roundup.rb', line 273

def wander_a_genome(wanderer, genome, master_probed_graph, options, report)
  # Create new finishm_graph with only probes from the ends of the scaffolds of this genome
  probe_indices = []
  genome.each_scaffold_end_numbered_probe{|probe| probe_indices.push(probe.number)}
  genome_graph = master_probed_graph.subgraph(probe_indices)

  num_scaffolds = genome.scaffolds.length

  all_connections = wanderer.probed_graph_to_connections(genome_graph, options)

  interpreter = Bio::FinishM::ConnectionInterpreter.new(all_connections, (0...num_scaffolds))
  connections = interpreter.doubly_single_contig_connections
  report.puts "Found #{connections.length} connections between contigs that can be used for scaffolding"
  unconnected_probes = interpreter.unconnected_probes
  report.puts "Found #{unconnected_probes.length} contig ends that did not connect to any others"
  unconnected_probes.each do |probe|
    report.puts "Did not connect to any other probes: #{probe.inspect}"
  end

  return interpreter.scaffolds(connections), all_connections, probe_indices
end