Class: Bio::FinishM::Primers::Checker

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

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/primers_check.rb', line 14

def add_options(optparse_object, options)
  optparse_object.banner = "\nUsage: finishm primers_check --primers <primers_file> [options]

Check that each pair of primers in the primers fil is compatible.
\n\n"

  options.merge!({
    :logger => 'stderr',
    :logger_trace_level => 'info',
    :melting_temperature_optimum => 56,
    :melting_temperature_tolerance => 2,
    :extra_global_primer3_options => {
      'PRIMER_MAX_POLY_X' => 4,
      'PRIMER_EXPLAIN_FLAG' => '1',
      },
    :persevere => false,
    })

  optparse_object.separator "Required arguments:\n\n"
  optparse_object.on("-p", "--primers PRIMERS_FILE", String, "A file of primers, newline separated [required]") do |arg|
    options[:primers_file] = arg
  end

  optparse_object.separator "\nOptional arguments:\n\n"
  optparse_object.on("--contig-universe FASTA_FILE", String, "All contigs in the mixture [default: unspecified (don't test this)]") do |arg|
    options[:contig_universe] = arg
  end
  optparse_object.on("--optimum-melting-temperature TEMPERATURE", Integer, "Primers aim for this melting temperature [default: default in primer3 (currently #{options[:melting_temperature_optimum]}C)]") do |arg|
    options[:melting_temperature_optimum] = arg.to_i
    raise Exception, " has to be greater than 0, found #{arg}" unless options[:melting_temperature_optimum] > 0
  end
  optparse_object.on("--primer3-options OPTION_LIST", "Give extra instructions to Primer3 [default <none>]. Acceptable values can be found in the primer3 manual e.g. 'PRIMER_MAX_POLY_X=4;PRIMER_MAX_SIZE=22' will specify those 2 parameters to primer3. Argument names are auto-capitalised so 'primer_max_poly_X=4;primer_max_size=22'is equivalent.") do |arg|
    options[:extra_global_primer3_options] = {}
    arg.split(';').each do |a2|
      splits = a2.split('=')
      unless splits.length == 2
        raise "Unexpected format of the --primer3-options flag, specifically couldn't parse this part: '#{a2}'"
      end
      options[:extra_global_primer3_options][splits[0].upcase]=splits[1]
    end
  end
end

#check_contig_universe(primers, contig_universe) ⇒ Object

Does any primer pairs amplify anything in a large set of background (universe) contigs? They shouldn’t.



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# File 'lib/finishm/primers_check.rb', line 156

def check_contig_universe(primers, contig_universe)
  num_hits = 0
  num_compared = 0

  primers.to_a.combination(2) do |array|
    primer1 = array[0]
    primer2 = array[1]
    num_compared += 1

    primer_set = Bio::Ipcress::PrimerSet.new primer1, primer2
    results = Bio::Ipcress.run primer_set, options[:contig_universe], ipcress_options

    if results.length > 0
      num_hits += 1
      log.warn "Found #{results.length} matches between #{primer1} and #{primer2} in the contig universe, expected none."
    end
  end

  log.debug "Tested #{num_compared} pairs of primers to see if anything else was hit, warnings above if any did so"
end

#check_primer_compatibilities(primers, primer3_options) ⇒ Object

Each pair of primers should be compatible



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# File 'lib/finishm/primers_check.rb', line 130

def check_primer_compatibilities(primers, primer3_options)
  # First just make sure that everything is ok here
  log.info "Double checking to make sure there is no incompatibilities between primer pairs"
  num_compared = 0
  primers.to_a.combination(2) do |array|
    primer1 = array[0]
    primer2 = array[1]

    compatible, result = Bio::Primer3.test_primer_compatibility(primer1, primer2, primer3_options, :return_result => true)
    num_compared += 1
    log.debug "Primer3 returned: #{result.inspect}" if log.debug?

    if compatible == false
      log.warn "Found an incompatibility between #{primer1} and #{primer2}"
      log.warn "Primer3 output was: #{result.inspect}"
      return false
    end
  end
  return num_compared
end

#run(options, argv) ⇒ Object 



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# File 'lib/finishm/primers_check.rb', line 72

def run(options, argv)
  Bio::Log::CLI.configure('bio-primer3')
  found_error = false

  # Read the primers in
  primers = []
  File.foreach(options[:primers_file]) do |line|
    line.strip!
    next if line.empty?
    unless line.match(/^[atgc]+$/i)
      raise "Malformed primer sequence '#{line}' found in the file - I'm just after one primer per line, that's all."
    end
    primers.push line
  end
  log.info "Read in #{primers.length} primers e.g. #{primers[0] }"
  raise "Need at least 2 primers!" unless primers.length >= 2


  primer3_options = options[:extra_global_primer3_options]
  primer3_options.merge!({
    'PRIMER_OPT_TM' => options[:melting_temperature_optimum],
    'PRIMER_MIN_TM' => options[:melting_temperature_optimum]-options[:melting_temperature_tolerance],
    'PRIMER_MAX_TM' => options[:melting_temperature_optimum]+options[:melting_temperature_tolerance],
    })

  # Check to make sure there is no incompatibilities primer vs primer:
  num_compared = check_primer_compatibilities(primers, primer3_options)
  if num_compared == false
    log.error "Found at least one incompatible primer set, giving up."
    exit 1
  end
  log.info "Validated #{num_compared} different pairs of primers, they don't seem to conflict with each other at all, according to primer3's check primers thing"


  # Check the contig universe
  if options[:contig_universe]
    log.info "in-silico PCR: testing the contig universe. This could probably be sped up by only making a single call to ipcress, but oh well."
    num_universe_hits = check_contig_universe(primers, options[:contig_universe])
    if num_universe_hits > 0
      log.warn "Found #{results.length} matches between #{primer1} and #{primer2} in the contig universe, expected none."
    else
      log.info "No sequence appears to be amplified with any two primers in the background set of contigs (this is a good thing)."
    end
  else
    log.info "Not checking to see if primers match any other contigs not targeted are picked up by these primers, because no universe of contigs was specified."
  end


  if found_error
    log.warn "At least one problem detected in the primer checking process"
  else
    log.warn "No primer issues detected"
  end
end

#validate_options(options, argv) ⇒ Object 



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# File 'lib/finishm/primers_check.rb', line 57

def validate_options(options, argv)
  if argv.length != 0
    return "Dangling argument(s) found e.g. #{argv[0] }"
  else
    [
      :primers_file,
      ].each do |sym|
        if options[sym].nil?
          return "No option found to specify #{sym}."
        end
      end
    return nil
  end
end