miga-base 0.7.26.0 → 1.0.0.1

data/utils/enveomics/Scripts/aai.rb

@@ -0,0 +1,419 @@
+#!/usr/bin/env ruby
+
+# @author  Luis M. Rodriguez-R
+# @license Artistic-2.0
+
+require 'optparse'
+require 'tmpdir'
+require 'zlib'
+has_rest_client = true
+has_sqlite3 = true
+begin
+  require 'rubygems'
+  require 'restclient'
+rescue LoadError
+  has_rest_client = false
+end
+begin
+  require 'sqlite3'
+rescue LoadError
+  has_sqlite3 = false
+end
+
+o = {
+  bits: 0, id: 20, len: 0, hits: 50, q: false, bin: '', program: 'blast+',
+  thr: 1, dec: 2, auto: false, lookupfirst: false, dbrbm: true, nucl: false,
+  len_fraction: 0.0, max_actg: 0.95
+}
+ARGV << '-h' if ARGV.size == 0
+OptionParser.new do |opts|
+  opts.banner = "
+Calculates the Average Amino Acid Identity between two genomes
+
+Usage: #{$0} [options]"
+  opts.separator ''
+  opts.separator 'Mandatory'
+  opts.on(
+    '-1', '--seq1 FILE',
+    'Path to the FastA file (.gz allowed) containing the genome 1 (proteins)'
+  ) { |v| o[:seq1] = v }
+  opts.on(
+    '-2', '--seq2 FILE',
+    'Path to the FastA file (.gz allowed) containing the genome 2 (proteins)'
+  ) { |v| o[:seq2] = v }
+  if has_rest_client
+    opts.separator '    Alternatively, you can supply the NCBI-acc of a ' +
+      'genome (nucleotides) with the format ncbi:CP014272 instead of files'
+  else
+    opts.separator '    Install rest-client to enable NCBI-acc support'
+  end
+  opts.separator ''
+  opts.separator 'Search Options'
+  opts.on(
+    '-l', '--len INT', Integer,
+    "Minimum alignment length (in residues).  By default: #{o[:len]}"
+  ) { |v| o[:len] = v }
+  opts.on(
+    '-L', '--len-fraction NUM', Float,
+    'Minimum alignment length as a fraction of the shorter sequence',
+    "(range 0-1).  By default: #{o[:len_fraction]}"
+  ) { |v| o[:len_fraction] = v }
+  opts.on(
+    '-i', '--id FLOAT', Float,
+    "Minimum alignment identity (in %).  By default: #{o[:id]}"
+  ) { |v| o[:id] = v }
+  opts.on(
+    '-s', '--bitscore FLOAT', Float,
+    "Minimum bit score (in bits).  By default: #{o[:bits]}"
+  ) { |v| o[:bits] = v }
+  opts.on(
+    '-n', '--hits INT', Integer,
+    "Minimum number of hits.  By default: #{o[:hits]}"
+  ) { |v| o[:hits] = v }
+  opts.on(
+    '-N', '--nucl',
+    'The input sequences are nucleotides (genes), not proteins'
+  ) { |v| o[:nucl] = v }
+  opts.on(
+    '--max-actg FLOAT', Float,
+    'Maximum fraction of ACTGN in the sequences before assuming nucleotides',
+    "By default: #{o[:max_actg]}"
+  ) { |v| o[:max_actg] = v }
+  opts.separator ''
+  opts.separator 'Software Options'
+  opts.on(
+    '-b', '--bin DIR',
+    'Path to the directory containing the binaries of the search program'
+  ) { |v| o[:bin] = v }
+  opts.on(
+    '-p', '--program STR',
+    'Search program to be used. One of: blast+ (default), blast, blat, diamond'
+  ) { |v| o[:program] = v }
+  opts.on(
+    '-t', '--threads INT', Integer,
+    "Number of parallel threads to be used.  By default: #{o[:thr]}"
+  ) { |v| o[:thr] = v }
+  opts.separator ''
+  opts.separator 'SQLite3 Options'
+  unless has_sqlite3
+    opts.separator '    Install sqlite3 gem to enable database support'
+  end
+  opts.on(
+    '-S', '--sqlite3 FILE',
+    'Path to the SQLite3 database to create (or update) with the results'
+  ) { |v| o[:sqlite3] = v }
+  opts.on(
+    '--name1 STR',
+    'Name of --seq1 to use in --sqlite3.  By default determined by filename'
+  ) { |v| o[:seq1name] = v }
+  opts.on(
+    '--name2 STR',
+    'Name of --seq2 to use in --sqlite3.  By default determined by filename'
+  ) { |v| o[:seq2name] = v }
+  opts.on(
+    '--[no-]save-rbm',
+    'Save (or don\'t save) the reciprocal best matches in the --sqlite3 db',
+    "By default: #{o[:dbrbm]}"
+  ) { |v| o[:dbrbm] = v }
+  opts.on(
+    '--lookup-first',
+    'Indicates if the AAI should be looked up first in the database',
+    'Requires --sqlite3, --auto, --name1, and --name2',
+    'Incompatible with --res, --tab, --out, and --rbm'
+  ) { |v| o[:lookupfirst] = v }
+  opts.separator ''
+  opts.separator 'Other Output Options'
+  opts.on(
+    '-d', '--dec INT', Integer,
+    "Decimal positions to report. By default: #{o[:dec]}"
+  ) { |v| o[:dec] = v }
+  opts.on(
+    '-R', '--rbm FILE',
+    'Saves a file with the reciprocal best matches'
+  ) { |v| o[:rbm] = v }
+  opts.on(
+    '-o', '--out FILE',
+    'Saves a file describing the alignments used for two-way AAI'
+  ) { |v| o[:out] = v }
+  opts.on(
+    '-r', '--res FILE', 'Saves a file with the final results'
+  ) { |v| o[:res] = v }
+  opts.on(
+    '-T', '--tab FILE',
+    'Saves a file with the final two-way results in a tab-delimited form',
+    'The columns are (in that order):',
+    'AAI, standard deviation, proteins used, proteins in the smallest genome'
+  ) { |v| o[:tab] = v }
+  opts.on(
+    '-a', '--auto',
+    'ONLY outputs the AAI value in STDOUT (or nothing, if calculation fails)'
+  ) { o[:auto] = true }
+  opts.on('-q', '--quiet', 'Run quietly (no STDERR output)') { o[:q] = true }
+  opts.on('-h', '--help', 'Display this screen') do
+    puts opts
+    exit
+  end
+  opts.separator ''
+end.parse!
+
+# Check input
+abort '-1 is mandatory' if o[:seq1].nil?
+abort '-2 is mandatory' if o[:seq2].nil?
+if o[:program] == 'diamond' && o[:nucl]
+  abort '-p diamond is incompatible with -N'
+end
+unless o[:sqlite3].nil? or has_sqlite3
+  abort 'SQLite3 requested (-S) but sqlite3 not supported: gem install sqlite3'
+end
+o[:bin] = o[:bin] + '/' if o[:bin].size > 0
+if o[:lookupfirst]
+  abort '--lookup-first requires --name1' if o[:seq1name].nil?
+  abort '--lookup-first requires --name2' if o[:seq2name].nil?
+  abort '--lookup-first needs --sqlite3' if o[:sqlite3].nil?
+  abort '--lookup-first requires --auto' unless o[:auto]
+  %w[res tab out rbm].each do |k|
+    abort "--lookup-first conflicts with --#{k}" unless o[k.to_sym].nil?
+  end
+end
+
+# Create SQLite3 file
+unless o[:sqlite3].nil?
+  $stderr.puts "Accessing SQLite3 file: #{o[:sqlite3]}." unless o[:q]
+  sqlite_db = SQLite3::Database.new o[:sqlite3]
+  sqlite_db.execute "create table if not exists rbm( seq1 varchar(256), " +
+    "seq2 varchar(256), id1 varchar(256), id2 varchar(256), id float, " +
+    "evalue float, bitscore float )"
+  sqlite_db.execute "create table if not exists aai( seq1 varchar(256), " +
+    "seq2 varchar(256), aai float, sd float, n int, omega int )"
+end
+
+# Look-up first
+if o[:lookupfirst]
+  val = sqlite_db.execute "select aai from aai where seq1=? and seq2=?",
+    [o[:seq1name], o[:seq2name]]
+  val = sqlite_db.execute "select aai from aai where seq1=? and seq2=?",
+    [o[:seq2name], o[:seq1name]] if val.empty?
+  unless val.empty?
+    puts val.first.first
+    exit
+  end
+end
+
+Dir.mktmpdir do |dir|
+  $stderr.puts "Temporal directory: #{dir}." unless o[:q]
+
+  # Create databases.
+  $stderr.puts "Creating databases." unless o[:q]
+  minfrg = nil
+  seq_names = []
+  seq_len = {}
+  actg_cnt = {}
+  ori_ids = {}
+  [:seq1, :seq2].each do |seq|
+    abort "GIs are no longer supported by NCBI. Please use NCBI-acc instead." if
+      /^gi:/.match(o[seq])
+    acc = /^ncbi:(\S+)/.match(o[seq])
+    unless acc.nil?
+      abort "NCBI-acc requested, but rest-client not supported.  First " +
+        "install gem rest-client." unless has_rest_client
+      abort "NCBI-acc are currently not supported with --nucl. Please use " +
+        "ani.rb instead." if o[:nucl]
+      $stderr.puts "  Downloading dataset from NCBI:#{acc[1]}." unless o[:q]
+      responseLink = RestClient.get(
+        "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi",
+        {params:{db:"protein",dbfrom:"nuccore",id:acc[1],idtype:"acc"}})
+      abort "Unable to reach NCBI EUtils, error code " +
+        responseLink.code.to_s + "." unless responseLink.code == 200
+      fromId = true
+      protIds = []
+      o[seq] = "#{dir}/ncbi-#{seq.to_s}.fa"
+      fo = File.open(o[seq], "w")
+      responseLink.to_str.each_line.grep(/\s<Id>/) do |ln|
+        idMatch = /<Id>(\S+)<\/Id>/.match(ln)
+        unless idMatch.nil?
+          protIds.push(idMatch[1]) unless fromId
+          fromId = false
+        end
+      end
+      response = RestClient.post(
+        'https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi',
+        db: 'protein', rettype: 'fasta', id: protIds.join(','), idtype: 'acc'
+      )
+      abort "Unable to reach NCBI EUtils, error code " +
+        response.code.to_s + "." unless response.code == 200
+      fo.puts response.to_str
+      fo.close
+      seq_names << ( o[ "#{seq}name".to_sym ].nil? ?
+        "ncbi:#{acc[1]}" :
+        o[ "#{seq}name".to_sym ])
+    else
+      seq_names << ( o[ "#{seq}name".to_sym ].nil? ?
+        File.basename(o[seq], ".*") :
+        o[ "#{seq}name".to_sym ])
+    end
+    $stderr.puts "  Reading FastA file: #{o[seq]}" unless o[:q]
+    unless o[:sqlite3].nil?
+      sqlite_db.execute "delete from rbm where seq1=? and seq2=?", seq_names
+      sqlite_db.execute "delete from aai where seq1=? and seq2=?", seq_names
+    end
+    ori_ids[seq] = [nil]
+    seq_len[seq] = [0]
+    actg_cnt[seq] = 0
+    seqs = 0
+    fi = File.extname(o[seq]) == '.gz' ?
+      Zlib::GzipReader.open(o[seq]) :
+      File.open(o[seq], 'r')
+    File.open("#{dir}/#{seq.to_s}.fa", 'w') do |fo|
+      fi.each_line do |ln|
+        if ln =~ /^>(\S+)/
+          seqs += 1
+          ori_ids[seq] << $1 unless o[:rbm].nil? and o[:sqlite3].nil?
+          seq_len[seq][seqs] = 0
+          fo.puts ">#{seqs}"
+        else
+          fo.puts ln
+          seq_len[seq][seqs] += ln.chomp.gsub(/[^A-Za-z]/,"").length
+          actg_cnt[seq] += ln.chomp.gsub(/[^ACTGNactgn]/,"").length
+        end
+      end
+    end
+    fi.close
+    unless o[:nucl]
+      actg_frx = actg_cnt[seq].to_f/seq_len[seq].inject(:+).to_f
+      abort "Input sequences appear to be nucleotides " +
+        "(ACTGN fraction: %.2f%%)." % (actg_frx*100) if actg_frx > o[:max_actg]
+    end
+    $stderr.puts "    File contains #{seqs} sequences." unless o[:q]
+    minfrg ||= seqs
+    minfrg = seqs if minfrg > seqs
+    case o[:program].downcase
+    when "blast"
+      `"#{o[:bin]}formatdb" -i "#{dir}/#{seq}.fa" \
+      -p #{o[:nucl] ? "F" : "T"}`
+    when "blast+"
+      `"#{o[:bin]}makeblastdb" -in "#{dir}/#{seq}.fa" \
+      -dbtype #{o[:nucl] ? "nucl" : "prot"}`
+    when "blat"
+      # Nothing to do
+    when "diamond"
+      `"#{o[:bin]}diamond" makedb --in "#{dir}/#{seq}.fa" \
+      --db "#{dir}/#{seq}.fa.dmnd" --threads "#{o[:thr]}" \
+      --quiet`
+    else
+      abort "Unsupported program: #{o[:program]}."
+    end
+  end
+
+  # Best-hits.
+  $stderr.puts "Running one-way comparisons." unless o[:q]
+  rbh = []
+  id2 = 0
+  sq2 = 0
+  n2  = 0
+  unless o[:out].nil?
+    fo = File.open(o[:out], "w")
+    fo.puts %w(identity aln.len mismatch gap.open evalue bitscore).join("\t")
+  end
+  res = File.open(o[:res], "w") unless o[:res].nil?
+  rbm = File.open(o[:rbm], "w") unless o[:rbm].nil?
+  [1,2].each do |i|
+    qry_seen = []
+    q = "#{dir}/seq#{i}.fa"
+    s = "#{dir}/seq#{i==1?2:1}.fa"
+    case o[:program].downcase
+    when "blast"
+      `"#{o[:bin]}blastall" -p blast#{o[:nucl] ? "n": "p"} -d "#{s}" \
+      -i "#{q}" -v 1 -b 1 -a #{o[:thr]} -m 8 -o "#{dir}/#{i}.tab"`
+    when "blast+"
+      `"#{o[:bin]}blast#{o[:nucl] ? "n" : "p"}" -db "#{s}" -query "#{q}" \
+      -max_target_seqs 1 -num_threads #{o[:thr]} -outfmt 6 \
+      -out "#{dir}/#{i}.tab"`
+    when "blat"
+      `"#{o[:bin]}blat" "#{s}" "#{q}" #{"-prot" unless o[:nucl]} -out=blast8 \
+      "#{dir}/#{i}.tab.uns"`
+      `sort -k 1 "#{dir}/#{i}.tab.uns" > "#{dir}/#{i}.tab"`
+    when "diamond"
+      `"#{o[:bin]}diamond" blastp --threads "#{o[:thr]}" --db "#{s}.dmnd" \
+      --query "#{q}" --sensitive --daa "#{dir}/#{i}.daa" --quiet \
+      && "#{o[:bin]}diamond" view --daa "#{dir}/#{i}.daa" --outfmt 6 \
+      --out "#{dir}/#{i}.tab" --quiet`
+    else
+      abort "Unsupported program: #{o[:program]}."
+    end
+    fh = File.open("#{dir}/#{i}.tab", "r")
+    id = 0
+    sq = 0
+    n  = 0
+    fh.each_line do |ln|
+      ln.chomp!
+      row = ln.split(/\t/)
+      next unless qry_seen[ row[0].to_i ].nil?
+      next if row[3].to_i < o[:len] and
+      next if row[2].to_f < o[:id]
+      next if row[11].to_f < o[:bits]
+      next if row[3].to_f/[
+          seq_len[i==1 ? :seq1 : :seq2][row[0].to_i],
+          seq_len[i==1 ? :seq2 : :seq1][row[1].to_i]
+        ].min < o[:len_fraction]
+      qry_seen[ row[0].to_i ] = 1
+      id += row[2].to_f
+      sq += row[2].to_f ** 2
+      n  += 1
+      if i==1
+        rbh[ row[0].to_i ] = row[1].to_i
+      else
+        if !rbh[ row[1].to_i ].nil? and rbh[ row[1].to_i ]==row[0].to_i
+          id2 += row[2].to_f
+          sq2 += row[2].to_f**2
+          n2  += 1
+          fo.puts [row[2..5],row[10..11]].join("\t") unless o[:out].nil?
+          rbm.puts [ori_ids[:seq1][row[1].to_i],
+            ori_ids[:seq2][row[0].to_i], row[2..5], row[8..9],
+            row[6..7], row[10..11]].join("\t") unless o[:rbm].nil?
+          sqlite_db.execute("insert into rbm values(?,?,?,?,?,?,?)",
+            seq_names + [ori_ids[:seq1][row[1].to_i],
+            ori_ids[:seq2][row[0].to_i], row[2], row[10], row[11]]
+            ) if not o[:sqlite3].nil? and o[:dbrbm]
+        end
+      end
+    end
+    fh.close
+    if n < o[:hits]
+      puts "Insuffient hits to estimate one-way AAI: #{n}." unless o[:auto]
+      res.puts "Insufficient hits to estimate one-way AAI: #{n}" unless
+        o[:res].nil?
+    else
+      printf "! One-way AAI %d: %.#{o[:dec]}f%% (SD: %.#{o[:dec]}f%%), " +
+        "from %i proteins.\n", i, id/n, (sq/n - (id/n)**2)**0.5, n unless
+        o[:auto]
+      res.puts sprintf "<b>One-way AAI %d:</b> %.#{o[:dec]}f%% " +
+        "(SD: %.#{o[:dec]}f%%), from %i proteins.<br/>", i, id/n,
+        (sq/n - (id/n)**2)**0.5, n unless o[:res].nil?
+    end
+  end
+  rbm.close unless o[:rbm].nil?
+  if n2 < o[:hits]
+    puts "Insufficient hits to estimate two-way AAI: #{n2}" unless o[:auto]
+    res.puts "Insufficient hits to estimate two-way AAI: #{n2}" unless
+      o[:res].nil?
+  else
+    printf "! Two-way AAI  : %.#{o[:dec]}f%% (SD: %.#{o[:dec]}f%%), from %i" +
+      " proteins.\n", id2/n2, (sq2/n2 - (id2/n2)**2)**0.5, n2 unless o[:auto]
+    res.puts sprintf "<b>Two-way AAI:</b> %.#{o[:dec]}f%% (SD: " +
+      "%.#{o[:dec]}f%%), from %i proteins.<br/>", id2/n2,
+      (sq2/n2 - (id2/n2)**2)**0.5, n2 unless o[:res].nil?
+    unless o[:tab].nil?
+      tab = File.open(o[:tab], "w")
+      tab.printf "%.#{o[:dec]}f\t%.#{o[:dec]}f\t%i\t%i\n", id2/n2,
+        (sq2/n2 - (id2/n2)**2)**0.5, n2, minfrg
+      tab.close
+    end
+    sqlite_db.execute("insert into aai values(?,?,?,?,?,?)",
+      seq_names + [id2/n2, (sq2/n2 - (id2/n2)**2)**0.5, n2, minfrg]) unless
+      o[:sqlite3].nil?
+    puts id2/n2 if o[:auto]
+  end
+  res.close unless o[:res].nil?
+  fo.close unless o[:out].nil?
+end
+

data/utils/enveomics/Scripts/ani.rb

@@ -0,0 +1,362 @@
+#!/usr/bin/env ruby
+
+# @author  Luis M. Rodriguez-R
+# @license Artistic-2.0
+
+require "optparse"
+require "tmpdir"
+has_rest_client = true
+has_sqlite3 = true
+begin
+  require "rubygems"
+  require "restclient"
+rescue LoadError
+  has_rest_client = false
+end
+begin
+  require "sqlite3"
+rescue LoadError
+  has_sqlite3 = false
+end
+
+o = {win:1000, step:200, id:70, len:700, correct:true, hits:50, q:false, bin:"",
+  program:"blast+", thr:1, dec:2, auto:false, lookupfirst:false,
+  dbregions:true, dbrbm: true, min_actg:0.95}
+ARGV << "-h" if ARGV.size==0
+OptionParser.new do |opts|
+  opts.banner = "
+Calculates the Average Nucleotide Identity between two genomes.
+
+Usage: #{$0} [options]"
+  opts.separator ""
+  opts.separator "Mandatory"
+  opts.on("-1", "--seq1 FILE",
+    "Path to the FastA file containing the genome 1."){ |v| o[:seq1] = v }
+  opts.on("-2", "--seq2 FILE",
+    "Path to the FastA file containing the genome 2."){ |v| o[:seq2] = v }
+  if has_rest_client
+    opts.separator "    Alternatively, you can supply a NCBI-acc with the " +
+      "format ncbi:CP014272 instead of files."
+  else
+    opts.separator "    Install rest-client to enable NCBI-acc support."
+  end
+  opts.separator ""
+  opts.separator "Search Options"
+  opts.on("-w", "--win INT",
+    "Window size in the ANI calculation (in bp).  By default: " +
+    "#{o[:win].to_s}."){ |v| o[:win] = v.to_i }
+  opts.on("-s", "--step INT",
+    "Step size in the ANI calculation (in bp).  By default: " +
+    "#{o[:step].to_s}."){ |v| o[:step] = v.to_i }
+  opts.on("-l", "--len INT",
+    "Minimum alignment length (in bp).  By default: #{o[:len]}."
+    ){ |v| o[:len] = v.to_i }
+  opts.on("-i", "--id NUM",
+    "Minimum alignment identity (in %).  By default: #{o[:id]}."
+    ){ |v| o[:id] = v.to_f }
+  opts.on("-n", "--hits INT",
+    "Minimum number of hits.  By default: #{o[:hits]}."
+    ){ |v| o[:hits] = v.to_i }
+  opts.on("-N", "--nocorrection",
+    "Report values without post-hoc correction."){ |v| o[:correct] = false }
+  opts.on("--min-actg FLOAT",
+    "Minimum fraction of ACTGN in the sequences before assuming proteins.",
+    "By default: #{o[:min_actg]}."
+    ){ |v| o[:min_actg] = v.to_f }
+  opts.separator ""
+  opts.separator "Software Options"
+  opts.on("-b", "--bin DIR",
+    "Path to the directory containing the binaries of the search program."
+    ){ |v| o[:bin] = v }
+  opts.on("-p", "--program STR",
+    "Search program to be used.  One of: blast+ (default), blast, blat."
+    ){ |v| o[:program] = v }
+  opts.on("-t", "--threads INT",
+    "Number of parallel threads to be used.  By default: #{o[:thr]}."
+    ){ |v| o[:thr] = v.to_i }
+  opts.separator ""
+  opts.separator "SQLite3 Options"
+  opts.on("-S", "--sqlite3 FILE",
+    "Path to the SQLite3 database to create (or update) with the results."
+    ){ |v| o[:sqlite3] = v }
+  opts.separator "    Install sqlite3 gem to enable database support." unless
+    has_sqlite3
+  opts.on("--name1 STR",
+    "Name of --seq1 to use in --sqlite3.  By default determined by filename."
+    ){ |v| o[:seq1name] = v }
+  opts.on("--name2 STR",
+    "Name of --seq2 to use in --sqlite3.  By default determined by filename."
+    ){ |v| o[:seq2name] = v }
+  opts.on("--[no-]save-regions",
+    "Save (or don't save) the fragments in the --sqlite3 database.",
+    "By default: #{o[:dbregions]}."){ |v| o[:dbregions] = !!v }
+  opts.on("--[no-]save-rbm",
+    "Save (or don't save) the reciprocal best matches in the --sqlite3 db.",
+    "By default: #{o[:dbrbm]}."){ |v| o[:dbrbm] = !!v }
+  opts.on("--lookup-first",
+    "Indicates if the ANI should be looked up first in the database.",
+    "Requires --sqlite3, --auto, --name1, and --name2.",
+    "Incompatible with --res, --tab, and --out."){ |v| o[:lookupfirst] = v }
+  opts.separator ""
+  opts.separator "Other Output Options"
+  opts.on("-d", "--dec INT",
+    "Decimal positions to report. By default: #{o[:dec]}"
+    ){ |v| o[:dec] = v.to_i }
+  opts.on("-o", "--out FILE",
+    "Saves a file describing the alignments used for two-way ANI."
+    ){ |v| o[:out] = v }
+  opts.on("-r", "--res FILE",
+    "Saves a file with the final results."){ |v| o[:res] = v }
+  opts.on("-T", "--tab FILE",
+    "Saves a file with the final two-way results in a tab-delimited form.",
+    "The columns are (in that order):",
+    "ANI, standard deviation, fragments used, fragments in the smallest genome."
+    ){ |v| o[:tab]=v }
+  opts.on("-a", "--auto",
+    "ONLY outputs the ANI value in STDOUT (or nothing, if calculation fails)."
+    ){ o[:auto] = true }
+  opts.on("-q", "--quiet", "Run quietly (no STDERR output)"){ o[:q] = true }
+  opts.on("-h", "--help", "Display this screen") do
+    puts opts
+    exit
+  end
+  opts.separator ""
+end.parse!
+abort "-1 is mandatory" if o[:seq1].nil?
+abort "-2 is mandatory" if o[:seq2].nil?
+abort "SQLite3 requested (-S) but sqlite3 not supported.  First install gem " +
+  "sqlite3." unless o[:sqlite3].nil? or has_sqlite3
+abort "Step size must be smaller than window size." if o[:step] > o[:win]
+o[:bin] = o[:bin]+"/" if o[:bin].size > 0
+if o[:lookupfirst]
+  abort "--lookup-first needs --sqlite3" if o[:sqlite3].nil?
+  abort "--lookup-first requires --auto" unless o[:auto]
+  abort "--lookup-first requires --name1" if o[:seq1name].nil?
+  abort "--lookup-first requires --name2" if o[:seq2name].nil?
+  abort "--lookup-first conflicts with --res" unless o[:res].nil?
+  abort "--lookup-first conflicts with --tab" unless o[:tab].nil?
+  abort "--lookup-first conflicts with --out" unless o[:out].nil?
+end
+
+# Create SQLite3 file
+unless o[:sqlite3].nil?
+  $stderr.puts "Accessing SQLite3 file: #{o[:sqlite3]}." unless o[:q]
+  sqlite_db = SQLite3::Database.new o[:sqlite3]
+  sqlite_db.execute "create table if not exists regions( " +
+    "seq varchar(256), id int, source varchar(256), `start` int," +
+    " `end` int )"
+  sqlite_db.execute "create table if not exists rbm( seq1 varchar(256), " +
+    "seq2 varchar(256), id1 int, id2 int, id float, evalue float, " +
+    "bitscore float )"
+  sqlite_db.execute "create table if not exists ani( seq1 varchar(256), " +
+    "seq2 varchar(256), ani float, sd float, n int, omega int )"
+end
+
+# Look-up first
+if o[:lookupfirst]
+  val = sqlite_db.execute "select ani from ani where seq1=? and seq2=?",
+    [o[:seq1name], o[:seq2name]]
+  val = sqlite_db.execute "select ani from ani where seq1=? and seq2=?",
+    [o[:seq2name], o[:seq1name]] if val.empty?
+  unless val.empty?
+    puts val.first.first
+    exit
+  end
+end
+
+Dir.mktmpdir do |dir|
+  $stderr.puts "Temporal directory: #{dir}." unless o[:q]
+
+  # Create databases.
+  $stderr.puts "Creating databases." unless o[:q]
+  minfrg = nil
+  seq_names = []
+  seq_len = {}
+  actg_cnt = {}
+  [:seq1, :seq2].each do |seq|
+    abort "GIs are no longer supported by NCBI. Please use NCBI-acc instead" if
+      /^gi:/.match(o[seq])
+    acc = /^ncbi:(\S+)/.match(o[seq])
+    if not acc.nil?
+      abort "NCBI-acc requested but rest-client not supported.  First " +
+        "install gem rest-client." unless has_rest_client
+      response = RestClient.get(
+        "https://eutils.ncbi.nlm.nih.gov/entrez/eutils/efetch.fcgi",
+        {params:{db:"nuccore",rettype:"fasta",id:acc[1],idtype:"acc"}})
+      abort "Unable to reach NCBI EUtils, error code " +
+        response.code.to_s + "." unless response.code == 200
+      o[seq] = "#{dir}/ncbi-#{seq.to_s}.fa"
+      fo = File.open(o[seq], "w")
+      fo.puts response.to_str
+      fo.close
+      seq_names << ( o[ "#{seq}name".to_sym ].nil? ?
+        "ncbi:#{acc[1]}" : o[ "#{seq}name".to_sym ] )
+    else
+      seq_names << ( o[ "#{seq}name".to_sym ].nil? ?
+        File.basename(o[seq], ".*") : o[ "#{seq}name".to_sym ] )
+    end
+    $stderr.puts "  Reading FastA file: #{o[seq]}" unless o[:q]
+    sqlite_db.execute("delete from regions where seq=?",
+      [seq_names.last]) unless o[:sqlite3].nil?
+    buffer = ""
+    frgs = 0
+    seq_len[seq] = 0
+    actg_cnt[seq] = 0
+    seqs = 0
+    disc = 0
+    seqn = ""
+    from = 1
+    fi = File.open(o[seq], "r")
+    fo = File.open("#{dir}/#{seq.to_s}.fa", "w")
+    fi.each_line do |ln|
+      if ln =~ /^>(\S+)/
+        seqs += 1
+        disc += buffer.size
+        buffer = ""
+        seqn = $1
+        from = 1
+      else
+        ln.gsub!(/[^A-Za-z]/, '')
+        seq_len[seq]  += ln.length
+        actg_cnt[seq] += ln.gsub(/[^ACTGNactgn]/,"").length
+        buffer = buffer + ln
+        while buffer.size > o[:win]
+          seq_i = buffer[0, o[:win]]
+          if seq_i =~ /^N+$/
+            disc += seq_i.size
+          else
+            frgs += 1
+            fo.puts ">#{frgs}"
+            fo.puts seq_i
+            sqlite_db.execute("insert into regions values(?,?,?,?,?)",
+              [seq_names.last, frgs, seqn, from, from+o[:win]]) if
+                not o[:sqlite3].nil? and o[:dbregions]
+          end
+          buffer = buffer[o[:step] .. -1]
+          from += o[:win]
+        end
+      end
+    end
+    fi.close
+    fo.close
+    actg_frx = actg_cnt[seq].to_f/seq_len[seq].to_f
+    abort "Input sequences appear to be proteins " +
+      "(ACTGN fraction: %.2f%%)." % (actg_frx*100) if actg_frx < o[:min_actg]
+    $stderr.puts "    Created #{frgs} fragments from #{seqs} sequences, " +
+      "discarded #{disc} bp." unless o[:q]
+    minfrg ||= frgs
+    minfrg = frgs if minfrg > frgs
+    case o[:program].downcase
+    when "blast"
+      `"#{o[:bin]}formatdb" -i "#{dir}/#{seq.to_s}.fa" -p F`
+    when "blast+"
+      `"#{o[:bin]}makeblastdb" -in "#{dir}/#{seq.to_s}.fa" -dbtype nucl`
+    when "blat"
+      # Nothing to do
+    else
+      abort "Unsupported program: #{o[:program]}."
+    end
+  end # [:seq1, :seq2].each
+
+  # Best-hits.
+  $stderr.puts "Running one-way comparisons." unless o[:q]
+  rbh = []
+  id2 = 0
+  sq2 = 0
+  n2  = 0
+  unless o[:sqlite3].nil?
+    sqlite_db.execute "delete from rbm where seq1=? and seq2=?", seq_names
+    sqlite_db.execute "delete from ani where seq1=? and seq2=?", seq_names
+  end
+  unless o[:out].nil?
+    fo = File.open(o[:out], "w")
+    fo.puts %w(identity aln.len mismatch gap.open evalue bitscore).join("\t")
+  end
+  res = File.open(o[:res], "w") unless o[:res].nil?
+  [1,2].each do |i|
+    qry_seen = []
+    q = "#{dir}/seq#{i}.fa"
+    s = "#{dir}/seq#{i==1?2:1}.fa"
+    case o[:program].downcase
+    when "blast"
+      `"#{o[:bin]}blastall" -p blastn -d "#{s}" -i "#{q}" \
+      -F F -v 1 -b 1 -a #{o[:thr]} -m 8 -o "#{dir}/#{i}.tab"`
+    when "blast+"
+      `"#{o[:bin]}blastn" -db "#{s}" -query "#{q}" \
+      -dust no -max_target_seqs 1 \
+      -num_threads #{o[:thr]} -outfmt 6 -out "#{dir}/#{i}.tab"`
+    when "blat"
+      `#{o[:bin]}blat "#{s}" "#{q}" -out=blast8 "#{dir}/#{i}.tab"`
+    else
+      abort "Unsupported program: #{o[:program]}."
+    end
+    fh = File.open("#{dir}/#{i}.tab", "r")
+    id = 0
+    sq = 0
+    n  = 0
+    fh.each_line do |ln|
+      ln.chomp!
+      row = ln.split(/\t/)
+      if qry_seen[ row[0].to_i ].nil? and row[3].to_i >= o[:len] and
+          row[2].to_f >= o[:id]
+        qry_seen[ row[0].to_i ] = 1
+        identity_corr = 100 - (100-row[2].to_f)/(o[:correct] ? 0.8621 : 1.0)
+        id += identity_corr
+        sq += identity_corr ** 2
+        n  += 1
+        if i==1
+          rbh[ row[0].to_i ] = row[1].to_i
+        else
+          if !rbh[ row[1].to_i ].nil? and rbh[ row[1].to_i ]==row[0].to_i
+            id2 += identity_corr
+            sq2 += identity_corr ** 2
+            n2  += 1
+            fo.puts [identity_corr,row[3..5],
+              row[10..11]].join("\t") unless o[:out].nil?
+            sqlite_db.execute("insert into rbm values(?,?,?,?,?,?,?)",
+              seq_names + [row[1], row[0], row[2], row[10], row[11]]
+              ) if not o[:sqlite3].nil? and o[:dbrbm]
+          end
+        end
+      end
+    end
+    fh.close
+    if n < o[:hits]
+      puts "Insuffient hits to estimate one-way ANI: #{n}." unless o[:auto]
+      res.puts "Insufficient hits to estimate one-way ANI: #{n}" unless
+        o[:res].nil?
+    else
+      printf "! One-way ANI %d: %.#{o[:dec]}f%% (SD: %.#{o[:dec]}f%%), " +
+        "from %i fragments.\n", i, id/n, (sq/n - (id/n)**2)**0.5, n unless
+        o[:auto]
+      res.puts sprintf "<b>One-way ANI %d:</b> %.#{o[:dec]}f%% " +
+        "(SD: %.#{o[:dec]}f%%), from %i fragments.<br/>", i, id/n,
+        (sq/n - (id/n)**2)**0.5, n unless o[:res].nil?
+    end
+  end # [1,2].each
+  if n2 < o[:hits]
+    puts "Insufficient hits to estimate two-way ANI: #{n2}" unless o[:auto]
+    res.puts "Insufficient hits to estimate two-way ANI: #{n2}" unless
+      o[:res].nil?
+  else
+    ani = id2/n2
+    ani_sd = (sq2/n2 - (id2/n2)**2)**0.5
+    printf "! Two-way ANI  : %.#{o[:dec]}f%% (SD: %.#{o[:dec]}f%%), " +
+      "from %i fragments.\n", ani, ani_sd, n2 unless o[:auto]
+    res.puts sprintf "<b>Two-way ANI:</b> %.#{o[:dec]}f%% " +
+      "(SD: %.#{o[:dec]}f%%), from %i fragments.<br/>",
+      ani, ani_sd, n2 unless o[:res].nil?
+    unless o[:tab].nil?
+      tab = File.open(o[:tab], "w")
+      tab.printf "%.#{o[:dec]}f\t%.#{o[:dec]}f\t%i\t%i\n",
+        ani, ani_sd, n2, minfrg
+      tab.close
+    end
+    sqlite_db.execute("insert into ani values(?,?,?,?,?,?)",
+      seq_names + [ani, ani_sd, n2, minfrg]) unless o[:sqlite3].nil?
+    puts ani if o[:auto]
+  end
+  res.close unless o[:res].nil?
+  fo.close unless o[:out].nil?
+end
+