miga-base 0.3.0.0 → 0.3.0.1

data/utils/enveomics/Scripts/VCF.KaKs.rb

@@ -0,0 +1,147 @@
+#!/usr/bin/env ruby
+
+# @author  Luis M. Rodriguez-R
+# @license Artistic-2.0
+
+$:.push File.expand_path("../lib", __FILE__)
+require "enveomics_rb/enveomics"
+require "enveomics_rb/vcf"
+
+o = {}
+OptionParser.new do |opt|
+  opt.banner = "
+  Estimates the Ka/Ks ratio from the SNPs in a VCF file. Ka and Ks are corrected
+  using pseudo-counts, but no corrections for multiple substitutions are
+  applied.
+  
+  Usage: #{$0} [options]".gsub(/^ +/,"")
+  opt.separator ""
+  opt.separator "Mandatory"
+  opt.on("-i", "--input FILE",
+    "Input file in Variant Call Format (VCF)."){ |v| o[:file] = v}
+  opt.on("-s", "--seqs FILE",
+    "Input gene sequences (nucleotides) in FastA format."){ |v| o[:seqs] = v}
+  opt.separator ""
+  opt.separator "Parameters"
+  opt.on("-f", "--syn-frx FLOAT",
+    "Fraction of synonymous substitutions. If passed, the number of sites are",
+    "estimated (not counted per gene), speeding up the computation ~10X."
+    ){ |v| o[:syn_frx] = v.to_f }
+  opt.on("-b", "--syn-bacterial-code",
+    "Sets --syn-frx to 0.760417, approximately the proportion of synonymous",
+    "substitutions in the bacterial code."){ o[:syn_frx] = 0.760417 }
+  opt.separator ""
+  opt.separator "Miscellaneous"
+  opt.on("-c", "--codon-file FILE",
+    "Output file including the codons of substitution variants."
+    ){ |v| o[:codon_file] = v }
+  opt.on("-h", "--help", "Display this screen.") do
+    puts opt
+    exit
+  end
+  opt.separator ""
+end.parse!
+
+abort "--input is mandatory" if o[:file].nil?
+abort "--seqs is mandatory"  if o[:seqs].nil?
+
+# Codon table (11. The Bacterial, Archaeal and Plant Plastid Code)
+# https://www.ncbi.nlm.nih.gov/Taxonomy/Utils/wprintgc.cgi#SG11
+t = {
+    AAs: "FFLLSSSSYY**CC*WLLLLPPPPHHQQRRRRIIIMTTTTNNKKSSRRVVVVAAAADDEEGGGG",
+ Starts: "---M------**--*----M------------MMMM---------------M------------",
+  Base1: "TTTTTTTTTTTTTTTTCCCCCCCCCCCCCCCCAAAAAAAAAAAAAAAAGGGGGGGGGGGGGGGG",
+  Base2: "TTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGGTTTTCCCCAAAAGGGG",
+  Base3: "TCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAGTCAG"
+}
+$codon_aa = {}
+$codon_st = {}
+(0 .. (t[:Base1].size-1)).each do |i|
+  cod = [:Base1, :Base2, :Base3].map{ |k| t[k][i] }.join
+  $codon_aa[cod] = t[:AAs][i]
+  $codon_st[cod] = t[:Starts][i]
+end
+
+##
+# Is the change +cod+ to +cod_alt+ synonymous? +start_codon+ indicates if the
+# codon the first in the gene.
+def syn?(cod, cod_alt, start_codon=false)
+  start_codon ?
+    ( $codon_st[cod] == $codon_st[cod_alt] ) :
+    ( $codon_aa[cod] == $codon_aa[cod_alt] )
+end
+
+##
+# Estimates the fraction of times that the substitutions in the sequence +seq+
+# result in synonymous mutations from those in position +pos+ by any of the
+# nucleotides in +alts+.
+def syn_fraction(seq, pos, alts)
+  cod_let = (pos-1)%3
+  cod_pos = (pos-1) - cod_let
+  cod = seq[cod_pos .. (cod_pos+2)]
+  syn = 0
+  cod_alts = alts.map do |alt|
+    cod_alt = "#{cod}"
+    cod_alt[cod_let] = alt
+    cod_alt
+  end
+  syn = cod_alts.map{ |i| syn?(cod, i, pos<=3) ? 1 : 0 }.inject(0,:+)
+  $codon_fh.puts [syn, cod, cod_alts.join(",")].join("\t") unless $codon_fh.nil?
+  syn.to_f/alts.size
+end
+
+# Read sequences
+seqs = {}
+File.open(o[:seqs], "r") do |fh|
+  id = ""
+  fh.each_line do |ln|
+    if ln =~ /^>(\S+)/
+      id = $1
+      seqs[id] = ""
+    else
+      seqs[id] += ln.chomp.gsub(/[^A-Za-z]/, "")
+    end
+  end
+end
+
+# Process variants
+$codon_fh = nil
+unless o[:codon_file].nil?
+  $codon_fh = File.open(o[:codon_file], "w")
+  $codon_fh.puts "#" + %w[Syn Ref Alt].join("\t")
+end
+vcf = VCF.new(o[:file])
+gen = {}
+vcf.each_variant do |v|
+  next if v.indel?
+  raise "REF doesn't match VCF:\n#{v}" unless seqs[v.chrom][v.pos-1] == v.ref
+  gen[v.chrom] ||= [0.0, 0.0]
+  alts = v.alt.split(",")
+  syn = syn_fraction(seqs[v.chrom], v.pos, alts)
+  gen[v.chrom][0] += 1.0-syn
+  gen[v.chrom][1] += syn
+end
+$codon_fh.close unless $codon_fh.nil?
+$codon_fh = nil
+
+# Ka/Ks
+puts "#" +
+  "SeqID KaKs Ka Ks NonSynSubs SynSubs NonSynSites SynSites".tr(" ","\t")
+gen.each do |k,v|
+  if o[:syn_frx].nil?
+    v[2,3] = [0.0,0.0]
+    (1 .. seqs[k].size).each do |pos|
+      alts = %w(A C T G) - [seqs[k][pos-1]]
+      syn = syn_fraction(seqs[k], pos, alts)
+      v[2] += 1.0-syn
+      v[3] += syn
+    end
+  else
+    v[2] = seqs[k].size.to_f*o[:syn_frx]
+    v[3] = seqs[k].size.to_f*(1.0-o[:syn_frx])
+  end
+  ka = (v[0] + 1) / (v[2] + 2)
+  ks = (v[1] + 1) / (v[3] + 2)
+  puts ([k, ka/ks, ka, ks] + v).join("\t")
+end
+

data/utils/enveomics/Scripts/VCF.SNPs.rb

@@ -0,0 +1,88 @@
+#!/usr/bin/env ruby
+
+# @author  Luis M. Rodriguez-R
+# @license Artistic-2.0
+
+$:.push File.expand_path(File.dirname(__FILE__) + "/lib")
+require "enveomics_rb/enveomics"
+require "enveomics_rb/vcf"
+
+o = {min_dp:4, max_dp:Float::INFINITY, min_ref_dp:2, min_alt_dp:2, min_qual:0.0,
+  indels:false, min_ic:0.0}
+OptionParser.new do |opt|
+  opt.banner = "
+  Counts the number of Single-Nucleotide Polymorphisms (SNPs) in a VCF file.
+  
+  Usage: #{$0} [options]".gsub(/^ +/,"")
+  opt.separator ""
+  opt.separator "Mandatory"
+  opt.on("-i", "--input FILE",
+    "Input file in Variant Call Format (VCF)."){ |v| o[:file] = v}
+  opt.separator ""
+  opt.separator "Parameters"
+  opt.on("-o", "--out FILE",
+    "Output (filtered) file in Variant Call Format (VCF)."){ |v| o[:out] = v}
+  opt.on("-m", "--min-dp INT",
+    "Minimum number of reads covering the position. By default: #{o[:min_dp]}."
+    ){ |v| o[:min_dp] = v.to_i }
+  opt.on("-M", "--max-dp INT",
+    "Maximum number of reads covering the position. By default: #{o[:max_dp]}."
+    ){ |v| o[:max_dp] = (v=="Infinity" ? Float::INFINITY : v.to_i) }
+  opt.on("-r", "--min-ref-dp INT",
+    "Minimum number of reads supporting allele REF. " +
+    "By default: #{o[:min_ref_dp]}."
+    ){ |v| o[:min_ref_dp] = v.to_i }
+  opt.on("-a", "--min-alt-dp INT",
+    "Minimum number of reads supporting allele ALT. " +
+    "By default: #{o[:min_alt_dp]}."
+    ){ |v| o[:min_alt_dp] = v.to_i }
+  opt.on("-q", "--min-quality FLOAT",
+    "Minimum quality of the position mapping. By default: #{o[:min_qual]}."
+    ){ |v| o[:max_dp] = v.to_f }
+  opt.on("-s", "--min-shannon FLOAT",
+    "Minimum information content (in bits, from 0 to 1). " +
+    "By default: #{o[:min_ic]}"){ |v| o[:min_ic] = v.to_f }
+  opt.on("--[no-]indels",
+    "Process (or ignore) indels. By default: ignore."
+    ){ |v| o[:indels] = v }
+  opt.on("-h", "--help", "Display this screen.") do
+    puts opt
+    exit
+  end
+  opt.separator ""
+end.parse!
+
+abort "--input is mandatory" if o[:file].nil?
+
+vcf = VCF.new(o[:file])
+c = 0
+dp = 0
+ref_dp = 0
+alt_dp = 0
+h = 0
+unless o[:out].nil?
+  ofh = File.open(o[:out], "w")
+  vcf.each_header{ |h| ofh.print h }
+end
+vcf.each_variant do |v|
+  next if v.indel? and not o[:indels]
+  next if v.dp < o[:min_dp]
+  next if v.dp > o[:max_dp]
+  next if v.ref_dp < o[:min_ref_dp]
+  next if v.alt_dp < o[:min_alt_dp]
+  next if v.qual < o[:min_qual]
+  next if v.shannon < o[:min_ic]
+  c += 1
+  dp += v.dp
+  ref_dp += v.ref_dp
+  alt_dp += v.alt_dp
+  h += v.shannon
+  ofh.print v.to_s unless o[:out].nil?
+end
+ofh.close unless o[:out].nil?
+
+puts "SNPs: #{c}", "Information content: #{h}",
+  "Average SNP depth: #{dp.to_f/c}",
+  "Average REF allele depth: #{ref_dp.to_f/c}",
+  "Average ALT allele depth: #{alt_dp.to_f/c}"
+

data/utils/enveomics/Scripts/aai.rb

@@ -0,0 +1,373 @@
+#!/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 = {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 containing the genome 1 (proteins)."
+    ){ |v| o[:seq1] = v }
+  opts.on("-2", "--seq2 FILE",
+    "Path to the FastA file 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",
+    "Minimum alignment length (in residues).  By default: #{o[:len]}."
+    ){ |v| o[:len] = v.to_i }
+  opts.on("-L", "--len-fraction NUM",
+    "Minimum alignment length as a fraction of the shorter sequence",
+    "(range 0-1).  By default: #{o[:len_fraction]}."
+    ){ |v| o[:len_fraction] = v.to_f }
+  opts.on("-i", "--id NUM",
+    "Minimum alignment identity (in %).  By default: #{o[:id]}."
+    ){ |v| o[:id] = v.to_f }
+  opts.on("-s", "--bitscore NUM",
+    "Minimum bit score (in bits).  By default: #{o[:bits]}."
+    ){ |v| o[:bits] = 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", "--nucl",
+    "The input sequences are nucleotides (genes), not proteins."
+    ){ |v| o[:nucl] = v }
+  opts.on("--max-actg FLOAT",
+    "Maximum fraction of ACTGN in the sequences before assuming nucleotides.",
+    "By default: #{o[:max_actg]}."
+    ){ |v| o[:max_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-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",
+    "Decimal positions to report. By default: #{o[:dec]}"
+    ){ |v| o[:dec] = v.to_i }
+  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!
+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
+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?
+  abort "--lookup-first conflicts with --rbm" unless o[:rbm].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 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])
+    if not 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:"nuccore",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], ".faa") :
+        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.open(o[seq], "r")
+    fo = File.open("#{dir}/#{seq.to_s}.fa", "w")
+    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(/[^ACTGN]/,"").length
+      end
+    end
+    fi.close
+    fo.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.to_s}.fa" \
+      -p #{o[:nucl] ? "F" : "T"}`
+    when "blast+"
+      `"#{o[:bin]}makeblastdb" -in "#{dir}/#{seq.to_s}.fa" \
+      -dbtype #{o[:nucl] ? "nucl" : "prot"}`
+    when "blat"
+      # Nothing to do
+    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"`
+    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
+