transrate 1.0.0.beta3 → 1.0.0.beta4

data/lib/transrate/read_metrics.rb

@@ -1,5 +1,7 @@
 module Transrate
 
+  class ReadMetricsError < TransrateError; end
+
   class ReadMetrics
 
     attr_reader :fragments, :fragments_mapping, :p_good_mapping
@@ -9,7 +11,7 @@ module Transrate
     attr_reader :read_length
 
     def initialize assembly
-      @assembly = assembly
+      @assembly = assembly # Transrate::Assembly
       @mapper = Snap.new
       @salmon = Salmon.new
       self.initial_values
@@ -60,7 +62,12 @@ module Transrate
         if !File.exist?(assigned_bam)
           assigned_bam = assign_and_quantify(bamfile, threads)
         end
-        File.rename(assigned_bam, final_bam)
+        if File.exist?(assigned_bam)
+          File.rename(assigned_bam, final_bam)
+        else
+          logger.error "Couldn't find #{assigned_bam} to rename"
+          raise ReadMetricsError
+        end
       end
       # analyse the final mappings
       analyse_read_mappings final_bam
@@ -116,6 +123,7 @@ module Transrate
     def analyse_expression salmon_output
       salmon_output.each_pair do |name, expr|
         contig_name = Bio::FastaDefline.new(name.to_s).entry_id
+        contig_name.gsub!(/;$/, '') # trim trailing semicolon
         contig = @assembly[contig_name]
         if expr[:eff_len]==0
           coverage = 0
@@ -188,6 +196,7 @@ module Transrate
 
     def populate_contig_data row
       name = Bio::FastaDefline.new(row[:name].to_s).entry_id
+      name.gsub!(/;$/, '') # trim trailing semicolon
       contig = @assembly[name]
       contig.p_seq_true = row[:p_seq_true]
       contig.uncovered_bases = row[:bases_uncovered]

data/lib/transrate/salmon.rb

@@ -17,6 +17,7 @@ module Transrate
     def run assembly, bamfile, threads=8
       assembly = assembly.file if assembly.is_a? Assembly
       output = "quant.sf"
+      sampled_bam = "postSample.bam"
       @fin_output = "#{File.basename assembly}_#{output}"
       unless File.exist? @fin_output
         salmon = Cmd.new build_command(assembly, bamfile, threads)
@@ -25,9 +26,13 @@ module Transrate
           logger.error salmon.stderr
           raise SalmonError.new("Salmon failed")
         end
+        unless File.exist?(sampled_bam)
+          logger.error salmon.stderr
+          raise SalmonError.new("#{sampled_bam} not created")
+        end
         File.rename(output, @fin_output)
       end
-      return 'postSample.bam'
+      return sampled_bam
     end
 
     def build_command assembly, bamfile, threads=4
@@ -39,7 +44,7 @@ module Transrate
       cmd << " --sampleOut"
       cmd << " --sampleUnaligned" # thanks Rob!
       cmd << " --output ."
-      cmd << " --useReadCompat"
+      cmd << " --useVBOpt"
       cmd << " --useErrorModel"
       cmd
     end
@@ -49,9 +54,14 @@ module Transrate
       File.open(file).each do |line|
         if line !~ /^#/
           line = line.chomp.split("\t")
+          unless line.length == 4
+            raise SalmonError.new("Salmon output file should have 4 columns " +
+              "but it had #{line.length}\n" +
+              "Please check you are using the correct version of Salmon")
+          end
           target = line[0]
           effective_length = line[1]
-          effective_count = line[4]
+          effective_count = line[3]
           tpm = line[2]
           expression[target] = {
             :eff_len => effective_length.to_i,

data/lib/transrate/score_optimiser.rb

@@ -6,6 +6,8 @@ module Transrate
   # while also minimising the number of low scoring contigs.
   class ScoreOptimiser
 
+    require 'csv'
+
     def initialize assembly, read_metrics
       @assembly = assembly
       @fragments = read_metrics.fragments
@@ -20,7 +22,7 @@ module Transrate
       @contig_score * (@good / @total.to_f)
     end
 
-    def optimal_score
+    def optimal_score(prefix='assembly')
       return [@optimal, @cutoff] unless @optimal.nil?
       product = 0
       good = 0
@@ -44,7 +46,10 @@ module Transrate
       end
       @optimal = 0
       @cutoff = 0
+      out = CSV.open("#{prefix}_score_optimisation.csv", 'w')
+      out << %w[cutoff assembly_score]
       cutoffscores.each do |c, score|
+        out << [c, score]
         if score > @optimal
           @optimal = score
           @cutoff = c

data/lib/transrate/snap.rb

@@ -41,8 +41,7 @@ module Transrate
       cmd
     end
 
-    def map_reads(file, left, right, insertsize: 200,
-                  insertsd: 50, outputname: nil, threads: 8)
+    def map_reads(file, left, right, outputname: nil, threads: 8)
       raise SnapError.new("Index not built") if !@index_built
 
       lbase = File.basename(left.split(",").first)

data/lib/transrate/transrater.rb

@@ -69,11 +69,11 @@ module Transrate
       return @score_optimiser.raw_score
     end
 
-    def assembly_optimal_score
+    def assembly_optimal_score prefix
       if !@score_optimiser
         @score_optimiser = ScoreOptimiser.new(@assembly, @read_metrics)
       end
-      return @score_optimiser.optimal_score
+      return @score_optimiser.optimal_score prefix
     end
 
     def assembly_metrics

data/lib/transrate/version.rb

@@ -11,7 +11,7 @@ module Transrate
     MAJOR = 1
     MINOR = 0
     PATCH = 0
-    BUILD = 'beta3'
+    BUILD = 'beta4'
 
     STRING = [MAJOR, MINOR, PATCH, BUILD].compact.join('.')
   end

data/test/data/sorghum_100.fa

@@ -1,6 +1,6 @@
 >Sb09g017110.1
 AGCGTGGGAAGGGAGATCACTGGAAGGGAGAAAGAAATGGAGCATGAAGAAGAGCTGCCTTCATCTTCTTCCTCCTTGGGTTACCTGATGCAGTGTAGGATCTGCCACGAGGAAGAGAACGAAGGGCGCGCGATCATGGAGTCTCCTTGTGGATGCTCCGGCTCTCTCAAGTATGCTCACAGGGGATGTGTGCAGAGATGGTGTGATGAGAAGGGGAGCACCCTCTGTGAGATTTGCCTTCAGAATTTCGAGCCAGGCTACACAATGCCTCCAAAGAAAACTCCGGCGATTGAAACTGCGGTCACTATCAGTGAACATGAGGACATGCAACCTTTGGAATCTCCGGAGGGCTCCATTGACGGTGCAGATTACACCAGGTGCTCCTACGCCGCAGATCAATGCGCCACATGGTGCCGGTCGCTGGCGATCACGTTCACCATTATGCTGTTGGCATGGCATCTGGTTGCAGTAGTGACGGTTGAAGCAGCAGATCACTGCGCGTTCAGTCTCCTGACAATGTACTTACTTCGTGCTGCTGGTATCCTGCTGCCGCTCTATGTTGTCATGCGGCTGATTCGCATCGTCCAGAATGGGCAGAGGCAGTATCGGTTGCAGCTGCTGGAGGACCAAAGAAGAAATGCATCAACTATATGAATTATATGCGTGACCACGAGAAGGACCAGCTAGTTATTAATATTCATTAAGCCAATGAATTTAAATGAATCTTATGGAAGGTTGTACAGATGTACATTATGTATTACTGGGGAATTTTTCAAACAGAACATCATTTGTAAACTTTCAGTATACATACCGTTGGTGCAGTGAAAAACAGCAGC
->Sb02g028080.1
+>Sb02g028080.1;
 ATGGGGACCCCTCTCCTCTTCCCCCTTCTCGTCACCCTCCAGCTGTTCACCGCCGCCTCCCCCGCGGTCGCGTCGTCGCACATCTCCGTCGTCATCTCGCAGTCGGGCCTCGACTTCGCCAAGGACCTGCTCGTGTCCCGTGCCGTCGCGACCCTCACGCCCCTGAACGTGCCGGACATCCAGAAGACCATGAGCACCGTCGTGGGCACCGTCCGCGTGGCCGCATCCGGGATTGTGCTCAACGGCCTCGCCGTCACCAACTCCACCGTCGCTATCGGGGACACGGGTGTTGTCGTGGCCGCCTCGTTGGCCAGAGCGAACCTCACCATGGAGTGGAACTACTCGTACAGCGCCTGGATTGTGACCATATCCGACAGCGGGAATGCTTCGATCCAGGTTGAAGGAATGGAGGTTGGTGTTTCCATGGTCATGAAGAATCAGAATGGATCTATCAAGCTGTCTGTTACAGAATGTAGCTGTAATATGGAGGACTTAGACATAACACTAAGTGGAGGAGCATCTTGGTTCTATCAAGTGTTTATAGATAGTTTCAGTAATCATATCAGATCATCAGTGGAAAATGCAATTGAGAACAAAGTAATGGAAGGTGCACTGAAGCTTGACTCTTTCCTGGGAAACCTTCCAAAGAAAATTGATCTTGATAGCGTTGCTGCAATGAATGTGACTTTTGTTAATGATCCACTATTCAAGAGCTCCTCTGTTGAGTTTGATATAGATGGCTTATTTATTCCATCGGATGAAACTGCTCCCGGAGACATGCTTCTTGGAAATACCCAATTTGCATTACCTCTTGGGAGCTCCTCGAGAATGCTTTGGATTTCATTGGATGAAGATGTTTTCAACTCCGTTTCAGCTCTCTACTTCAAGGCTGGTTTGCTGCAACGGATGGTGGACGAGGTTCCTGAACAGTTTCTTTTGAACACTGCTAGCTGGAGATTTTTGGTTCCTCGATTGTATCGAGAATATCCTGACGATGATATGCTACTGAATATCTCTGCAGTTTCGCCTCCCTCTGTGAGGATTAATGTGGGTAGAATTGATGCCACAGTTGACTTAGATGTCACAGTTAATGTCTTGGATTTTGGTGAGATAGTTCCAGTTGCATGCATATCAGTGTCGGTTGCTGTTTCTGGAGCTGCAGCGGTATCAGAGAATAATCTTGTTGGGAGAGTGAAATTGGATTATTTCTCATTTACCTTGAAATGGAGCAAAGTTGGCAAACTCCACACCAGTCTAGTGCAGACCGTGCTGAGGATTTTGCTGAAAAGTTTGTTTGTACCTTATGTGAACTCATATCTCGAGCAAGGCTTCCAGCTGCCCATCATCAAGGGATTCTCCGTCATAGATGCATATGTCCTCACTTCTTACTCAAGAATGATTGTTAGCTGCAATGTTGCGTTCCCTGAGCCAGAGGTTCTGTCTCCTATCCAAGAATCCAAGACCAACGAAGATTTGTCACATGAAGTTGGTTTGCTGATTGGATCTGCCAAAACTTGGCAGCCACCGATAACTAGTGTAAAATTCCTGTAAACAGTAACGCTATTCGTCGCAGTGTTTTGTTTTTAAAAATGTGTAAATAACTGTGCACTGTATATATGTATGTATGTATGTATGTATATATGCATATTAAATCAACAGATAGGAGCCATTCCGTAGCCTCTAAGTGGAATCGTTAGGTACTACAGTTTCCTCTATCCCAAATTATAAGCTGTTCTAGTTTTTT
 >Sb03g034100.1
 CTCACTCTCACACTACTCCTCCCCTCTCCGGCTCTCTGCCTCTGACGTCTGACCTCTCCTCCCCAACGGTGAGGCCGGCGCATTGCCGTTTCGAGCGCGGACACCGAGGGCTAGAACTAGAAGTGGCGGCGGTGCCAGGGCTCGGCGCTCGGTCGGCAATGGCGGGGCGGCTTATGCTGGCGGCGCTCCCTATTCTCCTCTTCTTATTGCTCGTCGGGCAATGCCACGGCGGCAAGATTGGCGTCTGCTACGGCCGCAACGCCGACGACCTGCCGGCGCCGGACAAGGTGGCGCAGCTAATCCAGCAGCAATCCATCAAGTACGTGCGCATCTACGACACCAACATCGACGTCATCAAGGCCTTCGCCAACACCGGCGTCGAGCTCATGGTCGGCGTCCCCAACTCCGACCTCCTCGCCTTCGCGCAGTACCAGTCCAACGTCGACACATGGCTCAAGAACAGCATTCTCCCCTACTACCCGGCCACCATGATCACCTACATCACCGTCGGCGCCGAGGTCACCGAGAGCCCCACCAACGTCTCCGCCCTCGTCGTGCCTGCCATGCGCAATGTGCACACCGCACTCAAGAAGGCCGGCCTGCACAAGAAGATCACCATCTCCAGCACCCACTCGCTCGGGATACTGTCACGGTCGTTCCCGCCGTCTGCTGGGGCGTTCAACAGCAGCTACGCCTACTTCTTGAAGCCTATGCTCGAGTTCCTTGTGGAGAATCAGGCGCCGTTCATGGTGGATTTATACCCCTACTATGCGTACCAGAACTCACCGAGCAATGTGTCCCTCAACTACGCCCTGTTCTCGCCACAGTCTCAGGATGTGATTGACCCAAACACTGGACTGGTTTACACTAACATGTTTGATGCCCAGGTTGATTCCATCTTCTTTGCGCTCATGGCTCTGAACTTCAAAACTCTGAAGATCATGATCACTGAGTCAGGGTGGCCAAACAAAGGGGCGGCCAAGGAGACTGGAGCCACTCCAGACAATGCTCAGACTTACAATACCAATTTGATACGCCATGTTGTTAATGACAGTGGCACGCCTGCGAAACCAGGGGAAGAAATTGATGTCTACATATTTTCATTGTTCAATGAGAACAGGAAACCTGGCATTGAGTCGGAGAGGAACTGGGGACTGTTTTTTCCTGATAAGAGCTCTATCTACAGCCTTGATTGGACGGGCCGAGGCAATGTGGATGTTATGACTGGAGCAAACATTACAAGTGCAAATGGTACCTGGTGTATTGCTTCAGCTAATGCATCAGAAACAGATCTGCAGAATGCCCTCAACTGGGCATGTGGTCCAGGCAACGTAGATTGCTCTGCCATTCAACCAAGCCAACCCTGCTACCAGCCGGACACTTTAGCTTCCCATGCTTCATATGCATTCAATAGCTACTACCAGCAAAATGGAGCCAACGTTGTGGCCTGTGACTTCAGTGGTGCGGGAATACGAACGACGAAAGATCCAAGTTACGACACTTGTGTCTATTTGGCTGCAGGCAATAAGATGAGCACAATGAATTCGACATCTCTTCCAGCTCAGAGCAACTCTGGTCCAGTTCCATGCGCCAAATACTTCACCACTTTCCTCCCCATGCTGGCCCCCGTGATGGCTGCAGTTATGCTGTGATCTATGGAAATGCTCCAGCTAGCCTCTGCAGATGTGGAGATGAAAGGTGAATTGCGTAATGCTGGTAACCAGCCGATGTTCTGTTTTGCTATGAGCAGTAGACTAGTAGTAGTCTAGTAGAGAGGCATATTATGCTGCTGTAGGAATTCTCTGGTCAGTTGAGATGTACATCGTCGCGCAGACAATATATATCAGCTGGCCTTAAGAACTCGATAACCTTTTCTGCTGTCTTTCG

data/test/data/test.sf

@@ -9,22 +9,22 @@
 # [ sampleOut ] => { }
 # [ useFragLenDist ] => { }
 # [ sampleUnaligned ] => { }
-# Name	Length	TPM	FPKM	NumReads
-scaffold1	1016	549.279	527.364	20690
-scaffold2	1439	598.782	574.892	31945
-scaffold3	783	408.072	391.791	11846
-scaffold4	893	441.382	423.772	14613
-scaffold5	622	494.487	474.758	11403
-scaffold6	2073	4.77214	4.58174	366.764
-scaffold7	1291	4.288	4.11692	205.236
-scaffold8	1355	17.9155	17.2007	900
-scaffold9	258	15.891	15.257	152
-scaffold10	1934	104.823	100.641	7516
-scaffold11	1922	23.9916	23.0344	1709.57
-scaffold12	1651	136.498	131.052	8355
-scaffold13	1834	360.757	346.363	24529.4
-scaffold14	580	6.13864	5.89373	132
-scaffold15	1539	4.8197	4.62741	275
-scaffold16	2302	26.1878	25.1429	2235
-scaffold17	543	1.98695	1.90767	40
-scaffold18	4121	25.4151	24.4011	3883
+# Name	Length	TPM	NumReads
+scaffold1	1016	549.279	20690
+scaffold2	1439	598.782	31945
+scaffold3	783	408.072	11846
+scaffold4	893	441.382	14613
+scaffold5	622	494.487	11403
+scaffold6	2073	4.77214	366.764
+scaffold7	1291	4.288	205.236
+scaffold8	1355	17.9155	900
+scaffold9	258	15.891	152
+scaffold10	1934	104.823	7516
+scaffold11	1922	23.9916	1709.57
+scaffold12	1651	136.498	8355
+scaffold13	1834	360.757	24529.4
+scaffold14	580	6.13864	132
+scaffold15	1539	4.8197	275
+scaffold16	2302	26.1878	2235
+scaffold17	543	1.98695	40
+scaffold18	4121	25.4151	3883

data/test/test_assembly.rb

@@ -28,9 +28,9 @@ class TestAssembly < Test::Unit::TestCase
           assert File.exist?("good.sorghum_100.fa"), "good output exists"
           assert File.exist?("bad.sorghum_100.fa"), "bad output"
           file_size = File.stat("good.sorghum_100.fa").size
-          assert_in_delta 81_000, file_size, 5000, "good file size"
+          assert_in_delta 86_119, file_size, 5000, "good file size"
           file_size = File.stat("bad.sorghum_100.fa").size
-          assert_in_delta 58_000, file_size, 5000, "bad file size"
+          assert_in_delta 53_000, file_size, 5000, "bad file size"
         end
       end
     end

data/test/test_bin.rb

@@ -116,6 +116,7 @@ class TestTransrateBin < Test::Unit::TestCase
       end
       assert_in_delta 137748, hash[:n_bases], 1000, "number of bases"
       assert_equal 1692, hash[:n50], "n50"
+      assert_equal 25006 + 223, hash[:fragments], "number of reads"
     end
 
     should "fail when one of multiple assemblies is missing" do

data/test/test_comp_metrics.rb

@@ -1,210 +1,381 @@
 require 'helper'
+require 'crb-blast'
+
+module Transrate
+  class ComparativeMetrics
+    attr_reader :assembly
+    attr_reader :reference
+    attr_reader :crbblast
+  end
+end
 
 module CRB_Blast
   class CRB_Blast
-    def change_hit(query_name, target_name, qstart, qend, tstart, tend, qlen, tlen)
-      hits = @reciprocals[query_name]
-      hits.each do |hit|
-        if hit.target == target_name
-          hit.qstart = qstart
-          hit.qend = qend
-          hit.tstart = tstart
-          hit.tend = tend
-          hit.qlen = qlen
-          hit.tlen = tlen
+    def add_missing
+      @missed.each do |query_id, missed|
+        missed.each do |hit|
+          @reciprocals[hit.query] ||= []
+          @reciprocals[hit.query] << hit
         end
       end
     end
+  end
+end
 
-    def add_hit(query_name, target_name, qstart, qend, tstart, tend, qlen, tlen)
-      @reciprocals[query_name] ||= []
-      list = Array.new(14)
-      list[0] = query_name
-      list[1] = target_name
-      list[6] = qstart
-      list[7] = qend
-      list[8] = tstart
-      list[9] = tend
-      list[12] = qlen
-      list[13] = tlen
-      @reciprocals[query_name] << Hit.new(list)
-    end
+class Tester
+  def self.testpath file
+    return File.join(File.dirname(__FILE__), 'data', file)
+  end
 
-    def remove_hit(query_name)
-      @reciprocals.delete(query_name)
-    end
+  def self.run_comp_metrics(query, target)
+    querypath = testpath(query)
+    targetpath = testpath(target)
+    @assembly = Transrate::Assembly.new(querypath)
+    @reference = Transrate::Assembly.new(targetpath)
+    @comp = Transrate::ComparativeMetrics.new(@assembly, @reference, 1)
+    @comp.run
+    return @comp
   end
 end
 
-class TestCompMetrics < Test::Unit::TestCase
+class TestCompMetrics2 < Test::Unit::TestCase
+
 
   context "ComparativeMetrics" do
 
     setup do
-      querypath = File.join(File.dirname(__FILE__),
-                            'data',
-                            'assembly.2.fa')
-      targetpath = File.join(File.dirname(__FILE__),
-                            'data',
-                            'Os.protein.2.fa')
-      @assembly = Transrate::Assembly.new(querypath)
-      @q_ids = @assembly.assembly.keys
-      @reference = Transrate::Assembly.new(targetpath)
-      @t_ids = @reference.assembly.keys
-      threads = 8
-      @comp = Transrate::ComparativeMetrics.new(@assembly, @reference, threads)
-    end
-
-    should "run metrics on assembly" do
-      Dir.mktmpdir do |tmpdir|
-        Dir.chdir tmpdir do
-          @comp.run
-          assert @comp.has_run
-        end
-      end
-    end
-
-    should "calculate reference coverage" do
-      crb = @comp.reciprocal_best_blast
-      # change the results so i know what i have
-      # qstart, qend, tstart, tend, qlen, tlen
-      #
-      # Q       |------------|
-      # T1 |-------------------------|
-      crb.change_hit("scaf_Os03g60760.1", "LOC_Os03g60760.1",
-                     1, 300, 101, 200, 300, 200) # 0.5
-      @reference["LOC_Os03g60760.1"].seq = "A"*200
-      #
-      # Q1   |----------|
-      # Q2                 |------------|
-      # T2  |-------------------------------|
-      crb.change_hit("scaf_Os10g39590.1", "LOC_Os10g39590.1",
-                     1, 150, 51, 100, 150, 200) # 0.25
-      crb.add_hit("scaf_Os10g39590.1", "LOC_Os10g39590.1",
-                     1, 150, 151, 200, 150, 200) # 0.25
-      @reference["LOC_Os10g39590.1"].seq = "A"*200
-      #
-      # adding first block [151..300]  scaf_Os09g38670.1
-      #    450 / 600.0
-      #    LOC_Os09g38670.1  0.75
-
-      #
-      #
-      # Q1           |-----------|
-      # Q2      |----------------------|
-      # T3  |-------------------------------|
-      crb.change_hit("scaf_Os09g38670.1", "LOC_Os09g38670.1",
-                     1, 150, 51, 100, 150, 200) # 0.25
-      crb.add_hit("scaf_Os09g38670.1", "LOC_Os09g38670.1",
-                     1, 450, 26, 175, 450, 200) # 0.75
-      @reference["LOC_Os09g38670.1"].seq = "A"*200
-
-      #
-      # Q1      |----------------------|
-      # Q2           |-----------|
-      # T4  |-------------------------------|
-      crb.change_hit("scaf_Os12g21920.1", "LOC_Os12g21920.1", #
-                     1, 450, 26, 175, 450, 200) # 0.75
-      crb.add_hit("scaf_Os12g21920.1", "LOC_Os12g21920.1",
-                     1, 150, 51, 100, 150, 200) # 0.25
-      @reference["LOC_Os12g21920.1"].seq = "A"*200
+    end
+
+    should "01 should run" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          comp = Tester.run_comp_metrics("test_contig_nc1.fa", "test_reference_nc1.fa")
+          assert comp.has_run
+        end
+      end
+    end
 
+    should "01-1n should get reference hits" do
+      # The reciprocals hash in crb blast has contig names as the key.
+      # In order to look up by the reference name we need to reverse this.
+      # Scan through the reciprocals and get this Hit objects and add them to
+      #   the @reference object for each reference sequence
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc1.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          assert_equal 1, comp.reference["reference1"].hits.size, "size of reference hits list"
+          assert_equal "contig1", comp.reference["reference1"].hits[0].query
+          assert_equal "reference1", comp.reference["reference1"].hits[0].target
+        end
+      end
+    end
 
-      #
-      # Q1    |------|
-      # Q2                      |--------|
-      # Q3       |-----------------|
-      # T5  |-------------------------------|
-      crb.change_hit("scaf_Os01g36294.1", "LOC_Os01g36294.1", #
-                     1, 300, 51, 100, 300, 400)
-      crb.add_hit("scaf_Os01g36294.1", "LOC_Os01g36294.1",
-                     1, 300, 200, 250, 300, 400)
-      crb.add_hit("scaf_Os01g36294.1", "LOC_Os01g36294.1",
-                     1, 300, 75, 225, 300, 400)
-      @reference["LOC_Os01g36294.1"].seq = "A"*400
+    should "01-1n get per contig reference coverage" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc1.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          assert_equal (2/3.0), comp.assembly["contig1"].reference_coverage
+        end
+      end
+    end
+
+    should "01-1a get per contig reference coverage on protein" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc1.fa")
+          target = Tester.testpath("test_reference_aa1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          assert_equal (2/3.0), comp.assembly["contig1"].reference_coverage
+        end
+      end
+    end
+
+    should "01e raise error because you can't have protein queries" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_reference_aa1.fa")
+          target = Tester.testpath("test_contig_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          assert_raise Transrate::TransrateError do
+            comp.per_query_contig_reference_coverage
+          end
+        end
+      end
+    end
+
+    should "02-2n calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+      # tmpdir = Dir.mktmpdir
+      # puts tmpdir
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc2.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          # answer should be 290/300.0
+          assert_equal 29/30.0, comp.reference["reference1"].reference_coverage
+        end
+      end
+    end
 
-      crb.change_hit("scaf_Os12g22750.1", "LOC_Os12g22750.1",
-                     1, 300, 101, 200, 300, 200) # 0.5 # 300/600
-      @reference["LOC_Os12g22750.1"].seq = "A"*200
+    should "02-3n calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc3.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          # answer should be 1.0000
+          assert_equal 1.00, comp.reference["reference1"].reference_coverage
+        end
+      end
+    end
 
-      crb.change_hit("scaf_Os02g55190.1", "LOC_Os02g55190.1",
-                     1, 300, 101, 200, 300, 200) # 0.5 # 300/600
-      @reference["LOC_Os02g55190.1"].seq = "A"*200
+    should "02-3a calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc3.fa")
+          target = Tester.testpath("test_reference_aa1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          # answer should be 1.0000
+          assert_equal 1.00, comp.reference["reference2"].reference_coverage
+        end
+      end
+    end
 
-      crb.change_hit("scaf_Os03g56500.1", "LOC_Os03g56500.1",
-                     1, 300, 101, 200, 300, 400) # 0.25
-      crb.change_hit("scaf_Os03g56500.2", "LOC_Os03g56500.1",
-                     1, 300, 201, 300, 300, 400) # 0.25 # 600 / 1200
-      @reference["LOC_Os03g56500.1"].seq = "A"*400
+    should "02-4n calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc4.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal 0.5, comp.reference["reference1"].reference_coverage
+        end
+      end
+    end
 
-      crb.change_hit("scaf_Os03g56724.1", "LOC_Os03g56724.1",
-                     1, 300, 101, 200, 300, 200) # 300/600 = 0.5
-      @reference["LOC_Os03g56724.1"].seq = "A"*200
+    should "02-4a calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc4.fa")
+          target = Tester.testpath("test_reference_aa1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal 0.5, comp.reference["reference2"].reference_coverage
+        end
+      end
+    end
 
-      crb.remove_hit("scaf_Os01g11360.1")
+    should "02-5a calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc5.fa")
+          target = Tester.testpath("test_reference_aa1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal (2/3.0), comp.reference["reference2"].reference_coverage
+        end
+      end
+    end
 
-      @reference["LOC_Os03g08270.3"].seq = "A"*200
-      @reference["LOC_Os10g41970.1"].seq = "A"*200
-      @reference["LOC_Os09g26780.1"].seq = "A"*200
-      @reference["LOC_Os12g24659.1"].seq = "A"*200
-      @reference["LOC_Os01g36410.1"].seq = "A"*200
-      @reference["LOC_Os12g22780.1"].seq = "A"*200
-      @reference["LOC_Os02g56470.1"].seq = "A"*200
-      @reference["LOC_Os03g30530.1"].seq = "A"*200
-      @reference["LOC_Os03g49850.1"].seq = "A"*200
-      @reference["LOC_Os01g11360.1"].seq = "A"*200
-      @reference["LOC_Os01g44140.1"].seq = "A"*200
+    should "02-5n calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc5.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal (2/3.0), comp.reference["reference1"].reference_coverage
+        end
+      end
+    end
 
-      assert_equal true, crb.target_is_prot, "target is prot"
-      assert_equal false, crb.query_is_prot, "query is prot"
-      # total_length of references should be 4400
+    should "02-6a calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc6.fa")
+          target = Tester.testpath("test_reference_aa1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal (1/3.0), comp.reference["reference2"].reference_coverage
+        end
+      end
+    end
 
-      cov = @comp.coverage crb
-      assert_equal 3600/13200.0, cov, "reference coverage"
+    should "02-6n calculate coverage for each reference sequence" do
+      Dir.mktmpdir do |tmpdir|
+        Dir.chdir tmpdir do
+          query = Tester.testpath("test_contig_nc6.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal (1/3.0), comp.reference["reference1"].reference_coverage
+        end
+      end
     end
 
-    should "calculate overlap amount" do
-      assert_equal 0.5, @comp.overlap_amount(201,500,101,400), "1"
-      assert_equal 0.5, @comp.overlap_amount(101,400,201,500), "2"
-      assert_equal 0.5, @comp.overlap_amount(201,400,101,500), "3"
-      assert_equal 0.5, @comp.overlap_amount(101,500,201,400), "4"
-    end
-
-    should "calculate number of contigs with crbblast hit" do
+    should "02-7a calculate coverage for each reference sequence" do
       Dir.mktmpdir do |tmpdir|
         Dir.chdir tmpdir do
-          @comp.run
-          assert_equal 11, @comp.comp_stats[:n_contigs_with_CRBB]
-          assert_equal 11/13.0, @comp.comp_stats[:p_contigs_with_CRBB]
+          query = Tester.testpath("test_contig_nc7.fa")
+          target = Tester.testpath("test_reference_aa1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal (1/3.0), comp.reference["reference2"].reference_coverage
         end
       end
     end
 
-    should "calculate number of reference sequences with crbblast hit" do
+    should "02-7n calculate coverage for each reference sequence" do
       Dir.mktmpdir do |tmpdir|
         Dir.chdir tmpdir do
-          @comp.run
-          assert_equal 10, @comp.comp_stats[:n_refs_with_CRBB]
-          assert_equal 0.5, @comp.comp_stats[:p_refs_with_CRBB]
+          query = Tester.testpath("test_contig_nc7.fa")
+          target = Tester.testpath("test_reference_nc1.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          crbblast.add_missing
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal (1/3.0), comp.reference["reference1"].reference_coverage
         end
       end
     end
 
-    should "calculate reference sequence coverage" do
-      # n&p of reference sequences covered to (25, 50, 75, 85, 95%)
-      # of their length by CRB-BLAST hit
+    should "03 calculate all metrics" do
       Dir.mktmpdir do |tmpdir|
         Dir.chdir tmpdir do
-          @comp.run
-          stats = @comp.comp_stats
-          assert_equal 10, stats[:cov25]
-          assert_equal 10, stats[:cov50]
-          assert_equal 7, stats[:cov75]
-          assert_equal 6, stats[:cov85]
-          assert_equal 3, stats[:cov95]
+          query = Tester.testpath("assembly.2.fa")
+          target = Tester.testpath("Os.protein.2.fa")
+          crbblast = CRB_Blast::CRB_Blast.new query, target
+          crbblast.run(1e-5, 1, true)
+          assembly = Transrate::Assembly.new(query)
+          reference = Transrate::Assembly.new(target)
+          comp = Transrate::ComparativeMetrics.new(assembly, reference, 1)
+          comp.get_reference_hits crbblast
+          comp.per_query_contig_reference_coverage
+          comp.per_target_contig_reference_coverage crbblast
+          assert_equal 11, comp.comp_stats[:CRBB_hits], "CRBB hits"
+          assert_equal 11, comp.comp_stats[:n_contigs_with_CRBB], "n_contigs_with_CRBB"
+          assert_equal 0.84615, comp.comp_stats[:p_contigs_with_CRBB].round(5), "p_contigs_with_CRBB"
+          assert_equal 0.55, comp.comp_stats[:rbh_per_reference], "rbh_per_reference"
+          assert_equal 10, comp.comp_stats[:n_refs_with_CRBB], "n_refs_with_CRBB"
+          assert_equal 0.5, comp.comp_stats[:p_refs_with_CRBB], "p_refs_with_CRBB"
+          assert_equal 10, comp.comp_stats[:cov25], "cov25"
+          assert_equal 10, comp.comp_stats[:cov50], "cov50"
+          assert_equal 7, comp.comp_stats[:cov75], "cov75"
+          assert_equal 6, comp.comp_stats[:cov85], "cov85"
+          assert_equal 3, comp.comp_stats[:cov95], "cov95"
+          assert_equal 0.5, comp.comp_stats[:p_cov25], "p_cov25"
+          assert_equal 0.5, comp.comp_stats[:p_cov50], "p_cov50"
+          assert_equal 0.35, comp.comp_stats[:p_cov75], "p_cov75"
+          assert_equal 0.3, comp.comp_stats[:p_cov85], "p_cov85"
+          assert_equal 0.15, comp.comp_stats[:p_cov95], "p_cov95"
+          assert_equal 0.37261, comp.comp_stats[:reference_coverage].round(5), "reference_coverage"
+
         end
       end
     end
 
+
+
   end
 end