bio-ngs 0.4.6.alpha.01 → 0.4.6.alpha.02

data/features/support/env.rb

@@ -0,0 +1,2 @@
+$LOAD_PATH << File.expand_path('../../../lib', __FILE__)
+require 'bio-ngs'

data/lib/bio-ngs.rb

@@ -24,9 +24,12 @@ require 'bio'
 require 'active_record'
 require 'sqlite3'
 
-# NGS classes
+#Generic classes
 require 'enumerable'
 require 'wrapper'
+require 'meta'
+
+# NGS classes
 require 'bio/ngs/utils'
 require 'bio/ngs/record'
 require 'bio/ngs/quality'
@@ -45,18 +48,29 @@ require 'bio/appl/ngs/sff_extract'
 require 'bio/appl/ngs/bcl2qseq' #TODO: FIX THIS BUGGY CODE in THOR TASK
 
 require 'bio/appl/ngs/cufflinks/iterators'
+require 'bio/appl/ngs/cufflinks/gtf/gtf_parser'
+require 'bio/appl/ngs/cufflinks/gtf/gtf'
+require 'bio/appl/ngs/cufflinks/gtf/transcript'
 require 'bio/appl/ngs/cufflinks'
 require 'bio/appl/ngs/samtools'
 require 'bio/appl/ngs/fastx'
 require 'bio/appl/ngs/blast'
 require 'bio/appl/ngs/bwa'
 
-# history 
+#Illumina utility for projects
+require 'bio/ngs/illumina/illumina'
+require 'bio/ngs/fs'
+
+# history
 Bio::Ngs::HISTORY_FILE = Dir.pwd+"/.task-history.yml"
 Bio::Ngs::Utils.extend_system_path
 
+
 # loading Tasks
-path = File.expand_path(File.dirname(__FILE__))
-Dir.glob(File.join(path,"tasks","*.thor")) do |thorfile|
-  Thor::Util.load_thorfile(thorfile)
+# TODO let the user define which tasks must be loaded, maybe a list of names
+if Bio::Ngs.const_defined?(:LoadBaseTasks) && Bio::Ngs.const_get(:LoadBaseTasks)==true
+  path = File.expand_path(File.dirname(__FILE__))
+  Dir.glob(File.join(path,"tasks","*.thor")) do |thorfile|
+    Thor::Util.load_thorfile(thorfile)
+  end
 end

data/lib/bio/appl/ngs/cufflinks.rb

@@ -7,10 +7,8 @@
 #
 #
 
-
-
 module Bio
-  module Ngs    
+  module Ngs
     module Cufflinks
       VERSION = "1.0.X"
       class << self
@@ -19,229 +17,258 @@ module Bio
         end
       end
 
+      module MarkCall
+        def mark
+          puts caller.first #elaborate the concept of tracking but not here please.
+        end
+      end
+
 
-      # cufflinks v1.3.0
-      # linked against Boost version 104000
-      # -----------------------------
-      # Usage:   cufflinks [options] <hits.sam>
-      # General Options:
-      #   -o/--output-dir              write all output files to this directory              [ default:     ./ ]
-      #   -p/--num-threads             number of threads used during analysis                [ default:      1 ]
-      #   --seed                       value of random number generator seed                 [ default:      0 ]
-      #   -G/--GTF                     quantitate against reference transcript annotations                      
-      #   -g/--GTF-guide               use reference transcript annotation to guide assembly                   
-      #   -M/--mask-file               ignore all alignment within transcripts in this file                     
-      #   -b/--frag-bias-correct       use bias correction - reference fasta required        [ default:   NULL ]
-      #   -u/--multi-read-correct      use 'rescue method' for multi-reads (more accurate)   [ default:  FALSE ]
-      #   --library-type               library prep used for input reads                     [ default:  below ]
-      # 
-      # Advanced Abundance Estimation Options:
-      #   -m/--frag-len-mean           average fragment length (unpaired reads only)         [ default:    200 ]
-      #   -s/--frag-len-std-dev        fragment length std deviation (unpaired reads only)   [ default:     80 ]
-      #   --upper-quartile-norm        use upper-quartile normalization                      [ default:  FALSE ]
-      #   --max-mle-iterations         maximum iterations allowed for MLE calculation        [ default:   5000 ]
-      #   --num-importance-samples     number of importance samples for MAP restimation      [ default:   1000 ]
-      #   --compatible-hits-norm       count hits compatible with reference RNAs only        [ default:  FALSE ]
-      #   --total-hits-norm            count all hits for normalization                      [ default:  TRUE  ]
-      # 
-      # Advanced Assembly Options:
-      #   -L/--label                   assembled transcripts have this ID prefix             [ default:   CUFF ]
-      #   -F/--min-isoform-fraction    suppress transcripts below this abundance level       [ default:   0.10 ]
-      #   -j/--pre-mrna-fraction       suppress intra-intronic transcripts below this level  [ default:   0.15 ]
-      #   -I/--max-intron-length       ignore alignments with gaps longer than this          [ default: 300000 ]
-      #   -a/--junc-alpha              alpha for junction binomial test filter               [ default:  0.001 ]
-      #   -A/--small-anchor-fraction   percent read overhang taken as 'suspiciously small'   [ default:   0.09 ]
-      #   --min-frags-per-transfrag    minimum number of fragments needed for new transfrags [ default:     10 ]
-      #   --overhang-tolerance         number of terminal exon bp to tolerate in introns     [ default:      8 ]
-      #   --max-bundle-length          maximum genomic length allowed for a given bundle     [ default:3500000 ]
-      #   --max-bundle-frags           maximum fragments allowed in a bundle before skipping [ default: 500000 ]
-      #   --min-intron-length          minimum intron size allowed in genome                 [ default:     50 ]
-      #   --trim-3-avgcov-thresh       minimum avg coverage required to attempt 3' trimming  [ default:     10 ]
-      #   --trim-3-dropoff-frac        fraction of avg coverage below which to trim 3' end   [ default:    0.1 ]
-      # 
-      # Advanced Reference Annotation Guided Assembly Options:
-      #   --no-faux-reads              disable tiling by faux reads                          [ default:  FALSE ]
-      #   --3-overhang-tolerance       overhang allowed on 3' end when merging with reference[ default:    600 ]
-      #   --intron-overhang-tolerance  overhang allowed inside reference intron when merging [ default:     30 ]
-      # 
-      # Advanced Program Behavior Options:
-      #   -v/--verbose                 log-friendly verbose processing (no progress bar)     [ default:  FALSE ]
-      #   -q/--quiet                   log-friendly quiet processing (no progress bar)       [ default:  FALSE ]
-      #   --no-update-check            do not contact server to check for update availability[ default:  FALSE ]
-      # 
-      # Supported library types:
-      #   ff-firststrand
-      #   ff-secondstrand
-      #   ff-unstranded
-      #   fr-firststrand
-      #   fr-secondstrand
-      #   fr-unstranded (default)
-      #   transfrags   
-      class Quantification
-
-        include Bio::Command::Wrapper
-        include Bio::Ngs::Cufflinks::Utils
-
-        set_program Bio::Ngs::Utils.binary("cufflinks")
-
-        add_option "output-dir", :type => :string, :aliases => '-o', :default => "./"
-        add_option "num-threads", :type => :numeric, :aliases => '-p', :default => 1
-        add_option "seed", :type => :numeric
-        add_option "GTF", :type => :string, :aliases => '-G'
-        add_option "GTF-guide", :type => :boolean, :aliases => '-g'
-        add_option "mask-file", :type => :string, :aliases => '-M'
-        add_option "frag-bias-correct", :type => :string, :aliases => '-b'
-        add_option "multi-read-correct", :type => :boolean, :aliases => '-u'
-        add_option "library-type", :type => :string
-        add_option "farg-len-mean", :type => :numeric, :aliases => '-m'#, :default => 200
-        add_option "frag-len-std-dev", :type => :numeric, :aliases => '-s'#, :default => 80
-        add_option "upper-quartile-norm", :type => :boolean
-        add_option "max-mle-iterations", :type => :numeric#, :default => 5000
-        add_option "num-importance-samples", :type => :numeric#, :default => 1000
-        add_option "compatible-hits-norm", :type => :boolean, :aliases => '-h'
-        add_option "total-hits-norm", :type => :boolean, :aliases => '-t'
-        add_option "label", :type => :string, :aliases => '-L', :default => "CUFF"
-        add_option "min-isoform-fraction", :type => :numeric, :aliases => '-F', :default => 0.15
-        add_option "pre-mrna-fraction", :type => :numeric, :aliases => '-j', :default => 0.15
-#deprecated        add_option "min-intron-fraction", :type => :numeric, :aliases => '-f', :default => 0.05
-        add_option "max-intron-length", :type => :numeric, :aliases => '-I', :default => 300000
-        add_option "junc-alpha", :type => :numeric, :aliases => '-a', :default => 0.01
-        add_option "small-anchor-fraction", :type => :numeric, :aliases => '-A', :default => 0.12
-        add_option "min-frags-per-transfrag", :type => :numeric#, :default => 10
-        add_option "overhang-tolerance", :type => :numeric#, :default => 8
-        add_option "max-bundle-length", :type => :numeric #, :default => 3500000
-        add_option "max-bundle-frags", :type => :numeric #, :default => 500000
-        add_option "min-intron-length", :type => :numeric#, :default => 50
-        add_option "trim-3-avgcov-thresh", :type => :numeric
-        add_option "trim-3-dropoff-frac", :type => :numeric
-        add_option "no-faux-reads", :type => :boolean
-        add_option "3-overhang-tolerance", :type => :numeric
-        add_option "intron-overhang-tolerance", :type => :numeric
-        add_option "verbose", :type => :boolean, :aliases => '-v'
-        add_option "quiet", :type => :boolean, :aliases => '-q'
-        
-#deprecated        add_option "min-map-qual", :type => :numeric, :aliases => '-Q', :default => 0
-#deprecated        add_option "reference-seq", :type => :string, :aliases => '-r'
-#deprecated        add_option "quartile-normalization", :type => :boolean, :aliases => '-N'
-
-        #TODO Check why with these defaults is not working properly
-
-
-        
-        add_iterator_for :genes
-        add_iterator_for :isoforms
-      end #Quantification  
-
-      # cuffdiff v1.3.0 (3022)
-      # -----------------------------
-      # Usage:   cuffdiff [options] <transcripts.gtf> <sample1_hits.sam> <sample2_hits.sam> [... sampleN_hits.sam]
-      #    Supply replicate SAMs as comma separated lists for each condition: sample1_rep1.sam,sample1_rep2.sam,...sample1_repM.sam
-      # General Options:
-      #   -o/--output-dir              write all output files to this directory              [ default:     ./ ]
-      #   --seed                       value of random number generator seed                 [ default:      0 ]
-      #   -T/--time-series             treat samples as a time-series                        [ default:  FALSE ]
-      #   -c/--min-alignment-count     minimum number of alignments in a locus for testing   [ default:   10 ]
-      #   --FDR                        False discovery rate used in testing                  [ default:   0.05 ]
-      #   -M/--mask-file               ignore all alignment within transcripts in this file  [ default:   NULL ]
-      #   -b/--frag-bias-correct       use bias correction - reference fasta required        [ default:   NULL ]
-      #   -u/--multi-read-correct      use 'rescue method' for multi-reads (more accurate)   [ default:  FALSE ]
-      #   -N/--upper-quartile-norm     use upper-quartile normalization                      [ default:  FALSE ]
-      #   -L/--labels                  comma-separated list of condition labels
-      #   -p/--num-threads             number of threads used during quantification          [ default:      1 ]
-      # 
-      # Advanced Options:
-      #   --library-type               Library prep used for input reads                     [ default:  below ]
-      #   -m/--frag-len-mean           average fragment length (unpaired reads only)         [ default:    200 ]
-      #   -s/--frag-len-std-dev        fragment length std deviation (unpaired reads only)   [ default:     80 ]
-      #   --num-importance-samples     number of importance samples for MAP restimation      [ default:   1000 ]
-      #   --num-bootstrap-samples      Number of bootstrap replications                      [ default:     20 ]
-      #   --bootstrap-fraction         Fraction of fragments in each bootstrap sample        [ default:    1.0 ]
-      #   --max-mle-iterations         maximum iterations allowed for MLE calculation        [ default:   5000 ]
-      #   --compatible-hits-norm       count hits compatible with reference RNAs only        [ default:   TRUE ]
-      #   --total-hits-norm            count all hits for normalization                      [ default:  FALSE ]
-      #   --poisson-dispersion         Don't fit fragment counts for overdispersion          [ default:  FALSE ]
-      #   -v/--verbose                 log-friendly verbose processing (no progress bar)     [ default:  FALSE ]
-      #   -q/--quiet                   log-friendly quiet processing (no progress bar)       [ default:  FALSE ]
-      #   --no-update-check            do not contact server to check for update availability[ default:  FALSE ]
-      #   --emit-count-tables          print count tables used to fit overdispersion         [ default:  FALSE ]
-      #   --max-bundle-frags           maximum fragments allowed in a bundle before skipping [ default: 500000 ]
-      # 
-      # Debugging use only:
-      #   --read-skip-fraction         Skip a random subset of reads this size               [ default:    0.0 ]
-      #   --no-read-pairs              Break all read pairs                                  [ default:  FALSE ]
-      #   --trim-read-length           Trim reads to be this long (keep 5' end)              [ default:   none ]
-      #   --cov-delta                  Maximum gap between bootstrap and IS                  [ default:   2.0  ]
-      # 
-      # Supported library types:
-      #   ff-firststrand
-      #   ff-secondstrand
-      #   ff-unstranded
-      #   fr-firststrand
-      #   fr-secondstrand
-      #   fr-unstranded (default)
-      #   transfrags
-      class Diff
-        include Bio::Command::Wrapper
-        include Bio::Ngs::Cufflinks::Utils
-
-        set_program Bio::Ngs::Utils.binary("cuffdiff")
-
-        add_option "output-dir", :type => :string, :aliases => '-o', :default => "./"
-        add_option "seed", :type => :numeric
-        add_option "time-series", :type => :boolean, :aliases => '-T'
-        add_option "min-alignment-count", :type => :numeric, :aliases => '-c'
-        add_option "FDR", :type => :numeric, :aliases => '-F'
-        #TODO:FIX        add_option "mask-file", :type => :string, :aliases => '-M'
-        #TODO:FIX        add_option "frag-bias-correct", :type => 
-        add_option "multi-read-correct", :type => :boolean, :aliases => '-u'
-        add_option "upper-quartile-norm", :type => :boolean, :aliases => 'N'
-        add_option "labels", :type => :array, :aliases => '-L'
-        add_option "num-threads", :type => :numeric, :aliases => '-p'
-        add_option "library-type", :type => :string, :aliases => '-l'
-        add_option "frag-len-mean", :type => :numeric, :aliases => '-m'
-        add_option "frag-len-std-dev", :type => :numeric, :aliases => '-s'
-        add_option "num-importance-samples", :type => :numeric, :aliases => '-i'
-        add_option "num-bootstrap-samples", :type => :numeric
-        add_option "bootstrap-fraction", :type => :numeric
-        add_option "max-mle-iterations", :type => :numeric, :aliases => '-e'
-        add_option "compatible-hits-norm", :type => :boolean, :aliases => '-h'
-        add_option "total-hits-norm", :type => :boolean, :aliases => '-t'
-        add_option "poisson-dispersion", :type => :boolean, :aliases => '-d'
-        add_option "verbose", :type => :boolean, :aliases => '-v'
-        add_option "quiet", :type => :boolean, :aliases => '-q'
-        add_option "no-update-check", :type => :boolean, :aliases => '-j'
-        add_option "emit-count-tables", :type => :boolean, :aliases => '-b'
-        add_option "max-bundle-frags", :type => :numeric
-        add_option "read-skip-fraction", :type => :numeric
-        add_option "no-read-pairs", :type => :numeric
-        add_option "trim-read-length", :type => :numeric
-        add_option "cov-delta", :type => :numeric
-        
-        #define iterators
-        add_iterator_for :genes
-        add_iterator_for :isoforms
-        add_iterator_for :cds
-        add_iterator_for :tss_groups
-
-        #Examples 
-        #Bio::Ngs::Cufflinks::Diff.isoforms("/Users/bonnalraoul/Desktop/RRep16giugno/DE_lane1-2-3-4-6-8/DE_lane1-2-3-4-6-8/isoform_exp.diff", "/Users/bonnalraoul/Desktop/RRep16giugno/COMPARE_lane1-2-3-4-6-8/COMPARE_lane1-2-3-4-6-8.combined.gtf",1.0,3,0.6,false,true)
-        #Bio::Ngs::Cufflinks::Diff.genes("/Users/bonnalraoul/Desktop/RRep16giugno/DE_lane1-2-3-4-6-8/DE_lane1-2-3-4-6-8/gene_exp.diff", "/Users/bonnalraoul/Desktop/RRep16giugno/COMPARE_lane1-2-3-4-6-8/COMPARE_lane1-2-3-4-6-8.combined.gtf",1.0,5,0.5,false,true)
-
-        class << self
-        
-          #Return the version of CuffDiff used to produce the output
-          def version(diff)
-            #cufflink_version_offset = Bio::Ngs::Cufflinks.version
-            f=File.open(diff,'r') 
-            header=f.readline #skip header
-            f.close            
-            cufflink_version_offset = case header.split.size
-            when 12
-              "0.9.X"
-            when 14
-              Bio::Ngs::Cufflinks.version #latest
-            end
-          end#version
+    # cufflinks v1.3.0
+    # linked against Boost version 104000
+    # -----------------------------
+    # Usage:   cufflinks [options] <hits.sam>
+    # General Options:
+    #   -o/--output-dir              write all output files to this directory              [ default:     ./ ]
+    #   -p/--num-threads             number of threads used during analysis                [ default:      1 ]
+    #   --seed                       value of random number generator seed                 [ default:      0 ]
+    #   -G/--GTF                     quantitate against reference transcript annotations
+    #   -g/--GTF-guide               use reference transcript annotation to guide assembly
+    #   -M/--mask-file               ignore all alignment within transcripts in this file
+    #   -b/--frag-bias-correct       use bias correction - reference fasta required        [ default:   NULL ]
+    #   -u/--multi-read-correct      use 'rescue method' for multi-reads (more accurate)   [ default:  FALSE ]
+    #   --library-type               library prep used for input reads                     [ default:  below ]
+    #
+    # Advanced Abundance Estimation Options:
+    #   -m/--frag-len-mean           average fragment length (unpaired reads only)         [ default:    200 ]
+    #   -s/--frag-len-std-dev        fragment length std deviation (unpaired reads only)   [ default:     80 ]
+    #   --upper-quartile-norm        use upper-quartile normalization                      [ default:  FALSE ]
+    #   --max-mle-iterations         maximum iterations allowed for MLE calculation        [ default:   5000 ]
+    #   --num-importance-samples     number of importance samples for MAP restimation      [ default:   1000 ]
+    #   --compatible-hits-norm       count hits compatible with reference RNAs only        [ default:  FALSE ]
+    #   --total-hits-norm            count all hits for normalization                      [ default:  TRUE  ]
+    #
+    # Advanced Assembly Options:
+    #   -L/--label                   assembled transcripts have this ID prefix             [ default:   CUFF ]
+    #   -F/--min-isoform-fraction    suppress transcripts below this abundance level       [ default:   0.10 ]
+    #   -j/--pre-mrna-fraction       suppress intra-intronic transcripts below this level  [ default:   0.15 ]
+    #   -I/--max-intron-length       ignore alignments with gaps longer than this          [ default: 300000 ]
+    #   -a/--junc-alpha              alpha for junction binomial test filter               [ default:  0.001 ]
+    #   -A/--small-anchor-fraction   percent read overhang taken as 'suspiciously small'   [ default:   0.09 ]
+    #   --min-frags-per-transfrag    minimum number of fragments needed for new transfrags [ default:     10 ]
+    #   --overhang-tolerance         number of terminal exon bp to tolerate in introns     [ default:      8 ]
+    #   --max-bundle-length          maximum genomic length allowed for a given bundle     [ default:3500000 ]
+    #   --max-bundle-frags           maximum fragments allowed in a bundle before skipping [ default: 500000 ]
+    #   --min-intron-length          minimum intron size allowed in genome                 [ default:     50 ]
+    #   --trim-3-avgcov-thresh       minimum avg coverage required to attempt 3' trimming  [ default:     10 ]
+    #   --trim-3-dropoff-frac        fraction of avg coverage below which to trim 3' end   [ default:    0.1 ]
+    #
+    # Advanced Reference Annotation Guided Assembly Options:
+    #   --no-faux-reads              disable tiling by faux reads                          [ default:  FALSE ]
+    #   --3-overhang-tolerance       overhang allowed on 3' end when merging with reference[ default:    600 ]
+    #   --intron-overhang-tolerance  overhang allowed inside reference intron when merging [ default:     30 ]
+    #
+    # Advanced Program Behavior Options:
+    #   -v/--verbose                 log-friendly verbose processing (no progress bar)     [ default:  FALSE ]
+    #   -q/--quiet                   log-friendly quiet processing (no progress bar)       [ default:  FALSE ]
+    #   --no-update-check            do not contact server to check for update availability[ default:  FALSE ]
+    #
+    # Supported library types:
+    #   ff-firststrand
+    #   ff-secondstrand
+    #   ff-unstranded
+    #   fr-firststrand
+    #   fr-secondstrand
+    #   fr-unstranded (default)
+    #   transfrags
+    class Quantification
+      include Bio::Command::Wrapper
+      include Bio::Ngs::Cufflinks::Utils
+
+      set_program Bio::Ngs::Utils.binary("cufflinks")
+
+      add_option "output-dir", :type => :string, :aliases => '-o', :default => "./"
+      add_option "num-threads", :type => :numeric, :aliases => '-p', :default => 1
+      add_option "seed", :type => :numeric
+      add_option "GTF", :type => :string, :aliases => '-G'
+      add_option "GTF-guide", :type => :string, :aliases => '-g'
+      add_option "mask-file", :type => :string, :aliases => '-M'
+      add_option "frag-bias-correct", :type => :string, :aliases => '-b'
+      add_option "multi-read-correct", :type => :boolean, :aliases => '-u'
+      add_option "library-type", :type => :string
+      add_option "farg-len-mean", :type => :numeric, :aliases => '-m'#, :default => 200
+      add_option "frag-len-std-dev", :type => :numeric, :aliases => '-s'#, :default => 80
+      add_option "upper-quartile-norm", :type => :boolean
+      add_option "max-mle-iterations", :type => :numeric#, :default => 5000
+      add_option "num-importance-samples", :type => :numeric#, :default => 1000
+      add_option "compatible-hits-norm", :type => :boolean, :aliases => '-h'
+      add_option "total-hits-norm", :type => :boolean, :aliases => '-t'
+      add_option "label", :type => :string, :aliases => '-L', :default => "CUFF"
+      add_option "min-isoform-fraction", :type => :numeric, :aliases => '-F', :default => 0.15
+      add_option "pre-mrna-fraction", :type => :numeric, :aliases => '-j', :default => 0.15
+      #deprecated        add_option "min-intron-fraction", :type => :numeric, :aliases => '-f', :default => 0.05
+      add_option "max-intron-length", :type => :numeric, :aliases => '-I', :default => 300000
+      add_option "junc-alpha", :type => :numeric, :aliases => '-a', :default => 0.01
+      add_option "small-anchor-fraction", :type => :numeric, :aliases => '-A', :default => 0.12
+      add_option "min-frags-per-transfrag", :type => :numeric#, :default => 10
+      add_option "overhang-tolerance", :type => :numeric#, :default => 8
+      add_option "max-bundle-length", :type => :numeric #, :default => 3500000
+      add_option "max-bundle-frags", :type => :numeric #, :default => 500000
+      add_option "min-intron-length", :type => :numeric#, :default => 50
+      add_option "trim-3-avgcov-thresh", :type => :numeric
+      add_option "trim-3-dropoff-frac", :type => :numeric
+      add_option "no-faux-reads", :type => :boolean
+      add_option "3-overhang-tolerance", :type => :numeric
+      add_option "intron-overhang-tolerance", :type => :numeric
+      add_option "verbose", :type => :boolean, :aliases => '-v'
+      add_option "quiet", :type => :boolean, :aliases => '-q'
+
+      #deprecated        add_option "min-map-qual", :type => :numeric, :aliases => '-Q', :default => 0
+      #deprecated        add_option "reference-seq", :type => :string, :aliases => '-r'
+      #deprecated        add_option "quartile-normalization", :type => :boolean, :aliases => '-N'
+
+      #TODO Check why with these defaults is not working properly
+
+
+
+      add_iterator_for :genes
+      add_iterator_for :isoforms
+
+
+
+
+    end #Quantification
+
+
+    class QuantificationDenovo  < Quantification
+      #set_program Bio::Ngs::Utils.binary("cufflinks")
+      delete_option "GTF"
+      #add_option "GTF-guide", :type => :string, :aliases => '-g'
+      #        add_alias "GTF", "GTF-guide"
+
+      # returns new trascripts from a gff3 file, it creates the file if doesn't exist
+      # gets only the brand new.
+      def get_new_transcripts(file=nil, type="gtf")
+        # TODO implement conversion to gff3
+        file||= "transcripts.#{type}"
+        # if type=="gtf"
+        # unless File.exists?(file)
+        #   to_gff3(File.dirname(File.absolute_path(file)))
+        # end
+        File.open()
+      end
+    end
+
+    # cuffdiff v1.3.0 (3022)
+    # -----------------------------
+    # Usage:   cuffdiff [options] <transcripts.gtf> <sample1_hits.sam> <sample2_hits.sam> [... sampleN_hits.sam]
+    #    Supply replicate SAMs as comma separated lists for each condition: sample1_rep1.sam,sample1_rep2.sam,...sample1_repM.sam
+    # General Options:
+    #   -o/--output-dir              write all output files to this directory              [ default:     ./ ]
+    #   --seed                       value of random number generator seed                 [ default:      0 ]
+    #   -T/--time-series             treat samples as a time-series                        [ default:  FALSE ]
+    #   -c/--min-alignment-count     minimum number of alignments in a locus for testing   [ default:   10 ]
+    #   --FDR                        False discovery rate used in testing                  [ default:   0.05 ]
+    #   -M/--mask-file               ignore all alignment within transcripts in this file  [ default:   NULL ]
+    #   -b/--frag-bias-correct       use bias correction - reference fasta required        [ default:   NULL ]
+    #   -u/--multi-read-correct      use 'rescue method' for multi-reads (more accurate)   [ default:  FALSE ]
+    #   -N/--upper-quartile-norm     use upper-quartile normalization                      [ default:  FALSE ]
+    #   -L/--labels                  comma-separated list of condition labels
+    #   -p/--num-threads             number of threads used during quantification          [ default:      1 ]
+    #
+    # Advanced Options:
+    #   --library-type               Library prep used for input reads                     [ default:  below ]
+    #   -m/--frag-len-mean           average fragment length (unpaired reads only)         [ default:    200 ]
+    #   -s/--frag-len-std-dev        fragment length std deviation (unpaired reads only)   [ default:     80 ]
+    #   --num-importance-samples     number of importance samples for MAP restimation      [ default:   1000 ]
+    #   --num-bootstrap-samples      Number of bootstrap replications                      [ default:     20 ]
+    #   --bootstrap-fraction         Fraction of fragments in each bootstrap sample        [ default:    1.0 ]
+    #   --max-mle-iterations         maximum iterations allowed for MLE calculation        [ default:   5000 ]
+    #   --compatible-hits-norm       count hits compatible with reference RNAs only        [ default:   TRUE ]
+    #   --total-hits-norm            count all hits for normalization                      [ default:  FALSE ]
+    #   --poisson-dispersion         Don't fit fragment counts for overdispersion          [ default:  FALSE ]
+    #   -v/--verbose                 log-friendly verbose processing (no progress bar)     [ default:  FALSE ]
+    #   -q/--quiet                   log-friendly quiet processing (no progress bar)       [ default:  FALSE ]
+    #   --no-update-check            do not contact server to check for update availability[ default:  FALSE ]
+    #   --emit-count-tables          print count tables used to fit overdispersion         [ default:  FALSE ]
+    #   --max-bundle-frags           maximum fragments allowed in a bundle before skipping [ default: 500000 ]
+    #
+    # Debugging use only:
+    #   --read-skip-fraction         Skip a random subset of reads this size               [ default:    0.0 ]
+    #   --no-read-pairs              Break all read pairs                                  [ default:  FALSE ]
+    #   --trim-read-length           Trim reads to be this long (keep 5' end)              [ default:   none ]
+    #   --cov-delta                  Maximum gap between bootstrap and IS                  [ default:   2.0  ]
+    #
+    # Supported library types:
+    #   ff-firststrand
+    #   ff-secondstrand
+    #   ff-unstranded
+    #   fr-firststrand
+    #   fr-secondstrand
+    #   fr-unstranded (default)
+    #   transfrags
+    class Diff
+      include Bio::Command::Wrapper
+      include Bio::Ngs::Cufflinks::Utils
+
+      set_program Bio::Ngs::Utils.binary("cuffdiff")
+
+      add_option "output-dir", :type => :string, :aliases => '-o', :default => "./"
+      add_option "seed", :type => :numeric
+      add_option "time-series", :type => :boolean, :aliases => '-T'
+      add_option "min-alignment-count", :type => :numeric, :aliases => '-c'
+      add_option "FDR", :type => :numeric, :aliases => '-F'
+      #TODO:FIX        add_option "mask-file", :type => :string, :aliases => '-M'
+      #TODO:FIX        add_option "frag-bias-correct", :type =>
+      add_option "multi-read-correct", :type => :boolean, :aliases => '-u'
+      add_option "upper-quartile-norm", :type => :boolean, :aliases => 'N'
+      add_option "labels", :type => :array, :aliases => '-L'
+      add_option "num-threads", :type => :numeric, :aliases => '-p'
+      add_option "library-type", :type => :string, :aliases => '-l'
+      add_option "frag-len-mean", :type => :numeric, :aliases => '-m'
+      add_option "frag-len-std-dev", :type => :numeric, :aliases => '-s'
+      add_option "num-importance-samples", :type => :numeric, :aliases => '-i'
+      add_option "num-bootstrap-samples", :type => :numeric
+      add_option "bootstrap-fraction", :type => :numeric
+      add_option "max-mle-iterations", :type => :numeric, :aliases => '-e'
+      add_option "compatible-hits-norm", :type => :boolean, :aliases => '-h'
+      add_option "total-hits-norm", :type => :boolean, :aliases => '-t'
+      add_option "poisson-dispersion", :type => :boolean, :aliases => '-d'
+      add_option "verbose", :type => :boolean, :aliases => '-v'
+      add_option "quiet", :type => :boolean, :aliases => '-q'
+      add_option "no-update-check", :type => :boolean, :aliases => '-j'
+      add_option "emit-count-tables", :type => :boolean, :aliases => '-b'
+      add_option "max-bundle-frags", :type => :numeric
+      add_option "read-skip-fraction", :type => :numeric
+      add_option "no-read-pairs", :type => :numeric
+      add_option "trim-read-length", :type => :numeric
+      add_option "cov-delta", :type => :numeric
+
+      #define iterators
+      add_iterator_for :genes
+      add_iterator_for :isoforms
+      add_iterator_for :cds
+      add_iterator_for :tss_groups
+
+      #Examples
+      #Bio::Ngs::Cufflinks::Diff.isoforms("/Users/bonnalraoul/Desktop/RRep16giugno/DE_lane1-2-3-4-6-8/DE_lane1-2-3-4-6-8/isoform_exp.diff", "/Users/bonnalraoul/Desktop/RRep16giugno/COMPARE_lane1-2-3-4-6-8/COMPARE_lane1-2-3-4-6-8.combined.gtf",1.0,3,0.6,false,true)
+      #Bio::Ngs::Cufflinks::Diff.genes("/Users/bonnalraoul/Desktop/RRep16giugno/DE_lane1-2-3-4-6-8/DE_lane1-2-3-4-6-8/gene_exp.diff", "/Users/bonnalraoul/Desktop/RRep16giugno/COMPARE_lane1-2-3-4-6-8/COMPARE_lane1-2-3-4-6-8.combined.gtf",1.0,5,0.5,false,true)
+
+      class << self
+
+        #Return the version of CuffDiff used to produce the output
+        def version(diff)
+          #cufflink_version_offset = Bio::Ngs::Cufflinks.version
+          f=File.open(diff,'r')
+          header=f.readline #skip header
+          f.close
+          cufflink_version_offset = case header.split.size
+          when 12
+            "0.9.X"
+          when 14
+            Bio::Ngs::Cufflinks.version #latest
+          end
+        end#version
 
 
         def offset_by_version(cufflinks_version)
@@ -252,7 +279,7 @@ module Bio
             1
           end
         end
-        
+
         #write a file with the information
         #See process_de for options available
         # Example: Bio::Ngs::Cufflinks::Diff.isoforms("/Users/bonnalraoul/Desktop/RRep16giugno/DEPopNormNOTh2s1NOTh17s1_lane1-2-3-4-6-8/isoform_exp.diff",
@@ -268,16 +295,16 @@ module Bio
         #See process_de for options available
         # Example: Bio::Ngs::Cufflinks::Diff.genes("/Users/bonnalraoul/Desktop/RRep16giugno/DEPopNormNOTh2s1NOTh17s1_lane1-2-3-4-6-8/gene_exp.diff",
         # "/Users/bonnalraoul/Desktop/RRep16giugno/COMPARE_PopNormNOTh2s1NOTh17s1_lane1-2-3-4-6-8/ComparepPopNormNOTh2s1NOTh17s1_lane1-2-3-4-6-8.combined.gtf",
-        # fold:0.5,min_samples:5,min_fpkm:0.5,z_scores:true, :regulated=>:up)        
+        # fold:0.5,min_samples:5,min_fpkm:0.5,z_scores:true, :regulated=>:up)
         def genes(diff, gtf, options={})
           process_de(diff, gtf, options) do |dict_info, diff_reference, gtf_kb, fpkm_values|
-#            puts diff_reference
-#            puts fpkm_values
+            #            puts diff_reference
+            #            puts fpkm_values
             # "#{dict_info[:winner].first}\t#{gtf_kb[diff_reference][:gene_name]}\t#{fpkm_values.join("\t")}"
-            #do not use th gtf kb 
+            #do not use th gtf kb
             "#{dict_info[:winner].first}\t#{dict_info[:gene_name]}\t#{fpkm_values.join("\t")}"
           end
-        end #genes          
+        end #genes
 
         private
         #Options hash
@@ -297,7 +324,7 @@ module Bio
           force_not_significative = options[:force_not_significative] || false
 
           #set up the kb if not available = pass an option with the path of the kb ?
-          gtf_kb = nil###### Bio::Ngs::Cufflinks::Compare.exists_kb?(gtf)  ? Bio::Ngs::Cufflinks::Compare.load_compare_kb(gtf) : Bio::Ngs::Cufflinks::Compare.build_compare_kb(gtf)            
+          gtf_kb = nil###### Bio::Ngs::Cufflinks::Compare.exists_kb?(gtf)  ? Bio::Ngs::Cufflinks::Compare.load_compare_kb(gtf) : Bio::Ngs::Cufflinks::Compare.build_compare_kb(gtf)
 
           #convert log2 fold value into natural log value (internally computed by cuffdiff)
           fold_log2 = fold
@@ -311,7 +338,7 @@ module Bio
 
           File.open(diff,'r') do |f|
             header=f.readline #skip header
-            
+
             test_id_idx = 0
             gene_name_idx = 2
             q_first_idx = 3 + cufflink_version_offset
@@ -320,76 +347,76 @@ module Bio
             fpkm_second_idx = 7 + cufflink_version_offset
             fold_idx = 8 + cufflink_version_offset
             significant_idx = 11 + cufflink_version_offset + (cufflink_version_offset==1 ? 1 : 0)
-            
+
             #Commenti:
             # per ogni riga del diff devo salvare il valore dei espressione di ogni test
             # quindi fpkm e se è significativo o meno
 
             f.each_line do |line|
               data=line.split
-              
+
               #fix comparison t-test, remove negative symbol e invert comparison: if fold change q1 vs q2 <0 abs(foldchange) & swaap q1,q2
-#              puts data[fold_idx].to_f
-#delete puts "#{data[fold_idx].to_f} #{data[fold_idx].to_f<0}"
+              #              puts data[fold_idx].to_f
+              #delete puts "#{data[fold_idx].to_f} #{data[fold_idx].to_f<0}"
               if data[fold_idx].to_f<0
                 data[fold_idx]=data[fold_idx][1..-1] #.sub(/-/,"")  remove the minus symbol from the number, the values q1, q2 and their fpkm will be reorganized into the data structure
-              else 
-#                puts "ciao"
+              else
+                #                puts "ciao"
                 data[fpkm_first_idx],data[fpkm_second_idx]=data[fpkm_second_idx],data[fpkm_first_idx]
                 data[q_first_idx],data[q_second_idx]=data[q_second_idx],data[q_first_idx]
-#delete                puts "#{q_first_idx},#{q_second_idx}"
+                #delete                puts "#{q_first_idx},#{q_second_idx}"
               end
-#delete                              puts "#{q_first_idx},#{q_second_idx}"
-#delete              puts "#{data[q_first_idx].to_sym} #{data[q_second_idx].to_sym}"
-#delete              puts "#{data[fpkm_first_idx].to_sym} #{data[fpkm_second_idx].to_sym}"
+              #delete                              puts "#{q_first_idx},#{q_second_idx}"
+              #delete              puts "#{data[q_first_idx].to_sym} #{data[q_second_idx].to_sym}"
+              #delete              puts "#{data[fpkm_first_idx].to_sym} #{data[fpkm_second_idx].to_sym}"
 
 
               #0 TCONS
               #4 name sample is the max diff for the item
               #5 name sample is the less diff for the item
-              #9 is the fold 
+              #9 is the fold
               dict_samples[data[q_first_idx]]
               dict_samples[data[q_second_idx]]
 
               #7 is the fpkm value of max pop/sample
               #8 is the fpkm value of min pop/sample
               k_reference = data[test_id_idx].to_sym #This can be TCONS if isoforms or XLOC if genes
-              
+
               unless dict[k_reference].key?(:values)
                 dict[k_reference][:values]={}
                 dict[k_reference][:gene_name]=data[gene_name_idx]
-              end              
+              end
               dict[k_reference][:values][data[q_first_idx].to_sym]=data[fpkm_first_idx].to_f unless dict[k_reference][:values].key?(data[q_first_idx].to_sym)
-              dict[k_reference][:values][data[q_second_idx].to_sym]=data[fpkm_second_idx].to_f unless dict[k_reference][:values].key?(data[q_second_idx].to_sym)              
-              
+              dict[k_reference][:values][data[q_second_idx].to_sym]=data[fpkm_second_idx].to_f unless dict[k_reference][:values].key?(data[q_second_idx].to_sym)
+
               if ((only_significative==true && data[significant_idx]=="yes") ||  ((data[significant_idx]=="yes"||force_not_significative) && data[fold_idx].to_f>=fold)) && data[fpkm_first_idx].to_f>=min_fpkm && data[fpkm_second_idx].to_f>=min_fpkm
-                
-               ###### puts data.join(" ") if k_reference == :XLOC_017497
+
+                ###### puts data.join(" ") if k_reference == :XLOC_017497
                 #TODO refactor: this can be done using lambda
                 k_sample = ""
                 if regulated==:up
 
                   k_sample = data[q_first_idx].to_sym
-#delete                  puts "#{k_sample} #{data[q_second_idx].to_sym}"
+                  #delete                  puts "#{k_sample} #{data[q_second_idx].to_sym}"
                   dict[k_reference][k_sample]<<data[q_second_idx].to_sym
-#delete                   puts "#{k_reference} #{q_first_idx}, #{q_second_idx}"
+                  #delete                   puts "#{k_reference} #{q_first_idx}, #{q_second_idx}"
                   k_sample
                 elsif regulated==:down
                   k_sample = data[q_second_idx].to_sym
                   dict[k_reference][k_sample]<<data[q_first_idx].to_sym
-                  k_sample                                    
+                  k_sample
                 end
-                
-             #delete   puts dict[k_reference].inspect if k_reference == :XLOC_017497
+
+                #delete   puts dict[k_reference].inspect if k_reference == :XLOC_017497
                 #delete puts dict.inspect
-                #store fpkm values as well for each pop/sample it should be 
+                #store fpkm values as well for each pop/sample it should be
                 if dict[k_reference][k_sample].size >= min_samples
                   (dict[k_reference][:winner] << k_sample).uniq!
                 end
-          #delete      puts dict[k_reference].inspect if k_reference == :XLOC_017497
+                #delete      puts dict[k_reference].inspect if k_reference == :XLOC_017497
               else
                 # k_reference = data[0].to_sym #This can be TCONS if isoforms or XLOC if genes
-                # 
+                #
                 # unless dict[k_reference].key?(:values)
                 #   dict[k_reference][:values]={}
                 # end
@@ -398,19 +425,19 @@ module Bio
                 # dict[k_reference][:values][data[q_second_idx].to_sym]=data[fpkm_second_idx].to_f unless dict[k_reference][:values].key?(data[q_second_idx].to_sym)
                 # #dict[k_reference][:values][data[4].to_sym]=data[7].to_f
               end
-#delete              puts dict[k_reference].inspect
-              
+              #delete              puts dict[k_reference].inspect
+
             end #each line
-            #example structure    
-            #{:TCONS_00086164=>{:q5=>[:q1, :q2, :q3, :q6]}, :TCONS_00086166=>{:q5=>[:q1, :q2, :q3, :q4, :q6]}  
+            #example structure
+            #{:TCONS_00086164=>{:q5=>[:q1, :q2, :q3, :q6]}, :TCONS_00086166=>{:q5=>[:q1, :q2, :q3, :q4, :q6]}
           end #file.open
 
           file_lines =[]
           dict.each do |diff_reference, dict_info|
-            
+
             if dict_info.key?(:winner)
               #puts dict_info.inspect
-              
+
               #BAD PERFORMANCES use lambda
               valz = case z_scores
               when true
@@ -419,13 +446,13 @@ module Bio
                 stdev = items.standard_deviation
                 items.map do |fpkm|
                   (fpkm-average)/stdev
-                end                                                    
+                end
               when false
                 dict_info[:values].sort.map{|sample| sample[1]}
               end #case
 
               #TODO generalize to isoforms and genes now only isoforms
-             # puts yield(dict_info, diff_reference, gtf_kb, valz) if diff_reference == :XLOC_017497
+              # puts yield(dict_info, diff_reference, gtf_kb, valz) if diff_reference == :XLOC_017497
               file_lines<< yield(dict_info, diff_reference, gtf_kb, valz) #fpkm_values
               #file_lines<<"#{dict_info[:winner].first}\t#{gtf_kb[diff_reference][:nearest_ref]}_#{gtf_kb[diff_reference][:gene_name]}\t#{valz.join("\t")}"
             else
@@ -451,42 +478,42 @@ module Bio
     # cuffcompare v1.0.2 (2335)
     # -----------------------------
     # Usage:
-    # cuffcompare [-r <reference_mrna.gtf>] [-R] [-T] [-V] [-s <seq_path>] 
-    #     [-o <outprefix>] [-p <cprefix>] 
+    # cuffcompare [-r <reference_mrna.gtf>] [-R] [-T] [-V] [-s <seq_path>]
+    #     [-o <outprefix>] [-p <cprefix>]
     #     {-i <input_gtf_list> | <input1.gtf> [<input2.gtf> .. <inputN.gtf>]}
-    # 
+    #
     #  Cuffcompare provides classification, reference annotation mapping and various
     #  statistics for Cufflinks transfrags.
     #  Cuffcompare clusters and tracks transfrags across multiple samples, writing
     #  matching transcripts (intron chains) into <outprefix>.tracking, and a GTF
-    #  file <outprefix>.combined.gtf containing a nonredundant set of transcripts 
+    #  file <outprefix>.combined.gtf containing a nonredundant set of transcripts
     #  across all input files (with a single representative transfrag chosen
     #  for each clique of matching transfrags across samples).
-    # 
+    #
     # Options:
     # -i provide a text file with a list of Cufflinks GTF files to process instead
     #    of expecting them as command line arguments (useful when a large number
     #    of GTF files should be processed)
-    # 
-    # -r  a set of known mRNAs to use as a reference for assessing 
+    #
+    # -r  a set of known mRNAs to use as a reference for assessing
     #     the accuracy of mRNAs or gene models given in <input.gtf>
-    # 
-    # -R  for -r option, reduce the set of reference transcripts to 
+    #
+    # -R  for -r option, reduce the set of reference transcripts to
     #     only those found to overlap any of the input loci
     # -M  discard (ignore) single-exon transfrags and reference transcripts
     # -N  discard (ignore) single-exon reference transcripts
-    # 
-    # -s  <seq_path> can be a multi-fasta file with all the genomic sequences or 
+    #
+    # -s  <seq_path> can be a multi-fasta file with all the genomic sequences or
     #     a directory containing multiple single-fasta files (one file per contig);
     #     lower case bases will be used to classify input transcripts as repeats
-    # 
+    #
     # -d  max distance (range) for grouping transcript start sites (100)
-    # -p  the name prefix to use for consensus transcripts in the 
+    # -p  the name prefix to use for consensus transcripts in the
     #     <outprefix>.combined.gtf file (default: 'TCONS')
     # -C  include the "contained" transcripts in the .combined.gtf file
     # -G  generic GFF input file(s) (do not assume Cufflinks GTF)
     # -T  do not generate .tmap and .refmap files for each input file
-    # -V  verbose processing mode (showing all GFF parsing warnings)      
+    # -V  verbose processing mode (showing all GFF parsing warnings)
     class Compare
       include Bio::Command::Wrapper
 
@@ -579,6 +606,145 @@ module Bio
         end #load_compare_kb
       end
     end #Compare
+
+    # cuffmerge takes two or more Cufflinks GTF files and merges them into a
+    # single unified transcript catalog.  Optionally, you can provide the script
+    # with a reference GTF, and the script will use it to attach gene names and other
+    # metadata to the merged catalog.
+
+    # Usage:
+    #     cuffmerge [Options] <assembly_GTF_list.txt>
+
+    # Options:
+    #     -h/--help                               Prints the help message and exits
+    #     -o                     <output_dir>     Directory where merged assembly will be written  [ default: ./merged_asm  ]
+    #     -g/--ref-gtf                            An optional "reference" annotation GTF.
+    #     -s/--ref-sequence      <seq_dir>/<seq_fasta> Genomic DNA sequences for the reference.
+    #     --min-isoform-fraction <0-1.0>          Discard isoforms with abundance below this       [ default:           0.05 ]
+    #     -p/--num-threads       <int>            Use this many threads to merge assemblies.       [ default:             1  ]
+    #     --keep-tmp                              Keep all intermediate files during merge
+    class Merge
+      include Bio::Command::Wrapper
+
+      set_program Bio::Ngs::Utils.binary("cuffmerge")
+
+      add_option "output-dir", :type => :string, :aliases => '-o', :default => "merged_asm"
+      add_option "ref-gtf", :type => :string, :aliases => '-g'
+      add_option "ref-sequence", :type => :string, :aliases => '-s'
+      add_option "min-isoform-fraction", :type => :numeric, :aliases => '-m'
+      add_option "num-threads", :type => :numeric, :aliases => '-p', :default => 6
+      add_option "keep-tmp", :type => :boolean, :aliases => 't'
+    end #Merge
+
+    # gffread <input_gff> [-g <genomic_seqs_fasta> | <dir>][-s <seq_info.fsize>]
+    #  [-o <outfile.gff>] [-t <tname>] [-r [[<strand>]<chr>:]<start>..<end> [-R]]
+    #  [-CTVNJMKQAFGUBHZWTOLE] [-w <exons.fa>] [-x <cds.fa>] [-y <tr_cds.fa>]
+    #  [-i <maxintron>]
+    #  Filters and/or converts GFF3/GTF2 records.
+    #  <input_gff> is a GFF file, use '-' if the GFF records will be given at stdin
+
+    #  Options:
+    #   -g  full path to a multi-fasta file with the genomic sequences
+    #       for all input mappings, OR a directory with single-fasta files
+    #       (one per genomic sequence, with file names matching sequence names)
+    #   -s  <seq_info.fsize> is a tab-delimited file providing this info
+    #       for each of the mapped sequences:
+    #       <seq-name> <seq-length> <seq-description>
+    #       (useful for -A option with mRNA/EST/protein mappings)
+    #   -i  discard transcripts having an intron larger than <maxintron>
+    #   -r  only show transcripts overlapping coordinate range <start>..<end>
+    #       (on chromosome/contig <chr>, strand <strand> if provided)
+    #   -R  for -r option, discard all transcripts that are not fully
+    #       contained within the given range
+    #   -U  discard single-exon transcripts
+    #   -C  coding only: discard mRNAs that have no CDS feature
+    #   -F  full GFF attribute preservation (all attributes are shown)
+    #   -G  only parse additional exon attributes from the first exon
+    #       and move them to the mRNA level (useful for GTF input)
+    #   -A  use the description field from <seq_info.fsize> and add it
+    #       as the value for a 'descr' attribute to the GFF record
+
+    #   -O  process also non-transcript GFF records (by default non-transcript
+    #       records are ignored)
+    #   -V  discard any mRNAs with CDS having in-frame stop codons
+    #   -H  for -V option, check and adjust the starting CDS phase
+    #       if the original phase leads to a translation with an
+    #       in-frame stop codon
+    #   -B  for -V option, single-exon transcripts are also checked on the
+    #       opposite strand
+    #   -N  discard multi-exon mRNAs that have any intron with a non-canonical
+    #       splice site consensus (i.e. not GT-AG, GC-AG or AT-AC)
+    #   -J  discard any mRNAs that either lack initial START codon
+    #       or the terminal STOP codon, or have an in-frame stop codon
+    #       (only print mRNAs with a fulll, valid CDS)
+
+    #   -M/--merge : cluster the input transcripts into loci, collapsing matching
+    #        transcripts (those with the same exact introns and fully contained)
+    #   -d <dupinfo> : for -M option, write collapsing info to file <dupinfo>
+    #   --cluster-only: same as --merge but without collapsing matching transcripts
+    #   -K  for -M option: also collapse shorter, fully contained transcripts
+    #       with fewer introns than the container
+    #   -Q  for -M option, remove the containment restriction:
+    #       (multi-exon transcripts will be collapsed if just their introns match,
+    #       while single-exon transcripts can partially overlap (80%))
+
+    #   -E  expose (warn about) duplicate transcript IDs and other potential
+    #       problems with the given GFF/GTF records
+    #   -Z  merge close exons into a single exon (for intron size<4)
+    #   -w  write a fasta file with spliced exons for each GFF transcript
+    #   -x  write a fasta file with spliced CDS for each GFF transcript
+    #   -W  for -w and -x options, also write for each fasta record the exon
+    #       coordinates projected onto the spliced sequence
+    #   -y  write a protein fasta file with the translation of CDS for each record
+    #   -L  Ensembl GTF to GFF3 conversion (implies -F; should be used with -m)
+    #   -m  <chr_replace> is a reference (genomic) sequence replacement table with
+    #       this format:
+    #       <original_ref_ID> <new_ref_ID>
+    #       GFF records on reference sequences that are not found among the
+    #       <original_ref_ID> entries in this file will be filtered out
+    #   -o  the "filtered" GFF records will be written to <outfile.gff>
+    #       (use -o- for printing to stdout)
+    #   -t  use <trackname> in the second column of each GFF output line
+    #   -T  -o option will output GTF format instead of GFF3
+    class GffRead
+      include Bio::Command::Wrapper
+
+      set_program Bio::Ngs::Utils.binary("gffread")
+      use_aliases
+
+      add_option "genomic-sequence", :type => :string, :aliases => '-g'
+      add_option "seq-info", :type => :string, :aliases => '-s'
+      add_option "discard-transcripts", :type => :numeric, :aliases => '-i'
+      add_option "orverlap-coords", :type => :string, :aliases => '-r'
+      add_option "discard-not-overlap", :type => :string, :aliases => '-R'
+      add_option "discard-single-exon", :type => :boolean, :aliases => '-U'
+      add_option "coding-only", :type => :boolean, :aliases => '-C'
+      add_option "full-attributes", :type => :boolean, :aliases => '-F'
+      add_option "partial-attributes", :type => :boolean, :aliases => '-G'
+      add_option "description-field", :type => :string, :aliases => '-A'
+      add_option "also-non-transcripts", :type => :boolean, :aliases => '-O'
+      add_option "discard-in-frame-stop", :type => :boolean, :aliases => '-V'
+      add_option "adjust-codon-phase", :type => :boolean, :aliases => '-H'
+      add_option "single-exon-check-opposite", :type => :boolean, :aliases => '-B'
+      add_option "discard-multi-exon", :type => :boolean, :aliases => '-N'
+      add_option "discard-wrong-codon", :type => :boolean, :aliases => '-J'
+      add_option "merge", :type => :boolean, :aliases => '-M'
+      add_option "output-collapsing", :type => :string, :aliases => '-d'
+      add_option "cluster-only", :type => :boolean, :aliases => '-c'
+      add_option "collaps-contained", :type => :boolean, :aliases => '-K'
+      add_option "remove-containment-restriction", :type => :boolean, :aliases => '-Q'
+      add_option "warnings", :type => :boolean, :aliases => '-E'
+      add_option "merge-close-exons", :type => :boolean, :aliases => '-Z'
+      add_option "write-exon-fasta", :type => :boolean, :aliases => '-w'
+      add_option "write-cds-fasta", :type => :boolean, :aliases => '-x'
+      add_option "write-coords", :type => :boolean, :aliases => '-W'
+      add_option "write-protein-fasta", :type => :boolean, :aliases => '-y'
+      add_option "ensembl-to-gff3", :type => :boolean, :aliases => '-L'
+      add_option "chr-replace", :type => :string, :aliases => '-m'
+      add_option "output", :type => :string, :aliases => '-o', :default => "outfile.gtf", :collapse=>true
+      add_option "track-name", :type => :string, :aliases => '-t'
+      add_option "output-gtf", :type => :boolean, :aliases => '-T'
+    end # GffRead
   end #Cufflinks
 end #Ngs
-end #Bio
+end #Bio