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1 <tool id="create_defusei_reference" name="Create DeFuse Reference" version="1.6.1">
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2 <description>create a defuse reference from Ensembl and UCSC sources</description>
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3 <requirements>
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4 <requirement type="package" version="0.6.1">defuse</requirement>
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5 <requirement type="package" version="0.1.18">samtools</requirement>
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6 <requirement type="package" version="1.0.0">bowtie</requirement>
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7 <requirement type="package" version="2013-05-09">gmap</requirement>
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8 <requirement type="package" version="latest">kent</requirement>
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9 </requirements>
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10 <command interpreter="command"> /bin/bash $shscript </command>
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11 <inputs>
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12 <param name="ensembl_genome_version" type="text" value="" label="Esembl Genome Version" help="Example: GRCh37"/>
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13 <param name="ensembl_version" type="integer" value="" label="Esembl Release Version" help="Example: 71"/>
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14 <param name="ucsc_genome_version" type="text" value="" label="UCSC Genome Version" help="Example: hg19"/>
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15 <param name="chromosomes" type="text" value="" label="Chromosomes" help="Example: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,X,Y,MT"/>
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16 <param name="mt_chromosome" type="text" value="MT" label="Mitochonrial Chromosome" />
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17 <param name="gene_sources" type="text" value="IG_C_gene,IG_D_gene,IG_J_gene,IG_V_gene,processed_transcript,protein_coding" label="Gene sources" />
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18 <param name="ig_gene_sources" type="text" value="IG_C_gene,IG_D_gene,IG_J_gene,IG_V_gene,IG_pseudogene" label="IG Gene sources" />
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19 <param name="rrna_gene_sources" type="text" value="Mt_rRNA,rRNA,rRNA_pseudogene" label="Ribosomal Gene sources" />
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20 </inputs>
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21 <outputs>
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22 <data format="txt" name="config_txt" label="${tool.name} on ${on_string}: config.txt"/>
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23 </outputs>
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24 <configfiles>
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25 <configfile name="defuse_config">
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26 #import ast
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27 #
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28 # Configuration file for defuse
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29 #
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30 # At a minimum, change all values enclused by []
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31 #
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32
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33 # Directory where the defuse code was unpacked
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34 ## Default location in the tool/defuse directory
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35 # source_directory = ${__root_dir__}/tools/defuse
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36 source_directory = __DEFUSE_PATH__
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37
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38 ensembl_version = $ensembl_version
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39 ensembl_genome_version = $ensembl_genome_version
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40 ucsc_genome_version = $ucsc_genome_version
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41
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42 # Directory where you want your dataset
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43 dataset_directory = $config_txt.extra_files_path
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44
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45 #raw
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46 # Input genome and gene models
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47 gene_models = $(dataset_directory)/Homo_sapiens.$(ensembl_genome_version).$(ensembl_version).gtf
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48 genome_fasta = $(dataset_directory)/Homo_sapiens.$(ensembl_genome_version).$(ensembl_version).dna.chromosomes.fa
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49
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50 # Repeat table from ucsc genome browser
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51 repeats_filename = $(dataset_directory)/repeats.txt
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52
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53 # EST info downloaded from ucsc genome browser
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54 est_fasta = $(dataset_directory)/est.fa
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55 est_alignments = $(dataset_directory)/intronEst.txt
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56
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57 # Unigene clusters downloaded from ncbi
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58 unigene_fasta = $(dataset_directory)/Hs.seq.uniq
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59 #end raw
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60
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61 # Paths to external tools
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62 samtools_bin = __SAMTOOLS_BIN__
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63 bowtie_bin = __BOWTIE_BIN__
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64 bowtie_build_bin = __BOWTIE_BUILD_BIN__
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65 blat_bin = __BLAT_BIN__
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66 fatotwobit_bin = __FATOTWOBIT_BIN__
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67 gmap_bin = __GMAP_BIN__
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68 gmap_setup_bin = __GMAP_SETUP_BIN__
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69 r_bin = __R_BIN__
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70 rscript_bin = __RSCRIPT_BIN__
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71
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72 #raw
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73 # Directory where you want your dataset
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74 gmap_index_directory = $(dataset_directory)/gmap
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75 #end raw
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76
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77 #raw
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78 # Dataset files
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79 dataset_prefix = $(dataset_directory)/defuse
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80 chromosome_prefix = $(dataset_prefix).dna.chromosomes
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81 exons_fasta = $(dataset_prefix).exons.fa
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82 cds_fasta = $(dataset_prefix).cds.fa
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83 cdna_regions = $(dataset_prefix).cdna.regions
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84 cdna_fasta = $(dataset_prefix).cdna.fa
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85 reference_fasta = $(dataset_prefix).reference.fa
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86 rrna_fasta = $(dataset_prefix).rrna.fa
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87 ig_gene_list = $(dataset_prefix).ig.gene.list
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88 repeats_regions = $(dataset_directory)/repeats.regions
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89 est_split_fasta1 = $(dataset_directory)/est.1.fa
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90 est_split_fasta2 = $(dataset_directory)/est.2.fa
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91 est_split_fasta3 = $(dataset_directory)/est.3.fa
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92 est_split_fasta4 = $(dataset_directory)/est.4.fa
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93 est_split_fasta5 = $(dataset_directory)/est.5.fa
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94 est_split_fasta6 = $(dataset_directory)/est.6.fa
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95 est_split_fasta7 = $(dataset_directory)/est.7.fa
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96 est_split_fasta8 = $(dataset_directory)/est.8.fa
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97 est_split_fasta9 = $(dataset_directory)/est.9.fa
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98
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99 # Fasta files with bowtie indices for prefiltering reads for concordantly mapping pairs
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100 prefilter1 = $(unigene_fasta)
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101
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102 # deFuse scripts and tools
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103 scripts_directory = $(source_directory)/scripts
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104 tools_directory = $(source_directory)/tools
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105 data_directory = $(source_directory)/data
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106 #end raw
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107
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108 #raw
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109 # Bowtie parameters
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110 bowtie_threads = 1
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111 bowtie_quals = --phred33-quals
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112 max_insert_size = 500
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113 #end raw
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114
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115 # Parameters for building the dataset
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116 chromosomes = $chromosomes
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117 mt_chromosome = $mt_chromosome
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118 gene_sources = $gene_sources
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119 ig_gene_sources = $ig_gene_sources
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120 rrna_gene_sources = $rrna_gene_sources
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121
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122 #raw
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123 # Blat sequences per job
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124 num_blat_sequences = 10000
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125
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126 # Minimum gene fusion range
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127 dna_concordant_length = 2000
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128
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129 # Trim length for discordant reads (split reads are not trimmed)
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130 discord_read_trim = 50
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131
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132 # Calculate extra annotations, fusion splice index and interrupted index
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133 calculate_extra_annotations = no
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134
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135 # Filtering parameters
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136 clustering_precision = 0.95
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137 span_count_threshold = 5
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138 percent_identity_threshold = 0.90
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139 split_min_anchor = 4
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140 splice_bias = 10
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141 positive_controls = $(data_directory)/controls.txt
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142 probability_threshold = 0.50
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143
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144 # Position density when calculating covariance
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145 covariance_sampling_density = 0.01
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146
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147 # Number of reads for each job in split
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148 reads_per_job = 1000000
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149
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150 # If you have command line 'mail' and wish to be notified
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151 mailto = andrew.mcpherson@gmail.com
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152
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153 # Remove temp files
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154 remove_job_files = yes
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155 remove_job_temp_files = yes
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156 #end raw
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157 </configfile>
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158 <configfile name="shscript">
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159 #!/bin/bash
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160 ## define some things for cheetah proccessing
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161 #set $ds = chr(36)
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162 #set $amp = chr(38)
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163 #set $gt = chr(62)
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164 #set $lt = chr(60)
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165 #set $echo_cmd = 'echo'
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166 ## Find the defuse.pl in the galaxy tool path
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167 #import Cheetah.FileUtils
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168 ## substitute pathnames into config file
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169 if `grep __DEFUSE_PATH__ $defuse_config ${gt} /dev/null`;then sed -i'.tmp' "s#__DEFUSE_PATH__#\${DEFUSE_PATH}#" $defuse_config; fi
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170 if `grep __SAMTOOLS_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} SAMTOOLS_BIN=`which samtools`;then sed -i'.tmp' "s#__SAMTOOLS_BIN__#\${SAMTOOLS_BIN}#" $defuse_config; fi
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171 if `grep __BOWTIE_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} BOWTIE_BIN=`which bowtie`;then sed -i'.tmp' "s#__BOWTIE_BIN__#\${BOWTIE_BIN}#" $defuse_config; fi
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172 if `grep __BOWTIE_BUILD_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} BOWTIE_BUILD_BIN=`which bowtie-build`;then sed -i'.tmp' "s#__BOWTIE_BUILD_BIN__#\${BOWTIE_BUILD_BIN}#" $defuse_config; fi
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173 if `grep __BLAT_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} BLAT_BIN=`which blat`;then sed -i'.tmp' "s#__BLAT_BIN__#\${BLAT_BIN}#" $defuse_config; fi
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174 if `grep __FATOTWOBIT_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} FATOTWOBIT_BIN=`which faToTwoBit`;then sed -i'.tmp' "s#__FATOTWOBIT_BIN__#\${FATOTWOBIT_BIN}#" $defuse_config; fi
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175 if `grep __GMAP_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} GMAP_BIN=`which gmap`;then sed -i'.tmp' "s#__GMAP_BIN__#\${GMAP_BIN}#" $defuse_config; fi
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176 if `grep __GMAP_SETUP_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} GMAP_SETUP_BIN=`which gmap_setup`;then sed -i'.tmp' "s#__GMAP_SETUP_BIN__#\${GMAP_SETUP_BIN}#" $defuse_config; fi
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177 if `grep __GMAP_INDEX_DIR__ $defuse_config ${gt} /dev/null` ${amp}${amp} GMAP_INDEX_DIR=`pwd`/gmap;then sed -i'.tmp' "s#__GMAP_INDEX_DIR__#\${GMAP_INDEX_DIR}#" $defuse_config; fi
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178 if `grep __R_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} R_BIN=`which R`;then sed -i'.tmp' "s#__R_BIN__#\${R_BIN}#" $defuse_config; fi
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179 if `grep __RSCRIPT_BIN__ $defuse_config ${gt} /dev/null` ${amp}${amp} RSCRIPT_BIN=`which Rscript`;then sed -i'.tmp' "s#__RSCRIPT_BIN__#\${RSCRIPT_BIN}#" $defuse_config; fi
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180
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181 ## copy config to output
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182 cp $defuse_config $config_txt
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183 ## make a data_dir and ln -s the input fastq
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184 mkdir -p $config_txt.extra_files_path
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185 ## run defuse.pl
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186 perl \${DEFUSE_PATH}/scripts/create_reference_dataset.pl -c $defuse_config
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187 </configfile>
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188 </configfiles>
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189
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190 <tests>
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191 </tests>
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192 <help>
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193 **DeFuse**
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194
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195 DeFuse_ is a software package for gene fusion discovery using RNA-Seq data. The software uses clusters of discordant paired end alignments to inform a split read alignment analysis for finding fusion boundaries. The software also employs a number of heuristic filters in an attempt to reduce the number of false positives and produces a fully annotated output for each predicted fusion.
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196
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197 Journal reference: http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.1001138
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198
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199 .. _DeFuse: http://sourceforge.net/apps/mediawiki/defuse/index.php?title=Main_Page
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200
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201 ------
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202
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203 **Inputs**
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204
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205 DeFuse requires 2 fastq files for paried reads, one with the left mate of the paired reads, and a second fastq with the the right mate of the paired reads (**with reads in the same order as in the first fastq dataset**).
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206
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207 If your fastq files have reads in different orders or include unpaired reads, you can preprocess them with **FASTQ interlacer** to create a single interlaced fastq dataset with only the paired reads and input that to **FASTQ de-interlacer** to separate the reads into a left fastq and right fastq.
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208
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209 DeFuse uses a Reference Dataset to search for gene fusions. The Reference Dataset is generated from the following sources in DeFuse_Version_0.4_:
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210 - genome_fasta from Ensembl
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211 - gene_models from Ensembl
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212 - repeats_filename from UCSC RepeatMasker rmsk.txt
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213 - est_fasta from UCSC
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214 - est_alignments from UCSC intronEst.txt
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215 - unigene_fasta from NCBI
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216
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217 .. _DeFuse_Version_0.4: http://sourceforge.net/apps/mediawiki/defuse/index.php?title=DeFuse_Version_0.4.2
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218
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219 ------
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220
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221 **Outputs**
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222
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223 The galaxy history will contain 5 outputs: the config.txt file that provides DeFuse with its parameters, the defuse.log which details what DeFuse has done and can be useful in determining any errors, and the 3 results files that defuse generates.
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224
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225 DeFuse generates 3 results files: results.txt, results.filtered.txt, and results.classify.txt. All three files have the same format, though results.classify.txt has a probability column from the application of the classifier to results.txt, and results.filtered.txt has been filtered according to the threshold probability as set in config.txt.
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226
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227 The file format is tab delimited with one prediction per line, and the following fields per prediction (not necessarily in this order):
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228
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229 - **Identification**
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230 - cluster_id : random identifier assigned to each prediction
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231 - library_name : library name given on the command line of defuse
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232 - gene1 : ensembl id of gene 1
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233 - gene2 : ensembl id of gene 2
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234 - gene_name1 : name of gene 1
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235 - gene_name2 : name of gene 2
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236 - **Evidence**
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237 - break_predict : breakpoint prediction method, denovo or splitr, that is considered most reliable
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238 - concordant_ratio : proportion of spanning reads considered concordant by blat
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239 - denovo_min_count : minimum kmer count across denovo assembled sequence
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240 - denovo_sequence : fusion sequence predicted by debruijn based denovo sequence assembly
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241 - denovo_span_pvalue : p-value, lower values are evidence the prediction is a false positive
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242 - gene_align_strand1 : alignment strand for spanning read alignments to gene 1
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243 - gene_align_strand2 : alignment strand for spanning read alignments to gene 2
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244 - min_map_count : minimum of the number of genomic mappings for each spanning read
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245 - max_map_count : maximum of the number of genomic mappings for each spanning read
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246 - mean_map_count : average of the number of genomic mappings for each spanning read
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247 - num_multi_map : number of spanning reads that map to more than one genomic location
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248 - span_count : number of spanning reads supporting the fusion
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249 - span_coverage1 : coverage of spanning reads aligned to gene 1 as a proportion of expected coverage
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250 - span_coverage2 : coverage of spanning reads aligned to gene 2 as a proportion of expected coverage
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251 - span_coverage_min : minimum of span_coverage1 and span_coverage2
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252 - span_coverage_max : maximum of span_coverage1 and span_coverage2
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253 - splitr_count : number of split reads supporting the prediction
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254 - splitr_min_pvalue : p-value, lower values are evidence the prediction is a false positive
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255 - splitr_pos_pvalue : p-value, lower values are evidence the prediction is a false positive
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256 - splitr_sequence : fusion sequence predicted by split reads
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257 - splitr_span_pvalue : p-value, lower values are evidence the prediction is a false positive
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258 - **Annotation**
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259 - adjacent : fusion between adjacent genes
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260 - altsplice : fusion likely the product of alternative splicing between adjacent genes
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261 - break_adj_entropy1 : di-nucleotide entropy of the 40 nucleotides adjacent to the fusion splice in gene 1
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262 - break_adj_entropy2 : di-nucleotide entropy of the 40 nucleotides adjacent to the fusion splice in gene 2
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263 - break_adj_entropy_min : minimum of break_adj_entropy1 and break_adj_entropy2
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264 - breakpoint_homology : number of nucleotides at the fusion splice that align equally well to gene 1 or gene 2
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265 - breakseqs_estislands_percident : maximum percent identity of fusion sequence alignments to est islands
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266 - cdna_breakseqs_percident : maximum percent identity of fusion sequence alignments to cdna
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267 - deletion : fusion produced by a genomic deletion
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268 - est_breakseqs_percident : maximum percent identity of fusion sequence alignments to est
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269 - eversion : fusion produced by a genomic eversion
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270 - exonboundaries : fusion splice at exon boundaries
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271 - expression1 : expression of gene 1 as number of concordant pairs aligned to exons
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272 - expression2 : expression of gene 2 as number of concordant pairs aligned to exons
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273 - gene_chromosome1 : chromosome of gene 1
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274 - gene_chromosome2 : chromosome of gene 2
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275 - gene_end1 : end position for gene 1
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276 - gene_end2 : end position for gene 2
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277 - gene_location1 : location of breakpoint in gene 1
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278 - gene_location2 : location of breakpoint in gene 2
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279 - gene_start1 : start of gene 1
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280 - gene_start2 : start of gene 2
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281 - gene_strand1 : strand of gene 1
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282 - gene_strand2 : strand of gene 2
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283 - genome_breakseqs_percident : maximum percent identity of fusion sequence alignments to genome
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284 - genomic_break_pos1 : genomic position in gene 1 of fusion splice / breakpoint
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285 - genomic_break_pos2 : genomic position in gene 2 of fusion splice / breakpoint
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286 - genomic_strand1 : genomic strand in gene 1 of fusion splice / breakpoint, retained sequence upstream on this strand, breakpoint is downstream
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287 - genomic_strand2 : genomic strand in gene 2 of fusion splice / breakpoint, retained sequence upstream on this strand, breakpoint is downstream
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288 - interchromosomal : fusion produced by an interchromosomal translocation
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289 - interrupted_index1 : ratio of coverage before and after the fusion splice / breakpoint in gene 1
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290 - interrupted_index2 : ratio of coverage before and after the fusion splice / breakpoint in gene 2
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291 - inversion : fusion produced by genomic inversion
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292 - orf : fusion combines genes in a way that preserves a reading frame
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293 - probability : probability produced by classification using adaboost and example positives/negatives (only given in results.classified.txt)
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294 - read_through : fusion involving adjacent potentially resulting from co-transcription rather than genome rearrangement
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295 - repeat_proportion1 : proportion of the spanning reads in gene 1 that span a repeat region
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296 - repeat_proportion2 : proportion of the spanning reads in gene 2 that span a repeat region
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297 - max_repeat_proportion : max of repeat_proportion1 and repeat_proportion2
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298 - splice_score : number of nucleotides similar to GTAG at fusion splice
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299 - num_splice_variants : number of potential splice variants for this gene pair
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300 - splicing_index1 : number of concordant pairs in gene 1 spanning the fusion splice / breakpoint, divided by number of spanning reads supporting the fusion with gene 2
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301 - splicing_index2 : number of concordant pairs in gene 2 spanning the fusion splice / breakpoint, divided by number of spanning reads supporting the fusion with gene 1
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302
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303
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304 **Example**
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305
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306 results.tsv::
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307
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308 cluster_id splitr_sequence splitr_count splitr_span_pvalue splitr_pos_pvalue splitr_min_pvalue adjacent altsplice break_adj_entropy1 break_adj_entropy2 break_adj_entropy_min break_predict breakpoint_homology breakseqs_estislands_percident cdna_breakseqs_percident concordant_ratio deletion est_breakseqs_percident eversion exonboundaries expression1 expression2 gene1 gene2 gene_align_strand1 gene_align_strand2 gene_chromosome1 gene_chromosome2 gene_end1 gene_end2 gene_location1 gene_location2 gene_name1 gene_name2 gene_start1 gene_start2 gene_strand1 gene_strand2 genome_breakseqs_percident genomic_break_pos1 genomic_break_pos2 genomic_strand1 genomic_strand2 interchromosomal interrupted_index1 interrupted_index2 inversion library_name max_map_count max_repeat_proportion mean_map_count min_map_count num_multi_map num_splice_variants orf read_through repeat_proportion1 repeat_proportion2 span_count span_coverage1 span_coverage2 span_coverage_max span_coverage_min splice_score splicing_index1 splicing_index2
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309 1169 GCTTACTGTATGCCAGGCCCCAGAGGGGCAACCACCCTCTAAAGAGAGCGGCTCCTGCCTCCCAGAAAGCTCACAGACTGTGGGAGGGAAACAGGCAGCAGGTGAAGATGCCAAATGCCAGGATATCTGCCCTGTCCTTGCTTGATGCAGCTGCTGGCTCCCACGTTCTCCCCAGAATCCCCTCACACTCCTGCTGTTTTCTCTGCAGGTTGGCAGAGCCCCATGAGGGCAGGGCAGCCACTTTGTTCTTGGGCGGCAAACCTCCCTGGGCGGCACGGAAACCACGGTGAGAAGGGGGCAGGTCGGGCACGTGCAGGGACCACGCTGCAGG|TGTACCCAACAGCTCCGAAGAGACAGCGACCATCGAGAACGGGCCATGATGACGATGGCGGTTTTGTCGAAAAGAAAAGGGGGAAATGTGGGGAAAAGCAAGAGAGATCAGATTGTTACTGTGTCTGTGTAGAAAGAAGTAGACATGGGAGACTCCATTTTGTTCTGTACTAAGAAAAATTCTTCTGCCTTGAGATTCGGTGACCCCACCCCCAACCCCGTGCTCTCTGAAACATGTGCTGTGTCCACTCAGGGTTGAATGGATTAAGGGCGGTGCGAGACGTGCTTT 2 0.000436307890680442 0.110748295953850 0.0880671602973091 N Y 3.19872427442695 3.48337348351473 3.19872427442695 splitr 0 0 0 0 Y 0 N N 0 0 ENSG00000105549 ENSG00000213753 + - 19 19 376013 59111168 intron upstream THEG AC016629.2 361750 59084870 - + 0 375099 386594 + - N 8.34107429512245 - N output_dir 82 0.677852348993289 40.6666666666667 1 11 1 N N 0.361271676300578 0.677852348993289 12 0.758602776578432 0.569678713445872 0.758602776578432 0.569678713445872 2 0.416666666666667 -
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310 3596 TGGGGGTTGAGGCTTCTGTTCCCAGGTTCCATGACCTCAGAGGTGGCTGGTGAGGTTATGACCTTTGCCCTCCAGCCCTGGCTTAAAACCTCAGCCCTAGGACCTGGTTAAAGGAAGGGGAGATGGAGCTTTGCCCCGACCCCCCCCCGTTCCCCTCACCTGTCAGCCCGAGCTGGGCCAGGGCCCCTAGGTGGGGAACTGGGCCGGGGGGCGGGCACAAGCGGAGGTGGTGCCCCCAAAAGGGCTCCCGGTGGGGTCTTGCTGAGAAGGTGAGGGGTTCCCGGGGCCGCAGCAGGTGGTGGTGGAGGAGCCAAGCGGCTGTAGAGCAAGGGGTGAGCAGGTTCCAGACCGTAGAGGCGGGCAGCGGCCACGGCCCCGGGTCCAGTTAGCTCCTCACCCGCCTCATAGAAGCGGGGTGGCCTTGCCAGGCGTGGGGGTGCTGCC|TTCCTTGGATGTGGTAGCCGTTTCTCAGGCTCCCTCTCCGGAATCGAACCCTGATTCCCCGTCACCCGTGGTCACCATGGTAGGCACGGCGACTACCATCGAAAGTTGATAGGGCAGACGTTCGAATGGGTCGTCGCCGCCACGGGGGGCGTGCGATCAGCCCGAGGTTATCTAGAGTCACCAAAGCCGCCGGCGCCCGCCCCCCGGCCGGGGCCGGAGAGGGGCTGACCGGGTTGGTTTTGATCTGATAAATGCACGCATCCCCCCCGCGAAGGGGGTCAGCGCCCGTCGGCATGTATTAGCTCTAGAATTACCACAGTTATCCAAGTAGGAGAGGAGCGAGCGACCAAAGGAACCATAACTGATTTAATGAGCCATTCGCAGTTTCACTGTACCGGCCGTGCGTACTTAGACATGCATGGCTTAATCTTTGAGACAAGCATATGCTACTGGCAGG 250 7.00711162298275e-72 0.00912124762512338 0.00684237452309549 N N 3.31745197152461 3.47233119514066 3.31745197152461 splitr 7 0.0157657657657656 0 0 N 0.0135135135135136 N N 0 0 ENSG00000156860 ENSG00000212932 - + 16 21 30682131 48111157 coding upstream FBRS RPL23AP4 30670289 48110676 + + 0.0157657657657656 30680678 9827473 - + Y - - N output_dir 2 1 1.11111111111111 1 1 1 N N 0 1 9 0.325530693397641 0.296465452915709 0.325530693397641 0.296465452915709 2 - -
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311
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312 </help>
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313 </tool>
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