annotate stringtie.xml @ 23:8eb8750070d4 draft

planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
author iuc
date Tue, 30 Apr 2024 11:53:08 +0000
parents ae618321f34a
children cbf488da3b2c
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1 <tool id="stringtie" name="StringTie" version="@TOOL_VERSION@+galaxy@VERSION_SUFFIX@" profile="22.05">
2
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2 <description>transcript assembly and quantification</description>
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3 <macros>
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4 <import>macros.xml</import>
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5 </macros>
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6 <xrefs>
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7 <xref type="bio.tools">stringtie</xref>
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8 </xrefs>
6
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9 <expand macro="requirements" />
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10 <expand macro="stdio" />
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11 <expand macro="version_command" />
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12 <command><![CDATA[
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13 #import re
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14 #set $compressed = ("bam","cram")
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15 mkdir -p ./special_de_output/sample1/ &&
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17 ## Get Guide GTF/GFF if selected
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18
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19 #if str($guide.use_guide) == 'yes':
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20 #if $guide.guide_source.guide_gff_select == "history":
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21 ln -s '$guide.guide_source.ref_hist' guide.gff &&
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22 #elif $guide.guide_source.guide_gff_select == "cached":
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23 ln -s '$guide.guide_source.ref_builtin.fields.path' guide.gff &&
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24 #end if
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25 #end if
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26
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27 #if $input_options.input_mode in ['short_reads','long_reads']:
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28 #if $input_options.input_bam.metadata.ftype == 'sam':
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29 samtools sort -@ \${GALAXY_SLOTS:-1} '$input_options.input_bam' -T "\${TMPDIR:-.}" | stringtie
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30 #else
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31 stringtie '$input_options.input_bam'
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32 #end if
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33 #if $input_options.input_mode == 'long_reads'
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34 -L
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35 -E $input_options.error_splice
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36 #end if
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37 #else
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38 #if $input_options.input_bam_short.file_ext in $compressed and $input_options.input_bam_long.file_ext in $compressed:
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39 stringtie '$input_options.input_bam_short' '$input_options.input_bam_long'
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40 #else if $input_options.input_bam_short.metadata.ftype == 'sam' and $input_options.input_bam_long.metadata.ftype in $compressed:
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41 samtools sort -@ \${GALAXY_SLOTS:-1} '$input_options.input_bam_short' -T "\${TMPDIR:-.}" -o short_sorted.sam
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42 && stringtie short_sorted.sam '$input_options.input_bam_long'
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43 #else if $input_options.input_bam_short.metadata.ftype in $compressed and $input_options.input_bam_long.metadata.ftype == 'sam':
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44 samtools sort -@ \${GALAXY_SLOTS:-1} '$input_options.input_bam_long' -T "\${TMPDIR:-.}" -o long_sorted.sam
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45 && stringtie'$input_options.input_bam_short' long_sorted.sam
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46 #else
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47 samtools sort -@ \${GALAXY_SLOTS:-1} '$input_options.input_bam_short' -T "\${TMPDIR:-.}" -o short_sorted.sam
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48 && samtools sort -@ \${GALAXY_SLOTS:-1} '$input_options.input_bam_long' -T "\${TMPDIR:-.}" -o long_sorted.sam
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49 && stringtie short_sorted.sam long_sorted.sam
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50 #end if
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51 -E $input_options.error_splice
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52 #end if
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53
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54 -o '$output_gtf'
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55 -p "\${GALAXY_SLOTS:-1}"
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56
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57 $rna_strandness
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58
12
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59 #if str($guide.use_guide) == 'yes':
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60 -G guide.gff
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61 #if $guide.coverage_file:
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62 -C '$coverage'
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63 #end if
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64 $guide.input_estimation
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65 #if $guide.special_outputs != 'no':
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66 -b ./special_de_output/sample1/
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67 #end if
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68 #end if
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69
12
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70 #if $adv.name_prefix:
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71 -l '$adv.name_prefix'
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72 #end if
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73 -f '$adv.fraction'
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74 -m '$adv.min_tlen'
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75 -a '$adv.min_anchor_len'
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76 -j '$adv.min_anchor_cov'
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77 -c '$adv.min_bundle_cov'
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78 -g '$adv.bdist'
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79 -M '$adv.bundle_fraction'
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80 $adv.disable_trimming
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81 $adv.multi_mapping
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82 #if $adv.point_features
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83 --ptf $adv.point_features
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84 #end if
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85 #if $adv.abundance_estimation:
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86 -A '$gene_abundance_estimation'
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87 #end if
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88 #if str($adv.omit_sequences).strip() != "":
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89 -x '$adv.omit_sequences'
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90 #end if
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91
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92 #if str($guide.use_guide) == 'yes':
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93 #if $guide.special_outputs.special_outputs_select == 'deseq2':
13
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94 #set escaped_element_identifier = re.sub('[^\w\-]', '_', str($input_bam.element_identifier))
12
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95 &&
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96 ln -s '$output_gtf' ./special_de_output/sample1/output.gtf
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97 &&
13
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98 TAB=\$(printf '\t')
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99 &&
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100 CR=\$(printf '\r')
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101 &&
12
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102 prepDE.py
13
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103 -i ./special_de_output/
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104 -g gene_counts.csv
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105 -t transcript_counts.csv
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106 -l $guide.special_outputs.read_length
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107 #if $guide.special_outputs.string:
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108 -s '$guide.special_outputs.string'
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109 #end if
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110 #if $guide.special_outputs.clustering:
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111 -c
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112 #if $guide.special_outputs.key:
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113 -k '$guide.special_outputs.key'
6
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114 #end if
13
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115 --legend '$legend'
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116 > /dev/null
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117 &&
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118 sed -i.bak -e "s/,/\${TAB}/g" -e "s/\${CR}//g" '$legend'
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119 #else
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120 > /dev/null
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121 #end if
12
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122
13
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123 ## Replace commas with tabs
12
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124 &&
13
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125 sed -i.bak -e "s/,/\${TAB}/g" -e "s/\${CR}//g" gene_counts.csv transcript_counts.csv
15
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126 ## Output header
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127 &&
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128 head -n 1 gene_counts.csv | sed -e 's/sample1/$escaped_element_identifier/' > '$gene_counts'
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129 &&
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130 head -n 1 transcript_counts.csv | sed -e 's/sample1/$escaped_element_identifier/' > '$transcript_counts'
13
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131 ## Sort count files on the first column
12
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132 &&
14
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133 tail -n +2 gene_counts.csv | sort -t"\${TAB}" -k1,1 >> '$gene_counts'
12
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134 &&
14
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135 tail -n +2 transcript_counts.csv | sort -t"\${TAB}" -k1,1 >> '$transcript_counts'
12
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136 #end if
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137 #end if
11
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138 ]]></command>
0
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139 <inputs>
18
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140 <conditional name="input_options">
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141 <param name="input_mode" type="select" label="Input options">
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142 <option value="short_reads">Short reads</option>
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143 <option value="long_reads">Long reads</option>
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144 <option value="mixed_reads">Mixed mode: short and long reads</option>
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145 </param>
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146 <when value="short_reads">
21
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147 <param name="input_bam" type="data" format="sam,bam,cram" label="Input short mapped reads" help="Input BAM/SAM/CRAM file containing the short reads you want to assemble into transcripts"/>
18
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148 </when>
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149 <when value="long_reads">
21
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150 <param name="input_bam" type="data" format="sam,bam,cram" label="Input long mapped reads" help="Input BAM/SAM/CRAM file containing the long reads you want to assemble into transcripts"/>
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151 <param name="error_splice" argument="-E" type="integer" min="0" max="50" value="25" label="Window around possibly erroneous splice sites" help="This option allows to define window around possibly erroneous splice sites from long reads to look out for correct splice sites. Default: 25"/>
18
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152 </when>
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153 <when value="mixed_reads">
21
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154 <param name="input_bam_short" type="data" format="sam,bam,cram" label="Input short mapped reads" help="Input BAM/SAM/CRAM file containing the short reads you want to assemble into transcripts"/>
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155 <param name="input_bam_long" type="data" format="sam,bam,cram" label="Input long mapped reads" help="Input BAM/SAM/CRAM file containing the long reads you want to assemble into transcripts"/>
23
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156 <param name="error_splice" argument="-E" type="integer" min="0" max="50" value="25" label="Window around possibly erroneous splice sites" help="This option allows to define window around possibly erroneous splice sites from long reads to look out for correct splice sites. Default: 25"/>
18
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157 </when>
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158 </conditional>
12
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159 <param name="rna_strandness" type="select" label="Specify strand information"
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160 help="Select 'Forward (FR)' if your reads are from a forward-stranded library, 'Reverse (RF)' if your reads are from a reverse-stranded library, or 'Unstranded' if your reads are not from a stranded library. See Help section below for more information. Default: Unstranded">
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161 <option value="" selected="true">Unstranded</option>
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162 <option value="--fr">Forward (FR)</option>
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163 <option value="--rf">Reverse (RF)</option>
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164 </param>
0
62d212192002 Imported from capsule None
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165 <conditional name="guide">
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166 <param name="use_guide" argument="-G" type="select" label="Use a reference file to guide assembly?" help="Use the reference annotation file (in GTF or GFF3 format) to guide the assembly process. The output will include expressed reference transcripts as well as any novel transcripts that are assembled. This option is required by option -e (Use Reference transcripts only), see below.">
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167 <option value="yes">Use reference GTF/GFF3</option>
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168 <option value="no" selected="true" >Do not use reference GTF/GFF3</option>
0
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169 </param>
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170 <when value="no" />
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171 <when value="yes">
12
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172 <conditional name="guide_source">
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173 <param name="guide_gff_select" type="select" label="Reference file">
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174 <option value="cached" selected="true">Use a built-in file</option>
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175 <option value="history">Use a file from history</option>
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176 </param>
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177 <when value="cached">
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178 <param name="ref_builtin" type="select" label="Use a built-in GTF" help="If the GTF file for your transcriptome of interest is not listed, contact your Galaxy administrator">
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179 <options from_data_table="gene_sets">
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180 <filter type="sort_by" column="2"/>
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181 <validator type="no_options" message="No GTF file is available."/>
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182 </options>
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183 </param>
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184 </when>
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185 <when value="history">
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186 <param name="ref_hist" type="data" format="gtf,gff3" label="GTF/GFF3 dataset to guide assembly"/>
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187 </when>
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188 </conditional>
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189 <param name="input_estimation" argument="-e" type="boolean" truevalue="-e" falsevalue="" checked="false" label="Use Reference transcripts only?" help="Limit the processing of read alignments to only estimate and output the assembled transcripts matching the reference transcripts given with the -G option. With this option, read bundles with no reference transcripts (novel transcripts) will be entirely skipped, which may provide a considerable speed boost when the given set of reference transcripts is limited to a set of target genes, for example. Default: No"/>
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190 <conditional name="special_outputs">
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191 <param name="special_outputs_select" type="select" label="Output files for differential expression?" help="Select to output additional files that can be used with Ballgown or DESeq2/edgeR. See Help section below for more information">
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192 <option value="ballgown">Ballgown</option>
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193 <option value="deseq2">DESeq2/edgeR/limma-voom</option>
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194 <option value="no" selected="true">No additional output</option>
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195 </param>
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196 <when value="ballgown" />
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197 <when value="deseq2">
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198 <param name="read_length" argument="--length" type="integer" min="0" value="75" label="Specify the average read length" help="Default: 75"/>
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199 <param name="clustering" argument="--cluster" type="boolean" truevalue="--cluster" falsevalue="" checked="false" label="Cluster overlapping genes" help="Choose whether to cluster genes with different gene IDs that overlap. Transcripts containing the geneID prefix will be ignored. Default: No"/>
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200 <param argument="--string" type="text" label="Prefix used for transcripts" help="If a different prefix was used for geneIDs assigned by StringTie than the default, specify it here. Only letters and numbers will be retained in this field. Default: MSTRG">
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201 <sanitizer>
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202 <valid initial="string.letters,string.digits"></valid>
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203 </sanitizer>
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204 </param>
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205 <param argument="--key" type="text" label="Prefix for clustering" help="If clustering, what prefix to use for geneIDs assigned by this script. Only letters and numbers will be retained in this field. Default: prepG">
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206 <sanitizer>
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207 <valid initial="string.letters,string.digits"></valid>
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208 </sanitizer>
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209 </param>
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210 </when>
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211 <when value="no" />
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212 </conditional>
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213 <param name="coverage_file" argument="-C" type="boolean" truevalue="-C" falsevalue="" checked="false" label="Output coverage file?" help="If StringTie is run with this option (requires -G), it returns a file with all the transcripts in the reference annotation that are fully covered, end to end, by reads. The output format is a GTF file as described below. Each line of the GTF corresponds to a gene or transcript in the reference annotation."/>
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214 </when>
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215 </conditional>
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216 <section name="adv" title="Advanced Options">
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217 <param name="abundance_estimation" argument="-A" type="boolean" truevalue="-A" falsevalue="" checked="false" label="Output gene abundance estimation file?" help="If selected, gene abundances will be reported in a tab-delimited file, see below for more information"/>
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218 <param name="omit_sequences" argument="-x" type="text" value="" label="Do not assemble any transcripts on these reference sequence(s)" help="Ignore all read alignments (and thus do not attempt to perform transcript assembly) on the specified reference sequences. This parameter can be a single reference sequence name (e.g. chrM) or a comma-delimited list of sequence names (e.g. chrM,chrX,chrY). This can speed up StringTie especially in the case of excluding the mitochondrial genome, whose genes may have very high coverage in some cases, even though they may be of no interest for a particular RNA-Seq analysis. The reference sequence names are case sensitive, they must match identically the names of chromosomes/contigs of the target genome against which the RNA-Seq reads were aligned in the first place."/>
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219 <param name="name_prefix" argument="-l" type="text" label="Name prefix for output transcripts" help="This prefix will be added to the name of the transcripts that are output. Only letters and numbers will be retained in this field. Default: STRG">
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220 <sanitizer>
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221 <valid initial="string.letters,string.digits"></valid>
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222 </sanitizer>
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223 </param>
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224 <param name="fraction" argument="-f" type="float" min="0.0" max="1.0" value="0.01" label="Minimum isoform fraction" help="Sets the minimum isoform abundance of the predicted transcripts as a fraction of the most abundant transcript assembled at a given locus. Lower abundance transcripts are often artifacts of incompletely spliced precursors of processed transcripts. Default: 0.05"/>
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225 <param name="min_tlen" argument="-m" type="integer" min="0" value="200" label="Minimum assembled transcript length" help="Sets the minimum length allowed for the predicted transcripts. Default: 200"/>
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226 <param name="min_anchor_len" argument="-a" type="integer" min="0" value="10" label="Minimum anchor length for junctions" help="Junctions that don't have spliced reads that align across them with at least this amount of bases on both sides are filtered out. Default: 10"/>
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227 <param name="min_anchor_cov" argument="-j" type="integer" min="0" value="1" label="Minimum junction coverage" help="There should be at least this many spliced reads that align across a junction (i.e. junction coverage). This number can be fractional, since some reads align in more than one place. A read that aligns in n places will contribute 1/n to the junction coverage. Default: 1"/>
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228 <param name="min_bundle_cov" argument="-c" type="integer" min="0" value="1" label="Minimum bundle reads per bp coverage to consider for assembly" help="Sets the minimum read coverage allowed for the predicted transcripts. A transcript with a lower coverage than this value is not shown in the output. Default: 2"/>
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229 <param name="bdist" argument="-g" type="integer" min="0" value="50" label="Gap between read mappings triggering a new bundle" help="Minimum locus gap separation value. Reads that are mapped closer than this distance are merged together in the same processing bundle. Default: 50 (bp)"/>
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230 <param name="bundle_fraction" argument="-M" type="float" min="0.0" max="1.0" value="1" label="Fraction of bundle allowed to be covered by multi-hit reads" help="Sets the maximum fraction of muliple-location-mapped reads that are allowed to be present at a given locus. Default: 0.95"/>
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231 <param name="disable_trimming" argument="-t" type="boolean" truevalue="-t" falsevalue="" checked="false" label="Disable trimming of predicted transcripts based on coverage" help="This parameter disables trimming at the ends of the assembled transcripts. By default StringTie adjusts the predicted transcript's start and/or stop coordinates based on sudden drops in coverage of the assembled transcript. Default: No"/>
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232 <param name="multi_mapping" argument="-u" type="boolean" truevalue="-u" falsevalue="" checked="false" label="Disable multi-mapping correction" help="Default: No"/>
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233 <param name="point_features" type="data" format="tabular" optional="true" label="Input point-features dataset" help="Loads a list of point-features from a text feature file to guide the transcriptome assembly. Accepted point features are transcription start sites (TSS) and polyadenylation sites (CPAS). There are four tab-delimited columns in the feature file. The first three define the location of the point feature on the cromosome (sequence name, coordinate and strand), and the last is the type of the feature (TSS or CPAS)."/>
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234 </section>
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235 </inputs>
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236 <outputs>
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237 <data name="output_gtf" format="gtf" label="${tool.name} on ${on_string}: Assembled transcripts"/>
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238 <data name="gene_abundance_estimation" format="tabular" label="${tool.name} on ${on_string}: Gene abundance estimates">
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239 <filter>adv['abundance_estimation']</filter>
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240 </data>
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241 <data name="coverage" format="gtf" label="${tool.name} on ${on_string}: Coverage">
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242 <filter>guide['use_guide'] == 'yes' and guide['coverage_file'] is True </filter>
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243 </data>
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244 <data name="exon_expression" format="tabular" from_work_dir="special_de_output/sample1/e_data.ctab"
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245 label="${tool.name} on ${on_string}: exon-level expression measurements">
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246 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'ballgown'</filter>
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247 </data>
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248 <data name="intron_expression" format="tabular" from_work_dir="special_de_output/sample1/i_data.ctab"
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249 label="${tool.name} on ${on_string}: intron-level expression measurements">
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250 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'ballgown'</filter>
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251 </data>
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252 <data name="transcript_expression" format="tabular" from_work_dir="special_de_output/sample1/t_data.ctab"
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253 label="${tool.name} on ${on_string}: transcript-level expression measurements">
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254 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'ballgown'</filter>
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255 </data>
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256 <data name="exon_transcript_mapping" format="tabular" from_work_dir="special_de_output/sample1/e2t.ctab"
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257 label="${tool.name} on ${on_string}: exon to transcript mapping">
10
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258 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'ballgown'</filter>
1
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parents: 0
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259 </data>
11
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260 <data name="intron_transcript_mapping" format="tabular" from_work_dir="special_de_output/sample1/i2t.ctab"
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261 label="${tool.name} on ${on_string}: intron to transcript mapping">
10
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262 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'ballgown'</filter>
1
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parents: 0
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263 </data>
12
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264 <data name="gene_counts" format="tabular" from_work_dir="special_de_output/sample1/gene_counts.tsv" label="${tool.name} on ${on_string}: Gene counts">
10
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265 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'deseq2'</filter>
1
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parents: 0
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266 </data>
12
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267 <data name="transcript_counts" format="tabular" from_work_dir="special_de_output/sample1/transcript_counts.tsv" label="${tool.name} on ${on_string}: Transcript counts">
10
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268 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'deseq2'</filter>
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269 </data>
12
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270 <data name="legend" format="tabular" from_work_dir="special_de_output/sample1/legend.tsv" label="${tool.name} on ${on_string}: legend">
10
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271 <filter>guide['use_guide'] == 'yes' and guide['special_outputs']['special_outputs_select'] == 'deseq2' and guide['special_outputs']['clustering'] is True</filter>
1
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272 </data>
0
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273 </outputs>
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diff changeset
274 <tests>
12
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275 <!--Ensure default GTF output works -->
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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276 <test expect_num_outputs="1">
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277 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
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278 <param name="input_mode" value="short_reads"/>
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279 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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280 </conditional>
23
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281 <output name="output_gtf" file="stringtie_out1_re.gtf" ftype="gtf" lines_diff="2"/>
0
62d212192002 Imported from capsule None
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282 </test>
12
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parents: 11
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283 <!--Ensure fraction option works -->
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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284 <test expect_num_outputs="1">
18
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parents: 17
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285 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
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286 <param name="input_mode" value="short_reads"/>
23
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287 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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288 </conditional>
23
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289 <param name="fraction" value="0.17"/>
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290 <output name="output_gtf" file="stringtie_out2_re.gtf" ftype="gtf" lines_diff="2"/>
1
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parents: 0
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291 </test>
12
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292 <!--Ensure guide option works -->
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293 <test expect_num_outputs="1">
18
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294 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
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295 <param name="input_mode" value="short_reads"/>
23
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296 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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297 </conditional>
23
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parents: 22
diff changeset
298 <param name="use_guide" value="yes"/>
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299 <param name="guide_gff_select" value="history"/>
12
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300 <param name="ref_hist" ftype="gtf" value="stringtie_in.gtf" />
23
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301 <output name="output_gtf" file="stringtie_out3_re.gtf" ftype="gtf" lines_diff="2"/>
12
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diff changeset
302 </test>
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parents: 11
diff changeset
303 <!--Ensure guide with fraction works -->
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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parents: 11
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304 <test expect_num_outputs="1">
18
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parents: 17
diff changeset
305 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
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306 <param name="input_mode" value="short_reads"/>
23
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diff changeset
307 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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308 </conditional>
23
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diff changeset
309 <param name="use_guide" value="yes"/>
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diff changeset
310 <param name="guide_gff_select" value="history"/>
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parents: 22
diff changeset
311 <param name="ref_hist" ftype="gtf" value="stringtie_in.gtf"/>
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parents: 22
diff changeset
312 <param name="fraction" value="0.17"/>
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parents: 22
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313 <output name="output_gtf" file="stringtie_out4_re.gtf" ftype="gtf" lines_diff="2"/>
1
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parents: 0
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314 </test>
12
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parents: 11
diff changeset
315 <!--Ensure coverage and output for Ballgown works -->
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316 <test expect_num_outputs="7">
18
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317 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
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318 <param name="input_mode" value="short_reads"/>
23
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319 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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320 </conditional>
23
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parents: 22
diff changeset
321 <param name="use_guide" value="yes"/>
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parents: 22
diff changeset
322 <param name="guide_gff_select" value="history"/>
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parents: 22
diff changeset
323 <param name="ref_hist" ftype="gtf" value="stringtie_in.gtf"/>
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diff changeset
324 <param name="special_outputs_select" value="ballgown"/>
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diff changeset
325 <param name="coverage_file" value="true"/>
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diff changeset
326 <output name="exon_expression" file="./ballgown/e_data.ctab" ftype="tabular"/>
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diff changeset
327 <output name="intron_expression" file="./ballgown/i_data.ctab" ftype="tabular"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
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diff changeset
328 <output name="transcript_expression" file="./ballgown/t_data.ctab" ftype="tabular"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
329 <output name="exon_transcript_mapping" file="./ballgown/e2t.ctab" ftype="tabular"/>
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diff changeset
330 <output name="intron_transcript_mapping" file="./ballgown/i2t.ctab" ftype="tabular"/>
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parents: 22
diff changeset
331 <output name="output_gtf" file="stringtie_out5.gtf" ftype="gtf" lines_diff="2"/>
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diff changeset
332 <output name="coverage" file="stringtie_out_coverage.gtf" ftype="gtf" lines_diff="2"/>
0
62d212192002 Imported from capsule None
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parents:
diff changeset
333 </test>
13
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parents: 12
diff changeset
334 <!--Ensure output for edgeR works -->
12
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parents: 11
diff changeset
335 <test expect_num_outputs="5">
18
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parents: 17
diff changeset
336 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
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parents: 17
diff changeset
337 <param name="input_mode" value="short_reads"/>
23
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diff changeset
338 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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parents: 17
diff changeset
339 </conditional>
23
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diff changeset
340 <param name="use_guide" value="yes"/>
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iuc
parents: 22
diff changeset
341 <param name="special_outputs_select" value="deseq2"/>
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342 <param name="input_estimation" value="true"/>
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343 <param name="guide_gff_select" value="history" />
23
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344 <param name="ref_hist" ftype="gtf" value="stringtie_in.gtf"/>
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345 <param name="coverage_file" value="true"/>
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346 <param name="clustering" value="true"/>
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diff changeset
347 <output name="gene_counts" file="gene_counts_edger.tsv" ftype="tabular"/>
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348 <output name="transcript_counts" file="transcript_counts_edger.tsv" ftype="tabular"/>
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diff changeset
349 <output name="legend" file="legend.tsv" ftype="tabular"/>
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350 <output name="output_gtf" file="stringtie_out6.gtf" ftype="gtf" lines_diff="2"/>
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351 <output name="coverage" file="stringtie_out_coverage.gtf" ftype="gtf" lines_diff="2"/>
10
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iuc
parents: 7
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352 </test>
12
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parents: 11
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353 <!--Ensure gene abundances output works -->
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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354 <test expect_num_outputs="2">
18
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parents: 17
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355 <conditional name="input_options">
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356 <param name="input_mode" value="short_reads"/>
23
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diff changeset
357 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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diff changeset
358 </conditional>
23
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parents: 22
diff changeset
359 <param name="use_guide" value="yes"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
360 <param name="guide_gff_select" value="history"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
361 <param name="ref_hist" ftype="gtf" value="stringtie_in.gtf"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
362 <param name="fraction" value="0.17"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
363 <param name="abundance_estimation" value="true"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
364 <output name="output_gtf" file="stringtie_out4_re.gtf" ftype="gtf" lines_diff="2"/>
16
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iuc
parents: 15
diff changeset
365 <output name="gene_abundance_estimation" file="stringtie_out7.tsv" ftype="tabular" lines_diff="2" />
5
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iuc
parents: 4
diff changeset
366 </test>
12
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iuc
parents: 11
diff changeset
367 <!--Ensure another fraction value works -->
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iuc
parents: 11
diff changeset
368 <test expect_num_outputs="1">
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iuc
parents: 17
diff changeset
369 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
370 <param name="input_mode" value="short_reads"/>
23
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parents: 22
diff changeset
371 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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parents: 17
diff changeset
372 </conditional>
23
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iuc
parents: 22
diff changeset
373 <param name="use_guide" value="yes"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
374 <param name="guide_gff_select" value="history"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
375 <param name="ref_hist" ftype="gtf" value="stringtie_in.gtf"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
376 <param name="fraction" value="0.15"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
377 <output name="output_gtf" file="stringtie_out8.gtf" ftype="gtf" lines_diff="2"/>
12
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iuc
parents: 11
diff changeset
378 </test>
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
diff changeset
379 <!--Ensure built-in GTFs work -->
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
diff changeset
380 <test expect_num_outputs="1">
18
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iuc
parents: 17
diff changeset
381 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
382 <param name="input_mode" value="short_reads"/>
23
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iuc
parents: 22
diff changeset
383 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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iuc
parents: 17
diff changeset
384 </conditional>
23
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iuc
parents: 22
diff changeset
385 <param name="use_guide" value="yes"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
386 <param name="guide_gff_select" value="cached"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
387 <param name="fraction" value="0.15"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
388 <output name="output_gtf" file="stringtie_out9.gtf" ftype="gtf" lines_diff="2"/>
18
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iuc
parents: 17
diff changeset
389 </test>
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
390 <!-- Test long reads input -->
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
391 <test expect_num_outputs="1">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
392 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
393 <param name="input_mode" value="long_reads"/>
23
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iuc
parents: 22
diff changeset
394 <param name="input_bam" ftype="bam" value="stringtie_in1.bam"/>
18
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iuc
parents: 17
diff changeset
395 </conditional>
23
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iuc
parents: 22
diff changeset
396 <param name="use_guide" value="yes"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
397 <param name="guide_gff_select" value="cached"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
398 <param name="fraction" value="0.15"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
399 <output name="output_gtf" file="stringtie_out10.gtf" ftype="gtf" lines_diff="2"/>
18
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iuc
parents: 17
diff changeset
400 </test>
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
401 <!-- Test error splice option -->
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
402 <test expect_num_outputs="1">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
403 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
404 <param name="input_mode" value="long_reads"/>
23
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iuc
parents: 22
diff changeset
405 <param name="input_bam" ftype="bam" value="long_reads.bam"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
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parents: 22
diff changeset
406 <param name="error_splice" value="30"/>
18
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iuc
parents: 17
diff changeset
407 </conditional>
23
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iuc
parents: 22
diff changeset
408 <param name="use_guide" value="yes"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
409 <param name="guide_gff_select" value="cached"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
410 <param name="fraction" value="0.15"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
411 <output name="output_gtf" file="stringtie_out11.gtf" ftype="gtf" lines_diff="2"/>
18
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iuc
parents: 17
diff changeset
412 </test>
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
413 <!-- Test mixed reads input -->
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
414 <test expect_num_outputs="1">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
415 <conditional name="input_options">
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
416 <param name="input_mode" value="mixed_reads"/>
23
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iuc
parents: 22
diff changeset
417 <param name="input_bam_short" ftype="bam" value="short_reads.bam"/>
8eb8750070d4 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 252f4cccd66513c985b3df9480d73c8b9f994520
iuc
parents: 22
diff changeset
418 <param name="input_bam_long" ftype="bam" value="long_reads.bam"/>
18
258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
diff changeset
419 </conditional>
22
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iuc
parents: 21
diff changeset
420 <output name="output_gtf" ftype="gtf">
ae618321f34a planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 12bd91ad0bc0a6471f34d1dd342c943f222a17d2
iuc
parents: 21
diff changeset
421 <assert_contents>
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iuc
parents: 21
diff changeset
422 <has_text text="5.043160"/>
ae618321f34a planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 12bd91ad0bc0a6471f34d1dd342c943f222a17d2
iuc
parents: 21
diff changeset
423 <has_text text="StringTie version @TOOL_VERSION@"/>
ae618321f34a planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 12bd91ad0bc0a6471f34d1dd342c943f222a17d2
iuc
parents: 21
diff changeset
424 <has_n_lines n="87"/>
ae618321f34a planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 12bd91ad0bc0a6471f34d1dd342c943f222a17d2
iuc
parents: 21
diff changeset
425 </assert_contents>
ae618321f34a planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 12bd91ad0bc0a6471f34d1dd342c943f222a17d2
iuc
parents: 21
diff changeset
426 </output>
5
f504b3b7e49d planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 65c9ce2fdc2b4ce892df8e456f18b859a85c410a
iuc
parents: 4
diff changeset
427 </test>
21
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428 <!-- Test cram input -->
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429 <test expect_num_outputs="1">
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430 <conditional name="input_options">
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431 <param name="input_mode" value="mixed_reads"/>
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432 <param name="input_bam_short" ftype="cram" value="stringtie_in.cram"/>
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433 <param name="input_bam_long" ftype="cram" value="stringtie_in.cram"/>
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434 </conditional>
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435 <output name="output_gtf" ftype="gtf">
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436 <assert_contents>
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437 <has_text text="gi|251831106|ref|NC_012920.1|"/>
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438 <has_text text="StringTie version @TOOL_VERSION@"/>
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439 </assert_contents>
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440 </output>
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441 </test>
0
62d212192002 Imported from capsule None
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442 </tests>
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443 <help><![CDATA[
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444
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445 .. class:: infomark
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446
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447 **What it does**
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448
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449 StringTie_ is a fast and highly efficient assembler of RNA-Seq alignments into potential transcripts. It uses a novel network flow algorithm as well as an optional *de novo* assembly step to assemble and quantitate full-length transcripts representing multiple splice variants for each gene locus. Its input can include not only the alignments of raw reads used by other transcript assemblers, but also alignments of longer sequences that have been assembled from those reads. In order to identify differentially expressed genes between experiments, StringTie's output can be processed by specialized software like Ballgown_, Cuffdiff_ or other programs (DESeq2_, edgeR_, limma_ etc.).
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450
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451 -----
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452
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453 **Inputs**
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454
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455 StringTie takes as input a BAM (or SAM) file of paired-end RNA-seq reads, which must be sorted by genomic location (coordinate position). This file contains spliced read alignments and can be produced directly by programs such as HISAT2_. We recommend using HISAT2 as it is a fast and accurate alignment program. Every spliced read alignment (i.e. an alignment across at least one junction) in the input BAM file must contain the tag XS to indicate the genomic strand that produced the RNA from which the read was sequenced. Alignments produced by HISAT2 (when run with --dta option) already include this tag, but if you use a different read mapper you should check that this XS tag is included for spliced alignments.
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456
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457 *NOTE: be sure to run HISAT2 with the --dta option for alignment (under Spliced alignment options), or your results will suffer.*
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458
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459 Also note that if your reads are from a stranded library, you need to choose the appropriate setting under **Specify strand information** above. As, if Forward (FR) is selected, StringTie will assume the reads are from a --fr library, while if Reverse (RF) is selected, StringTie will assume the reads are from a --rf library, otherwise it is assumed that the reads are from an unstranded library (The widely-used, although now deprecated, TopHat had a similar --library-type option, where fr-firststrand corresponded to RF; fr-secondstrand corresponded to FR). If you don't know whether your reads are from are a stranded library or not, you could use the tool **RSeQC Infer Experiment** to try to determine.
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460
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461 As an option, a reference annotation file in `GTF/GFF3`_ format can be provided to StringTie. In this case, StringTie will prefer to use these "known" genes from the annotation file, and for the ones that are expressed it will compute coverage, TPM and FPKM values. It will also produce additional transcripts to account for RNA-seq data that aren't covered by (or explained by) the annotation. Note that if option -e is not used the reference transcripts need to be fully covered by reads in order to be included in StringTie's output. In that case, other transcripts assembled from the data by StringTie and not present in the reference file will be printed as well.
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462
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463 *NOTE: we highly recommend that you provide annotation if you are analyzing a genome that is well-annotated, such as human, mouse, or other model organisms.*
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464
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465 -----
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466
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467 **Outputs**
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468
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469 StringTie's primary output is
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470
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471 * a GTF file containing the **Assembled transcripts**
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472
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473 Optionally, it can output
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474
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475 * a TSV (tab-delimited) file of **Gene abundances**
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476
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477 If a reference GTF/GFF3 file is used as a guide, StringTie can also output:
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478
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479 * a GTF file containing all **fully-covered reference transcripts** in the provided reference file that are covered end-to-end by reads
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480 * Files (tables) for **Ballgown** and/or **DESeq2/edgeR/limma-voom**, which can use them to estimate differential expression
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481
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482
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483 **StringTie's primary GTF output**
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484
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485 The primary output of StringTie is a Gene Transfer Format (GTF) file that contains details of the transcripts that StringTie assembles from RNA-Seq data. GTF is an extension of GFF (Gene Finding Format, also called General Feature Format), and is very similar to GFF2 and GFF3. The field definitions for the 9 columns of GTF output can be found at the `Ensembl site here`_. The following is an example of a transcript assembled by StringTie as shown in a GTF file:
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486
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487 =========== ========== =========== ========= ======= ========= ========== ========= ===========================================================================================
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488 **seqname** **source** **feature** **start** **end** **score** **strand** **frame** **attributes**
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489 ----------- ---------- ----------- --------- ------- --------- ---------- --------- -------------------------------------------------------------------------------------------
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490 chrX StringTie transcript 281394 303355 1000 \+ . gene_id "ERR188044.1"; transcript_id "ERR188044.1.1"; reference_id "NM_018390"; ref_gene_id
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491 "NM_018390"; ref_gene_name "PLCXD1"; cov "101.256691"; FPKM "530.078918"; TPM "705.667908";
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492 chrX StringTie exon 281394 281684 1000 \+ . gene_id "ERR188044.1"; transcript_id "ERR188044.1.1"; exon_number "1"; reference_id
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493 "NM_018390"; ref_gene_id "NM_018390"; ref_gene_name "PLCXD1"; cov "116.270836";
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494 =========== ========== =========== ========= ======= ========= ========== ========= ===========================================================================================
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495
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496
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497 * **seqname**: Denotes the chromosome, contig, or scaffold for this transcript. Here the assembled transcript is on chromosome X.
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498
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499 * **source**: The source of the GTF file. Since this example was produced by StringTie, this column simply shows 'StringTie'.
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500
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501 * **feature**: Feature type (e.g., exon, transcript, mRNA, 5'UTR).
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502
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503 * **start**: Start position of the feature (exon, transcript, etc), using a 1-based index.
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504
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505 * **end**: End position of the feature, using a 1-based index.
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506
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507 * **score**: A confidence score for the assembled transcript. Currently this field is not used, and StringTie reports a constant value of 1000 if the transcript has a connection to a read alignment bundle.
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508
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509 * **strand**: If the transcript resides on the forward strand, '+'. If the transcript resides on the reverse strand, '-'.
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510
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511 * **frame**: Frame or phase of CDS features. StringTie does not use this field and simply records a ".".
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512
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513 * **attributes**: A semicolon-separated list of tag-value pairs, providing additional information about each feature. Depending on whether an instance is a transcript or an exon and on whether the transcript matches the reference annotation file provided by the user, the content of the attributes field will differ. The following list describes the possible attributes shown in this column:
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514
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515 * *gene_id*: A unique identifier for a single gene and its child transcript and exons based on the alignments' file name.
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516
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517 * *transcript_id*: A unique identifier for a single transcript and its child exons based on the alignments' file name.
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518
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519 * *exon_number*: A unique identifier for a single exon, starting from 1, within a given transcript.
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520
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521 * *reference_id*: The transcript_id in the reference annotation (optional) that the instance matched.
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522
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523 * *ref_gene_id*: The gene_id in the reference annotation (optional) that the instance matched.
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524
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525 * *ref_gene_name*: The gene_name in the reference annotation (optional) that the instance matched.
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526
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527 * *cov*: The average per-base coverage for the transcript or exon.
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528
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529 * *FPKM*: Fragments per kilobase of transcript per million read pairs. This is the number of pairs of reads aligning to this feature, normalized by the total number of fragments sequenced (in millions) and the length of the transcript (in kilobases).
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530
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531 * *TPM*: Transcripts per million. This is the number of transcripts from this particular gene normalized first by gene length, and then by sequencing depth (in millions) in the sample. A detailed explanation and a comparison of TPM and FPKM can be found here_, and TPM was defined `by B. Li and C. Dewey here`_.
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532
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533
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534 **Gene abundances in tab-delimited format**
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535
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536 If StringTie is run with the -A option, it returns a file containing gene abundances. The tab-delimited gene abundances output file has nine fields; below is an example of a gene abundance file produced by StringTie using reference annotation:
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537
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538 =========== ============= ============= ========== ========= ======= ============ ======== ========
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539 **Gene ID** **Gene Name** **Reference** **Strand** **Start** **End** **Coverage** **FPKM** **TPM**
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540 ----------- ------------- ------------- ---------- --------- ------- ------------ -------- --------
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541 NM_000451 SHOX chrX \+ 624344 646823 0.000000 0.000000 0.000000
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542 NM_006883 SHOX chrX \+ 624344 659411 0.000000 0.000000 0.000000
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543 =========== ============= ============= ========== ========= ======= ============ ======== ========
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544
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545 * **Gene ID**: The gene identifier comes from the reference annotation provided with the -G option. If no reference is provided this field is replaced with the name prefix for output transcripts (-l).
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546 * **Gene Name**: This field contains the gene name in the reference annotation provided with the -G option. If no reference is provided this field is populated with '-'.
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547 * **Reference**: Name of the reference sequence that was used in the alignment of the reads. Equivalent to the 3rd column in the .SAM alignment.
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548 * **Strand**: '+' denotes that the gene is on the forward strand, '-' for the reverse strand.
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549 * **Start**: Start position of the gene (1-based index).
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550 * **End**: End position of the gene (1-based index).
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551 * **Coverage**: Per-base coverage of the gene.
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552 * **FPKM**: normalized expression level in FPKM units (see previous section).
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553 * **TPM**: normalized expression level in RPM units (see previous section).
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554
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555 **Fully covered transcripts matching the reference annotation transcripts (in GTF format)**
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556
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557 If StringTie is run with the use reference guide option (-G), it will also return a file with all the transcripts in the reference annotation that are fully covered, end to end, by reads. The output format is a GTF file as described above. Each line of the GTF is corresponds to a gene or transcript in the reference annotation.
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558
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559 **Ballgown Input Table Files**
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560
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561 An option to output files for Ballgown can be selected under **Output files for differential expression?** above. If selected, StringTie will return Ballgown input table files containing coverage data for the reference transcripts given with the -G option. These tables have these specific names: (1) e2t.ctab, (2) e_data.ctab, (3) i2t.ctab, (4) i_data.ctab, and (5) t_data.ctab. A detailed description of each of these five required inputs to Ballgown can be found at `this link`. With this option StringTie can be used as a direct replacement of the tablemaker program included with the Ballgown distribution.
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562
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563
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564 **DESeq2/edgeR/limma-voom Input Table Files**
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565
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566 DESeq2_, edgeR_ and limma_ are three popular Bioconductor_ packages for analyzing differential expression, which take as input a matrix of read counts mapped to particular genomic features (e.g., genes). This read count information can be extracted directly from the files generated by StringTie (run with the -e parameter) by selecting DESeq2/edgeR/limma-voom under **Output files for differential expression?** above. This uses the StringTie helper script ``prepDE.py`` to convert the GTF output from StringTie into two tab-delimited (TSV) files, containing the count matrices for genes and transcripts, using the coverage values found in the output of StringTie -e.
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567
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568 -----
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569
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570 **More Information**
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571
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572 *Evaluating transcript assemblies:*
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573 A simple way of getting more information about the transcripts assembled by StringTie (summary of gene and transcript counts, novel vs. known etc.), or even performing basic tracking of assembled isoforms across multiple RNA-Seq experiments, is to use the **gffcompare** program. Basic usage information for this program can be found on the `GFF utilities page`_.
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574
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575 *Differential expression analysis:*
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576
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577 Together with HISAT and Ballgown (or DESeq2/edgeR/limma-voom), StringTie can be used for estimating differential expression across multiple RNA-Seq samples and generating plots and differential expression tables as described in our `protocol paper`_ and shown in a diagram in the `StringTie manual here`_.
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578
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579 Our recommended workflow includes the following steps:
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580
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581 1. For each RNA-Seq sample, map the reads to the genome with HISAT2 using the --dta option. It is highly recommended to use the reference annotation information when mapping the reads, which can be either embedded in the genome index (built with the --ss and --exon options, see HISAT2 manual), or provided separately at run time (using the --known-splicesite-infile option of HISAT2). The SAM output of each HISAT2 run must be sorted and converted to BAM using samtools as explained above.
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582
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583 2. For each RNA-Seq sample, use this StringTie tool to assemble the read alignments obtained in the previous step; it is recommended to run StringTie with the -G option if the reference annotation is available.
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584
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585 3. Run the separate **StringTie merge** tool in order to generate a non-redundant set of transcripts observed in all the RNA-Seq samples assembled previously. ``StringTie merge`` takes as input a list of all the assembled transcripts files (in GTF format) previously obtained for each sample, as well as a reference annotation file (-G option) if available.
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586
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587 4. For each RNA-Seq sample, run this StringTie tool selecting to output files for Ballgown (or DESeq2/edgeR/limma-voom), which will generate tables of transcript and gene estimated abundances (count files). The option -e (*Use Reference transcripts only*) is not required but is recommended for this run in order to produce more accurate abundance estimations of the input transcripts. Each StringTie run in this step will take as input the sorted read alignments (BAM file) obtained in step 1 for the corresponding sample and the -G option with the merged transcripts (GTF file) generated by ``stringtie merge`` in step 3. Please note that this is the only case where the -G option is not used with a reference annotation, but with the global, merged set of transcripts as observed across all samples. (This step is the equivalent of the *Tablemaker* step described in the original Ballgown pipeline.)
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588
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589 5. Ballgown (or DESeq2/edgeR/limma-voom) can now be used to load the coverage tables generated in the previous step and perform various statistical analyses for differential expression, generate plots etc.
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590
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591 An alternate, faster differential expression analysis workflow can be pursued if there is no interest in novel isoforms (i.e. assembled transcripts present in the samples but missing from the reference annotation), or if only a well known set of transcripts of interest are targeted by the analysis. This simplified protocol has only 3 steps (depicted in the `StringTie manual here`_) as it bypasses the individual assembly of each RNA-Seq sample and the "transcript merge" step. This simplified workflow attempts to directly estimate and analyze the expression of a known set of transcripts as given in the reference annotation file.
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62d212192002 Imported from capsule None
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parents:
diff changeset
592
2
520e0988ec1c planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit b03fd81ed11744f119608d4884c24fa182eb605a
iuc
parents: 1
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593 .. _StringTie: http://ccb.jhu.edu/software/stringtie/
12
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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parents: 11
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594 .. _Ballgown: https://www.biorxiv.org/content/early/2014/09/05/003665
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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595 .. _Cuffdiff: http://cole-trapnell-lab.github.io/cufflinks/cuffdiff/
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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596 .. _DESeq2: https://bioconductor.org/packages/release/bioc/html/DESeq2.html
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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597 .. _edgeR: https://bioconductor.org/packages/release/bioc/html/edgeR.html
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dd4df992d93d planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit a834a41c94d184df80e45ffa2339723826a075b1
iuc
parents: 14
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598 .. _limma: https://bioconductor.org/packages/release/bioc/html/limma.html
12
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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599 .. _Bioconductor: https://www.bioconductor.org/
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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600 .. _SAM: http://samtools.github.io/hts-specs/SAMv1.pdf
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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601 .. _HISAT2: http://ccb.jhu.edu/software/hisat2
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258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
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602 .. _`GTF/GFF3`: http://ccb.jhu.edu/software/stringtie/gff.shtml
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76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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parents: 11
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603 .. _`this link`: https://github.com/alyssafrazee/ballgown#ballgown-readable-expression-output
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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604 .. _`Ensembl site here`: http://useast.ensembl.org/info/website/upload/gff.html
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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605 .. _here: http://www.rna-seqblog.com/rpkm-fpkm-and-tpm-clearly-explained/
76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
iuc
parents: 11
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606 .. _`by B. Li and C. Dewey here`: http://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-12-323
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258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
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607 .. _`GFF utilities page`: http://ccb.jhu.edu/software/stringtie/gff.shtml#gffcompare
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76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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608 .. _`protocol paper`: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5032908/
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258d696dbd7e "planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 999b45e8bcf810f871cda58cb66573f7d6ae37f3"
iuc
parents: 17
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609 .. _`StringTie manual here`: http://ccb.jhu.edu/software/stringtie/index.shtml?t=manual
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76d290331481 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit 11ee7ac206d41894c0b6a11f2439aaea490824f0
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parents: 11
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610
11
6e45b443ef1f planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit d46e597732cda927a633e133c14dd4ece39f5edf
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parents: 10
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611 ]]></help>
6
d0bb240d7525 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit acdc3dfe5fd6317ae57899aa0e83130dff7460e7
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parents: 5
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612 <expand macro="citations" />
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a305d75e13f2 planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/stringtie commit e811a7887db870f4f94f620f52bce656c8d5ba23
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613 </tool>