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1 <tool id="featurecounts" name="featureCounts" version="1.4.6.p5" profile="16.04">
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2 <description>Measure gene expression in RNA-Seq experiments from SAM or BAM files.</description>
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3 <requirements>
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4 <requirement type="package" version="1.4.6p5">subread</requirement>
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5 </requirements>
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6
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7 <version_command>featureCounts -v 2>&1 | grep .</version_command>
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8 <command><![CDATA[
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9 ## Check whether all alignments are from the same type (bam || sam)
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10 featureCounts
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11 #if $gtf_source.ref_source=="history":
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12 -a "$gtf_source.reference_gene_sets"
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13 #else:
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14 -a "$gtf_source.reference_gene_sets_builtin.fields.path"
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15 #end if
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16
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17 -o "output"
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18 -T \${GALAXY_SLOTS:-2}
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19
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20 -t "$extended_parameters.gff_feature_type"
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21 -g "$extended_parameters.gff_feature_attribute"
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22 $extended_parameters.summarization_level
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23 $extended_parameters.contribute_to_multiple_features
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24 -s $extended_parameters.strand_specificity
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25 $extended_parameters.multimapping_enabled.multimapping_counts
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26
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27 #if str($extended_parameters.multimapping_enabled.multimapping_counts) == " -M"
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28 $extended_parameters.multimapping_enabled.fraction
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29 #end if
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30
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31 -Q $extended_parameters.mapping_quality
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32 $extended_parameters.largest_overlap
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33 --minOverlap $extended_parameters.min_overlap
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34 $extended_parameters.read_reduction
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35 $extended_parameters.primary
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36 $extended_parameters.ignore_dup
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37
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38 #if str($extended_parameters.read_extension_5p) != "0"
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39 --readExtension5 $extended_parameters.read_extension_5p
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40 #end if
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41
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42 #if str($extended_parameters.read_extension_3p) != "0"
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43 --readExtension3 $extended_parameters.read_extension_3p
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44 #end if
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45
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46 $pe_parameters.fragment_counting_enabled.fragment_counting
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47 #if str($pe_parameters.fragment_counting_enabled.fragment_counting) == " -p"
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48 $pe_parameters.fragment_counting_enabled.check_distance_enabled.check_distance
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49 #if str($pe_parameters.fragment_counting_enabled.check_distance_enabled.check_distance) == " -P"
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50 -d $pe_parameters.fragment_counting_enabled.check_distance_enabled.minimum_fragment_length
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51 -D $pe_parameters.fragment_counting_enabled.check_distance_enabled.maximum_fragment_length
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52 #end if
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53 #end if
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54
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55 $pe_parameters.only_both_ends
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56 -S $pe_parameters.orientation
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57 $pe_parameters.exclude_chimerics
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58
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59 "${alignment}"
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60
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61 ## Removal of comment and column-header line
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62 && grep -v "^#" "output" | tail -n+2 > body.txt
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63
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64 ## Set the right columns for the tabular formats
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65 #if $format.value == "tabdel_medium"
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66 && cut -f 1,7 body.txt > expression_matrix.txt
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67
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68 ## Paste doesn't allow a non ordered list of columns: -f 1,7,8,6 will only return columns 1,7 and 8
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69 ## Thus the gene length column (last column) has to be added separately
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70 && cut -f 6 body.txt > gene_lengths.txt
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71 && paste expression_matrix.txt gene_lengths.txt > expression_matrix.txt.bak
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72 && mv -f expression_matrix.txt.bak "${output_medium}"
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73 #elif $format.value == "tabdel_short"
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74 && cut -f 1,7 body.txt > "${output_short}"
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75 #else
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76 && cp body.txt "${output_full}"
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77 #end if
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78
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79
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80 #if str($include_feature_length_file) == "true"
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81 && cut -f 1,6 body.txt > "${output_feature_lengths}"
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82 #end if
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83
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84 && tail -n+2 "output.summary" > "${output_summary}"
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85
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86 ]]></command>
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87 <inputs>
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88 <param name="alignment"
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89 type="data"
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90 multiple="false"
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91 format="bam,sam"
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92 label="Alignment file"
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93 help="The input alignment file(s) where the gene expression has to be counted. The file can have a SAM or BAM format; but ALL files must be in the same format" />
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94
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95 <conditional name="gtf_source">
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96 <param name="ref_source" type="select" label="Gene annotation file">
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97 <option value="cached">locally cached</option>
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98 <option value="history">in your history</option>
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99 </param>
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100 <when value="cached">
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101 <param name="reference_gene_sets_builtin" type="select" label="Using locally cached annotation" help="If the annotation file you require is not listed here, please contact the Galaxy administrator">
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102 <options from_data_table="gene_sets">
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103 <filter type="sort_by" column="1" />
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104 <validator type="no_options" message="No annotations are available." />
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105 </options>
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106 </param>
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107 </when>
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108 <when value="history">
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109 <param name="reference_gene_sets"
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110 format="gff,gtf,gff3"
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111 type="data"
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112 label="Gene annotation file"
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113 help="The program assumes that the provided annotation file is in GTF format. Make sure that the gene annotation file corresponds to the same reference genome as used for the alignment" />
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114 </when>
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115 </conditional>
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116
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117 <param name="format"
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118 type="select"
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119 label="Output format"
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120 help="The output format will be tabular, select the preferred columns here">
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121 <option value="tabdel_short" selected="true">Gene-ID "\t" read-count (DESeq2 IUC wrapper compatible)</option>
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122 <option value="tabdel_medium">Gene-ID "\t" read-count "\t" gene-length</option>
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123 <option value="tabdel_full">featureCounts 1.4.0+ default (includes regions provided by the GTF file)</option>
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124 </param>
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125
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126 <param name="include_feature_length_file"
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127 type="boolean"
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128 truevalue="true"
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129 falsevalue="false"
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130 checked="false"
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131 label="Create gene-length file"
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132 help="Creates a tabular file that contains the effective (nucleotides used for counting reads) length of the feature; might be useful for estimating FPKM/RPKM" />
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133
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134
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135 <section name="pe_parameters" title="Options for paired-end reads">
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136 <conditional name="fragment_counting_enabled">
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137
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138 <param name="fragment_counting"
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139 type="select"
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140 argument="-p"
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141 checked="true"
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142 label="Count fragments instead of reads"
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143 help="If specified, fragments (or templates) will be counted instead of reads.">
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144 <option value="" selected="true">Disabled; all reads/mates will be counted individually</option>
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145 <option value=" -p">Enabled; fragments (or templates) will be counted instead of reads</option>
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146 </param>
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147
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148 <when value=" -p">
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149 <conditional name="check_distance_enabled">
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150 <param name="check_distance"
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151 type="boolean"
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152 truevalue=" -P"
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153 falsevalue=""
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154 argument="-P"
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155 label="Check paired-end distance"
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156 help="If specified, paired-end distance will be checked when assigning fragments to meta-features or features. This option is only applicable when -p (Count fragments instead of reads) is specified. The distance thresholds should be specified using -d and -D (minimum and maximum fragment/template length) options." />
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157 <when value=" -P">
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158 <param name="minimum_fragment_length"
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159 type="integer"
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160 value="50"
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161 argument="-d"
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162 label="Minimum fragment/template length." />
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163 <param name="maximum_fragment_length"
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164 type="integer"
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165 value="600"
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166 argument="-D"
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167 label="Maximum fragment/template length." />
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168 </when>
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169 <when value="" />
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170 </conditional>
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171 </when>
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172 <when value="" />
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173 </conditional>
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174
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175 <param name="only_both_ends"
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176 type="boolean"
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177 truevalue=" -B"
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178 falsevalue=""
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179 argument="-B"
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180 label="Only allow fragments with both reads aligned"
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181 help="If specified, only fragments that have both ends successfully aligned will be considered for summarization. This option is only applicable for paired-end reads." />
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182
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183 <param name="orientation"
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184 type="select"
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185 label="Orientation of the two read from the same pair"
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186 argument="-S"
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187 help="Default is 'fr'">
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188 <option value="fr" selected="true">Forward, Reverse (fr)</option>
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189 <option value="ff">Forward, Forward (ff)</option>
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190 <option value="rf">Reverse, Forward (rf)</option>
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191 </param>
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192
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193 <param name="exclude_chimerics"
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194 type="boolean"
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195 truevalue=" -C"
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196 falsevalue=""
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197 argument="-C"
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198 checked="true"
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199 label="Exclude chimeric fragments"
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200 help="If specified, the chimeric fragments (those fragments that have their two ends aligned to different chromosomes) will NOT be included for summarization. This option is only applicable for paired-end read data." />
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201 </section>
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202
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203 <section name="extended_parameters" title="Advanced options">
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204 <param name="gff_feature_type"
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205 type="text"
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206 value="exon"
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207 argument="-t"
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208 label="GFF feature type filter"
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209 help="Specify the feature type. Only rows which have the matched matched feature type in the provided GTF annotation file will be included for read counting. `exon' by default." />
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210
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211 <param name="gff_feature_attribute"
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212 type="text"
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213 value="gene_id"
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214 argument="-g"
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215 label="GFF gene identifier"
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216 help="Specify the attribute type used to group features (eg. exons) into meta-features (eg. genes), when GTF annotation is provided. `gene_id' by default. This attribute type is usually the gene identifier. This argument is useful for the meta-feature level summarization." />
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217
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218 <param name="summarization_level"
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219 type="boolean"
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220 truevalue=" -f"
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221 falsevalue=""
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222 argument="-f"
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223 label="On feature level"
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224 help="If specified, read summarization will be performed at the feature level. By default (-f is not specified), the read summarization is performed at the meta-feature level." />
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225
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226 <param name ="contribute_to_multiple_features"
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227 type="boolean"
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228 truevalue=" -O"
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229 falsevalue=""
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230 argument="-O"
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231 label="Allow read to contribute to multiple features"
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232 help="If specified, reads (or fragments if -p is specified) will be allowed to be assigned to more than one matched meta- feature (or matched feature if -f is specified)" />
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233
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234 <param name="strand_specificity"
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235 type="select"
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236 label="Strand specificity of the protocol"
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237 argument="-s"
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238 help="Indicate if strand-specific read counting should be performed.">
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239 <option value="0" selected="true">Unstranded</option>
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240 <option value="1">Stranded (forwards)</option>
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241 <option value="2">Stranded (reverse)</option>
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242 </param>
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243
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244 <conditional name="multimapping_enabled">
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245 <param name="multimapping_counts"
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246 type="select"
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247 argument="-M"
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248 label="Count multi-mapping reads/fragments"
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249 help="If specified, multi-mapping reads/fragments will be counted (ie. a multi-mapping read will be counted up to N times if it has N reported mapping locations). The program uses the `NH' tag to find multi-mapping reads.">
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250 <option value="" selected="true">Disabled; multi-mapping reads are excluded (default)</option>
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251 <option value=" -M">Enabled; multi-mapping reads are included</option>
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252 </param>
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253 <when value=" -M">
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254 <param name="fraction"
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255 type="boolean"
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256 truevalue="--fraction"
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257 falsevalue=""
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258 argument="--fraction"
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259 label="Assign fractions to multimapping reads"
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260 help="If specified, a fractional count 1/n will be generated for each multi-mapping read, where n is the number of alignments (indica- ted by 'NH' tag) reported for the read. This option must be used together with the '-M' option." />
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261 </when>
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262 <when value="" />
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263 </conditional>
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264
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265 <param name="mapping_quality"
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266 type="integer"
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267 value="12"
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268 argument="-Q"
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269 label="Minimum mapping quality per read"
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270 help="The minimum mapping quality score a read must satisfy in order to be counted. For paired-end reads, at least one end should satisfy this criteria. 12 by default." />
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271
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272 <param name="largest_overlap"
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273 type="boolean"
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274 truevalue=" --largestOverlap"
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275 falsevalue=""
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276 argument="--largestOverlap"
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277 label="Largest overlap"
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278 help="If specified, reads (or fragments) will be assigned to the target that has the largest number of overlapping bases" />
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279
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280 <param name="min_overlap"
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281 type="integer"
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282 value="1"
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283 argument="--minOverlap"
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284 label="Minimum overlap"
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285 help="Specify the minimum required number of overlapping bases between a read (or a fragment) and a feature. 1 by default. If a negative value is provided, the read will be extended from both ends." />
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286
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287 <param name="read_extension_5p"
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288 type="integer"
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289 value="0"
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290 argument="--readExtension5"
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291 label="Read 5' extension"
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292 help="Reads are extended upstream by ... bases from their 5' end" />
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293
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294 <param name="read_extension_3p"
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295 type="integer"
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296 value="0"
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297 argument="--readExtension3"
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298 label="Read 3' extension"
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299 help="Reads are extended upstream by ... bases from their 3' end" />
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300
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301 <param name="read_reduction"
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302 type="select"
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303 label="Reduce read to single position"
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304 argument="--read2pos"
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305 help="The read is reduced to its 5' most base or 3'most base. Read summarization is then performed based on thesingle base which the read is reduced to.">
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306 <option value="" selected="true">Leave the read as it is</option>
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307 <option value="--read2pos 5">Reduce it to the 5' end</option>
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308 <option value="--read2pos 3">Reduce it to the 3' end</option>
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309 </param>
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310
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311 <param name="primary"
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312 type="boolean"
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313 truevalue=" --primary"
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314 falsevalue=""
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315 argument="--primary"
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316 label="Only count primary alignments"
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317 help="If specified, only primary alignments will be counted. Primaryand secondary alignments are identified using bit 0x100 in theFlag field of SAM/BAM files. All primary alignments in a datasetwill be counted no matter they are from multi-mapping reads ornot ('-M' is ignored)." />
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318
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319 <param name="ignore_dup"
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320 type="boolean"
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321 truevalue=" --ignoreDup"
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322 falsevalue=""
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323 argument="--ignoreDup"
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324 label="Ignore reads marked as duplicate"
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325 help="If specified, reads that were marked asduplicates will be ignored. Bit Ox400 in FLAG field of SAM/BAMfile is used for identifying duplicate reads. In paired enddata, the entire read pair will be ignored if at least one endis found to be a duplicate read." />
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326
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327 <param name="count_split_alignments_only"
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328 type="boolean"
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329 truevalue=" --countSplitAlignmentsOnly"
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330 falsevalue=""
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331 argument="--countSplitAlignmentsOnly"
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332 label="Ignore reads marked as duplicate"
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333 help="If specified, only split alignments (CIGARstrings containing letter `N') will be counted. All the otheralignments will be ignored. An example of split alignments isthe exon-spanning reads in RNA-seq data." />
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334 </section>
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335 </inputs>
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336 <outputs>
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337 <data format="tabular"
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338 name="output_medium"
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339 label="${tool.name} on ${on_string}">
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340 <filter>format == "tabdel_medium"</filter>
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341 <actions>
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342 <action name="column_names" type="metadata" default="Geneid,${alignment.name},Length" />
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343 </actions>
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344 </data>
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345
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346 <data format="tabular"
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347 name="output_short"
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348 label="${tool.name} on ${on_string}">
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349 <filter>format == "tabdel_short"</filter>
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350 <actions>
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351 <action name="column_names" type="metadata" default="Geneid,${alignment.name}" />
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352 </actions>
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353 </data>
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354
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355 <data format="tabular"
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356 name="output_full"
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357 label="${tool.name} on ${on_string}: count table">
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358 <filter>format == "tabdel_full"</filter>
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359 <actions>
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360 <action name="column_names" type="metadata" default="Geneid,Chr,Start,End,Strand,Length,${alignment.name}" />
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361 </actions>
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362 </data>
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363
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364 <data format="tabular"
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365 name="output_summary"
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366 hidden="true"
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367 label="${tool.name} on ${on_string}: summary">
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368 <actions>
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369 <action name="column_names" type="metadata" default="Status,${alignment.name}" />
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370 </actions>
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371 </data>
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372
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373 <data format="tabular"
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374 name="output_feature_lengths"
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375 label="${tool.name} on ${on_string}: feature lengths">
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376 <filter>include_feature_length_file</filter>
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377 <actions>
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378 <action name="column_names" type="metadata" default="Feature,Length" />
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379 </actions>
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380 </data>
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381 </outputs>
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382 <tests>
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383 <test>
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384 <param name="alignment" value="featureCounts_input1.bam" ftype="bam" />
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385 <param name="reference_gene_sets" value="featureCounts_guide.gff" ftype="gff" />
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386 <param name="format" value="tabdel_short" />
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387 <param name="include_feature_length_file" value="true"/>
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388 <param name="ref_source" value="history" />
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389 <output name="output" file="output_1_short.tab"/>
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390 <output name="output_summary" file="output_1_summary.tab"/>
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391 </test>
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392 <test>
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393 <param name="alignment" value="featureCounts_input1.bam" ftype="bam" />
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394 <param name="reference_gene_sets" value="featureCounts_guide.gff" ftype="gff" />
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395 <param name="format" value="tabdel_medium" />
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396 <param name="include_feature_length_file" value="true"/>
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397 <param name="ref_source" value="history" />
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398 <output name="output" file="output_1_medium.tab"/>
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399 <output name="output_summary" file="output_1_summary.tab"/>
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400 </test>
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401 <test>
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402 <param name="alignment" value="featureCounts_input1.bam" ftype="bam" />
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403 <param name="reference_gene_sets" value="featureCounts_guide.gff" ftype="gff" />
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404 <param name="format" value="tabdel_full" />
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405 <param name="include_feature_length_file" value="true"/>
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406 <param name="ref_source" value="history" />
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407 <output name="output" file="output_1_full.tab"/>
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408 <output name="output_summary" file="output_1_summary.tab"/>
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409 <output name="output_feature_lengths" file="output_feature_lengths.tab"/>
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410 </test>
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411
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412 <test>
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413 <param name="alignment" value="featureCounts_input1.bam" ftype="bam" />
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414 <param name="reference_gene_sets" value="featureCounts_guide.gff" ftype="gff" />
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415 <param name="format" value="tabdel_short" />
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416 <param name="include_feature_length_file" value="true"/>
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417 <param name="ref_source" value="history" />
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418 <output name="output" file="output_2_short.tab"/>
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419 <output name="output_summary" file="output_2_summary.tab"/>
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420 </test>
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421 <test>
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422 <param name="alignment" value="featureCounts_input1.bam" ftype="bam" />
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423 <param name="reference_gene_sets" value="featureCounts_guide.gff" ftype="gff" />
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424 <param name="format" value="tabdel_medium" />
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425 <param name="include_feature_length_file" value="true"/>
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426 <param name="ref_source" value="history" />
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427 <output name="output" file="output_2_medium.tab"/>
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428 <output name="output_summary" file="output_2_summary.tab"/>
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429 </test>
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430 <test>
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431 <param name="alignment" value="featureCounts_input1.bam" ftype="bam" />
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432 <param name="reference_gene_sets" value="featureCounts_guide.gff" ftype="gff" />
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433 <param name="format" value="tabdel_full" />
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434 <param name="include_feature_length_file" value="true"/>
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435 <param name="ref_source" value="history" />
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436 <output name="output" file="output_2_full.tab"/>
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437 <output name="output_summary" file="output_2_summary.tab"/>
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438 <output name="output_feature_lengths" file="output_feature_lengths.tab"/>
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439 </test>
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440 </tests>
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441
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442 <help><![CDATA[
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443 featureCounts
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444 #############
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445
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446 Overview
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447 --------
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448 FeatureCounts is a light-weight read counting program written entirely in the C programming language. It can be used to count both gDNA-seq and RNA-seq reads for genomic features in in SAM/BAM files.
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449
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450 Input formats
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451 -------------
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452 Alignments should be provided in either:
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453
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454 - SAM format, http://samtools.sourceforge.net/samtools.shtml#5
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455 - BAM format
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456
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457 Gene regions should be provided in the GFF/GTF format:
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458
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459 - http://genome.ucsc.edu/FAQ/FAQformat.html#format3
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460 - http://www.ensembl.org/info/website/upload/gff.html
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461
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462 Output format
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463 -------------
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464 FeatureCounts produces a table containing counted reads, per gene, per row. Optionally the last column can be set to be the effective gene-length. These tables are compatible with the DESeq2 Galaxy wrapper by IUC. Column names are added as metadata object.
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465 ]]></help>
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466 <citations>
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467 <citation type="doi">10.1093/bioinformatics/btt656</citation>
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468 </citations>
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469 </tool>
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