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1 <tool id ="pyReadCounters" name="pyReadCounters" force_history_refresh="True">
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2 <requirements>
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3 <requirement type="package">pyCRAC</requirement>
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4 </requirements>
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5 <command interpreter="perl">
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6 pyReadCounters.pl
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7 -f $ftype.input
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8 --file_type $ftype.file_type
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9 --gtf $addGTF.gtf
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10 #if ($ftype.file_type == "novo" or $ftype.file_type == "sam") and $ftype.disc.discard == "discard":
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11 --discarded $discarded
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12 #end if#
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13 #if ($ftype.file_type == "novo" or $ftype.file_type == "sam") and $ftype.addAlignOpt.alignoptions == "edit":
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14 --alignOpt
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15 --align_quality $ftype.addAlignOpt.align_quality
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16 --align_score $ftype.addAlignOpt.align_score
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17 #if int($ftype.addAlignOpt.max) > 0:
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18 --max $ftype.addAlignOpt.max
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19 #end if#
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20 --distance $ftype.addAlignOpt.d
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21 --length $ftype.addAlignOpt.length
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22 $ftype.addAlignOpt.unique
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23 $ftype.addAlignOpt.blocks
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24 $ftype.addAlignOpt.mutations
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25 #end if#
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26 #if $addOpt.options == "edit":
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27 --options
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28 --range $addOpt.range
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29 $addOpt.ignore
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30 --overlap $addOpt.overlap
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31 #end if#
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32
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33 --stats $stats
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34 --hittable $hittable
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35 --intronUTRoverlap $intronUTRoverlap
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36
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37 #if $ftype.file_type == "novo" or $ftype.file_type == "sam":
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38 --countoutput $countoutput
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39 #end if#
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40
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41 --id $stats.id
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42 </command>
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43 <version_command>/usr/local/bin/pyReadCounters.py --version</version_command>
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44 <inputs>
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45 <conditional name="addGTF">
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46 <param name="gtfFile" type="select" label="Choose GTF File from">
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47 <option value="default" selected="true">Defaults</option>
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48 <option value="other">History</option>
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49 </param>
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50 <when value="default">
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51 <param name="gtf" type="select" label="GTF File --gtf" help="GTF file containing gene ID co-ordinates">
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52 <options from_data_table="pycrac_gtf"/>
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53 </param>
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54 </when>
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55 <when value="other">
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56 <param format="GTF" name="gtf" type="data" label="GTF File --gtf" help="GTF file containing gene ID co-ordinates"/>
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57 </when>
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58 </conditional>
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59 <conditional name="ftype">
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60 <param name="file_type" type="select" label="Input File Type --file_type" help="Use .novo or .sam input files">
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61 <option value="novo" selected="true">Novo</option>
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62 <option value="sam">Sam/Bam</option>
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63 <option value="gtf">GTF</option>
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64 </param>
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65 <when value="novo">
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66 <param format="tabular" name="input" type="data" label="Input File --input_file" help="Alignment file of type .novo" />
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67 <conditional name="disc">
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68 <param name="discard" type="select" label="Print discarded reads to a separate file">
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69 <option value="" selected="true">OFF</option>
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70 <option value="discard">ON</option>
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71 </param>
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72 <when value="discard">
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73 </when>
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74 <when value="">
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75 </when>
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76 </conditional>
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77 <conditional name="addAlignOpt">
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78 <param name="alignoptions" type="select" label="Alignment Options">
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79 <option value="default" selected="true">Default</option>
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80 <option value="edit">Edit</option>
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81 </param>
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82 <when value="edit">
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83 <param name="mutations" type="select" label="Option for selecting type of mutations to report --mutations" help="cross-linking sites are often highlighted by deletions and/or substitutions in the reads. You can use this option to select specific mutations that you want to have reported in the GTF output file.">
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84 <option value="" selected="true">Off</option>
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85 <option value="--mutations delsonly">deletions</option>
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86 <option value="--mutations subsonly">substitutions</option>
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87 <option value="--mutations TC">T->C substitutions</option>
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88 <option value="--mutations nomuts">no mutations</option>
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89 </param>
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90 <param format="integer" name="align_quality" type="integer" label="Align Quality --align_quality " value="0" size="5" >
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91 <validator type="in_range" min="0" message="Please enter a value >= 0"/>
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92 </param>
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93 <param format="integer" name="align_score" type="integer" label="Align Score --align_score " value="0" size="5" >
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94 <validator type="in_range" min="0" message="Please enter a value >= 0"/>
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95 </param>
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96 <param format="integer" name="max" type="integer" label="Mapped reads to read from input file --max" help="Set to 0 to align all reads." value="0" size="10" >
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97 <validator type="in_range" min="0" max="100000000" message="Please enter a value between 1 and 100000000 or 0 to align all reads"/>
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98 </param>
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99 <param format="integer" name="d" type="integer" label="Distance --distance " value="1000" size="6" help="Set the maximum number of bp allowed between two non-overlapping paired reads">
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100 <validator type="in_range" min="1" message="Please enter a value >= 0"/>
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101 </param>
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102 <param format="integer" name="length" type="integer" label="Set the maximum length of reads --length" value="1000" size="7" help="Set the read length threshold between 15 and 1000">
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103 <validator type="in_range" min="15" max="1000" message="Please enter a value between 15 and 1000"/>
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104 </param>
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105 <param name="unique" type="select" label="Remove reads with multiple alignment locations --unique">
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106 <option value="" selected="true">OFF</option>
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107 <option value="--unique">ON</option>
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108 </param>
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109 <param name="blocks" type="select" label="Only count reads with same start and end coords once --blocks">
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110 <option value="" selected="true">OFF</option>
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111 <option value="--blocks">ON</option>
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112 </param>
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113 </when>
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114 <when value="default">
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115 </when>
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116 </conditional>
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117 </when>
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118 <when value="sam">
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119 <param format="sam,bam" name="input" type="data" label="Input File --input_file" help="Alignment file of type .sam or .bam" />
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120 <conditional name="disc">
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121 <param name="discard" type="select" label="Print discarded reads to a separate file">
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122 <option value="" selected="true">OFF</option>
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123 <option value="discard">ON</option>
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124 </param>
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125 <when value="discard">
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126 </when>
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127 <when value="">
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128 </when>
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129 </conditional>
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130 <conditional name="addAlignOpt">
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131 <param name="alignoptions" type="select" label="Alignment Options">
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132 <option value="default" selected="true">Default</option>
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133 <option value="edit">Edit</option>
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134 </param>
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135 <when value="edit">
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136 <param name="mutations" type="select" label="Option for selecting type of mutations to report --mutations" help="cross-linking sites are often highlighted by deletions and/or substitutions in the reads. You can use this option to select specific mutations that you want to have reported in the GTF output file.">
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137 <option value="" selected="true">Off</option>
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138 <option value="--mutations delsonly">deletions</option>
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139 <option value="--mutations subsonly">substitutions</option>
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140 <option value="--mutations TC">T->C mutations</option>
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141 <option value="--mutations nomuts">no mutations</option>
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142 </param>
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143 <param format="integer" name="align_quality" type="integer" label="Align Quality --align_quality " value="0" size="5" >
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144 <validator type="in_range" min="0" message="Please enter a value >= 0"/>
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145 </param>
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146 <param format="integer" name="align_score" type="integer" label="Align Score --align_score " value="0" size="5" >
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147 <validator type="in_range" min="0" message="Please enter a value >= 0"/>
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148 </param>
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149 <param format="integer" name="max" type="integer" label="Mapped reads to read from input file --max" help="Set to 0 to align all reads." value="0" size="10" >
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150 <validator type="in_range" min="0" max="100000000" message="Please enter a value between 1 and 100000000 or 0 to align all reads"/>
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151 </param>
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152 <param format="integer" name="d" type="integer" label="Distance --distance " value="1000" size="6" help="Set the maximum number of bp allowed between two non-overlapping paired reads">
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153 <validator type="in_range" min="1" message="Please enter a value >= 0"/>
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154 </param>
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155 <param format="integer" name="length" type="integer" label="Set the maximum length of reads --length" value="1000" size="7" help="Set the read length threshold between 15 and 1000">
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156 <validator type="in_range" min="15" max="1000" message="Please enter a value between 15 and 1000"/>
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157 </param>
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158 <param name="unique" type="select" label="Remove reads with multiple alignment locations --unique">
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159 <option value="" selected="true">OFF</option>
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160 <option value="--unique">ON</option>
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161 </param>
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162 <param name="blocks" type="select" label="Only count reads with same start and end coords once --blocks">
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163 <option value="" selected="true">OFF</option>
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164 <option value="--blocks">ON</option>
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165 </param>
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166 </when>
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167 <when value="default">
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168 </when>
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169 </conditional>
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170 </when>
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171 <when value="gtf">
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172 <param format="gtf" name="input" type="data" label="Input File --input_file" help="File of type .gtf" />
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173 </when>
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174 </conditional>
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175 <conditional name="addOpt">
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176 <param name="options" type="select" label="Standard Options">
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177 <option value="default" selected="true">Default</option>
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178 <option value="edit">Edit</option>
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179 </param>
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180 <when value="edit">
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181 <param format="integer" name="range" type="integer" label="Range --range" value="0" size="5" help="Manually set the length of the 5' and 3' UTRs 0>50000">
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182 <validator type="in_range" min="0" max="50000" message="Please enter a value between 0 and 50000"/>
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183 </param>
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184 <param name="ignore" type="select" label="Ignore strand information? --ignorestrand">
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185 <option value="" selected="true">No</option>
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186 <option value="--ignorestrand">Yes</option>
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187 </param>
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188 <param format="integer" name="overlap" type="integer" label="Overlap --overlap" value="1" size="5" help="Sets the number of nucleotides a read has to overlap with a gene before it is considered a hit. ">
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189 <validator type="in_range" min="1" message="Please enter a positive integer"/>
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190 </param>
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191 </when>
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192 <when value="default">
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193 </when>
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194 </conditional>
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195 <param name="label" type="text" format="txt" size="30" value="pyReadCounters" label="Enter output file label -o" />
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196 </inputs>
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197 <outputs>
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198 <data format="tabular" name="stats" label="${label.value}_file_statistics.txt"/>
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199 <data format="tabular" name="hittable" label="${label.value}_hittable.txt"/>
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200 <data format="gtf" name="intronUTRoverlap" label="${label.value}_intron_and_UTR_overlap.txt"/>
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201 <data format="gtf" name="countoutput" label="${label.value}_count_output.gtf">
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202 <filter>ftype['file_type'] == "novo" or ftype['file_type'] == "sam"</filter>
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203 </data>
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204 <data format="txt" name="discarded" label="${label.value}_discarded.txt">
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205 <filter>(ftype['file_type'] == "novo" or ftype['file_type'] == "sam") and ftype['disc']['discard'] == "discard"</filter>
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206 </data>
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207 </outputs>
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208 <help>
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209
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210 .. class:: infomark
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211
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212 **pyReadCounters**
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213
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214 pyReadCounters is part of the pyCRAC_ package. Produces a gene hittable file, two GTF output files showing to which genomic features the reads overlap.
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215 Finally the tool produces a read statistics file that provides information about the complexity of your dataset.
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216
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217 **Output file examples**
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218
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219 A hittable file::
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220
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221 # generated by pyReadCounters version 1.1.0, Mon Apr 16 20:34:22 2012
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222 # /usr/local/bin/pyReadCounters.py -f RNAseq_data.novo -c 1 --unique
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223 # total number of reads 12534556
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224 # total number of paired reads 10947376
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225 # total number of single reads 483095
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226 # total number of mapped reads: 11430471
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227 # total number of overlapping genomic features 7019550
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228 # sense 5960669
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229 # anti-sense 1058881
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230 # feature sense_overlap anti-sense_overlap number of reads
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231
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232 ## protein_coding 3190701
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233 YEF3 49930 3629 24221
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234 PMA1 32621 2650 21776
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235 COX1 24559 1037 15174
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236 TFP1 21539 1689 13506
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237 HSC82 21177 1458 12729
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238 ADH1 20245 1467 11351
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239 AI5_ALPHA 20022 918 13101
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240 AI4 19390 886 12638
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241 AI3 17823 798 11473
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242 AI2 17590 790 11297
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243 RPL10 16822 1113 8797
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244 ENO2 16336 1125 8913
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245 TEF1 15578 1333 5450
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246
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247 An example of a GTF 'count_output' file::
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248
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249 ##gff-version 2
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250 # generated by Counters version 1.2.0, Tue Jan 8 22:47:29 2013
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251 # pyReadCounters.py -f PAR_CLIP_unique.novo --mutations=TC -v
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252 # total number of reads: 2455251
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253 # total number of paired reads: 0
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254 # total number of single reads: 2455251
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255 # total number of mapped reads: 2455251
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256 # total number of overlapping genomic features: 5153943
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257 # sense: 2640600
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258 # anti-sense: 2513343
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259 chrXIV reads exon 661572 661605 2 + . gene_id "INT_0_6716,YNR016C"; gene_name "INT_0_6716,ACC1"; # 661596S;
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260 chrXIV reads exon 661720 661738 1 + . gene_id "INT_0_6716,YNR016C"; gene_name "INT_0_6716,ACC1"; # 661726S;
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261 chrXIV reads exon 661839 661878 4 + . gene_id "INT_0_6716,YNR016C"; gene_name "INT_0_6716,ACC1"; # 661875S;
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262
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263 This output file also reports whether a read contains a mutation.
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264
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265 For example::
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266
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267 # 661596S
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268
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269 Indicates that the read had a nucleotide substitution ("S") at genomic coordinate 661596. The chromosome name can be found in the first column.
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270
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271 .. _pyCRAC: http://sandergranneman.bio.ed.ac.uk/Granneman_Lab/pyCRAC_software.html
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272
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273 ------
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274
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275 **Parameter list**
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276
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277 File input options::
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278
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279 -f FILE, --input_file=FILE
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280 provide the path to your novo, SAM/BAM or gtf data
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281 file. Default is standard input. Make sure to specify
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282 the file type of the file you want to have analyzed
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283 using the --file_type option!
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284 -o OUTPUT_FILE, --output_file=OUTPUT_FILE
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285 Use this flag to override the standard file names. Do
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286 NOT add an extension.
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287 --file_type=FILE_TYPE
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288 use this option to specify the file type (i.e.
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289 'novo','sam' or 'gtf'). This will tell the program
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290 which parsers to use for processing the files. Default
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291 = 'novo'
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292 --gtf=annotation_file.gtf
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293 type the path to the gtf annotation file that you want
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294 to use
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295
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296 Common pyCRAC options::
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297
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298 --ignorestrand
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299 To ignore strand information and all reads overlapping
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300 with genomic features will be considered sense reads.
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301 Useful for analysing ChIP or RIP data
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302 --overlap=1
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303 sets the number of nucleotides a read has to overlap
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304 with a gene before it is considered a hit. Default =
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305 1 nucleotide
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306 -r 100, --range=100
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307 allows you to add regions flanking the genomic
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308 feature. If you set '-r 50' or '--range=50', then the
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309 program will add 50 nucleotides to each feature on
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310 each side regardless of whether the GTF file has genes
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311 with annotated UTRs
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312
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313 Options for SAM/BAM and Novo files::
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314
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315 --mutations=delsonly
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316 Use this option to only track mutations that are of
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317 interest. For CRAC data this is usually deletions
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318 (--mutations=delsonly). For PAR-CLIP data this is
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319 usually T-C mutations (--mutations=TC). Other options
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320 are\: do not report any mutations: --mutations=nomuts.
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321 Only report specific base mutations, for example only
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322 in T's, C's and G's :--mutations=[TCG]. The brackets
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323 are essential. Other nucleotide combinations are also
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324 possible
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325 --align_quality=100, --mapping_quality=100
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326 with these options you can set the alignment quality
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327 (Novoalign) or mapping quality (SAM) threshold. Reads
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328 with qualities lower than the threshold will be
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329 ignored. Default = 0
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330 --align_score=100
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331 with this option you can set the alignment score
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332 threshold. Reads with alignment scores lower than the
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333 threshold will be ignored. Default = 0
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334 --unique
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335 with this option reads with multiple alignment
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336 locations will be removed. Default = Off
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337 --blocks
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338 with this option reads with the same start and end
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339 coordinates on a chromosome will be counted as one
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340 cDNA. Default = Off
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341 -m 100000, --max=100000
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342 maximum number of mapped reads that will be analyzed.
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343 Default = All
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344 -d 1000, --distance=1000
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345 this option allows you to set the maximum number of
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346 base-pairs allowed between two non-overlapping paired
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347 reads. Default = 1000
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348 --discarded=FILE
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349 prints the lines from the alignments file that were
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350 discarded by the parsers. This file contains reads
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351 that were unmapped (NM), of poor quality (i.e. QC) or
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352 paired reads that were mapped to different chromosomal
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353 locations or were too far apart on the same
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354 chromosome. Useful for debugging purposes
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355 -l 100, --length=1000
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356 to set read length threshold. Default = 1000
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357
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358 </help>
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359 </tool>
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