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1 <tool id="pileup_parser" name="Filter pileup" version="1.0.2">>
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2 <description>on coverage and SNPs</description>
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3 <command interpreter="perl">
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4 #if $pileup_type.type_select == "six" #pileup_parser.pl $input "3" "5" "6" "4" $qv_cutoff $cvrg_cutoff $snps_only $interval "2" $out_file1 $diff $qc_base
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5 #elif $pileup_type.type_select == "ten" #pileup_parser.pl $input "3" "9" "10" "8" $qv_cutoff $cvrg_cutoff $snps_only $interval "2" $out_file1 $diff $qc_base
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6 #elif $pileup_type.type_select == "manual" #pileup_parser.pl $input $pileup_type.ref_base_column $pileup_type.read_bases_column $pileup_type.read_qv_column $pileup_type.cvrg_column $qv_cutoff $cvrg_cutoff $snps_only $interval $pileup_type.coord_column $out_file1 $diff $qc_base
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7 #end if#
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8 </command>
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9 <inputs>
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10 <param name="input" type="data" format="tabular" label="Select dataset"/>
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11 <conditional name="pileup_type">
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12 <param name="type_select" type="select" label="which contains" help="See "Types of pileup datasets" below for examples">
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13 <option value="six" selected="true">Pileup with six columns (simple)</option>
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14 <option value="ten">Pileup with ten columns (with consensus)</option>
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15 <option value="manual">Set columns manually</option>
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16 </param>
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17 <when value="manual">
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18 <param name="ref_base_column" label="Select column with reference base" type="data_column" numerical="false" data_ref="input" />
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19 <param name="read_bases_column" label="Select column with read bases" type="data_column" numerical="false" data_ref="input" help="something like this: ..,a.."/>
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20 <param name="read_qv_column" label="Select column with base qualities" type="data_column" numerical="false" data_ref="input" help="something like this: IIIGIAI"/>
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21 <param name="cvrg_column" label="Select column with coverage" type="data_column" numerical="true" data_ref="input" />
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22 <param name="coord_column" label="Select coordinate column" type="data_column" numerical="true" data_ref="input" />
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23 </when>
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24 <when value="six">
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25 </when>
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26 <when value="ten">
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27 </when>
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28 </conditional>
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29 <param name="qv_cutoff" label="Do not consider read bases with quality lower than" type="integer" value="20" help="No variants with quality below this value will be reported"/>
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30 <param name="cvrg_cutoff" label="Do not report positions with coverage lower than" type="integer" value="3" help="Pileup lines with coverage lower than this value will be skipped"/>
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31 <param name="snps_only" label="Only report variants?" type="select" help="See "Examples 1 and 2" below for explanation">
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32 <option value="No">No</option>
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33 <option value="Yes" selected="true">Yes</option>
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34 </param>
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35 <param name="interval" label="Convert coordinates to intervals?" type="select" help="See "Output format" below for explanation">
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36 <option value="No" selected="true">No</option>
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37 <option value="Yes">Yes</option>
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38 </param>
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39 <param name="diff" label="Print total number of differences?" type="select" help="See "Example 3" below for explanation">
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40 <option value="No" selected="true">No</option>
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41 <option value="Yes">Yes</option>
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42 </param>
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43 <param name="qc_base" label="Print quality and base string?" type="select" help="See "Example 4" below for explanation">
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44 <option value="No">No</option>
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45 <option value="Yes" selected="true">Yes</option>
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46 </param>
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47
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48 </inputs>
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49 <outputs>
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50 <data format="tabular" name="out_file1">
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51 <change_format>
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52 <when input="interval" value="Yes" format="interval" />
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53 </change_format>
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54 </data>
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55 </outputs>
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56 <tests>
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57 <test>
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58 <param name="input" value="pileup_parser.6col.pileup"/>
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59 <output name="out_file1" file="pileup_parser.6col.20-3-yes-yes.pileup.out"/>
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60 <param name="type_select" value="six"/>
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61 <param name="qv_cutoff" value="20" />
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62 <param name="cvrg_cutoff" value="3" />
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63 <param name="snps_only" value="Yes"/>
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64 <param name="interval" value="Yes" />
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65 <param name="diff" value="No" />
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66 <param name="qc_base" value="Yes" />
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67 </test>
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68 <test>
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69 <param name="input" value="pileup_parser.6col.pileup"/>
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70 <output name="out_file1" file="pileup_parser.6col.20-3-yes-no.pileup.out"/>
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71 <param name="type_select" value="six"/>
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72 <param name="qv_cutoff" value="20" />
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73 <param name="cvrg_cutoff" value="3" />
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74 <param name="snps_only" value="Yes"/>
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75 <param name="interval" value="No" />
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76 <param name="diff" value="No" />
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77 <param name="qc_base" value="Yes" />
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78 </test>
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79 <test>
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80 <param name="input" value="pileup_parser.6col.pileup"/>
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81 <output name="out_file1" file="pileup_parser.6col.20-3-no-no.pileup.out"/>
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82 <param name="type_select" value="six"/>
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83 <param name="qv_cutoff" value="20" />
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84 <param name="cvrg_cutoff" value="3" />
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85 <param name="snps_only" value="No"/>
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86 <param name="interval" value="No" />
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87 <param name="diff" value="No" />
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88 <param name="qc_base" value="Yes" />
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89 </test>
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90 <test>
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91 <param name="input" value="pileup_parser.10col.pileup"/>
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92 <output name="out_file1" file="pileup_parser.10col.20-3-yes-yes.pileup.out"/>
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93 <param name="type_select" value="ten"/>
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94 <param name="qv_cutoff" value="20" />
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95 <param name="cvrg_cutoff" value="3" />
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96 <param name="snps_only" value="Yes"/>q
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97 <param name="interval" value="Yes" />
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98 <param name="diff" value="No" />
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99 <param name="qc_base" value="Yes" />
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100 </test>
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101 <test>
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102 <param name="input" value="pileup_parser.10col.pileup"/>
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103 <output name="out_file1" file="pileup_parser.10col.20-3-yes-yes.pileup.out"/>
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104 <param name="type_select" value="manual"/>
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105 <param name="ref_base_column" value="3"/>
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106 <param name="read_bases_column" value="9"/>
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107 <param name="read_qv_column" value="10"/>
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108 <param name="cvrg_column" value="8"/>
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109 <param name="coord_column" value="2"/>
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110 <param name="qv_cutoff" value="20" />
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111 <param name="cvrg_cutoff" value="3" />
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112 <param name="snps_only" value="Yes"/>
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113 <param name="interval" value="Yes" />
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114 <param name="diff" value="No" />
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115 <param name="qc_base" value="Yes" />
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116 </test>
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117 <test>
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118 <param name="input" value="pileup_parser.10col.pileup"/>
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119 <output name="out_file1" file="pileup_parser.10col.20-3-yes-yes-yes-yes.pileup.out"/>
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120 <param name="type_select" value="manual"/>
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121 <param name="ref_base_column" value="3"/>
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122 <param name="read_bases_column" value="9"/>
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123 <param name="read_qv_column" value="10"/>
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124 <param name="cvrg_column" value="8"/>
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125 <param name="coord_column" value="2"/>
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126 <param name="qv_cutoff" value="20" />
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127 <param name="cvrg_cutoff" value="3" />
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128 <param name="snps_only" value="Yes"/>
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129 <param name="interval" value="Yes" />
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130 <param name="diff" value="Yes" />
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131 <param name="qc_base" value="Yes" />
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132 </test>
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133 <test>
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134 <param name="input" value="pileup_parser.10col.pileup"/>
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135 <output name="out_file1" file="pileup_parser.10col.20-3-yes-yes-yes-no.pileup.out"/>
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136 <param name="type_select" value="manual"/>
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137 <param name="ref_base_column" value="3"/>
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138 <param name="read_bases_column" value="9"/>
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139 <param name="read_qv_column" value="10"/>
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140 <param name="cvrg_column" value="8"/>
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141 <param name="coord_column" value="2"/>
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142 <param name="qv_cutoff" value="20" />
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143 <param name="cvrg_cutoff" value="3" />
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144 <param name="snps_only" value="Yes"/>
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145 <param name="interval" value="Yes" />
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146 <param name="diff" value="Yes" />
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147 <param name="qc_base" value="No" />
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148 </test>
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149
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150
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151 </tests>
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152 <help>
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153
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154 **What it does**
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155
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156 Allows one to find sequence variants and/or sites covered by a specified number of reads with bases above a set quality threshold. The tool works on six and ten column pileup formats produced with *samtools pileup* command. However, it also allows you to specify columns in the input file manually. The tool assumes the following:
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157
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158 - the quality scores follow phred33 convention, where input qualities are ASCII characters equal to the Phred quality plus 33.
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159 - the pileup dataset was produced by the *samtools pileup* command (although you can override this by setting column assignments manually).
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160
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161 --------
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162
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163 **Types of pileup datasets**
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164
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165 The descriptions of the following pileup formats are largely based on information that can be found on the SAMTools_ documentation page. The 6- and 10-column variants are described below.
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166
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167 .. _SAMTools: http://samtools.sourceforge.net/pileup.shtml
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168
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169 **Six column pileup**::
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170
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171 1 2 3 4 5 6
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172 ---------------------------------
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173 chrM 412 A 2 ., II
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174 chrM 413 G 4 ..t, IIIH
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175 chrM 414 C 4 ..Ta III2
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176 chrM 415 C 4 TTTt III7
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177
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178 where::
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179
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180 Column Definition
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181 ------- ----------------------------
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182 1 Chromosome
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183 2 Position (1-based)
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184 3 Reference base at that position
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185 4 Coverage (# reads aligning over that position)
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186 5 Bases within reads
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187 6 Quality values (phred33 scale, see Galaxy wiki for more)
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188
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189 **Ten column pileup**
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190
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191 The `ten-column`__ pileup incorporates additional consensus information generated with the *-c* option of the *samtools pileup* command::
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192
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193
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194 1 2 3 4 5 6 7 8 9 10
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195 ------------------------------------------------
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196 chrM 412 A A 75 0 25 2 ., II
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197 chrM 413 G G 72 0 25 4 ..t, IIIH
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198 chrM 414 C C 75 0 25 4 ..Ta III2
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199 chrM 415 C T 75 75 25 4 TTTt III7
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200
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201 where::
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202
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203 Column Definition
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204 ------- ----------------------------
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205 1 Chromosome
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206 2 Position (1-based)
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207 3 Reference base at that position
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208 4 Consensus bases
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209 5 Consensus quality
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210 6 SNP quality
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211 7 Maximum mapping quality
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212 8 Coverage (# reads aligning over that position)
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213 9 Bases within reads
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214 10 Quality values (phred33 scale, see Galaxy wiki for more)
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215
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216
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217 .. __: http://samtools.sourceforge.net/cns0.shtml
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218
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219 ------
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220
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221 **The output format**
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222
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223 The tool modifies the input dataset in two ways:
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224
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225 1. It appends five columns to the end of every reported line:
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226
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227 - Number of **A** variants
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228 - Number of **C** variants
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229 - Number of **G** variants
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230 - Number of **T** variants
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231 - Number of read bases covering this position, where quality is equal to or higher than the value set by **Do not consider read bases with quality lower than** option.
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232
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233 Optionally, if **Print total number of differences?** is set to **Yes**, the tool will append the sixth column with the total number of deviants (see below).
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234
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235 2. If **Convert coordinates to intervals?** is set to **Yes**, the tool replaces the position column (typically the second column) with a pair of tab-delimited start/end values.
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236
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237 For example, if you are calling variants with base quality above 20 on this dataset::
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238
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239 chrM 412 A 2 ., II
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240 chrM 413 G 4 ..t, III2
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241 chrM 414 C 4 ..Ta III2
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242 chrM 415 C 4 TTTt III7
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243
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244 you will get::
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245
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246 chrM 413 G 4 ..t, IIIH 0 0 2 1 3
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247 chrM 414 C 4 ..Ta III2 1 1 0 1 3
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248 chrM 415 C 4 TTTt III7 0 0 0 4 4
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249
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250 where::
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251
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252 Column Definition
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253 ------- ----------------------------
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254 1 Chromosome
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255 2 Position (1-based)
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256 3 Reference base at that position
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257 4 Coverage (# reads aligning over that position)
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258 5 Bases within reads where
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259 6 Quality values (phred33 scale, see Galaxy wiki for more)
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260 7 Number of A variants
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261 8 Number of C variants
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262 9 Number of G variants
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263 10 Number of T variants
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264 11 Quality adjusted coverage:
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265 12 Number of read bases (i.e., # of reads) with quality above the set threshold
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266 13 Total number of deviants (if Convert coordinates to intervals? is set to yes)
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267
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268 if **Print total number of differences?** is set to **Yes**, you will get::
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269
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270 chrM 413 G 4 ..t, IIIH 0 0 2 1 3 1
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271 chrM 414 C 4 ..Ta III2 1 2 0 1 3 2
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272 chrM 415 C 4 TTTt III7 0 0 0 4 4 0
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273
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274 Note the additional column 13, that contains the number of deviant reads (e.g., there are two deviants, T and a, for position 414).
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275
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276
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277 Finally, if **Convert coordinates to intervals?** is set to **Yes**, you will get one additional column with the end coordinate::
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278
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279 chrM 412 413 G 4 ..t, III2 0 0 2 1 3
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280 chrM 414 415 C 4 ..Ta III2 1 2 0 1 3
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281 chrM 414 415 C 4 TTTt III7 0 0 0 4 4
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282
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283 where::
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284
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285 Column Definition
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286 ------- ----------------------------
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287 1 Chromosome
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288 2 Start position (0-based)
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289 3 End position (1-based)
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290 4 Reference base at that position
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291 5 Coverage (# reads aligning over that position)
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292 6 Bases within reads
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293 7 Quality values (phred33 scale, see Galaxy wiki for more)
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294 8 Number of A variants
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295 9 Number of C variants
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296 10 Number of G variants
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297 11 Number of T variants
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298 12 Quality adjusted coverage
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299 13 Total number of deviants (if Convert coordinates to intervals? is set to yes)
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300
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301
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302 Note that in this case the coordinates of SNPs were converted to intervals, where the start coordinate is 0-based and the end coordinate in 1-based using the UCSC Table Browser convention.
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303
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304 Although three positions have variants in the original file (413, 414, and 415), only 413 and 415 are reported because the quality values associated with these two SNPs are above the threshold of 20. In the case of 414 the **a** allele has a quality value of 17 ( ord("2")-33 ), and is therefore not reported. Note that five columns have been added to each of the reported lines::
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305
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306 chrM 413 G 4 ..t, IIIH 0 0 2 1 3
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307
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308 Here, there is one variant, **t**. Because the fourth column represents **T** counts, it is incremented by 1. The last column shows that at this position, three reads have bases above the quality threshold of 20.
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309
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310 -----
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311
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312 **Example 1**: Just variants
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313
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314 In this mode, the tool only outputs the lines from the input datasets where at least one read contains a sequence variant with quality above the threshold set by the **Do not consider read bases with quality lower than** option. For example, suppose one has a pileup dataset like the following::
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315
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316 chrM 412 A 2 ., II
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317 chrM 413 G 4 ..t, III2
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318 chrM 414 C 4 ..Ta III2
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319 chrM 415 C 4 TTTt III7
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320
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321 To call all variants (with no restriction by coverage) with quality above phred value of 20, we will need to set the parameters as follows:
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322
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323 .. image:: ./static/images/pileup_parser_help1.png
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324
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325 Running the tool with these parameters will return::
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326
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327 chrM 413 G 4 ..t, IIIH 0 0 0 1 3
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328 chrM 414 C 4 ..Ta III2 0 2 0 1 3
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329 chrM 415 C 4 TTTt III7 0 0 0 4 4
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330
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331 **Note** that position 414 is not reported because the *a* variant has associated quality value of 17 (because ord('2')-33 = 17) and is below the phred threshold of 20 set by the **Count variants with quality above this value** parameter.
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332
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333 -----
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334
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335 **Example 2**: Report everything
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336
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337 In addition to calling variants, it is often useful to know the quality adjusted coverage. Running the tool with these parameters:
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338
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339 .. image:: ./static/images/pileup_parser_help2.png
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340
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341 will report everything from the original file::
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342
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343 chrM 412 A 2 ., II 2 0 0 0 2
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344 chrM 413 G 4 ..t, III2 0 0 2 1 3
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345 chrM 414 C 4 ..Ta III2 0 2 0 1 3
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346 chrM 415 C 4 TTTt III7 0 0 0 4 4
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347
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348 Here, you can see that although the total coverage at position 414 is 4 (column 4), the quality adjusted coverage is 3 (last column). This is because only three out of four reads have bases with quality above the set threshold of 20 (the actual qualities are III2 or, after conversion, 40, 40, 40, 17).
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349
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350 One can use the last column of this dataset to filter out (using Galaxy's **Filter** tool) positions where quality adjusted coverage (last column) is below a set threshold.
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351
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352 ------
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353
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354 **Example 3**: Report everything and print total number of differences
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355
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356 If you set the **Print total number of differences?** to **Yes** the tool will print an additional column with the total number of reads where a devinat base is above the quality threshold. So, seetiing parametrs like this:
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357
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358 .. image:: ./static/images/pileup_parser_help3.png
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359
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360 will produce this::
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361
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362 chrM 412 A 2 ., II 2 0 0 0 2 0
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363 chrM 413 G 4 ..t, III2 0 0 2 1 3 1
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364 chrM 414 C 4 ..Ta III2 0 2 0 1 3 1
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365 chrM 415 C 4 TTTt III7 0 0 0 4 4 0
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366
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367
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368 -----
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369
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370 **Example 4**: Report everything, print total number of differences, and ignore qualities and read bases
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371
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372 Setting **Print quality and base string?** to **Yes** as shown here:
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373
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374 .. image:: ./static/images/pileup_parser_help4.png
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375
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376 will produce this::
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377
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378 chrM 412 A 2 2 0 0 0 2 0
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379 chrM 413 G 4 0 0 2 1 3 1
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380 chrM 414 C 4 0 2 0 1 3 1
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381 chrM 415 C 4 0 0 0 4 4 0
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382
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383
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384
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385
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386 </help>
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387 </tool>
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