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1 <tool id="bismark_bowtie2" name="Bismark" version="0.14.3">
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18
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2 <!-- Wrapper compatible with Bismark version 0.10 -->
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9
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3 <description>bisulfite mapper (bowtie2)</description>
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4 <!--<version_command>bismark version</version_command>-->
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5 <requirements>
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18
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6 <requirement type="package" version="0.1.19">samtools</requirement>
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22
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7 <requirement type="package" version="2.2.5">bowtie2</requirement>
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9
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8 </requirements>
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21
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9 <stdio>
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10 <exit_code range="1:" />
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11 <exit_code range=":-1" />
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12 <regex match="Error:" />
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13 <regex match="Exception:" />
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14 </stdio>
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9
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15 <command interpreter="python">
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21
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16 <![CDATA[
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17 bismark_wrapper.py
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21
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18
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19 ## Change this to accommodate the number of threads you have available.
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18
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20 --num-threads "\${GALAXY_SLOTS:-24}"
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21
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22
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22 --bismark_path $__tool_directory__
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23
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24 --bowtie2
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25
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26 ##
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27 ## Bismark Genome Preparation, if desired.
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28 ##
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29
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30 ## Handle reference file.
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31 #if $refGenomeSource.genomeSource == "history":
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32 --own-file=$refGenomeSource.ownFile
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33 #else:
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34 --indexes-path ${refGenomeSource.index.fields.path}
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35 #end if
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36
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37
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38 ##
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39 ## Input parameters
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40 ##
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41
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42 #if $singlePaired.sPaired == "single":
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43 --single-paired $singlePaired.input_singles
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44
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45 #if $singlePaired.input_singles.ext == "fastqillumina":
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46 --phred64-quals
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47 --fastq
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48 #elif $singlePaired.input_singles.ext == "fastqsanger":
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49 --fastq
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50 #elif $singlePaired.input_singles.ext == "fasta":
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51 --fasta
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52 #end if
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53 #else:
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54 --mate-paired
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55 #set $mate1 = list()
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56 #set $mate2 = list()
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57 #for $mate_pair in $singlePaired.mate_list
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58 $mate1.append( str($mate_pair.input_mate1) )
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59 $mate2.append( str($mate_pair.input_mate2) )
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60 #end for
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61
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62 --mate1 #echo ','.join($mate1)
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63 --mate2 #echo ','.join($mate2)
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64
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21
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65 #for $mate_pair in $singlePaired.mate_list:
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66 #if $mate_pair.input_mate1.ext == "fastqillumina":
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67 --phred64-quals
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68 --fastq
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69 #elif $mate_pair.input_mate1.ext == "fastqsanger":
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70 --fastq
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71 #elif $mate_pair.input_mate1.ext == "fasta":
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72 --fasta
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73 #end if
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74 #break
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75 #end for
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76
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77 -I $singlePaired.minInsert
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78 -X $singlePaired.maxInsert
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79 #end if
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80
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81 #if $sort_bam:
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82 --sort-bam
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83 #end if
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84
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85 ## for now hardcode the value for the required memory per thread in --best mode
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86 --chunkmbs 512
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87
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88
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89 #if $params.settingsType == "custom":
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90
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91 ## default 20
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92 --seed-len $params.seed_len
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93 ## default 0
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94 --seed-mismatches $params.seed_mismatches
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95 ## default 15
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96 --seed-extention-attempts $params.seed_extention_attempts
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97 ## default 2
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98 --max-reseed $params.max_reseed
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99
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100 ## default 70
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101 ##--maqerr $params.maqerr
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102
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103 ## default unlimited
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104 #if $params.qupto != 0:
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105 --qupto $params.qupto
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106 #end if
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107 #if $params.skip_reads != 0:
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108 --skip-reads $params.skip_reads
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109 #end if
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110
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111 ## if set, disable the original behaviour
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112 $params.no_mixed
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113 ## if set, disable the original behaviour
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114 $params.no_discordant
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115
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116 #if $params.bismark_stdout:
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117 --stdout $output_stdout
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118 #end if
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119
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120 #if $params.isReportOutput:
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121 --output-report-file $report_file
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122 #end if
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123
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124 #end if
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125
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126 ##
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127 ## Output parameters.
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128 ##
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129 --output $output
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130 ##$suppress_header
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131
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132 #if str( $singlePaired.sPaired ) == "single"
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133 #if $output_unmapped_reads_l
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134 --output-unmapped-reads $output_unmapped_reads_l
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135 #end if
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136 #if $output_suppressed_reads_l
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137 --output-suppressed-reads $output_suppressed_reads_l
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138 #end if
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139 #else
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140 #if $output_unmapped_reads_l and $output_unmapped_reads_r
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141 --output-unmapped-reads-l $output_unmapped_reads_l
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142 --output-unmapped-reads-r $output_unmapped_reads_r
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143 #end if
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144 #if $output_suppressed_reads_l and $output_suppressed_reads_l
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145 --output-suppressed-reads-l $output_suppressed_reads_l
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146 --output-suppressed-reads-r $output_suppressed_reads_r
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147 #end if
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148 #end if
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149
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150 ]]>
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151 </command>
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152 <inputs>
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153 <conditional name="refGenomeSource">
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154 <param name="genomeSource" type="select" label="Will you select a reference genome from your history or use a built-in index?" help="Built-ins were indexed using default options">
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155 <option value="indexed">Use a built-in index</option>
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156 <option value="history">Use one from the history</option>
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157 </param>
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158 <when value="indexed">
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159 <param name="index" type="select" label="Select a reference genome" help="If your genome of interest is not listed, contact your Galaxy admin">
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160 <options from_data_table="bowtie2_indexes">
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161 <filter type="sort_by" column="2"/>
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162 <validator type="no_options" message="No indexes are available for the selected input dataset"/>
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163 </options>
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164 </param>
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165 </when> <!-- build-in -->
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166 <when value="history">
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167 <param name="ownFile" type="data" format="fasta" metadata_name="dbkey" label="Select the reference genome" />
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168 </when> <!-- history -->
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169 </conditional> <!-- refGenomeSource -->
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170
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171 <!-- Input Parameters -->
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172 <conditional name="singlePaired">
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173 <param name="sPaired" type="select" label="Is this library mate-paired?">
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174 <option value="single">Single-end</option>
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175 <option value="paired">Paired-end</option>
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176 </param>
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177 <when value="single">
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178 <param name="input_singles" type="data" format="fastqsanger,fastqillumina,fastq,fasta" label="FASTQ/FASTA file" help="FASTQ or FASTA files." />
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179 </when>
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180 <when value="paired">
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181 <repeat name="mate_list" title="Paired End Pairs" min="1">
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182 <param name="input_mate1" type="data" format="fastqsanger,fastqillumina,fastq,fasta" label="Mate pair 1" help="FASTQ or FASTA files." />
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183 <param name="input_mate2" type="data" format="fastqsanger,fastqillumina,fastq,fasta" label="Mate pair 2" help="FASTQ or FASTA files." />
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184 </repeat>
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185 <param name="minInsert" type="integer" value="0" label="Minimum insert size for valid paired-end alignments" />
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186 <param name="maxInsert" type="integer" value="500" label="Maximum insert size for valid paired-end alignments" />
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187 </when>
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188 </conditional>
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189
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190 <param name="sort_bam" type="boolean" truevalue="true" falsevalue="false" checked="False" label="Sort BAM file by chromosomal position (not compatibile with methylation extractor)"/>
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191
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192 <conditional name="params">
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193 <param name="settingsType" type="select" label="Bismark settings to use" help="You can use the default settings or set custom values for any of Bismark's parameters.">
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194 <option value="default">Use Defaults</option>
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195 <option value="custom">Full parameter list</option>
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196 </param>
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197 <when value="default" />
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198 <!-- Full/advanced params. -->
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199 <when value="custom">
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200 <!-- -N -->
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201 <param name="seed_mismatches" type="integer" value="0" label="Number of mismatches to be allowed in a seed alignment during multiseed alignment" />
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202 <!-- -L -->
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203 <param name="seed_len" type="integer" value="20" label="Length of the seed substrings to align during multiseed alignment" />
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204 <!--
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205 <param name="maqerr" type="integer" value="70" label="Maximum permitted total of quality values at all mismatched read positions throughout the entire alignment, not just in the 'seed'." />
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206 -->
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207 <!-- -D -->
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208 <param name="seed_extention_attempts" type="integer" value="15" label="How many consecutive seed extension attempts can fail before Bowtie 2 moves on" />
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209 <!-- -R -->
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210 <param name="max_reseed" type="integer" value="2" label="Maximum number of times Bowtie 2 will re-seed reads with repetitive seeds" />
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211
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212 <param name="qupto" type="integer" value="0" label="Only aligns the first N reads or read pairs from the input" help="Default is 0 and means 'no-limit'." />
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213 <param name="skip_reads" type="integer" value="0" label="Skip (i.e. do not align) the first N reads or read pairs from the input" />
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214
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215 <param name="no_discordant" type="boolean" truevalue="--no-discordant" falsevalue="" checked="false" label="Disable looking for discordant alignments if it cannot find any concordant alignments" help="" />
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216 <param name="no_mixed" type="boolean" truevalue="--no-mixed" falsevalue="" checked="false" label="Disable Bowtie 2's behaviour to try to find alignments for the individual mates" help="" />
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217
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218 <param name="suppressed_read_file" type="boolean" truevalue="true" falsevalue="false" checked="false" label="Write ambiguous reads to an extra output file" help="Write all reads which produce more than one valid alignment with the same number of lowest mismatches or other reads that fail to align uniquely." />
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219 <param name="unmapped_read_file" type="boolean" truevalue="true" falsevalue="false" checked="false" label="Write all reads that could not be aligned to a file" />
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220 <!-- output Options -->
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221 <param name="bismark_stdout" type="boolean" truevalue="true" falsevalue="false" checked="false" label="Write the bismark output and summary information to an extra file" />
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222 <param name="isReportOutput" type="boolean" truevalue="true" falsevalue="false" checked="false" label="Offer all report files concatenated in one file" />
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223
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224 <!--end output options -->
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225 </when> <!-- full -->
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226 </conditional> <!-- params -->
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227 <!--
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228 <param name="suppress_header" type="boolean" truevalue="..suppress-header" falsevalue="" checked="false" label="Suppress the header in the output SAM file" help="Bowtie produces SAM with several lines of header information by default." />
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229 -->
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230 </inputs>
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231
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232
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233 <outputs>
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234 <data format="txt" name="report_file" label="${tool.name} on ${on_string}: Report">
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235 <filter>
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236 ((
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237 params['settingsType'] == "custom" and
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238 params['isReportOutput'] is True
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239 ))
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240 </filter>
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241 </data>
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242 <data format="txt" name="output_stdout" label="${tool.name} on ${on_string}: Summary">
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243 <filter>
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244 ((
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245 params['settingsType'] == "custom" and
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246 params['bismark_stdout'] is True
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247 ))
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248 </filter>
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249 </data>
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250
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251 <data format="bam" name="output" label="${tool.name} on ${on_string}: mapped reads">
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252 <actions>
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253 <conditional name="refGenomeSource.genomeSource">
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254 <when value="indexed">
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255 <action type="metadata" name="dbkey">
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256 <option type="from_data_table" name="bowtie2_indexes" column="1" offset="0">
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257 <filter type="param_value" column="0" value="#" compare="startswith" keep="False"/>
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258 <filter type="param_value" ref="refGenomeSource.index" column="0"/>
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259 </option>
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260 </action>
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261 </when>
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262 <when value="history">
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263 <action type="metadata" name="dbkey">
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264 <option type="from_param" name="refGenomeSource.ownFile" param_attribute="dbkey" />
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265 </action>
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266 </when>
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267 </conditional>
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268 </actions>
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269 </data>
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270
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271 <data format="fastq" name="output_suppressed_reads_l" label="${tool.name} on ${on_string}: suppressed reads (L)">
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272 <filter>
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273 ((
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274 params['settingsType'] == "custom" and
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275 params['suppressed_read_file'] is True
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276 ))
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277 </filter>
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278 <actions>
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279 <conditional name="singlePaired.sPaired">
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280 <when value="single">
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281 <action type="format">
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282 <option type="from_param" name="singlePaired.input_singles" param_attribute="ext" />
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283 </action>
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284 </when>
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285 <when value="paired">
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286 <!--action type="format">
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287 <option type="from_param" name="singlePaired.mate_list[0].input_mate1" param_attribute="ext" />
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288 </action-->
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289 </when>
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290 </conditional>
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291 </actions>
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292 </data>
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293
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294 <data format="fastq" name="output_suppressed_reads_r" label="${tool.name} on ${on_string}: suppressed reads (R)">
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295 <filter>
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296 ((
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297 singlePaired['sPaired'] == "paired" and
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298 params['settingsType'] == "custom" and
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299 params['suppressed_read_file'] is True
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300 ))
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301 </filter>
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9
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302 <actions>
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303 <conditional name="singlePaired.sPaired">
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304 <when value="single">
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305 <action type="format">
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306 <option type="from_param" name="singlePaired.input_singles" param_attribute="ext" />
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307 </action>
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308 </when>
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309 <when value="paired">
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21
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310 <!--action type="format">
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311 <option type="from_param" name="singlePaired.mate_list[0].input_mate1" param_attribute="ext" />
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21
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312 </action-->
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9
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313 </when>
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314 </conditional>
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315 </actions>
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316 </data>
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317
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318 <!-- Outout unmapped reads -->
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319 <data format="fastq" name="output_unmapped_reads_l" label="${tool.name} on ${on_string}: unmapped reads (L)">
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320 <filter>
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321 ((
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322 params['settingsType'] == "custom" and
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323 params['unmapped_read_file'] is True
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324 ))
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325 </filter>
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326 <actions>
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327 <conditional name="singlePaired.sPaired">
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328 <when value="single">
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329 <action type="format">
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330 <option type="from_param" name="singlePaired.input_singles" param_attribute="ext" />
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331 </action>
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332 </when>
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333 <when value="paired">
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21
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334 <!--action type="format">
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18
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335 <option type="from_param" name="singlePaired.mate_list[0].input_mate1" param_attribute="ext" />
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21
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336 </action-->
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9
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337 </when>
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338 </conditional>
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339 </actions>
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340 </data>
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18
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341
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9
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342 <data format="fastq" name="output_unmapped_reads_r" label="${tool.name} on ${on_string}: unmapped reads (R)">
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21
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343 <filter>
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344 ((
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345 singlePaired['sPaired'] == "paired" and
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346 params['settingsType'] == "custom" and
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347 params['unmapped_read_file'] is True
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348 ))
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349 </filter>
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9
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350 <actions>
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351 <conditional name="singlePaired.sPaired">
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352 <when value="single">
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353 <action type="format">
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354 <option type="from_param" name="singlePaired.input_singles" param_attribute="ext" />
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355 </action>
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356 </when>
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357 <when value="paired">
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21
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358 <!--action type="format">
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18
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359 <option type="from_param" name="singlePaired.mate_list[0].input_mate1" param_attribute="ext" />
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21
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360 </action-->
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9
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361 </when>
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362 </conditional>
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363 </actions>
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364 </data>
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365 </outputs>
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366
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367 <tests>
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368 </tests>
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369
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370 <help>
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371 <![CDATA[
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372
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373 **What it does**
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374
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375 Bismark_ is a bisulfite mapper and methylation caller. Bismark takes in FastA or FastQ files and aligns the
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376 reads to a specified bisulfite genome. Sequence reads are transformed into a bisulfite converted forward strand
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377 version (C->T conversion) or into a bisulfite treated reverse strand (G->A conversion of the forward strand).
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378 Each of these reads are then aligned to bisulfite treated forward strand index of a reference genome
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379 (C->T converted) and a bisulfite treated reverse strand index of the genome (G->A conversion of the
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380 forward strand, by doing this alignments will produce the same positions). These instances of Bowtie 2
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381 are run in parallel. The sequence file(s) are then read in again sequence by sequence to pull out the original
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382 sequence from the genome and determine if there were any protected C's present or not.
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383
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384 .. _Bismark: http://www.bioinformatics.babraham.ac.uk/projects/bismark/
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385
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386 As of version 0.7.0 Bismark will only run 2 alignment threads for OT and OB in parallel, the 4 strand mode can be
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387 re-enabled by using non_directional mode.
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388
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389 It is developed by Krueger F and Andrews SR. at the Babraham Institute. Krueger F, Andrews SR. (2011) Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics, 27, 1571-2.
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390
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391 ------
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392
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393 **Know what you are doing**
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394
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395 .. class:: warningmark
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396
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397 There is no such thing (yet) as an automated gearshift in short read mapping. It is all like stick-shift driving in San Francisco. In other words = running this tool with default parameters will probably not give you meaningful results. A way to deal with this is to **understand** the parameters by carefully reading the `documentation`__ and experimenting. Fortunately, Galaxy makes experimenting easy.
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398
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399 .. __: http://www.bioinformatics.babraham.ac.uk/projects/bismark/
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400
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401
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402 .. class:: warningmark
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403
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404 Make sure all your input reads are in the correct and same format. If thats not the case please adjust/convert the filetype with galaxy's build-in converters.
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405
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9
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406 ------
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407
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408 **Input formats**
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409
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410 Bismark accepts files in either Sanger FASTQ format (galaxy type *fastqsanger*), Illumina FASTQ format (galaxy type *fastqillumina*) or FASTA format (galaxy type *fasta*). Use the FASTQ Groomer to prepare your files.
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411
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412 ------
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413
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414 **A Note on Built-in Reference Genomes**
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415
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416 The default variant for all genomes is "Full", defined as all primary chromosomes (or scaffolds/contigs) including mitochondrial plus associated unmapped, plasmid, and other segments. When only one version of a genome is available in this tool, it represents the default "Full" variant. Some genomes will have more than one variant available. The "Canonical Male" or sometimes simply "Canonical" variant contains the primary chromosomes for a genome. For example a human "Canonical" variant contains chr1-chr22, chrX, chrY, and chrM. The "Canonical Female" variant contains the primary chromosomes excluding chrY.
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417
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418 ------
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419
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420 The final output of Bismark is in SAM format by default.
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421
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422 **Outputs**
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423
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424 The output is in SAM format, and has the following columns::
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425
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426 Column Description
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427 -------- --------------------------------------------------------
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428 1 QNAME seq-ID
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429 2 FLAG this flag tries to take the strand a bisulfite read
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430 originated from into account
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431 (this is different from ordinary DNA alignment flags!)
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432 3 RNAME chromosome
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433 4 POS start position
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434 5 MAPQ always 255
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435 6 CIGAR extended CIGAR string
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436 7 MRNM Mate Reference sequence NaMe ('=' if same as RNAME)
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437 8 MPOS 1-based Mate POSition
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438 9 ISIZE Inferred insert SIZE
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439 10 SEQ query SEQuence on the same strand as the reference
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440 11 QUAL Phred33 scale
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441 12 NM-tag edit distance to the reference)
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442 13 XX-tag base-by-base mismatches to the reference.
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443 This does not include indels.
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444 14 XM-tag methylation call string
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445 15 XR-tag read conversion state for the alignment
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446 16 XG-tag genome conversion state for the alignment
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447
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448
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449 Each read of paired-end alignments is written out in a separate line in the above format.
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450
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451
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452 It looks like this (scroll sideways to see the entire example)::
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453
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454 QNAME FLAG RNAME POS MAPQ CIAGR MRNM MPOS ISIZE SEQ QUAL OPT
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455 HWI-EAS91_1_30788AAXX:1:1:1761:343 4 * 0 0 * * 0 0 AAAAAAANNAAAAAAAAAAAAAAAAAAAAAAAAAAACNNANNGAGTNGNNNNNNNGCTTCCCACAGNNCTGG hhhhhhh;;hhhhhhhhhhh^hOhhhhghhhfhhhgh;;h;;hhhh;h;;;;;;;hhhhhhghhhh;;Phhh
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456 HWI-EAS91_1_30788AAXX:1:1:1578:331 4 * 0 0 * * 0 0 GTATAGANNAATAAGAAAAAAAAAAATGAAGACTTTCNNANNTCTGNANNNNNNNTCTTTTTTCAGNNGTAG hhhhhhh;;hhhhhhhhhhhhhhhhhhhhhhhhhhhh;;h;;hhhh;h;;;;;;;hhhhhhhhhhh;;hhVh
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457
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458 -------
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459
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460 **Bismark settings**
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461
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462 All of the options have a default value. You can change any of them. If any Bismark function is missing please contact the tool author or your Galaxy admin.
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463
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464 ------
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465
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466 **Bismark parameter list**
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467
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468 This is an exhaustive list of Bismark options.
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469
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470 Input::
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471
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472 --singles A comma- or space-separated list of files containing the reads to be aligned (e.g.
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473 lane1.fq,lane2.fq lane3.fq). Reads may be a mix of different lengths. Bismark will
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474 produce one mapping result and one report file per input file.
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475
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476 -1 mates1 Comma-separated list of files containing the #1 mates (filename usually includes
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477 "_1"), e.g. flyA_1.fq,flyB_1.fq). Sequences specified with this option must
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478 correspond file-for-file and read-for-read with those specified in mates2.
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479 Reads may be a mix of different lengths. Bismark will produce one mapping result
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480 and one report file per paired-end input file pair.
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481
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482 -2 mates2 Comma-separated list of files containing the #2 mates (filename usually includes
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483 "_2"), e.g. flyA_1.fq,flyB_1.fq). Sequences specified with this option must
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484 correspond file-for-file and read-for-read with those specified in mates1.
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485 Reads may be a mix of different lengths.
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486
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487 -q/--fastq The query input files (specified as mate1,mate2 or singles are FASTQ
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488 files (usually having extension .fg or .fastq). This is the default. See also
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489 --solexa-quals.
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490
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491 -f/--fasta The query input files (specified as mate1,mate2 or singles are FASTA
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492 files (usually havin extension .fa, .mfa, .fna or similar). All quality values
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493 are assumed to be 40 on the Phred scale.
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494
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495 -s/--skip INT Skip (i.e. do not align) the first INT reads or read pairs from the input.
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496
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497 -u/--upto INT Only aligns the first INT reads or read pairs from the input. Default: no limit.
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498
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499 --phred33-quals FASTQ qualities are ASCII chars equal to the Phred quality plus 33. Default: on.
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500
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501 --phred64-quals FASTQ qualities are ASCII chars equal to the Phred quality plus 64. Default: off.
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502
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503 --solexa-quals Convert FASTQ qualities from solexa-scaled (which can be negative) to phred-scaled
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504 (which can't). The formula for conversion is:
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505 phred-qual = 10 * log(1 + 10 ** (solexa-qual/10.0)) / log(10). Used with -q. This
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506 is usually the right option for use with (unconverted) reads emitted by the GA
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507 Pipeline versions prior to 1.3. Works only for Bowtie 1. Default: off.
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508
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509 --solexa1.3-quals Same as --phred64-quals. This is usually the right option for use with (unconverted)
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510 reads emitted by GA Pipeline version 1.3 or later. Default: off.
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511
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512
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513 Alignment::
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514
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515 -n/--seedmms INT The maximum number of mismatches permitted in the "seed", i.e. the first L base pairs
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516 of the read (where L is set with -l/--seedlen). This may be 0, 1, 2 or 3 and the
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517 default is 1. This option is only available for Bowtie 1 (for Bowtie 2 see -N).
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518
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519 -l/--seedlen The "seed length"; i.e., the number of bases of the high quality end of the read to
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520 which the -n ceiling applies. The default is 28. Bowtie (and thus Bismark) is faster for
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521 larger values of -l. This option is only available for Bowtie 1 (for Bowtie 2 see -L).
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522
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523 -e/--maqerr INT Maximum permitted total of quality values at all mismatched read positions throughout
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524 the entire alignment, not just in the "seed". The default is 70. Like Maq, bowtie rounds
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525 quality values to the nearest 10 and saturates at 30. This value is not relevant for
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526 Bowtie 2.
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527
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528 --chunkmbs INT The number of megabytes of memory a given thread is given to store path descriptors in
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529 --best mode. Best-first search must keep track of many paths at once to ensure it is
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530 always extending the path with the lowest cumulative cost. Bowtie tries to minimize the
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531 memory impact of the descriptors, but they can still grow very large in some cases. If
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532 you receive an error message saying that chunk memory has been exhausted in --best mode,
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533 try adjusting this parameter up to dedicate more memory to the descriptors. This value
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534 is not relevant for Bowtie 2. Default: 512.
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535
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536 -I/--minins INT The minimum insert size for valid paired-end alignments. E.g. if -I 60 is specified and
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537 a paired-end alignment consists of two 20-bp alignments in the appropriate orientation
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538 with a 20-bp gap between them, that alignment is considered valid (as long as -X is also
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539 satisfied). A 19-bp gap would not be valid in that case. Default: 0.
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540
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541 -X/--maxins INT The maximum insert size for valid paired-end alignments. E.g. if -X 100 is specified and
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542 a paired-end alignment consists of two 20-bp alignments in the proper orientation with a
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543 60-bp gap between them, that alignment is considered valid (as long as -I is also satisfied).
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544 A 61-bp gap would not be valid in that case. Default: 500.
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545
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546
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547
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548 Output::
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549
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550 --non_directional The sequencing library was constructed in a non strand-specific manner, alignments to all four
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551 bisulfite strands will be reported. Default: OFF.
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552
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553 (The current Illumina protocol for BS-Seq is directional, in which case the strands complementary
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554 to the original strands are merely theoretical and should not exist in reality. Specifying directional
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555 alignments (which is the default) will only run 2 alignment threads to the original top (OT)
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556 or bottom (OB) strands in parallel and report these alignments. This is the recommended option
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557 for sprand-specific libraries).
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558
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559 --sam-no-hd Suppress SAM header lines (starting with @). This might be useful when very large input files are
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560 split up into several smaller files to run concurrently and the output files are to be merged.
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561
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562 --quiet Print nothing besides alignments.
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563
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564 --vanilla Performs bisulfite mapping with Bowtie 1 and prints the 'old' output (as in Bismark 0.5.X) instead
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565 of SAM format output.
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566
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567 -un/--unmapped Write all reads that could not be aligned to a file in the output directory. Written reads will
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568 appear as they did in the input, without any translation of quality values that may have
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569 taken place within Bowtie or Bismark. Paired-end reads will be written to two parallel files with _1
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570 and _2 inserted in their filenames, i.e. _unmapped_reads_1.txt and unmapped_reads_2.txt. Reads
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571 with more than one valid alignment with the same number of lowest mismatches (ambiguous mapping)
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572 are also written to _unmapped_reads.txt unless the option --ambiguous is specified as well.
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573
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574 --ambiguous Write all reads which produce more than one valid alignment with the same number of lowest
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575 mismatches or other reads that fail to align uniquely to a file in the output directory.
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576 Written reads will appear as they did in the input, without any of the translation of quality
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577 values that may have taken place within Bowtie or Bismark. Paired-end reads will be written to two
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578 parallel files with _1 and _2 inserted in theit filenames, i.e. _ambiguous_reads_1.txt and
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579 _ambiguous_reads_2.txt. These reads are not written to the file specified with --un.
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580
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581 -o/--output_dir DIR Write all output files into this directory. By default the output files will be written into
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582 the same folder as the input file(s). If the specified folder does not exist, Bismark will attempt
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583 to create it first. The path to the output folder can be either relative or absolute.
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584
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585 --temp_dir DIR Write temporary files to this directory instead of into the same directory as the input files. If
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586 the specified folder does not exist, Bismark will attempt to create it first. The path to the
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587 temporary folder can be either relative or absolute.
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588
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589 ------
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590
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591 Bowtie 2 alignment options::
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592
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593 -N INT Sets the number of mismatches to allowed in a seed alignment during multiseed alignment.
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594 Can be set to 0 or 1. Setting this higher makes alignment slower (often much slower)
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595 but increases sensitivity. Default: 0. This option is only available for Bowtie 2 (for
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596 Bowtie 1 see -n).
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597
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598 -L INT Sets the length of the seed substrings to align during multiseed alignment. Smaller values
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599 make alignment slower but more senstive. Default: the --sensitive preset of Bowtie 2 is
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600 used by default, which sets -L to 20. This option is only available for Bowtie 2 (for
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601 Bowtie 1 see -l).
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602
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603 --ignore-quals When calculating a mismatch penalty, always consider the quality value at the mismatched
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604 position to be the highest possible, regardless of the actual value. I.e. input is treated
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605 as though all quality values are high. This is also the default behavior when the input
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606 doesn't specify quality values (e.g. in -f mode). This option is invariable and on by default.
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607
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608
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609 Bowtie 2 paired-end options::
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610
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611 --no-mixed This option disables Bowtie 2's behavior to try to find alignments for the individual mates if
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612 it cannot find a concordant or discordant alignment for a pair. This option is invariable and
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613 and on by default.
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614
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615 --no-discordant Normally, Bowtie 2 looks for discordant alignments if it cannot find any concordant alignments.
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616 A discordant alignment is an alignment where both mates align uniquely, but that does not
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617 satisfy the paired-end constraints (--fr/--rf/--ff, -I, -X). This option disables that behavior
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618 and it is on by default.
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619
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620
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621 Bowtie 2 effort options::
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622
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623 -D INT Up to INT consecutive seed extension attempts can "fail" before Bowtie 2 moves on, using
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624 the alignments found so far. A seed extension "fails" if it does not yield a new best or a
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625 new second-best alignment. Default: 15.
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626
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627 -R INT INT is the maximum number of times Bowtie 2 will "re-seed" reads with repetitive seeds.
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628 When "re-seeding," Bowtie 2 simply chooses a new set of reads (same length, same number of
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629 mismatches allowed) at different offsets and searches for more alignments. A read is considered
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630 to have repetitive seeds if the total number of seed hits divided by the number of seeds
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631 that aligned at least once is greater than 300. Default: 2.
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632
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633
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634 Bowtie 2 Scoring options::
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635
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636 --score_min "func" Sets a function governing the minimum alignment score needed for an alignment to be considered
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637 "valid" (i.e. good enough to report). This is a function of read length. For instance, specifying
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638 L,0,-0.2 sets the minimum-score function f to f(x) = 0 + -0.2 * x, where x is the read length.
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639 See also: setting function options at http://bowtie-bio.sourceforge.net/bowtie2. The default is
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640 L,0,-0.2.
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641
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642
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643 Bowtie 2 Reporting options::
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644
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645 --most_valid_alignments INT This used to be the Bowtie 2 parameter -M. As of Bowtie 2 version 2.0.0 beta7 the option -M is
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646 deprecated. It will be removed in subsequent versions. What used to be called -M mode is still the
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647 default mode, but adjusting the -M setting is deprecated. Use the -D and -R options to adjust the
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648 effort expended to find valid alignments.
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649
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650 For reference, this used to be the old (now deprecated) description of -M:
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651 Bowtie 2 searches for at most INT+1 distinct, valid alignments for each read. The search terminates when it
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652 can't find more distinct valid alignments, or when it finds INT+1 distinct alignments, whichever
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653 happens first. Only the best alignment is reported. Information from the other alignments is used to
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21
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654 estimate mapping quality and to set SAM optional fields, such as AS:i and XS:i. Increasing -M makes
|
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9
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655 Bowtie 2 slower, but increases the likelihood that it will pick the correct alignment for a read that
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656 aligns many places. For reads that have more than INT+1 distinct, valid alignments, Bowtie 2 does not
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657 guarantee that the alignment reported is the best possible in terms of alignment score. -M is
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658 always used and its default value is set to 10.
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659
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21
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660 ]]>
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9
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661 </help>
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21
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662 <citations>
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663 <citation type="doi">10.1093/bioinformatics/btr167</citation>
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664 </citations>
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665 </tool>
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