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1 <tool id="FLASH" name="FLASH" version="1.3.0">
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2 <description>merge paired-end reads from fragments that are shorter than twice the length of reads</description>
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3 <command interpreter="bash">
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4 FLASH.sh $extendedFrags $notCombined1 $notCombined2 $interNotCombined $readsAndPairs $log_file -o out -t 4
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5 #if $min_overlap
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6 -m $min_overlap
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7 #end if
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8 #if $max_overlap
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9 -M $max_overlap
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10 #else
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11 -M 250
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12 #end if
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13 #if $outputs.output_type == "Interleaved_fastq"
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14 --interleaved-output
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15 #else if $outputs.output_type == "tab"
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16 -To
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17 #end if
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18 #if $options.options_select == "advanced"
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19 #if $options.max_mismatch_density
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20 -x $options.max_mismatch_density
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21 #end if
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22 #if $options.phred_offset
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23 -p $options.phred_offset
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24 #end if
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25 #if $options.read_length
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26 -r $options.read_length
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27 #end if
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28 #if $options.fragment_length
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29 -f $options.fragment_length
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30 #end if
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31 #if $options.fragment_stdev
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32 -s $options.fragment_stdev
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33 #end if
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34 #if $options.cap_mismatch_quals
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35 $options.cap_mismatch_quals
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36 #end if
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37 #if $options.quiet
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38 $options.quiet
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39 #end if
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40 #end if
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41
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42 #if $input_type.sPaired == "paired":
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43 $input_type.pInput1 $input_type.pInput2
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44 #elif $input_type.sPaired == "collections":
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45 $input_type.fastq_collection.forward $input_type.fastq_collection.reverse
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46 #end if
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47
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48 </command>
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49 <inputs>
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50 <conditional name="input_type">
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51 <param name="sPaired" type="select" label="Single Pair or Collection">
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52 <option value="collections">Paired-end Collections</option>
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53 <option value="paired">Paired-end</option>
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54 </param>
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55 <when value="paired">
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56 <param name="pInput1" type="data" format="fastq,fastqsanger,fastqillumina,fastqsolexa" label="Forward FASTQ file" help="Must have ASCII encoded quality scores"/>
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57 <param name="pInput2" type="data" format="fastq,fastqsanger,fastqillumina,fastqsolexa" label="Reverse FASTQ file" help="File format must match the Forward FASTQ file"/>
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58 </when>
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59 <when value="collections">
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60 <param name="fastq_collection" type="data_collection" label="Paired-end Fastq collection" help="" optional="false" format="txt" collection_type="paired" />
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61 </when>
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62 </conditional>
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63
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64 <param name="min_overlap" type="integer" label="Minimum overlap" optional="true"/>
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65 <param name="max_overlap" type="integer" label="Maximum overlap" value="250" optional="true"/>
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66 <conditional name="outputs">
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67 <param name="output_type" type="select" label="Output type">
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68 <option value="Non-interleaved_fastq">Non-interleaved fastq</option>
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69 <option value="Interleaved_fastq">Interleaved fastq</option>
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70 <option value="tab">Tab-deliminated</option>
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71 </param>
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72 </conditional>
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73 <conditional name="options">
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74 <param name="options_select" type="select" label="Options Type">
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75 <option value="basic">Basic</option>
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76 <option value="advanced">Advanced</option>
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77 </param>
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78 <when value="advanced">
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79 <param name="max_mismatch_density" type="float" label="Maximum mismatch density" optional="true"/>
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80 <param name="phred_offset" type="select" label="Phred-offset" optional="true">
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81 <option value="33">33</option>
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82 <option value="64">64</option>
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83 </param>
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84 <param name="read_length" type="integer" label="Average read length" optional="true"/>
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85 <param name="fragment_length" type="integer" label="Fragment length" optional="true"/>
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86 <param name="fragment_stdev" type="integer" label="Fragment length standard deviation" optional="true"/>
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87 <param name="cap_mismatch_quals" type="boolean" label="Cap mismatch quality scores" truevalue="--cap-mismatch-quals" optional="true"/>
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88 <!--<param name="compress" type="boolean" label="Compress output files with gzip" optional="true"/>
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89 <param name="compress_prog" type="text" label="Compression program" optional="true"/>
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90 <param name="compress_prog_args" type="text" label="Compression program arguments" optional="true"/> <~~~~~~~~Phil says the compression options aren't needed-->
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91 <param name="quiet" type="boolean" label="Do not print informational messages" truevalue="-q" optional="true"/>
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92 </when>
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93 </conditional>
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94 </inputs>
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95 <outputs>
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96 <data format="fastqsanger" name="extendedFrags" label="Merged reads">
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97 <filter>outputs['output_type'] != "tab"</filter>
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98 </data>
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99 <data format="fastqsanger" name="notCombined1" label="Read 1 of mate pairs not merged">
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100 <filter>outputs['output_type'] == "Non-interleaved_fastq"</filter>
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101 </data>
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102 <data format="fastqsanger" name="notCombined2" label="Read 2 of mate pairs not merged">
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103 <filter>outputs['output_type'] == "Non-interleaved_fastq"</filter>
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104 </data>
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105 <data format="fastqsanger" name="interNotCombined" label="Interleaved non-combined pairs">
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106 <filter>outputs['output_type'] == "Interleaved_fastq"</filter>
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107 </data>
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108 <data format="tabular" name="readsAndPairs" label="Merged and non-merged pairs">
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109 <filter>outputs['output_type'] == "tab"</filter>
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110 </data>
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111 <data format="txt" name="log_file" label="Log file"/>
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112 <!-- <data format="txt" name="numericHistogram" label="Numeric histogram of merged read lengths"/>
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113 <data format="txt" name="visualHistogram" label="Visual histogram of merged read lengths"/>-->
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114 </outputs>
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115 <requirements>
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116 <requirement type="package" version="1.2.9">FLASH</requirement>
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117 </requirements>
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118 <help>
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119 ----------------------------------------------------------------------------
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120 DESCRIPTION
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121 ----------------------------------------------------------------------------
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122
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123 FLASH (Fast Length Adjustment of SHort reads) is an accurate and fast tool
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124 to merge paired-end reads that were generated from DNA fragments whose
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125 lengths are shorter than twice the length of reads. Merged read pairs result
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126 in unpaired longer reads, which are generally more desired in genome
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127 assembly and genome analysis processes.
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128
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129 Briefly, the FLASH algorithm considers all possible overlaps at or above a
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130 minimum length between the reads in a pair and chooses the overlap that
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131 results in the lowest mismatch density (proportion of mismatched bases in
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132 the overlapped region). Ties between multiple overlaps are broken by
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133 considering quality scores at mismatch sites. When building the merged
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134 sequence, FLASH computes a consensus sequence in the overlapped region.
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135 More details can be found in the original publication
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136 (http://bioinformatics.oxfordjournals.org/content/27/21/2957.full).
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137
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138 Limitations of FLASH include:
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139 - FLASH cannot merge paired-end reads that do not overlap.
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140 - FLASH cannot merge read pairs that have an outward orientation, either
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141 due to being "jumping" reads or due to excessive trimming.
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142 - FLASH is not designed for data that has a significant amount of indel
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143 errors (such as Sanger sequencing data). It is best suited for Illumina
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144 data.
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145
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146 ----------------------------------------------------------------------------
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147 MANDATORY INPUT
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148 ----------------------------------------------------------------------------
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149
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150 The most common input to FLASH is two FASTQ files containing read 1 and read 2
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151 of each mate pair, respectively, in the same order.
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152
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153 Alternatively, you may provide one FASTQ file, which may be standard input,
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154 containing paired-end reads in either interleaved FASTQ (see the
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155 --interleaved-input option) or tab-delimited (see the --tab-delimited-input
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156 option) format. In all cases, gzip compressed input is autodetected. Also,
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157 in all cases, the PHRED offset is, by default, assumed to be 33; use the
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158 --phred-offset option to change it.
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159
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160 ----------------------------------------------------------------------------
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161 OUTPUT
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162 ----------------------------------------------------------------------------
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163
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164 The default output of FLASH consists of the following files:
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165
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166 - out.extendedFrags.fastq The merged reads.
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167 - out.notCombined_1.fastq Read 1 of mate pairs that were not merged.
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168 - out.notCombined_2.fastq Read 2 of mate pairs that were not merged.
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169 - out.hist Numeric histogram of merged read lengths.
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170 - out.histogram Visual histogram of merged read lengths.
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171
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172 FLASH also logs informational messages to standard output. These can be
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173 redirected to a file, as in the following example:
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174
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175 $ flash reads_1.fq reads_2.fq | tee flash.log
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176
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177 In addition, FLASH supports several features affecting the output:
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178
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179 - Writing the merged reads directly to standard output (--to-stdout)
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180 - Writing gzip compressed output files (-z) or using an external
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181 compression program (--compress-prog)
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182 - Writing the uncombined read pairs in interleaved FASTQ format
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183 (--interleaved-output)
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184 - Writing all output reads to a single file in tab-delimited format
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185 (--tab-delimited-output)
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186
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187 ----------------------------------------------------------------------------
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188 OPTIONS
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189 ----------------------------------------------------------------------------
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190
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191 -m, --min-overlap=NUM The minimum required overlap length between two
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192 reads to provide a confident overlap. Default:
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193 10bp.
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194
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195 -M, --max-overlap=NUM Maximum overlap length expected in approximately
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196 90% of read pairs. It is by default set to 70bp,
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197 which works well for 100bp reads generated from a
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198 180bp library, assuming a normal distribution of
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199 fragment lengths. Overlaps longer than the maximum
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200 overlap parameter are still considered as good
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201 overlaps, but the mismatch density (explained below)
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202 is calculated over the first max_overlap bases in
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203 the overlapped region rather than the entire
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204 overlap. Default: 70bp, or calculated from the
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205 specified read length, fragment length, and fragment
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206 length standard deviation.
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207
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208 -x, --max-mismatch-density=NUM
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209 Maximum allowed ratio between the number of
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210 mismatched base pairs and the overlap length.
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211 Two reads will not be combined with a given overlap
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212 if that overlap results in a mismatched base density
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213 higher than this value. Note: Any occurence of an
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214 'N' in either read is ignored and not counted
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215 towards the mismatches or overlap length. Our
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216 experimental results suggest that higher values of
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217 the maximum mismatch density yield larger
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218 numbers of correctly merged read pairs but at
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219 the expense of higher numbers of incorrectly
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220 merged read pairs. Default: 0.25.
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221
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222 -p, --phred-offset=OFFSET
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223 The smallest ASCII value of the characters used to
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224 represent quality values of bases in FASTQ files.
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225 It should be set to either 33, which corresponds
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226 to the later Illumina platforms and Sanger
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227 platforms, or 64, which corresponds to the
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228 earlier Illumina platforms. Default: 33.
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229
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230 -r, --read-len=LEN
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231
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232 -f, --fragment-len=LEN
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233
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234 -s, --fragment-len-stddev=LEN
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235 Average read length, fragment length, and fragment
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236 standard deviation. These are convenience parameters
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237 only, as they are only used for calculating the
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238 maximum overlap (--max-overlap) parameter.
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239 The maximum overlap is calculated as the overlap of
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240 average-length reads from an average-size fragment
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241 plus 2.5 times the fragment length standard
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242 deviation. The default values are -r 100, -f 180,
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243 and -s 18, so this works out to a maximum overlap of
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244 65 bp. If --max-overlap is specified, then the
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245 specified value overrides the calculated value.
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246
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247 If you do not know the standard deviation of the
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248 fragment library, you can probably assume that the
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249 standard deviation is 10% of the average fragment
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250 length.
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251
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252 --cap-mismatch-quals Cap quality scores assigned at mismatch locations
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253 to 2. This was the default behavior in FLASH v1.2.7
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254 and earlier. Later versions will instead calculate
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255 such scores as the
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256 absolute value of the difference in quality scores,
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257 but at least 2. Essentially, the new behavior
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258 prevents a low quality base call that is likely a
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259 sequencing error from significantly bringing down
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260 the quality of a high quality, likely correct base
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261 call.
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262
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263 --interleaved-input Instead of requiring files MATES_1.FASTQ and
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264 MATES_2.FASTQ, allow a single file MATES.FASTQ that
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265 has the paired-end reads interleaved. Specify "-"
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266 to read from standard input.
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267
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268 --interleaved-output Write the uncombined pairs in interleaved FASTQ
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269 format.
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270
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271 -I, --interleaved Equivalent to specifying both --interleaved-input
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272 and --interleaved-output.
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273
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274 -Ti, --tab-delimited-input
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275 Assume the input is in tab-delimited format
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276 rather than FASTQ, in the format described below in
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277 '--tab-delimited-output'. In this mode you should
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278 provide a single input file, each line of which must
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279 contain either a read pair (5 fields) or a single
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280 read (3 fields). FLASH will try to combine the read
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281 pairs. Single reads will be written to the output
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282 file as-is if also using --tab-delimited-output;
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283 otherwise they will be ignored. Note that you may
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284 specify "-" as the input file to read the
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285 tab-delimited data from standard input.
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286
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287 -To, --tab-delimited-output
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288 Write output in tab-delimited format (not FASTQ).
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289 Each line will contain either a combined pair in the
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290 format 'tag <tab> seq <tab> qual' or an uncombined
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291 pair in the format 'tag <tab> seq_1 <tab> qual_1
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292 <tab> seq_2 <tab> qual_2'.
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293
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294 -o, --output-prefix=PREFIX
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295 Prefix of output files. Default: "out".
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296
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297 -d, --output-directory=DIR
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298 Path to directory for output files. Default:
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299 current working directory.
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300
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301 -c, --to-stdout
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302 Write the combined reads to standard output. In
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303 this mode, with FASTQ output (the default) the
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304 uncombined reads are discarded. With tab-delimited
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305 output, uncombined reads are included in the
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306 tab-delimited data written to standard output.
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307 In both cases, histogram files are not written,
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308 and informational messages are sent to standard
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309 error rather than to standard output.
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310
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311 --suffix=SUFFIX, --output-suffix=SUFFIX
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312 Use SUFFIX as the suffix of the output files
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313 after ".fastq". A dot before the suffix is assumed,
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314 unless an empty suffix is provided. Default:
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315 nothing; or 'gz' if -z is specified; or PROG if
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316 --compress-prog=PROG is specified.
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317
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318 -t, --threads=NTHREADS Set the number of worker threads. This is in
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319 addition to the I/O threads. Default: number of
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320 processors. Note: if you need FLASH's output to
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321 appear deterministically or in the same order as
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322 the original reads, you must specify -t 1
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323 (--threads=1).
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324
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325 -q, --quiet Do not print informational messages.
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326
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327 -h, --help Display this help and exit.
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328
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329 -v, --version Display version.
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330 </help>
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331 </tool>
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