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1 <tool id="amplicon_analysis_pipeline" name="Amplicon Analysis Pipeline" version="1.3.5.0">
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2 <description>analyse 16S rRNA data from Illumina Miseq paired-end reads</description>
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3 <requirements>
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4 <requirement type="package" version="1.3.5">amplicon_analysis_pipeline</requirement>
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5 </requirements>
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6 <stdio>
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7 <exit_code range="1:" />
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8 </stdio>
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9 <command><![CDATA[
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10
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11 ## Convenience variable for pipeline name
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12 #set $pipeline_name = $pipeline.pipeline_name
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13
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14 ## Set the reference database name
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15 #if str( $pipeline_name ) == "DADA2"
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16 #set reference_database_name = "silva"
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17 #else
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18 #set reference_database = $pipeline.reference_database
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19 #if $reference_database == "-S"
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20 #set reference_database_name = "silva"
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21 #else if $reference_database == "-H"
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22 #set reference_database_name = "homd"
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23 #else
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24 #set reference_database_name = "gg"
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25 #end if
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26 #end if
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27
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28 ## Run the amplicon analysis pipeline wrapper
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29 python $__tool_directory__/amplicon_analysis_pipeline.py
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30 ## Set options
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31 #if str( $forward_pcr_primer ) != ""
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32 -g "$forward_pcr_primer"
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33 #end if
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34 #if str( $reverse_pcr_primer ) != ""
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35 -G "$reverse_pcr_primer"
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36 #end if
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37 #if str( $trimming_threshold ) != ""
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38 -q $trimming_threshold
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39 #end if
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40 #if str( $sliding_window_length ) != ""
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41 -l $sliding_window_length
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42 #end if
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43 #if str( $minimum_overlap ) != ""
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44 -O $minimum_overlap
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45 #end if
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46 #if str( $minimum_length ) != ""
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47 -L $minimum_length
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48 #end if
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49 -P $pipeline_name
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50 -r \${AMPLICON_ANALYSIS_REF_DATA_PATH-ReferenceData}
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51 #if str( $pipeline_name ) != "DADA2"
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52 ${reference_database}
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53 #end if
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54 #if str($categories_file_in) != 'None'
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55 -c "${categories_file_in}"
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56 #end if
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57 ## Input files
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58 "${metatable_file_in}"
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59 ## FASTQ pairs
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60 #if str($input_type.pairs_or_collection) == "collection"
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61 #set fastq_pairs = $input_type.fastq_collection
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62 #else
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63 #set fastq_pairs = $input_type.fastq_pairs
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64 #end if
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65 #for $fq_pair in $fastq_pairs
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66 "${fq_pair.name}" "${fq_pair.forward}" "${fq_pair.reverse}"
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67 #end for
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68 &&
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69
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70 ## Collect outputs
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71 cp Metatable_log/Metatable_mod.txt "${metatable_mod}" &&
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72 #if str( $pipeline_name ) == "Vsearch"
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73 # Vsearch-specific
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74 cp ${pipeline_name}_OTU_tables/multiplexed_linearized_dereplicated_mc2_repset_nonchimeras_tax_OTU_table.biom "${tax_otu_table_biom_file}" &&
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75 cp Multiplexed_files/${pipeline_name}_pipeline/multiplexed_linearized_dereplicated_mc2_repset_nonchimeras_OTUs.fasta "${dereplicated_nonchimera_otus_fasta}" &&
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76 cp QUALITY_CONTROL/Reads_count.txt "$read_counts_out" &&
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77 #else
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78 # DADA2-specific
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79 cp ${pipeline_name}_OTU_tables/DADA2_tax_OTU_table.biom "${tax_otu_table_biom_file}" &&
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80 cp ${pipeline_name}_OTU_tables/seqs.fa "${dereplicated_nonchimera_otus_fasta}" &&
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81 #end if
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82 cp ${pipeline_name}_OTU_tables/otus.tre "${otus_tre_file}" &&
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83 cp RESULTS/${pipeline_name}_${reference_database_name}/OTUs_count.txt "${otus_count_file}" &&
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84 cp RESULTS/${pipeline_name}_${reference_database_name}/table_summary.txt "${table_summary_file}" &&
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85 cp fastqc_quality_boxplots.html "${fastqc_quality_boxplots_html}" &&
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86
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87 ## OTU table heatmap
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88 cp RESULTS/${pipeline_name}_${reference_database_name}/Heatmap.pdf "${heatmap_otu_table_pdf}"" &&
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89
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90 ## HTML outputs
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91
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92 ## Phylum genus barcharts
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93 mkdir $phylum_genus_dist_barcharts_html.files_path &&
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94 cp -r RESULTS/${pipeline_name}_${reference_database_name}/phylum_genus_charts/charts $phylum_genus_dist_barcharts_html.files_path &&
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95 cp -r RESULTS/${pipeline_name}_${reference_database_name}/phylum_genus_charts/raw_data $phylum_genus_dist_barcharts_html.files_path &&
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96 cp RESULTS/${pipeline_name}_${reference_database_name}/phylum_genus_charts/bar_charts.html "${phylum_genus_dist_barcharts_html}" &&
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97
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98 ## Beta diversity weighted 2d plots
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99 mkdir $beta_div_even_weighted_2d_plots.files_path &&
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100 cp -r RESULTS/${pipeline_name}_${reference_database_name}/beta_div_even/weighted_2d_plot/* $beta_div_even_weighted_2d_plots.files_path &&
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101 cp RESULTS/${pipeline_name}_${reference_database_name}/beta_div_even/weighted_2d_plot/weighted_unifrac_pc_2D_PCoA_plots.html "${beta_div_even_weighted_2d_plots}" &&
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102
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103 ## Beta diversity unweighted 2d plots
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104 mkdir $beta_div_even_unweighted_2d_plots.files_path &&
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105 cp -r RESULTS/${pipeline_name}_${reference_database_name}/beta_div_even/unweighted_2d_plot/* $beta_div_even_unweighted_2d_plots.files_path &&
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106 cp RESULTS/${pipeline_name}_${reference_database_name}/beta_div_even/unweighted_2d_plot/unweighted_unifrac_pc_2D_PCoA_plots.html "${beta_div_even_unweighted_2d_plots}" &&
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107
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108 ## Alpha diversity rarefaction plots
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109 mkdir $alpha_div_rarefaction_plots.files_path &&
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110 cp RESULTS/${pipeline_name}_${reference_database_name}/Alpha_diversity/rarefaction_curves/rarefaction_plots.html $alpha_div_rarefaction_plots &&
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111 cp -r RESULTS/${pipeline_name}_${reference_database_name}/Alpha_diversity/rarefaction_curves/average_plots $alpha_div_rarefaction_plots.files_path &&
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112
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113 ## DADA2 error rate plots
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114 #if str($pipeline_name) == "DADA2"
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115 mkdir $dada2_error_rate_plots.files_path &&
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116 cp DADA2_OTU_tables/Error_rate_plots/error_rate_plots.html $dada2_error_rate_plots &&
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117 cp -r DADA2_OTU_tables/Error_rate_plots/*.pdf $dada2_error_rate_plots.files_path &&
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118 #end if
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119
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120 ## Categories data
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121 #if str($categories_file_in) != 'None'
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122 ## Alpha diversity boxplots
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123 mkdir $alpha_div_boxplots.files_path &&
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124 cp alpha_diversity_boxplots.html "$alpha_div_boxplots" &&
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125 cp RESULTS/${pipeline_name}_${reference_database_name}/Alpha_diversity/Alpha_diversity_boxplot/Categories_shannon/*.pdf $alpha_div_boxplots.files_path &&
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126 #end if
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127
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128 ## Pipeline outputs (log files etc)
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129 mkdir $log_files.files_path &&
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130 cp Amplicon_analysis_pipeline.log $log_files.files_path &&
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131 cp pipeline.log $log_files.files_path &&
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132 cp Pipeline_outputs.txt $log_files.files_path &&
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133 cp Metatable_log/Metatable.html $log_files.files_path &&
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134 cp pipeline_outputs.html "$log_files"
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135 ]]></command>
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136 <inputs>
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137 <param name="title" type="text" value="test" size="25"
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138 label="Title" help="Optional text that will be added to the output dataset names" />
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139 <param type="data" name="metatable_file_in" format="tabular"
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140 label="Input Metatable.txt file" />
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141 <param type="data" name="categories_file_in" format="txt"
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142 label="Input Categories.txt file" optional="true"
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143 help="(optional)" />
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144 <conditional name="input_type">
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145 <param name="pairs_or_collection" type="select"
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146 label="Input FASTQ type">
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147 <option value="pairs_of_files">Pairs of datasets</option>
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148 <option value="collection" selected="true">Dataset pairs in a collection</option>
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149 </param>
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150 <when value="collection">
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151 <param name="fastq_collection" type="data_collection"
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152 format="fastqsanger,fastq" collection_type="list:paired"
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153 label="Collection of FASTQ forward and reverse (R1/R2) pairs"
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154 help="Each FASTQ pair will be treated as one sample; the name of each sample will be taken from the first column of the Metatable file " />
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155 </when>
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156 <when value="pairs_of_files">
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157 <repeat name="fastq_pairs" title="Input fastq pairs" min="1">
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158 <param type="text" name="name" value=""
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159 label="Final name for FASTQ pair" />
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160 <param type="data" name="fastq_r1" format="fastqsanger,fastq"
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161 label="FASTQ with forward reads (R1)" />
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162 <param type="data" name="fastq_r2" format="fastqsanger,fastq"
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163 label="FASTQ with reverse reads (R2)" />
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164 </repeat>
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165 </when>
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166 </conditional>
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167 <param type="text" name="forward_pcr_primer" value=""
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168 label="Forward PCR primer sequence"
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169 help="Optional; must not include barcode or adapter sequence (-g)" />
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170 <param type="text" name="reverse_pcr_primer" value=""
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171 label="Reverse PCR primer sequence"
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172 help="Optional; must not include barcode or adapter sequence (-G)" />
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173 <param type="integer" name="trimming_threshold" value="20"
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174 label="Threshold quality below which read will be trimmed"
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175 help="Phred score; default is 20 (-q)" />
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176 <param type="integer" name="minimum_overlap" value="10"
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177 label="Minimum overlap in bp between forward and reverse reads"
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178 help="Default is 10 (-O)" />
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179 <param type="integer" name="minimum_length" value="200"
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180 label="Minimum length in bp to keep sequence after overlapping"
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181 help="Default is 200 (-L)" />
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182 <param type="integer" name="sliding_window_length" value="10"
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183 label="Minimum length in bp to retain a read after trimming"
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184 help="Supplied to Sickle; default is 10 (-l)" />
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185 <conditional name="pipeline">
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186 <param type="select" name="pipeline_name"
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187 label="Pipeline to use for analysis">
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188 <option value="Vsearch" selected="true" >Vsearch</option>
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189 <option value="DADA2">DADA2</option>
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190 </param>
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191 <when value="Vsearch">
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192 <param type="select" name="reference_database"
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193 label="Reference database">
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194 <option value="" selected="true">GreenGenes</option>
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195 <option value="-S">Silva</option>
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196 <option value="-H">Human Oral Microbiome Database (HOMD)</option>
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197 </param>
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198 </when>
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199 <when value="DADA2">
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200 </when>
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201 </conditional>
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202 </inputs>
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203 <outputs>
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204 <data format="tabular" name="metatable_mod"
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205 label="${tool.name}:${title} Metatable_mod.txt" />
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206 <data format="tabular" name="read_counts_out"
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207 label="${tool.name} (${pipeline.pipeline_name}):${title} read counts">
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208 <filter>pipeline['pipeline_name'] == 'Vsearch'</filter>
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209 </data>
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210 <data format="biom" name="tax_otu_table_biom_file"
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211 label="${tool.name} (${pipeline.pipeline_name}):${title} tax OTU table (biom format)" />
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212 <data format="tabular" name="otus_tre_file"
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213 label="${tool.name} (${pipeline.pipeline_name}):${title} otus.tre" />
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214 <data format="html" name="phylum_genus_dist_barcharts_html"
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215 label="${tool.name} (${pipeline.pipeline_name}):${title} phylum genus dist barcharts HTML" />
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216 <data format="tabular" name="otus_count_file"
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217 label="${tool.name} (${pipeline.pipeline_name}):${title} OTUs count file" />
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218 <data format="tabular" name="table_summary_file"
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219 label="${tool.name} (${pipeline.pipeline_name}):${title} table summary file" />
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220 <data format="fasta" name="dereplicated_nonchimera_otus_fasta"
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221 label="${tool.name} (${pipeline.pipeline_name}):${title} multiplexed linearized dereplicated mc2 repset nonchimeras OTUs FASTA" />
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222 <data format="html" name="fastqc_quality_boxplots_html"
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223 label="${tool.name} (${pipeline.pipeline_name}):${title} FastQC per-base quality boxplots HTML" />
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224 <data format="pdf" name="heatmap_otu_table_pdf"
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225 label="${tool.name} (${pipeline.pipeline_name}):${title} heatmap OTU table PDF" />
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226 <data format="html" name="beta_div_even_weighted_2d_plots"
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227 label="${tool.name} (${pipeline.pipeline_name}):${title} beta diversity weighted 2D plots HTML" />
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228 <data format="html" name="beta_div_even_unweighted_2d_plots"
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229 label="${tool.name} (${pipeline.pipeline_name}):${title} beta diversity unweighted 2D plots HTML" />
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230 <data format="html" name="alpha_div_rarefaction_plots"
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231 label="${tool.name} (${pipeline.pipeline_name}):${title} alpha diversity rarefaction plots HTML" />
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232 <data format="html" name="dada2_error_rate_plots"
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233 label="${tool.name} (${pipeline.pipeline_name}):${title} DADA2 error rate plots">
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234 <filter>pipeline['pipeline_name'] == 'DADA2'</filter>
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235 </data>
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236 <data format="html" name="alpha_div_boxplots"
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237 label="${tool.name} (${pipeline.pipeline_name}):${title} alpha diversity boxplots">
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238 <filter>categories_file_in is not None</filter>
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239 </data>
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240 <data format="html" name="log_files"
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241 label="${tool.name} (${pipeline.pipeline_name}):${title} log files" />
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242 </outputs>
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243 <tests>
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244 </tests>
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245 <help><![CDATA[
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246
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247 What it does
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248 ------------
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249
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250 This pipeline has been designed for the analysis of 16S rRNA data from
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251 Illumina Miseq (Casava >= 1.8) paired-end reads.
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252
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253 Usage
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254 -----
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255
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256 1. Preparation of the mapping file and format of unique sample id
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257 *****************************************************************
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258
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259 Before using the amplicon analysis pipeline it would be necessary to
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260 follow the steps as below to avoid analysis failures and ensure samples
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261 are labelled appropriately. Sample names for the labelling are derived
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262 from the fastq files names that are generated from the sequencing. The
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263 labels will include everything between the beginning of the name and
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264 the sample number (from C11 to S19 in Fig. 1)
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265
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266 .. image:: Pipeline_description_Fig1.png
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267 :height: 46
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268 :width: 382
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269
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270 **Figure 1**
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271
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272 If analysing 16S data from multiple runs:
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273
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274 The samples from different runs may have identical IDs. For example,
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275 when sequencing the same samples twice, by chance, these could be at
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276 the same position in both the runs. This would cause the fastq files
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277 to have exactly the same IDs (Fig. 2).
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278
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279 .. image:: Pipeline_description_Fig2.png
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280 :height: 100
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281 :width: 463
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282
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283 **Figure 2**
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284
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285 In case of identical sample IDs the pipeline will fail to run and
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286 generate an error at the beginning of the analysis.
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287
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288 To avoid having to change the file names, before uploading the files,
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289 ensure that the samples IDs are not repeated.
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290
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291 2. To upload the file
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292 *********************
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293
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294 Click on **Get Data/Upload File** from the Galaxy tool panel on the
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295 left hand side.
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296
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297 From the pop-up window, choose how to upload the file. The
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298 **Choose local file** option can be used for files up to 4Gb. Fastq files
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299 from Illumina MiSeq will rarely be bigger than 4Gb and this option is
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300 recommended.
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301
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302 After choosing the files click **Start** to begin the upload. The window can
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303 now be closed and the files will be uploaded onto the Galaxy server. You
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304 will see the progress on the ``HISTORY`` panel on the right
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305 side of the screen. The colour will change from grey (queuing), to yellow
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306 (uploading) and finally green (uploaded).
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307
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308 Once all the files are uploaded, click on the operations on multiple
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309 datasets icon and select the fastq files that need to be analysed.
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310 Click on the tab **For all selected...** and on the option
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311 **Build List of Dataset pairs** (Fig. 3).
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312
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313 .. image:: Pipeline_description_Fig3.png
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314 :height: 247
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315 :width: 586
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316
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317 **Figure 3**
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318
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319 Change the filter parameter ``_1`` and ``_2`` to be ``_R1`` and ``_R2``.
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320 The fastq files forward R1 and reverse R2 should now appear in the
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321 corresponding columns.
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322
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323 Select **Autopair**. This creates a collection of paired fastq files for
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324 the forward and reverse reads for each sample. The name of the pairs will
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325 be the ones used by the pipeline. You are free to change the names at this
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326 point as long as they are the same used in the Metatable file
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327 (see section 3).
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328
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329 Name the collection and click on **create list**. This reduces the time
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330 required to input the forward and reverse reads for each individual sample.
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331
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332 3. Create the Metatable files
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333 *****************************
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334
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335 Metatable.txt
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336 ~~~~~~~~~~~~~
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337
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338 Click on the list of pairs you just created to see the name of the single
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339 pairs. The name of the pairs will be the ones used by the pipeline,
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340 therefore, these are the names that need to be used in the Metatable file.
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341
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342 The Metatable file has to be in QIIME format. You can find a description
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343 of it on QIIME website http://qiime.org/documentation/file_formats.html
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344
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345 EXAMPLE::
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346
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347 #SampleID BarcodeSequence LinkerPrimerSequence Disease Gender Description
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348 Mock-RUN1 TAAGGCGAGCGTAAGA PsA Male Control
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349 Mock-RUN2 CGTACTAGGCGTAAGA PsA Male Control
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350 Mock-RUN3 AGGCAGAAGCGTAAGA PsC Female Control
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351
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352 Briefly: the column ``LinkerPrimerSequence`` is empty but it cannot be
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353 deleted. The header is very important. ``#SampleID``, ``Barcode``,
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354 ``LinkerPrimerSequence`` and ``Description`` are mandatory. Between
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355 ``LinkerPrimerSequence`` and ``Description`` you can add as many columns
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356 as you want. For every column a PCoA plot will be created (see
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357 **Results** section). You can create this file in Excel and it will have
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358 to be saved as ``Text(Tab delimited)``.
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359
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360 During the analysis the Metatable.txt will be checked to ensure that the
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361 file has the correct format. If necessary, this will be modified and will
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362 be available as Metatable_corrected.txt in the history panel. If you are
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363 going to use the metatable file for any other statistical analyses,
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364 remember to use the ``Metatable_mod.txt`` one, otherwise the sample
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365 names might not match!
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366
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367 Categories.txt (optional)
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368 ~~~~~~~~~~~~~~~~~~~~~~~~~
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369
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370 This file is required if you want to get box plots for comparison of
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371 alpha diversity indices (see **Results** section). The file is a list
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372 (without header and IN ONE COLUMN) of categories present in the
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373 Metatable.txt file. THE NAMES YOU ARE USING HAVE TO BE THE SAME AS THE
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374 ONES USED IN THE METATABLE.TXT. You can create this file in Excel and
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375 will have to be saved as ``Text(Tab delimited)``.
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376
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377 EXAMPLE::
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378
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379 Disease
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380 Gender
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381
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382 Metatable and categories files can be uploaded using Get Data as done
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383 with the fatsq files.
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384
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385 4. Analysis
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386 ***********
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387
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388 Under **Amplicon_Analysis_Pipeline**
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389
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390 * **Title** Name to distinguish between the runs. It will be shown at
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391 the beginning of each output file name.
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392
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393 * **Input Metatable.txt file** Select the Metatable.txt file related to
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394 this analysis
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395
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396 * **Input Categories.txt file (Optional)** Select the Categories.txt file
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397 related to this analysis
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398
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399 * **Input FASTQ type** select *Dataset pairs in a collection* and, then,
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400 the collection of pairs you created earlier.
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401
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402 * **Forward/Reverse PCR primer sequence** if the PCR primer sequences
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403 have not been removed from the MiSeq during the fastq creation, they
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404 have to be removed before the analysis. Insert the PCR primer sequence
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405 in the corresponding field. DO NOT include any barcode or adapter
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406 sequence. If the PCR primers have been already trimmed by the MiSeq,
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407 and you include the sequence in this field, this would lead to an error.
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408 Only include the sequences if still present in the fastq files.
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409
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410 * **Threshold quality below which reads will be trimmed** Choose the
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411 Phred score used by Sickle to trim the reads at the 3’ end.
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412
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413 * **Minimum length to retain a read after trimming** If the read length
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414 after trimming is shorter than a user defined length, the read, along
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415 with the corresponding read pair, will be discarded.
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416
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417 * **Minimum overlap in bp between forward and reverse reads** Choose the
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418 minimum basepair overlap used by Pandaseq to assemble the reads.
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419 Default is 10.
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420
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421 * **Minimum length in bp to keep a sequence after overlapping** Choose the
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422 minimum sequence length used by Pandaseq to keep a sequence after the
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423 overlapping. This depends on the expected amplicon length. Default is
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424 380 (used for V3-V4 16S sequencing; expected length ~440bp)
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425
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426 * **Pipeline to use for analysis** Choose the pipeline to use for OTU
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427 clustering and chimera removal. The Galaxy tool supports the ``Vsearch``
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428 and ``DADA2`` pipelines.
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429
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430 * **Reference database** Choose between ``GreenGenes``, ``Silva`` or
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431 ``HOMD`` (Human Oral Microbiome Database) for taxa assignment.
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432
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433 Click on **Execute** to start the analysis.
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434
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435 5. Results
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436 **********
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437
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438 Results are entirely generated using QIIME scripts. The results will
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439 appear in the History panel when the analysis is completed.
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440
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441 The following outputs are captured:
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442
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443 * **Vsearch_tax_OTU_table.biom|DADA2_tax_OTU_table.biom (biom format)**
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444 The OTU table in BIOM format (http://biom-format.org/)
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445
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446 * **otus.tre** Phylogenetic tree constructed using ``make_phylogeny.py``
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447 (fasttree) QIIME script (http://qiime.org/scripts/make_phylogeny.html)
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448
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449 * **Phylum_genus_dist_barcharts_HTML** HTML file with bar charts at
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450 Phylum, Genus and Species level
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451 (http://qiime.org/scripts/summarize_taxa.html and
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452 http://qiime.org/scripts/plot_taxa_summary.html)
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453
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454 * **OTUs_count_file** Summary of OTU counts per sample
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455 (http://biom-format.org/documentation/summarizing_biom_tables.html)
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456
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457 * **Table_summary_file** Summary of sequences counts per sample
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458 (http://biom-format.org/documentation/summarizing_biom_tables.html)
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459
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460 * **multiplexed_linearized_dereplicated_mc2_repset_nonchimeras_OTUs.fasta|seqs.fa**
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461 Fasta file with OTU sequences (Vsearch|DADA2)
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462
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463 * **Heatmap_PDF** OTU heatmap in PDF format
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464 (http://qiime.org/1.8.0/scripts/make_otu_heatmap_html.html )
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465
|
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466 * **Vsearch_beta_diversity_weighted_2D_plots_HTML** PCoA plots in HTML
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467 format using weighted Unifrac distance measure. Samples are grouped
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468 by the column names present in the Metatable file. The samples are
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469 firstly rarefied to the minimum sequencing depth
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470 (http://qiime.org/scripts/beta_diversity_through_plots.html )
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471
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472 * **Vsearch_beta_diversity_unweighted_2D_plots_HTML** PCoA plots in HTML
|
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473 format using Unweighted Unifrac distance measure. Samples are grouped
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474 by the column names present in the Metatable file. The samples are
|
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475 firstly rarefied to the minimum sequencing depth
|
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476 (http://qiime.org/scripts/beta_diversity_through_plots.html )
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477
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478 Code availability
|
|
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479 -----------------
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480
|
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481 **Code is available at** https://github.com/MTutino/Amplicon_analysis
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|
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482
|
|
|
483 Credits
|
|
|
484 -------
|
|
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485
|
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486 Pipeline author: Mauro Tutino
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487
|
|
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488 Galaxy tool: Peter Briggs
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489
|
|
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490 ]]></help>
|
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|
491 <citations>
|
|
|
492 <citation type="bibtex">
|
|
|
493 @misc{githubAmplicon_analysis,
|
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|
494 author = {Tutino, Mauro},
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495 year = {2017},
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|
|
496 title = {Amplicon Analysis Pipeline},
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497 publisher = {GitHub},
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498 journal = {GitHub repository},
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499 url = {https://github.com/MTutino/Amplicon_analysis},
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500 }</citation>
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501 </citations>
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502 </tool>
|
