Mercurial > repos > lparsons > htseq_count
annotate htseq-count.xml @ 16:227f9d3f0e32 draft
Updated HTSeq package to version 0.6.1, fixed input format string, updated dependency definitions
| author | lparsons |
|---|---|
| date | Fri, 11 Apr 2014 15:05:28 -0400 |
| parents | 3ffe4e2572a7 |
| children | d5edaf8dc974 |
| rev | line source |
|---|---|
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227f9d3f0e32
Updated HTSeq package to version 0.6.1, fixed input format string, updated dependency definitions
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1 <tool id="htseq_count" name="htseq-count" version="0.4"> |
| 0 | 2 <description> - Count aligned reads in a BAM file that overlap features in a GFF file</description> |
| 3 <version_command>htseq-count -h | grep version | sed 's/^\(.*\)*\(version .*\)\./\2/'</version_command> | |
| 4 <requirements> | |
| 13 | 5 <requirement type="package" version="1.7.1">numpy</requirement> |
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6 <requirement type="package" version="0.6.1">htseq</requirement> |
| 13 | 7 <requirement type="package" version="0.1.19">samtools</requirement> |
| 0 | 8 </requirements> |
| 9 <command> | |
| 10 ##set up input files | |
| 11 #set $reference_fasta_filename = "localref.fa" | |
| 12 #if $samout_conditional.samout: | |
| 13 #if str( $samout_conditional.reference_source.reference_source_selector ) == "history": | |
| 14 ln -s "${samout_conditional.reference_source.ref_file}" "${reference_fasta_filename}" && | |
| 15 samtools faidx "${reference_fasta_filename}" 2>&1 || echo "Error running samtools faidx for htseq-count" >&2 && | |
| 16 #else: | |
| 17 #set $reference_fasta_filename = str( $samout_conditional.reference_source.ref_file.fields.path ) | |
| 18 #end if | |
| 19 #end if | |
| 20 htseq-count | |
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Updated HTSeq package to version 0.6.1, fixed input format string, updated dependency definitions
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21 --format=$samfile.extension |
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227f9d3f0e32
Updated HTSeq package to version 0.6.1, fixed input format string, updated dependency definitions
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22 --order=pos |
| 0 | 23 --mode=$mode |
| 24 --stranded=$stranded | |
| 25 --minaqual=$minaqual | |
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26 --type=$featuretype |
| 0 | 27 --idattr=$idattr |
| 28 #if $samout_conditional.samout: | |
| 29 --samout=$__new_file_path__/${samoutfile.id}_tmp | |
| 30 #end if | |
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31 $samfile |
| 0 | 32 $gfffile |
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33 | awk '{if ($1 ~ "no_feature|ambiguous|too_low_aQual|not_aligned|alignment_not_unique") print $0 | "cat 1>&2"; else print $0}' > $counts 2>$othercounts |
| 0 | 34 #if $samout_conditional.samout: |
| 35 && samtools view -Su -t ${reference_fasta_filename}.fai $__new_file_path__/${samoutfile.id}_tmp | samtools sort -o - sorted > $samoutfile | |
| 36 #end if</command> | |
| 37 <inputs> | |
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227f9d3f0e32
Updated HTSeq package to version 0.6.1, fixed input format string, updated dependency definitions
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38 <param format="sam,bam" name="samfile" type="data" label="Aligned SAM/BAM File"/> |
| 0 | 39 <param format="gff" name="gfffile" type="data" label="GFF File"/> |
| 40 <param name="mode" type="select" label="Mode"> | |
| 41 <help>Mode to handle reads overlapping more than one feature.</help> | |
| 42 <option value="union" selected="true">Union</option> | |
| 43 <option value="intersection-strict">Intersection (strict)</option> | |
| 44 <option value="intersection-nonempty">Intersection (nonempty)</option> | |
| 45 </param> | |
| 46 <param name="stranded" type="select" label="Stranded"> | |
| 47 <help>Specify whether the data is from a strand-specific assay. 'Reverse' means yes with reversed strand interpretation.</help> | |
| 48 <option value="yes" selected="true">Yes</option> | |
| 49 <option value="no">No</option> | |
| 50 <option value="reverse">Reverse</option> | |
| 51 </param> | |
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52 <param name="minaqual" type="integer" value="10" label="Minimum alignment quality"> |
| 0 | 53 <help>Skip all reads with alignment quality lower than the given minimum value</help> |
| 54 </param> | |
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55 <param name="featuretype" type="text" value="exon" label="Feature type"> |
| 0 | 56 <help>Feature type (3rd column in GFF file) to be used. All features of other types are ignored. The default, suitable for RNA-Seq and Ensembl GTF files, is exon.</help> |
| 57 </param> | |
| 58 <param name="idattr" type="text" value="gene_id" label="ID Attribute"> | |
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59 <help>GFF attribute to be used as feature ID. Several GFF lines with the same feature ID will be considered as parts of the same feature. The feature ID is used to identity the counts in the output table. All features of the specified type MUST have a value for this attribute. The default, suitable for RNA-SEq and Ensembl GTF files, is gene_id.</help> |
| 0 | 60 </param> |
| 61 <conditional name="samout_conditional"> | |
| 62 <param name="samout" type="boolean" value="False" truevalue="True" falsevalue="False" label="Additional BAM Output"> | |
| 63 <help>Write out all SAM alignment records into an output BAM file, annotating each line with its assignment to a feature or a special counter (as an optional field with tag ‘XF’).</help> | |
| 64 </param> | |
| 65 <when value="True"> | |
| 66 <conditional name="reference_source"> | |
| 67 <param name="reference_source_selector" type="select" label="Choose the source for the reference list"> | |
| 68 <option value="cached">Locally cached</option> | |
| 69 <option value="history">History</option> | |
| 70 </param> | |
| 71 <when value="cached"> | |
| 72 <param name="ref_file" type="select" label="Using reference genome"> | |
| 73 <options from_data_table="sam_fa_indexes"> | |
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74 <filter type="data_meta" key="dbkey" ref="samfile" column="1"/> |
| 0 | 75 </options> |
| 76 <validator type="no_options" message="A built-in reference genome is not available for the build associated with the selected input file"/> | |
| 77 </param> | |
| 78 </when> | |
| 79 <when value="history"> <!-- FIX ME!!!! --> | |
| 80 <param name="ref_file" type="data" format="fasta" label="Using reference file" /> | |
| 81 </when> | |
| 82 </conditional> | |
| 83 </when> | |
| 84 </conditional> | |
| 85 </inputs> | |
| 86 | |
| 87 <outputs> | |
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88 <data format="tabular" name="counts" metadata_source="samfile" label="${tool.name} on ${on_string}"/> |
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89 <data format="tabular" name="othercounts" metadata_source="samfile" label="${tool.name} on ${on_string} (no feature)"/> |
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90 <data format="bam" name="samoutfile" metadata_source="samfile" label="${tool.name} on ${on_string} (BAM)"> |
| 0 | 91 <filter>samout_conditional['samout']</filter> |
| 92 </data> | |
| 93 </outputs> | |
| 94 | |
| 95 <stdio> | |
| 96 <exit_code range="1:" level="fatal" description="Unknown error occurred" /> | |
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97 <regex match="htseq-count: command not found" source="stderr" level="fatal" description="The HTSeq python package is not properly installed, contact Galaxy administrators" /> |
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98 <regex match="samtools: command not found" source="stderr" level="fatal" description="The samtools package is not properly installed, contact Galaxy administrators" /> |
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99 <regex match="Error: Feature (.+) does not contain a '(.+)' attribute" source="both" level="fatal" description="Error parsing the GFF file, at least one feature of the specified 'Feature type' does not have a value for the specified 'ID Attribute'" /> |
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100 <regex match="Error occured in line (\d+) of file" source="stderr" level="fatal" description="Unknown error parsing the GFF file" /> |
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parents:
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diff
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101 <regex match="Error" source="stderr" level="fatal" description="Unknown error occured" /> |
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102 <regex match="Warning: Read (.+) claims to have an aligned mate which could not be found. \(Is the SAM file properly sorted\?\)" source="stderr" level="warning" description="PAIRED DATA MISSING OR NOT PROPERLY SORTED. Try reruning and selecting the paired-end option. See stderr output of this dataset for more information." /> |
| 0 | 103 </stdio> |
| 104 | |
| 105 <tests> | |
| 106 <test> | |
| 107 <param name="samfile" value="htseq-test.sam" /> | |
| 108 <param name="gfffile" value="htseq-test.gff" /> | |
| 109 <param name="samout" value="False" /> | |
| 110 <output name="counts" file="htseq-test_counts.tsv" /> | |
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111 <output name="othercounts" file="htseq-test_othercounts.tsv" /> |
| 0 | 112 </test> |
| 113 <test> | |
| 114 <param name="samfile" value="htseq-test.bam" /> | |
| 115 <param name="gfffile" value="htseq-test.gff" /> | |
| 116 <param name="samout" value="False" /> | |
| 117 <output name="counts" file="htseq-test_counts.tsv" /> | |
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118 <output name="othercounts" file="htseq-test_othercounts.tsv" /> |
| 0 | 119 </test> |
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120 <test> |
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121 <param name="samfile" value="htseq-test-paired.bam" /> |
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122 <param name="singlepaired" value="paired" /> |
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123 <param name="gfffile" value="htseq-test.gff" /> |
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124 <param name="samout" value="False" /> |
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125 <output name="counts" file="htseq-test-paired_counts.tsv" /> |
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126 <output name="othercounts" file="htseq-test-paired_othercounts.tsv" /> |
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127 </test> |
| 0 | 128 <!-- Seems to be an issue setting the $reference_fasta_filename variable during test |
| 129 <test> | |
| 130 <param name="samfile" value="htseq-test.sam" /> | |
| 131 <param name="gfffile" value="htseq-test.gff" /> | |
| 132 <param name="samout" value="True" /> | |
| 133 <param name="reference_source_selector" value="history" /> | |
| 134 <param name="ref_file" value="htseq-test_reference.fasta" /> | |
| 135 <output name="counts" file="htseq-test_counts.tsv" /> | |
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136 <output name="othercounts" file="htseq-test_othercounts.tsv" /> |
| 0 | 137 <output name="samoutfile" file="htseq-test_samout.bam" /> |
| 138 </test> | |
| 139 --> | |
| 140 </tests> | |
| 141 | |
| 142 <help> | |
| 143 Overview | |
| 144 -------- | |
| 145 | |
| 146 This tool takes an alignment file in SAM or BAM format and feature file in GFF format | |
| 147 and calculates the number of reads mapping to each feature. It uses the *htseq-count* | |
| 148 script that is part of the HTSeq python module. See | |
| 149 http://www-huber.embl.de/users/anders/HTSeq/doc/count.html for details. | |
| 150 | |
| 151 A feature is an interval (i.e., a range of positions) on a chromosome or a union of | |
| 152 such intervals. In the case of RNA-Seq, the features are typically genes, where | |
| 153 each gene is considered here as the union of all its exons. One may also consider | |
| 154 each exon as a feature, e.g., in order to check for alternative splicing. For | |
| 155 comparative ChIP-Seq, the features might be binding regions from a pre-determined | |
| 156 list. | |
| 157 | |
| 158 | |
| 159 Overlap Modes | |
| 160 ------------- | |
| 161 | |
| 162 Special care must be taken to decide how to deal with reads that overlap more than one feature. | |
| 163 | |
| 164 The htseq-count script allows to choose between three modes: *union*, *intersection-strict*, and *intersection-nonempty*. | |
| 165 | |
| 166 The following figure illustrates the effect of these three modes: | |
| 167 | |
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168 .. image:: count_modes.png |
| 0 | 169 |
| 11 | 170 |
| 0 | 171 Strandedness |
| 172 ------------ | |
| 173 | |
| 174 **Important**: The default for strandedness is yes. If your RNA-Seq data has not been made with a strand-specific protocol, this causes half of the reads to be lost. Hence, make sure to set the option Stranded to 'No' unless you have strand-specific data! | |
| 175 | |
| 11 | 176 |
| 0 | 177 Output |
| 178 ------ | |
| 179 | |
| 180 The script outputs a table with counts for each feature, followed by the special counters, which count reads that were not counted for any feature for various reasons, namely | |
| 181 | |
| 182 - *no_feature*: reads which could not be assigned to any feature (set S as described above was empty). | |
| 183 | |
| 184 - *ambiguous*: reads which could have been assigned to more than one feature and hence were not counted for any of these (set S had mroe than one element). | |
| 185 | |
| 186 - *too_low_aQual*: reads which were not counted due to the -a option, see below | |
| 187 | |
| 188 - *not_aligned*: reads in the SAM file without alignment | |
| 189 | |
| 190 - *alignment_not_unique*: reads with more than one reported alignment. These reads are recognized from the NH optional SAM field tag. (If the aligner does not set this field, multiply aligned reads will be counted multiple times.) | |
| 191 | |
| 192 | |
| 193 Options Summary | |
| 194 --------------- | |
| 195 | |
| 196 Usage: htseq-count [options] sam_file gff_file | |
| 197 | |
| 198 This script takes an alignment file in SAM format and a feature file in GFF | |
| 199 format and calculates for each feature the number of reads mapping to it. See | |
| 200 http://www-huber.embl.de/users/anders/HTSeq/doc/count.html for details. | |
| 201 | |
| 202 Options: | |
| 203 -h, --help show this help message and exit | |
| 204 -m MODE, --mode=MODE mode to handle reads overlapping more than one | |
| 205 feature(choices: union, intersection-strict, | |
| 206 intersection-nonempty; default: union) | |
| 207 -s STRANDED, --stranded=STRANDED | |
| 208 whether the data is from a strand-specific assay. | |
| 209 Specify 'yes', 'no', or 'reverse' (default: yes). | |
| 210 'reverse' means 'yes' with reversed strand | |
| 211 interpretation | |
| 212 -a MINAQUAL, --minaqual=MINAQUAL | |
| 213 skip all reads with alignment quality lower than the | |
| 214 given minimum value (default: 0) | |
| 215 -t FEATURETYPE, --type=FEATURETYPE | |
| 216 feature type (3rd column in GFF file) to be used, all | |
| 217 features of other type are ignored (default, suitable | |
| 218 for Ensembl GTF files: exon) | |
| 219 -i IDATTR, --idattr=IDATTR | |
| 220 GFF attribute to be used as feature ID (default, | |
| 221 suitable for Ensembl GTF files: gene_id) | |
| 222 -o SAMOUT, --samout=SAMOUT | |
| 223 write out all SAM alignment records into an output SAM | |
| 224 file called SAMOUT, annotating each line with its | |
| 225 feature assignment (as an optional field with tag | |
| 226 'XF') | |
| 227 -q, --quiet suppress progress report and warnings | |
| 228 | |
| 229 Written by Simon Anders (sanders@fs.tum.de), European Molecular Biology | |
| 230 Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General | |
| 231 Public License v3. Part of the 'HTSeq' framework. | |
| 232 </help> | |
| 233 </tool> |