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author lparsons
date Tue, 04 Sep 2012 13:45:36 -0400
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1 <tool id="htseq_count" name="htseq-count" version="0.1">
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>
5 <requirement type="package" version="0.5.3p9">htseq</requirement>
6 <requirement type="package" version="0.1.18">samtools</requirement>
7 </requirements>
8 <command>
9 ##set up input files
10 #set $reference_fasta_filename = "localref.fa"
11 #if $samout_conditional.samout:
12 #if str( $samout_conditional.reference_source.reference_source_selector ) == "history":
13 ln -s "${samout_conditional.reference_source.ref_file}" "${reference_fasta_filename}" &amp;&amp;
14 samtools faidx "${reference_fasta_filename}" 2&gt;&amp;1 || echo "Error running samtools faidx for htseq-count" &gt;&amp;2 &amp;&amp;
15 #else:
16 #set $reference_fasta_filename = str( $samout_conditional.reference_source.ref_file.fields.path )
17 #end if
18 #end if
19
20 #if $samfile.extension == "bam":
21 samtools view $samfile |
22 #end if
23 htseq-count
24 --mode=$mode
25 --stranded=$stranded
26 --minaqual=$minaqual
27 --type=$type
28 --idattr=$idattr
29 #if $samout_conditional.samout:
30 --samout=$__new_file_path__/${samoutfile.id}_tmp
31 #end if
32 #if $samfile.extension == "bam":
33 -
34 #else
35 $samfile
36 #end if
37 $gfffile
38 &gt; $counts
39 #if $samout_conditional.samout:
40 &amp;&amp; samtools view -Su -t ${reference_fasta_filename}.fai $__new_file_path__/${samoutfile.id}_tmp | samtools sort -o - sorted > $samoutfile
41 #end if</command>
42 <inputs>
43 <param format="sam, bam" name="samfile" type="data" label="Aligned SAM File">
44 <help>Paired-End data must be sorted by QUERY NAME, use Picard Read Mate Fixer and Query name sort order before using this tool on paired data</help>
45 </param>
46 <param format="gff" name="gfffile" type="data" label="GFF File"/>
47 <param name="mode" type="select" label="Mode">
48 <help>Mode to handle reads overlapping more than one feature.</help>
49 <option value="union" selected="true">Union</option>
50 <option value="intersection-strict">Intersection (strict)</option>
51 <option value="intersection-nonempty">Intersection (nonempty)</option>
52 </param>
53 <param name="stranded" type="select" label="Stranded">
54 <help>Specify whether the data is from a strand-specific assay. 'Reverse' means yes with reversed strand interpretation.</help>
55 <option value="yes" selected="true">Yes</option>
56 <option value="no">No</option>
57 <option value="reverse">Reverse</option>
58 </param>
59 <param name="minaqual" type="integer" value="0" label="Minimum alignment quality">
60 <help>Skip all reads with alignment quality lower than the given minimum value</help>
61 </param>
62 <param name="type" type="text" value="exon" label="Feature type">
63 <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>
64 </param>
65 <param name="idattr" type="text" value="gene_id" label="ID Attribute">
66 <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. The default, suitable for RNA-SEq and Ensembl GTF files, is gene_id.</help>
67 </param>
68 <conditional name="samout_conditional">
69 <param name="samout" type="boolean" value="False" truevalue="True" falsevalue="False" label="Additional BAM Output">
70 <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>
71 </param>
72 <when value="True">
73 <conditional name="reference_source">
74 <param name="reference_source_selector" type="select" label="Choose the source for the reference list">
75 <option value="cached">Locally cached</option>
76 <option value="history">History</option>
77 </param>
78 <when value="cached">
79 <param name="ref_file" type="select" label="Using reference genome">
80 <options from_data_table="sam_fa_indexes">
81 <filter type="data_meta" key="dbkey" ref="samfile" column="3"/>
82 </options>
83 <validator type="no_options" message="A built-in reference genome is not available for the build associated with the selected input file"/>
84 </param>
85 </when>
86 <when value="history"> <!-- FIX ME!!!! -->
87 <param name="ref_file" type="data" format="fasta" label="Using reference file" />
88 </when>
89 </conditional>
90 </when>
91 </conditional>
92 </inputs>
93
94 <outputs>
95 <data format="tabular" name="counts" label="${tool.name} on ${on_string}"/>
96 <data format="bam" name="samoutfile" label="${tool.name} on ${on_string} (BAM)">
97 <filter>samout_conditional['samout']</filter>
98 </data>
99 </outputs>
100
101 <stdio>
102 <exit_code range="1:" level="fatal" description="Unknown error occurred" />
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" />
111 </test>
112 <test>
113 <param name="samfile" value="htseq-test.bam" />
114 <param name="gfffile" value="htseq-test.gff" />
115 <param name="samout" value="False" />
116 <output name="counts" file="htseq-test_counts.tsv" />
117 </test>
118 <!-- Seems to be an issue setting the $reference_fasta_filename variable during test
119 <test>
120 <param name="samfile" value="htseq-test.sam" />
121 <param name="gfffile" value="htseq-test.gff" />
122 <param name="samout" value="True" />
123 <param name="reference_source_selector" value="history" />
124 <param name="ref_file" value="htseq-test_reference.fasta" />
125 <output name="counts" file="htseq-test_counts.tsv" />
126 <output name="samoutfile" file="htseq-test_samout.bam" />
127 </test>
128 -->
129 </tests>
130
131 <help>
132 Overview
133 --------
134
135 This tool takes an alignment file in SAM or BAM format and feature file in GFF format
136 and calculates the number of reads mapping to each feature. It uses the *htseq-count*
137 script that is part of the HTSeq python module. See
138 http://www-huber.embl.de/users/anders/HTSeq/doc/count.html for details.
139
140 A feature is an interval (i.e., a range of positions) on a chromosome or a union of
141 such intervals. In the case of RNA-Seq, the features are typically genes, where
142 each gene is considered here as the union of all its exons. One may also consider
143 each exon as a feature, e.g., in order to check for alternative splicing. For
144 comparative ChIP-Seq, the features might be binding regions from a pre-determined
145 list.
146
147 **Paired-end Data MUST be sorted by QUERY NAME first**
148
149 This tool requires that paired-end data be sorted by query name, which is NOT the default for Galaxy. Using the Picard Paired Read Mate Fixer with Query name sort FIRST is required for paired end data.
150
151
152 Overlap Modes
153 -------------
154
155 Special care must be taken to decide how to deal with reads that overlap more than one feature.
156
157 The htseq-count script allows to choose between three modes: *union*, *intersection-strict*, and *intersection-nonempty*.
158
159 The following figure illustrates the effect of these three modes:
160
161 .. image:: /static/images/count_modes.png
162 :width: 500
163
164 Strandedness
165 ------------
166
167 **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!
168
169 Output
170 ------
171
172 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
173
174 - *no_feature*: reads which could not be assigned to any feature (set S as described above was empty).
175
176 - *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).
177
178 - *too_low_aQual*: reads which were not counted due to the -a option, see below
179
180 - *not_aligned*: reads in the SAM file without alignment
181
182 - *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.)
183
184
185 Options Summary
186 ---------------
187
188 Usage: htseq-count [options] sam_file gff_file
189
190 This script takes an alignment file in SAM format and a feature file in GFF
191 format and calculates for each feature the number of reads mapping to it. See
192 http://www-huber.embl.de/users/anders/HTSeq/doc/count.html for details.
193
194 Options:
195 -h, --help show this help message and exit
196 -m MODE, --mode=MODE mode to handle reads overlapping more than one
197 feature(choices: union, intersection-strict,
198 intersection-nonempty; default: union)
199 -s STRANDED, --stranded=STRANDED
200 whether the data is from a strand-specific assay.
201 Specify 'yes', 'no', or 'reverse' (default: yes).
202 'reverse' means 'yes' with reversed strand
203 interpretation
204 -a MINAQUAL, --minaqual=MINAQUAL
205 skip all reads with alignment quality lower than the
206 given minimum value (default: 0)
207 -t FEATURETYPE, --type=FEATURETYPE
208 feature type (3rd column in GFF file) to be used, all
209 features of other type are ignored (default, suitable
210 for Ensembl GTF files: exon)
211 -i IDATTR, --idattr=IDATTR
212 GFF attribute to be used as feature ID (default,
213 suitable for Ensembl GTF files: gene_id)
214 -o SAMOUT, --samout=SAMOUT
215 write out all SAM alignment records into an output SAM
216 file called SAMOUT, annotating each line with its
217 feature assignment (as an optional field with tag
218 'XF')
219 -q, --quiet suppress progress report and warnings
220
221 Written by Simon Anders (sanders@fs.tum.de), European Molecular Biology
222 Laboratory (EMBL). (c) 2010. Released under the terms of the GNU General
223 Public License v3. Part of the 'HTSeq' framework.
224 </help>
225 </tool>