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1 <tool id="readthrough_analysis" name="Stop_supp">
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2 <description>To analyse Ribo-seq alignments for the detection of stop codon readthrough events</description>
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3 <requirements>
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4 <requirement type="package">samtools</requirement>
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5 <requirement type="python-module">HTseq</requirement>
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6 <requirement type="python-module">pysam</requirement>
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7 <requirement type="python-module">csv</requirement>
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8 <requirement type="python-module">Bio</requirement>
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9 </requirements>
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10 <command interpreter="python">
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11 metagene_readthrough.py --gff $gff --fasta $fasta --bam $mapping --dirout=$output,$output.files_path --extend $ext
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12
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13 </command>
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14
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15 <inputs>
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16 <param name="gff" type="data" label="Reference annotation file (GFF)" format="gff" />
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17 <param name="fasta" type="data" label="Reference genome in Fasta format" format="fasta" />
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18 <param name="mapping" type="data" label="Bam File" format="bam" />
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19 <param name="ext" type="integer" label="Length of 3’ UTR extension downstream the annotated stop codon (in bp)" value="300" />
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20 </inputs>
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21
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22 <outputs>
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23 <data format="html" name="output" label="[RP]Readthrough results on ${on_string}"/>
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24 </outputs>
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25
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26 <help>
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27 Summary
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28 -------
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29 This tool uses Ribo-seq data (BAM file) to extract genes displaying potential stop codon readthrough events from a reference annotation file (`GFF3`_).
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30
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31 C-terminal protein extensions were identified as previously described (Dunn J.G. et al, 2013). Only uniquely mapped footprints with a size in the 25 to 34 range are considered.
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32 A gene is considered to display readthrough if:
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33
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34 i) It is covered by more than 128 footprints.
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35
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36 ii) There are footprints after the stop codon.
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37
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38 iii) There are footprints overlapping the next in-frame stop codon.
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39
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40 iv) There is no methionine codon in the next five codons downstream from the official stop codon of the CDS.
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41
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42 v) Coverage is homogeneous within the extension.
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43
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44 Stop codon readthrough was estimated by calculating the ratio of the number of footprints in the C-terminal extension to that in the CDS. Ribosome density footprints were estimated in RPKM (reads per kilobase per million). We controlled for variability due to stop codon peaks, by excluding footprints mapping to stop codons from the calculation of RPKM.
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45
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46 .. _GFF3: http://gmod.org/wiki/GFF3
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47
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48
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49 Output
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50 -------
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51 This tool produces an html file with plots for each gene displaying readthrough.
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52
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53
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54 Dependances
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55 ------------
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56
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57 .. class:: warningmark
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58
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59 This tool depends on Python (>=2.7) and following packages : numpy 1.8.0, Biopython 1.58, matplotlib 1.3.1, HTSeq and pysam. Samtools is used for bam manipulation.
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61 </help>
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62 </tool>
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