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comparison RPKM_saturation.xml @ 40:1e66f05a23aa

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author lparsons
date Wed, 23 Jul 2014 10:44:50 -0400
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39:1b769e35cd8e 40:1e66f05a23aa
1 <tool id="rseqc_RPKM_saturation" name="RPKM Saturation" version="2.3.9">
2 <description>calculates raw count and RPKM values for transcript at exon, intron, and mRNA level</description>
3 <requirements>
4 <requirement type="package" version="3.0.1">R</requirement>
5 <requirement type="package" version="1.7.1">numpy</requirement>
6 <requirement type="package" version="2.3.9">rseqc</requirement>
7 </requirements>
8 <command> RPKM_saturation.py -i $input -o output -r $refgene
9
10 #if str($strand_type.strand_specific) == "pair"
11 -d
12 #if str($strand_type.pair_type) == "sd"
13 '1++,1--,2+-,2-+'
14 #else
15 '1+-,1-+,2++,2--'
16 #end if
17 #end if
18
19 #if str($strand_type.strand_specific) == "single"
20 -d
21 #if str($strand_type.single_type) == "s"
22 '++,--'
23 #else
24 '+-,-+'
25 #end if
26 #end if
27
28 -l $percentileFloor -u $percentileCeiling -s $percentileStep -c $rpkmCutoff
29
30 </command>
31 <stdio>
32 <exit_code range="1:" level="fatal" description="An error occured during execution, see stderr and stdout for more information" />
33 <regex match="[Ee]rror" source="both" description="An error occured during execution, see stderr and stdout for more information" />
34 </stdio>
35 <inputs>
36 <param name="input" type="data" format="bam" label="input bam/sam file" />
37 <param name="refgene" type="data" format="bed" label="Reference gene model" />
38 <conditional name="strand_type">
39 <param name="strand_specific" type="select" label="Strand-specific?" value="None">
40 <option value="none">None</option>
41 <option value="pair">Pair-End RNA-seq</option>
42 <option value="single">Single-End RNA-seq</option>
43 </param>
44 <when value="pair">
45 <param name="pair_type" type="select" display="radio" label="Pair-End Read Type (format: mapped --> parent)" value="sd">
46 <option value="sd"> read1 (positive --> positive; negative --> negative), read2 (positive --> negative; negative --> positive)</option>
47 <option value="ds">read1 (positive --> negative; negative --> positive), read2 (positive --> positive; negative --> negative)</option>
48 </param>
49 </when>
50 <when value="single">
51 <param name="single_type" type="select" display="radio" label="Single-End Read Type (format: mapped --> parent)" value="s">
52 <option value="s">positive --> positive; negative --> negative</option>
53 <option value="d">positive --> negative; negative --> positive</option>
54 </param>
55 </when>
56 <when value="none"></when>
57 </conditional>
58 <param name="percentileFloor" type="integer" value="5" label="Begin sampling from this percentile (default=5)" />
59 <param name="percentileCeiling" type="integer" value="100" label="End sampling at this percentile (default=100)" />
60 <param name="percentileStep" type="integer" value="5" label="Sampling step size (default=5)" />
61 <param name="rpkmCutoff" type="text" value="0.01" label="Ignore transcripts with RPKM smaller than this number (default=0.01)" />
62 </inputs>
63 <outputs>
64 <data format="xls" name="outputxls" from_work_dir="output.eRPKM.xls" label="${tool.name} on ${on_string} (RPKM XLS)"/>
65 <data format="xls" name="outputrawxls" from_work_dir="output.rawCount.xls" label="${tool.name} on ${on_string} (Raw Count XLS)"/>
66 <data format="txt" name="outputr" from_work_dir="output.saturation.r" label="${tool.name} on ${on_string} (R Script)"/>
67 <data format="pdf" name="outputpdf" from_work_dir="output.saturation.pdf" label="${tool.name} on ${on_string} (PDF)"/>
68 </outputs>
69 <help>
70 RPKM_saturation.py
71 ++++++++++++++++++
72
73 The precision of any sample statitics (RPKM) is affected by sample size (sequencing depth);
74 \'resampling\' or \'jackknifing\' is a method to estimate the precision of sample statistics by
75 using subsets of available data. This module will resample a series of subsets from total RNA
76 reads and then calculate RPKM value using each subset. By doing this we are able to check if
77 the current sequencing depth was saturated or not (or if the RPKM values were stable or not)
78 in terms of genes' expression estimation. If sequencing depth was saturated, the estimated
79 RPKM value will be stationary or reproducible. By default, this module will calculate 20
80 RPKM values (using 5%, 10%, ... , 95%,100% of total reads) for each transcripts.
81
82 In the output figure, Y axis is "Percent Relative Error" or "Percent Error" which is used
83 to measures how the RPKM estimated from subset of reads (i.e. RPKMobs) deviates from real
84 expression level (i.e. RPKMreal). However, in practice one cannot know the RPKMreal. As a
85 proxy, we use the RPKM estimated from total reads to approximate RPKMreal.
86
87 .. image:: http://rseqc.sourceforge.net/_images/RelativeError.png
88 :height: 80 px
89 :width: 400 px
90 :scale: 100 %
91
92 Inputs
93 ++++++++++++++
94
95 Input BAM/SAM file
96 Alignment file in BAM/SAM format.
97
98 Reference gene model
99 Gene model in BED format.
100
101 Strand sequencing type (default=none)
102 See Infer Experiment tool if uncertain.
103
104 Options
105 ++++++++++++++
106
107 Skip Multiple Hit Reads
108 Use Multiple hit reads or use only uniquely mapped reads.
109
110 Only use exonic reads
111 Renders program only used exonic (UTR exons and CDS exons) reads, otherwise use all reads.
112
113 Output
114 ++++++++++++++
115
116 1. output..eRPKM.xls: RPKM values for each transcript
117 2. output.rawCount.xls: Raw count for each transcript
118 3. output.saturation.r: R script to generate plot
119 4. output.saturation.pdf:
120
121 .. image:: http://rseqc.sourceforge.net/_images/saturation.png
122 :height: 600 px
123 :width: 600 px
124 :scale: 80 %
125
126 - All transcripts were sorted in ascending order according to expression level (RPKM). Then they are divided into 4 groups:
127 1. Q1 (0-25%): Transcripts with expression level ranked below 25 percentile.
128 2. Q2 (25-50%): Transcripts with expression level ranked between 25 percentile and 50 percentile.
129 3. Q3 (50-75%): Transcripts with expression level ranked between 50 percentile and 75 percentile.
130 4. Q4 (75-100%): Transcripts with expression level ranked above 75 percentile.
131 - BAM/SAM file containing more than 100 million alignments will make module very slow.
132 - Follow example below to visualize a particular transcript (using R console)::
133
134 pdf("xxx.pdf") #starts the graphics device driver for producing PDF graphics
135 x &lt;- seq(5,100,5) #resampling percentage (5,10,15,...,100)
136 rpkm &lt;- c(32.95,35.43,35.15,36.04,36.41,37.76,38.96,38.62,37.81,38.14,37.97,38.58,38.59,38.54,38.67, 38.67,38.87,38.68, 38.42, 38.23) #Paste RPKM values calculated from each subsets
137 scatter.smooth(x,100*abs(rpkm-rpkm[length(rpkm)])/(rpkm[length(rpkm)]),type="p",ylab="Precent Relative Error",xlab="Resampling Percentage")
138 dev.off() #close graphical device
139
140 .. image:: http://rseqc.sourceforge.net/_images/saturation_eg.png
141 :height: 600 px
142 :width: 600 px
143 :scale: 80 %
144
145 -----
146
147 About RSeQC
148 +++++++++++
149
150 The RSeQC_ package provides a number of useful modules that can comprehensively evaluate high throughput sequence data especially RNA-seq data. "Basic modules" quickly inspect sequence quality, nucleotide composition bias, PCR bias and GC bias, while "RNA-seq specific modules" investigate sequencing saturation status of both splicing junction detection and expression estimation, mapped reads clipping profile, mapped reads distribution, coverage uniformity over gene body, reproducibility, strand specificity and splice junction annotation.
151
152 The RSeQC package is licensed under the GNU GPL v3 license.
153
154 .. image:: http://rseqc.sourceforge.net/_static/logo.png
155
156 .. _RSeQC: http://rseqc.sourceforge.net/
157
158
159 </help>
160 </tool>