Mercurial > repos > mkim8 > decornai
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/FETCH_HEAD Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,1 @@ +eac6dbe543d206136426a5e86c0c981305cd0655 branch 'master' of https://github.com/QBRC/offtarget
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/HEAD Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,1 @@ +ref: refs/heads/master
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/ORIG_HEAD Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,1 @@ +855c6e9684f9e373d1ed470f18a5df6f1062f86d
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/config Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,13 @@ +[core] + bare = false + repositoryformatversion = 0 + filemode = false + symlinks = false + ignorecase = true + logallrefupdates = true +[remote "origin"] + url = https://github.com/QBRC/offtarget.git + fetch = +refs/heads/*:refs/remotes/origin/* +[branch "master"] + remote = origin + merge = refs/heads/master
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/description Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,1 @@ +Unnamed repository; edit this file 'description' to name the repository.
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/hooks/README.sample Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,5 @@ +#!/bin/sh +# +# Place appropriately named executable hook scripts into this directory +# to intercept various actions that git takes. See `git help hooks` for +# more information.
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/info/exclude Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,2 @@ +# File patterns to ignore; see `git help ignore` for more information. +# Lines that start with '#' are comments.
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/logs/HEAD Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,4 @@ +0000000000000000000000000000000000000000 855c6e9684f9e373d1ed470f18a5df6f1062f86d Min Soo <mins.kim@utsouthwestern.edu> 1394126673 -0600 commit (initial): Galaxy code +855c6e9684f9e373d1ed470f18a5df6f1062f86d 05f3c10c767726d9384dd0e7db30ca5ea09ac78d Min Soo <mins.kim@utsouthwestern.edu> 1394126781 -0600 pull --progress --rebase --prune: checkout 05f3c10c767726d9384dd0e7db30ca5ea09ac78d +05f3c10c767726d9384dd0e7db30ca5ea09ac78d 05f3c10c767726d9384dd0e7db30ca5ea09ac78d Min Soo <mins.kim@utsouthwestern.edu> 1394126783 -0600 rebase finished: returning to refs/heads/master +05f3c10c767726d9384dd0e7db30ca5ea09ac78d eac6dbe543d206136426a5e86c0c981305cd0655 Min Soo <mins.kim@utsouthwestern.edu> 1394126813 -0600 commit: Add executables and demo input data
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/logs/refs/heads/master Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,3 @@ +0000000000000000000000000000000000000000 855c6e9684f9e373d1ed470f18a5df6f1062f86d Min Soo <mins.kim@utsouthwestern.edu> 1394126673 -0600 commit (initial): Galaxy code +855c6e9684f9e373d1ed470f18a5df6f1062f86d 05f3c10c767726d9384dd0e7db30ca5ea09ac78d Min Soo <mins.kim@utsouthwestern.edu> 1394126783 -0600 rebase finished: refs/heads/master onto 05f3c10c767726d9384dd0e7db30ca5ea09ac78d +05f3c10c767726d9384dd0e7db30ca5ea09ac78d eac6dbe543d206136426a5e86c0c981305cd0655 Min Soo <mins.kim@utsouthwestern.edu> 1394126813 -0600 commit: Add executables and demo input data
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/logs/refs/remotes/origin/master Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,3 @@ +0000000000000000000000000000000000000000 855c6e9684f9e373d1ed470f18a5df6f1062f86d Min Soo <mins.kim@utsouthwestern.edu> 1394126682 -0600 update by push +855c6e9684f9e373d1ed470f18a5df6f1062f86d 05f3c10c767726d9384dd0e7db30ca5ea09ac78d unknown <mkim8@SW.(none)> 1394126776 -0600 pull --progress --rebase --prune: fast-forward +05f3c10c767726d9384dd0e7db30ca5ea09ac78d eac6dbe543d206136426a5e86c0c981305cd0655 Min Soo <mins.kim@utsouthwestern.edu> 1394126833 -0600 update by push
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/objects/41/2eeda78dc9de1186c2e0e1526764af82ab3431 Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,2 @@ +xuN1 YԭCXn@:!QbAW?, 9qUD]\&xBcB Qv֭ +O.0A<%+1pr.̅߯`@od XT?)' o"a+AK6k8V4y&ofk)M?Z7} \ No newline at end of file
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/refs/heads/master Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,1 @@ +eac6dbe543d206136426a5e86c0c981305cd0655
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.git/refs/remotes/origin/master Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,1 @@ +eac6dbe543d206136426a5e86c0c981305cd0655
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.gitattributes Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,22 @@ +# Auto detect text files and perform LF normalization +* text=auto + +# Custom for Visual Studio +*.cs diff=csharp +*.sln merge=union +*.csproj merge=union +*.vbproj merge=union +*.fsproj merge=union +*.dbproj merge=union + +# Standard to msysgit +*.doc diff=astextplain +*.DOC diff=astextplain +*.docx diff=astextplain +*.DOCX diff=astextplain +*.dot diff=astextplain +*.DOT diff=astextplain +*.pdf diff=astextplain +*.PDF diff=astextplain +*.rtf diff=astextplain +*.RTF diff=astextplain
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/.gitignore Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,215 @@ +################# +## Eclipse +################# + +*.pydevproject +.project +.metadata +bin/ +tmp/ +*.tmp +*.bak +*.swp +*~.nib +local.properties +.classpath +.settings/ +.loadpath + +# External tool builders +.externalToolBuilders/ + +# Locally stored "Eclipse launch configurations" +*.launch + +# CDT-specific +.cproject + +# PDT-specific +.buildpath + + +################# +## Visual Studio +################# + +## Ignore Visual Studio temporary files, build results, and +## files generated by popular Visual Studio add-ons. + +# User-specific files +*.suo +*.user +*.sln.docstates + +# Build results + +[Dd]ebug/ +[Rr]elease/ +x64/ +build/ +[Bb]in/ +[Oo]bj/ + +# MSTest test Results +[Tt]est[Rr]esult*/ +[Bb]uild[Ll]og.* + +*_i.c +*_p.c +*.ilk +*.meta +*.obj +*.pch +*.pdb +*.pgc +*.pgd +*.rsp +*.sbr +*.tlb +*.tli +*.tlh +*.tmp +*.tmp_proj +*.log +*.vspscc +*.vssscc +.builds +*.pidb +*.log +*.scc + +# Visual C++ cache files +ipch/ +*.aps +*.ncb +*.opensdf +*.sdf +*.cachefile + +# Visual Studio profiler +*.psess +*.vsp +*.vspx + +# Guidance Automation Toolkit +*.gpState + +# ReSharper is a .NET coding add-in +_ReSharper*/ +*.[Rr]e[Ss]harper + +# TeamCity is a build add-in +_TeamCity* + +# DotCover is a Code Coverage Tool +*.dotCover + +# NCrunch +*.ncrunch* +.*crunch*.local.xml + +# Installshield output folder +[Ee]xpress/ + +# DocProject is a documentation generator add-in +DocProject/buildhelp/ +DocProject/Help/*.HxT +DocProject/Help/*.HxC +DocProject/Help/*.hhc +DocProject/Help/*.hhk +DocProject/Help/*.hhp +DocProject/Help/Html2 +DocProject/Help/html + +# Click-Once directory +publish/ + +# Publish Web Output +*.Publish.xml +*.pubxml + +# NuGet Packages Directory +## TODO: If you have NuGet Package Restore enabled, uncomment the next line +#packages/ + +# Windows Azure Build Output +csx +*.build.csdef + +# Windows Store app package directory +AppPackages/ + +# Others +sql/ +*.Cache +ClientBin/ +[Ss]tyle[Cc]op.* +~$* +*~ +*.dbmdl +*.[Pp]ublish.xml +*.pfx +*.publishsettings + +# RIA/Silverlight projects +Generated_Code/ + +# Backup & report files from converting an old project file to a newer +# Visual Studio version. Backup files are not needed, because we have git ;-) +_UpgradeReport_Files/ +Backup*/ +UpgradeLog*.XML +UpgradeLog*.htm + +# SQL Server files +App_Data/*.mdf +App_Data/*.ldf + +############# +## Windows detritus +############# + +# Windows image file caches +Thumbs.db +ehthumbs.db + +# Folder config file +Desktop.ini + +# Recycle Bin used on file shares +$RECYCLE.BIN/ + +# Mac crap +.DS_Store + + +############# +## Python +############# + +*.py[co] + +# Packages +*.egg +*.egg-info +dist/ +build/ +eggs/ +parts/ +var/ +sdist/ +develop-eggs/ +.installed.cfg + +# Installer logs +pip-log.txt + +# Unit test / coverage reports +.coverage +.tox + +#Translations +*.mo + +#Mr Developer +.mr.developer.cfg
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/README.md Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,9 @@ +offtarget +========= + +DecoRNAi + +High-throughput RNAi screening has been widely used in a spectrum of biomedical research and made it possible to study functional genomics. However, a challenge for authentic biological interpretation of large-scale siRNA or shRNA-mediated loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA and shRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling that can result from short regions (~6 nucleotides) of oligonucleotide complementarity to many different mRNAs. To help identify and correct miRNA-mimic off-target effects, we have developed DecoRNAi (deconvolution analysis of RNAi screening data) for automated quantitation and annotation of microRNA-like off-target effects in primary RNAi screening data sets. DecoRNAi can effectively identify and correct off-target effects from primary screening data and provide data visualization for study and publication. DecoRNAi contains pre-computed seed sequence families for 3 commonly employed commercial siRNA libraries. For custom collections, the tool will compute seed sequence membership from a user-supplied reagent sequence table. All parameters are tunable and output files include global data visualization, the identified seed family associations, the siRNA pools containing off-target seed families, corrected z-scores and the potential miRNAs with phenotypes of interest. + +Author Rui Zhong. For any suggestions or inquiries please contact rui.zhong@utsouthwestern.edu +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/offtarget.xml Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,77 @@ +<tool id="sirna_offtarget" name="DecoRNAi"> + <description></description> + <command interpreter="sh">offtarget_wrapper.sh $input_file $seed $strand $lambda $library $str $sig $output</command> + <inputs> + <param name="input_file" type="data" format="txt" label="Input File" help="CSV File containing responce variables and siRNA sequence data."/> + <param name="strand" type="select" label="Strand Orientation for Analysis" help="Specify which strand orientation for Analysis."> + <option value="sense">Sense</option> + <option value="antisense">AntiSense</option> + <option value="both" selected="true">Both</option> + </param> + <param name="lambda" type="float" value="0.001" label="Lambda Value" help="Penalty parameter used in model."/> + <param name="seed" type="integer" value="2" label="Seed" min="1" max="14" help="Range: 1-14 Specify the seed region to be used."/> + <param name="library" type="select" label="siRNA Library to use" help="Specify siRNA library. Default is custom which requires user input sequences."> + <option value="custom" selected="true">Custom</option> + <option value="ambion">Ambion</option> + <option value="new_dharmacon">Dharmacon 2005</option> + <option value="old_dharmacon">Dharmacon 2009</option> + </param> + <param name="str" type="integer" value="1" label="Strength of Seed-Linked Effect." min="1" help="Specify cutoff for strength of seed-linked effect. Must be positive value."/> + <param name="sig" type="float" value="0.01" label="Significance P-Value" help="Specify cutoff for significance (P-value)."/> + </inputs> + <outputs> + <data format="zip" name="output" /> + </outputs> + + <requirements> + <requirement type="package" version="2.15.1">R</requirement> + </requirements> + + <help> + +**Description** + +High-throughput RNAi screening has been widely used in a spectrum of biomedical research and made it possible to study functional genomics. However, a challenge for authentic biological interpretation of large-scale siRNA or shRNA-mediated loss-of-function studies is the biological pleiotropy resulting from multiple modes of action of siRNA and shRNA reagents. A major confounding feature of these reagents is the microRNA-like translational quelling that can result from short regions (~6 nucleotides) of oligonucleotide complementarity to many different mRNAs. To help identify and correct miRNA-mimic off-target effects, we have developed DecoRNAi (deconvolution analysis of RNAi screening data) for automated quantitation and annotation of microRNA-like off-target effects in primary RNAi screening data sets. DecoRNAi can effectively identify and correct off-target effects from primary screening data and provide data visualization for study and publication. DecoRNAi contains pre-computed seed sequence families for 3 commonly employed commercial siRNA libraries. For custom collections, the tool will compute seed sequence membership from a user-supplied reagent sequence table. All parameters are tunable and output files include global data visualization, the identified seed family associations, the siRNA pools containing off-target seed families, corrected z-scores and the potential miRNAs with phenotypes of interest. + + +------ + +**Manual** + +Manual.pdf_ + +.. _Manual.pdf: http://galaxy.qbrc.org/static/DecoRNAi_Manual.pdf + + +------ + +**Parameters** + +*Input File* +Name of input file for analysis. This file should contain Gene ID (for example, Gene Entrez ID name), normalized screening data and sense strand siRNA sequences. Default format is a csv (comma separated value) file, in which the first column contains Gene ID name, the second column contains normalized screening data and the following columns are the sense strand siRNA sequences (one sequence per column, i.e., for example, there would be 4 separate sequence columns if 4 oligos are present in a pool). See the user’s manual for details. + +*Strand* +For identification and quantification of off-target effects, DecoRNAi can employ sense strand only, antisense strand only or both strands. The default setting is using both strands. + +*Lambda* +Penalty parameter in the model for identification of off-target effects. Default is 0.001. + +*Seed* +Specify which hexamer is used to define the seed sequence for analysis. For the most part, a siRNA oligo contains 21 nt. We can therefore assign any of 14 different hexamers as the seed sequence. For example, 1 means nucleotides 5’ 1~6 hexamer and 2 means nucleotides 5’ 2~7 hexamer and etc. Default is 2. + +*Library* +Users can specify the siRNA library for analysis. Seed families are pre-computed for the Dharmacon siGenome (version history 0), Dharmacon siGenome (version history 2), and Ambion. Gene Entrez ID is necessary to map between input data and stored sequences. Users can also upload custom library-wide sequence information for each oligonucleotide or processed siRNA, in which case Gene ID is free of format and type. Default is Custom. + +*Strength of seed-linked effect* +Users can specify the cutoff for strength of seed-linked effect. Must be positive value and default is 1. A smaller value will select more off-target seed families. + +*Significance (P value)* +Users can specify the cutoff for significance (P value). Default is 0.01. In summary report, False Discovery Rate (FDR) will be provided to control multiple testing issues. . + + +------ + +**Author** +Rui Zhong. For any suggestions or inquiries please contact rui.zhong@utsouthwestern.edu + </help> +</tool>
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/offtarget_v7.R Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,575 @@ +library(methods) +library(lattice) +suppressMessages(library(Matrix)) +suppressMessages(library(ClassComparison)) +suppressMessages(library(glmnet)) +options(warn=-1) + +####################### +### arguments input ### +####################### + +arg=commandArgs(TRUE) + +InputFile=arg[1] # must be given by user including both + # response variable and siRNA sequences + +Seed=as.integer(arg[2]) # Name: "Seed" + # specify the seed region to be used in analysis + # an interger out of 1~14. + # default is 2, which means 2~7 hexamer is used + +Strand=arg[3] # Name:"Strand Orientation for Analysis" + # specify which strand is used in analysis + # must be given by user, options are "sense", + #"antisense" or "both" + +Lambda=as.numeric(arg[4]) # Name:"Lambda Value" + # penalty parameter in LASSO regression + # default = 0.001, which can be changed by user + # in order to perform differennt LASSO estimation + +Library=arg[5] # Name:"siRNA Library to use" + # user has to specify which siRNA library will be + # used in analysis can be "Custom", "Ambion", "New Dharmacon" + # or "Old Dharmacon". Default is "Custom" + +### new parameter ### + +Str=as.numeric(arg[6]) # Name:"Strength of seed-linked effect“ + # specify cutoff for strenth of seed-linked effet, must be positive + # default is 1 + +Sig=as.numeric(arg[7]) # Name:"Significance (P value)" + # specify cutoff for significance (p value) + # default is 0.01 + +################# +### functions ### +################# + +# uppercase a sequence and transform T to U ### + +TtoU=function(x){ + x=as.character(x) + x=toupper(x) + x=chartr("T", "U", x) + return(x) +} + +# reverse a sequence # + +StrReverse <- function(x){ + x=as.character(x) + x=sapply(lapply(strsplit(x, NULL), rev), paste, collapse="") + return(x) +} + +# generate complementary sequence # + +DuplexReverse=function(x) +{ + x=as.character(x) + x=gsub("U","T",x) + x=gsub("A","U",x) + x=gsub("T","A",x) + x=gsub("G","g",x) + x=gsub("C","G",x) + x=gsub("g","C",x) + x=StrReverse(x) + return(x) +} + +# check if input contains AUGC only # + +SeqCheck=function(x){ + x=as.character(x) + x=strsplit(x, "") + x=unlist(x) + if(sum(x %in% c("A", "U", "G", "C")) == length(x) ){ + return(TRUE) + } + else{ + return(FALSE) + } +} + +# extract seed sequence from siRNA # + +SeedExtract=function(x, start=2, end=7){ + x=as.character(x) + x=substr(x, start, end) + return(x) +} + +# examine if a pool contain a seed # + +SeedExist=function(dfr, s){ + dfr=data.frame(dfr) + s=as.character(s) + return(as.numeric(apply(dfr == s, 1, sum))) +} + +# seed analysis # + +SeedAnalysis=function(Z, data, start=2, end=7){ + + print("Generating seed.dfr ... ... ") + data=data.frame(data) + seed.dfr=data + for(i in 1:ncol(seed.dfr)){ + seed.dfr[, i]=as.character(SeedExtract(seed.dfr[, i], start=start, end=end)) + } + colnames(seed.dfr)=paste("seed", 1:ncol(seed.dfr), sep=".") + print("Generating seed.table ... ... ") + seed.table=data.frame(table(unlist(seed.dfr))) + names(seed.table)=c("seed", "family.size") + + seed.table$ks.p.value=rep(NA, length(seed.table$seed)) + y=Z + for(i in 1:length(seed.table$seed)){ + s=SeedExist(seed.dfr, seed.table$seed[i]) + seed.table$ks.p.value[i]= + ks.test(y[s > 0], y[s== 0])$p.value + print(paste("seed ", i)) + } + + return(list(Z=Z, data=data, seed.table=seed.table, seed.dfr=seed.dfr)) +} + +# offtarget analysis # + +OfftargetAnalysis=function(object, s=0.001){ + + seed.dfr=object$seed.dfr + seed.table=object$seed.table + y=object$Z + + print("Generating Matrix X ... ... ") + s.sub=as.character(seed.table$seed) + x=matrix(0, length(y), length(s.sub)) + colnames(x)=as.character(s.sub) + + for(i in 1:length(s.sub)){ + n=as.character(s.sub[i]) + x[, n]=SeedExist(seed.dfr, n) + print(paste("Matrix X seed ", i)) + } + + print("Fitting ... ... ") + gc() + + fit=glmnet(x, y) + + print("offtarget analysis") + offtarget=data.frame(as.matrix(coef(fit, s=s))) + offtarget$seed=rownames(offtarget) + offtarget=offtarget[, c(2, 1)] + names(offtarget)[2]="coef" + rownames(offtarget)=NULL + + offtarget=merge(offtarget, seed.table, by="seed", all.x=T) + y.fit=as.numeric(predict(fit, x, s=s)) + y.corrected=y-y.fit + print("Off-target analysis Done!") + + return(list(fit=fit, X=x, offtarget=offtarget, Z.corrected=y.corrected)) + + +} + +# generating data frame of siRNA sequence # + +SeqGenerate=function(SeqDat){ + SenseSeq=data.frame(apply(SeqDat, 2, TtoU)) + AntiSeq=data.frame(apply(SenseSeq, 2, DuplexReverse)) + for(i in 1:ncol(SenseSeq)){ + AntiSeq[, i]=DuplexReverse(SenseSeq[, i]) + } + BothSeq=cbind(SenseSeq, AntiSeq) + names(BothSeq)=paste("duplex", 1:ncol(BothSeq), sep=".") + output=list(SenseSeq=SenseSeq, AntiSeq=AntiSeq, BothSeq=BothSeq) + return(output) +} + +# DataAnalysis # + +DataAnalysis=function(Z, Seq, SeedStart, Strand, Lambda){ + + SeedEnd=SeedStart+5 + + analysis=list() + + if(Strand == 'sense'){ + Seq=Seq$SenseSeq + analysis$seed.analysis=SeedAnalysis(Z=Z, data=Seq, start=SeedStart, end=SeedEnd) + analysis$offtarget.analysis=OfftargetAnalysis(analysis$seed.analysis, s=Lambda) + } + else if(Strand == "antisense"){ + Seq=Seq$AntiSeq + analysis$seed.analysis=SeedAnalysis(Z=Z, data=Seq, start=SeedStart, end=SeedEnd) + analysis$offtarget.analysis=OfftargetAnalysis(analysis$seed.analysis, s=Lambda) + } + else if(Strand == 'both'){ + Seq=Seq$BothSeq + analysis$seed.analysis=SeedAnalysis(Z=Z, data=Seq, start=SeedStart, end=SeedEnd) + analysis$offtarget.analysis=OfftargetAnalysis(analysis$seed.analysis, s=Lambda) + } + return(analysis) +} + +# new functions # + +# FDR calculation # + +fdr.bum=function(p.value, Sig){ + p.sum=attributes(summary(Bum(p.value), tau=Sig))$estimates + f=p.sum[4]/(p.sum[2]+p.sum[4]) + f=as.numeric(f) + return(f) +} + +# load miRNA database # + +load("/home/galaxy/galaxy-dist/tools/offtarget/miRNA.RData") + +################## +### data input ### +################## + +# data input # + +SeedStart=Seed +inputfile=read.csv(InputFile, header=F) +id=inputfile[, 1] +Z=as.numeric(inputfile[, 2]) + +# Z score checking # + +if(sum(is.na(Z)) > 0){ + print("Missing Value in Experimental Readouts!") + quit() +} + +if(sum(Z == "") > 0){ + print("Missing Value in Experimental Readouts!") + quit() +} + +##################### +### data analysis ### +##################### + +### Custom ### + +if(Library == "custom"){ + + ### check if user selected wrong "Library" parameter ### + if(ncol(inputfile) < 3){ + print("siRNA sequences not available and please check your Library selection") + quit() + } + ################################################################## + + SeqDat=inputfile[, 3:ncol(inputfile)] + SeqDat=data.frame(SeqDat) + + if(sum(is.na(unlist(SeqDat))) > 0){ + print("Missing Value in siRNA sequences!") + quit() + } + + if(sum(unlist(SeqDat) == "") > 0){ + print("Missing Value in siRNA sequences!") + quit() + } + + Seq=SeqGenerate(SeqDat) + + SenseSeq=Seq$SenseSeq + Check=data.frame(apply(SenseSeq, 1, SeqCheck)) + + if(sum(Check == FALSE) > 0){ + print("Non-Sequence Value in siRNA sequences!") + quit() + } + else{ + analysis=DataAnalysis(Z, Seq, SeedStart, Strand, Lambda) + } +} + +### Ambion ### + +if(Library == "ambion"){ + + id=as.integer(id) + + if(sum(is.na(id)) > 0){ + print("Missing Value in Gene ID!") + quit() + } + + if(sum(id == "") > 0){ + print("Missing Value in Gene ID!") + quit() + } + + load("/home/galaxy/galaxy-dist/tools/offtarget/Ambion.RData") + id.ambion=unique(ambion$id) + + if(length(id[!(id %in% id.ambion)]) > 0){ + write.csv(c("No Matching siRNA Sequences with Below Gene ID and Removed from Analysis", + id[!(id %in% id.ambion)]), "unmatched.id.csv") + } + + Dat=data.frame(id=id, z=Z) + Dat=merge(Dat, ambion, by="id", sort=F) + Z=Dat$z + id=Dat$id + SeqDat=Dat[, 6:8] + Seq=SeqGenerate(SeqDat) + analysis=DataAnalysis(Z, Seq, SeedStart, Strand, Lambda) + +} + +### New Dhar ### + +if(Library == "new_dharmacon"){ + + id=as.integer(id) + + if(sum(is.na(id)) > 0){ + print("Missing Value in Gene ID!") + quit() + } + + if(sum(id == "") > 0){ + print("Missing Value in Gene ID!") + quit() + } + + load("/home/galaxy/galaxy-dist/tools/offtarget/New_Dhar.RData") + id.new.dhar=unique(new.dhar$id) + + if(length(id[!(id %in% id.new.dhar)]) > 0){ + write.csv(c("No Matching siRNA Sequences with Below Gene ID and Removed from Analysis", + id[!(id %in% id.new.dhar)]), "unmatched.id.csv") + } + + Dat=data.frame(id=id, z=Z) + Dat=merge(Dat, new.dhar, by="id", sort=F) + Z=Dat$z + id=Dat$id + SeqDat=Dat[, 7:10] + Seq=SeqGenerate(SeqDat) + analysis=DataAnalysis(Z, Seq, SeedStart, Strand, Lambda) + +} + +### Old Dhar ### + +if(Library == "old_dharmacon"){ + + id=as.integer(id) + + if(sum(is.na(id)) > 0){ + print("Missing Value in Gene ID!") + quit() + } + + if(sum(id == "") > 0){ + print("Missing Value in Gene ID!") + quit() + } + + load("/home/galaxy/galaxy-dist/tools/offtarget/Old_Dhar.RData") + id.old.dhar=unique(old.dhar$id) + + if(length(id[!(id %in% id.old.dhar)]) > 0){ + write.csv(c("No Matching siRNA Sequences with Below Gene ID and Removed from Analysis", + id[!(id %in% id.old.dhar)]), "unmatched.id.csv") + } + + Dat=data.frame(id=id, z=Z) + Dat=merge(Dat, old.dhar, by="id", sort=F) + Z=Dat$z + id=Dat$id + SeqDat=Dat[, 7:10] + Seq=SeqGenerate(SeqDat) + analysis=DataAnalysis(Z, Seq, SeedStart, Strand, Lambda) + +} + +###################### +### results output ### +###################### + +# code chagned below # + +# generating output files # + +duplex.dfr=analysis$seed.analysis$data +names(duplex.dfr)=paste("duplex", 1:length(names(duplex.dfr)), sep=".") +seed.dfr=analysis$seed.analysis$seed.dfr +offtarget=analysis$offtarget.analysis$offtarget +Z.corrected=analysis$offtarget.analysis$Z.corrected +output.corr=cbind(ID=id, Z.Score=Z, Corrected.Z.Score=Z.corrected, + duplex.dfr, seed.dfr) + + +print("outputing results... ...") + +if(Library == 'custom'){ + + print("outputing corrected Z score... ...") + write.csv(output.corr, "CorrectedZScore.csv", row.names=F) ### export corrected z score + + print("outputing summary... ...") + write.csv(offtarget, "Seed_Famliy_Summary.csv", row.names=FALSE) ### output seed family summary table + + print("outputing offtarget seed families... ...") + + off.target=offtarget$seed[offtarget$ks.p.value < Sig & ( + offtarget$coef < -Str | offtarget$coef > Str)] # select off target by custom setup + + write.csv(offtarget[offtarget$seed %in% off.target, ], + "Off-Target_Seed_Families.csv", row.names=FALSE) ### export off-target seed families + + print("outputing miRNAs... ...") + if(nrow(miRNA[miRNA$Seed.sequence %in% off.target, ]) == 0){ + write.csv("No miRNA detected", "miRNA.csv", row.names=F) + fe="No" + } ### export miRNAs table sharing common seed + + if(nrow(miRNA[miRNA$Seed.sequence %in% off.target, ]) != 0){ + write.csv(miRNA[miRNA$Seed.sequence %in% off.target, ], "miRNA.csv", row.names=F) + fe="Yes" + } ### export miRNAs talbe sharing common seed + + if(length(off.target) > 0){ + for(i in 1:length(off.target)){ + + n=off.target[i] + si=SeedExist(seed.dfr, n) + si.offtarget=output.corr[si == 1, -3] + si.offtarget$off.target.seed=n + si.offtarget=si.offtarget[order(si.offtarget$Z.Score), ] + print(paste(n, "siRNA pools ... ...")) + write.csv(si.offtarget, paste("siRNAsPool_OffTargetSeedFamily_", n, ".csv", sep=""), row.names=F) + + } + } +}else{ + + print("outputing corrected Z score... ...") + write.csv(output.corr[, 1:3], "CorrectedZScore.csv", row.names=F) + + print("outputing summary... ...") + write.csv(offtarget, "Seed_Famliy_Summary.csv", row.names=FALSE) ### output seed family summary table + + print("outputing offtarget seed families... ...") + + off.target=offtarget$seed[offtarget$ks.p.value < Sig & ( + offtarget$coef < -Str | offtarget$coef > Str)] # select off target by custom setup + + write.csv(offtarget[offtarget$seed %in% off.target, ], + "Off-Target_Seed_Families.csv", row.names=FALSE) ### export off-target seed families + + print("outputing miRNAs... ...") + if(nrow(miRNA[miRNA$Seed.sequence %in% off.target, ]) == 0){ + write.csv("No miRNA detected", "miRNA.csv", row.names=F) + fe="No" + } ### export miRNAs table sharing common seed + + if(nrow(miRNA[miRNA$Seed.sequence %in% off.target, ]) != 0){ + write.csv(miRNA[miRNA$Seed.sequence %in% off.target, ], "miRNA.csv", row.names=F) + fe="Yes" + } ### export miRNAs talbe sharing common seed + + if(length(off.target) > 0){ + for(i in 1:length(off.target)){ + + n=off.target[i] + si=SeedExist(seed.dfr, n) + si.offtarget=output.corr[si == 1, -3] + si.offtarget$off.target.seed=n + si.offtarget=si.offtarget[order(si.offtarget$Z.Score), ] + print(paste(n, "siRNA pools ... ...")) + write.csv(si.offtarget, paste("siRNAsPool_OffTargetSeedFamily_", n, ".csv", sep=""), row.names=F) + + } + } +} + + +# generating output figures # + +print("outputing figures... ...") + +jpeg(file="SeedFamilyOffTarget.jpeg", height=600, width=800) +par(mar=c(5, 5, 2, 2)) +plot(offtarget$coef, -log10(offtarget$ks.p.value), + pch=20, col="blue", yaxt="n", cex.axis=1.5, cex.lab=2, + xlab="Strength of seed-linked effect", ylab="Significance (P value)") +p.log=ceiling(-log10(min(offtarget$ks.p.value, na.rm=T))/2) +p.range=10^(0:-p.log*2) +axis(2, p.range, + at=-log(p.range, 10), + cex.axis=1.5) +off.target=offtarget$seed[offtarget$coef < -Str & offtarget$ks.p.value < Sig] +nn=length(off.target) +points(offtarget$coef[offtarget$seed %in% off.target], + -log10(offtarget$ks.p.value[offtarget$seed %in% off.target]), + col="red", pch=20) +off.target=offtarget$seed[offtarget$coef > Str & offtarget$ks.p.value < Sig] +np=length(off.target) +points(offtarget$coef[offtarget$seed %in% off.target], + -log10(offtarget$ks.p.value[offtarget$seed %in% off.target]), + col="green", pch=20) +abline(v=-Str, col="red", lwd=3) +abline(v=Str, col="green", lwd=3) +abline(h=-log10(Sig), col="blue", lwd=3) +dev.off() + +# generating legend # + +jpeg(file="SeedFamilyOffTargetLegend.jpeg", width=500, height=100) +par(mar=c(0, 0, 0, 0)) +plot(c(0, 2), c(0.8, 1.2), type="n", bty="n", yaxt="n", xaxt='n') +points(c(0, 0), c(1.1, 0.9), col=c('red', "green"), pch=20, cex=2) +text(1, 1.1, paste('N: ', nn, " (Strength < ", -Str, ", P value < ", Sig, ")", sep=""), cex=2) +text(1, 0.9, paste('N: ', np, " (Strength > ", Str, ", P value < ", Sig, ")", sep=""), cex=2) +dev.off() + +# new output # + +# generating summary # + +print("Ansysis Summary ... ...") + +print("p value ... ...") +p.value=offtarget$ks.p.value[offtarget$coef < -Str | offtarget$coef > Str] + +print("FDR ... ...") +f=fdr.bum(p.value, Sig) +print("rounding ... ...") +f=round(f, digits=3) +print(f) +print("FDR done ... ...") + + +print("off.sum ... ...") +off.sum=matrix(rep("", 30), 15, 2) +off.sum[1,1]="Analysis is successfully done!" +off.sum[3,1]="Parameters set up" +off.sum[4:15, 1]=c("Strand Orientation for Analysis:", "Lambda value:", "Seed:", + "siRNA Library:", "Strength of seed-linked effect:", "P value:", "", + "Analysis summary", + "Number of negative off-target seed families:", + "Number of positive off-target seed families:", "FDR:", + "Identify miRNA with phenotypical effects:") +off.sum[4:15, 2]=c(Strand, Lambda, Seed, Library, Str, Sig, "", "", nn, np, f, fe) +write.table(off.sum, "Analysis_Summary.csv", row.names=F, col.names=F, sep=",") + +print("Analysis done ... ...") +
--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/offtarget_wrapper.sh Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,11 @@ +R --vanilla --slave --args $1 $2 $3 $4 $5 $6 $7 < /home/galaxy/galaxy-dist/tools/offtarget/offtarget_v7.R > dump + +if [ -f ${__tool_data_path__}CorrectedZScore.csv ] + then + mv dump log.txt + zip temp ${__tool_data_path__}*.* + mv ${__tool_data_path__}temp.zip $8 + else + cat dump >&2 +fi +
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--- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/offtarget/tool_dependencies.xml Thu Mar 13 16:50:41 2014 -0400 @@ -0,0 +1,23 @@ +<?xml version="1.0"?> +<tool_dependency> + <package name="R" version="2.15.1"> + <install version="1.0"> + <actions> + <action type="download_by_url">http://CRAN.R-project.org/src/base/R-2/R-2.15.1.tar.gz</action> + <action type="shell_command">./configure --prefix=$INSTALL_DIR</action> + <action type="shell_command">make</action> + <action type="set_environment"> + <environment_variable name="PATH" action="prepend_to">$INSTALL_DIR/bin</environment_variable> + </action> + <action type="setup_r_environment"> + <package>http://cran.us.r-project.org/src/contrib/lattice_0.20-27.tar.gz</package> + <package>http://cran.r-project.org/src/contrib/Matrix_1.1-2-2.tar.gz</package> + <package>http://cran.r-project.org/src/contrib/glmnet_1.9-5.tar.gz</package> + + + </action> + </actions> + </install> + <readme>You need a FORTRAN compiler or perhaps f2c in addition to a C compiler to build R.</readme> + </package> +</tool_dependency> \ No newline at end of file