# HG changeset patch # User fubar # Date 1374890698 14400 # Node ID 91e77df18a7fb3af6c0597885f43c87a09c9c5c0 # Parent 6431647290573e50d1567c7a093e2d1680291a76 Uploaded diff -r 643164729057 -r 91e77df18a7f rgedgeR/rgToolFactory.py --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/rgedgeR/rgToolFactory.py Fri Jul 26 22:04:58 2013 -0400 @@ -0,0 +1,599 @@ +# rgToolFactory.py +# see https://bitbucket.org/fubar/galaxytoolfactory/wiki/Home +# +# copyright ross lazarus (ross stop lazarus at gmail stop com) May 2012 +# +# all rights reserved +# Licensed under the LGPL +# suggestions for improvement and bug fixes welcome at https://bitbucket.org/fubar/galaxytoolfactory/wiki/Home +# +# july 2013 +# added ability to combine images and individual log files into html output +# just make sure there's a log file foo.log and it will be output +# together with all images named like "foo_*.pdf +# otherwise old format for html +# +# January 2013 +# problem pointed out by Carlos Borroto +# added escaping for <>$ - thought I did that ages ago... +# +# August 11 2012 +# changed to use shell=False and cl as a sequence + +# This is a Galaxy tool factory for simple scripts in python, R or whatever ails ye. +# It also serves as the wrapper for the new tool. +# +# you paste and run your script +# Only works for simple scripts that read one input from the history. +# Optionally can write one new history dataset, +# and optionally collect any number of outputs into links on an autogenerated HTML page. + +# DO NOT install on a public or important site - please. + +# installed generated tools are fine if the script is safe. +# They just run normally and their user cannot do anything unusually insecure +# but please, practice safe toolshed. +# Read the fucking code before you install any tool +# especially this one + +# After you get the script working on some test data, you can +# optionally generate a toolshed compatible gzip file +# containing your script safely wrapped as an ordinary Galaxy script in your local toolshed for +# safe and largely automated installation in a production Galaxy. + +# If you opt for an HTML output, you get all the script outputs arranged +# as a single Html history item - all output files are linked, thumbnails for all the pdfs. +# Ugly but really inexpensive. +# +# Patches appreciated please. +# +# +# long route to June 2012 product +# Behold the awesome power of Galaxy and the toolshed with the tool factory to bind them +# derived from an integrated script model +# called rgBaseScriptWrapper.py +# Note to the unwary: +# This tool allows arbitrary scripting on your Galaxy as the Galaxy user +# There is nothing stopping a malicious user doing whatever they choose +# Extremely dangerous!! +# Totally insecure. So, trusted users only +# +# preferred model is a developer using their throw away workstation instance - ie a private site. +# no real risk. The universe_wsgi.ini admin_users string is checked - only admin users are permitted to run this tool. +# + +import sys +import shutil +import subprocess +import os +import time +import tempfile +import optparse +import tarfile +import re +import shutil +import math + +progname = os.path.split(sys.argv[0])[1] +myversion = 'V000.2 June 2012' +verbose = False +debug = False +toolFactoryURL = 'https://bitbucket.org/fubar/galaxytoolfactory' + +def timenow(): + """return current time as a string + """ + return time.strftime('%d/%m/%Y %H:%M:%S', time.localtime(time.time())) + +html_escape_table = { + "&": "&", + ">": ">", + "<": "<", + "$": "\$" + } + +def html_escape(text): + """Produce entities within text.""" + return "".join(html_escape_table.get(c,c) for c in text) + +def cmd_exists(cmd): + return subprocess.call("type " + cmd, shell=True, + stdout=subprocess.PIPE, stderr=subprocess.PIPE) == 0 + + +class ScriptRunner: + """class is a wrapper for an arbitrary script + """ + + def __init__(self,opts=None,treatbashSpecial=True): + """ + cleanup inputs, setup some outputs + + """ + self.useGM = cmd_exists('gm') + self.useIM = cmd_exists('convert') + self.useGS = cmd_exists('gs') + self.treatbashSpecial = treatbashSpecial + if opts.output_dir: # simplify for the tool tarball + os.chdir(opts.output_dir) + self.thumbformat = 'jpg' + self.opts = opts + self.toolname = re.sub('[^a-zA-Z0-9_]+', '', opts.tool_name) # a sanitizer now does this but.. + self.toolid = self.toolname + self.myname = sys.argv[0] # get our name because we write ourselves out as a tool later + self.pyfile = self.myname # crude but efficient - the cruft won't hurt much + self.xmlfile = '%s.xml' % self.toolname + s = open(self.opts.script_path,'r').readlines() + s = [x.rstrip() for x in s] # remove pesky dos line endings if needed + self.script = '\n'.join(s) + fhandle,self.sfile = tempfile.mkstemp(prefix=self.toolname,suffix=".%s" % (opts.interpreter)) + tscript = open(self.sfile,'w') # use self.sfile as script source for Popen + tscript.write(self.script) + tscript.close() + self.indentedScript = '\n'.join([' %s' % x for x in s]) # for restructured text in help + self.escapedScript = '\n'.join([html_escape(x) for x in s]) + if opts.output_dir: # may not want these complexities + self.tlog = os.path.join(self.opts.output_dir,"%s_runner.log" % self.toolname) + art = '%s.%s' % (self.toolname,opts.interpreter) + artpath = os.path.join(self.opts.output_dir,art) # need full path + artifact = open(artpath,'w') # use self.sfile as script source for Popen + artifact.write(self.script) + artifact.close() + self.cl = [] + self.html = [] + a = self.cl.append + a(opts.interpreter) + if self.treatbashSpecial and opts.interpreter in ['bash','sh']: + a(self.sfile) + else: + a('-') # stdin + a(opts.input_tab) + a(opts.output_tab) + self.outFormats = 'tabular' # TODO make this an option at tool generation time + self.inputFormats = 'tabular' # TODO make this an option at tool generation time + self.test1Input = '%s_test1_input.xls' % self.toolname + self.test1Output = '%s_test1_output.xls' % self.toolname + self.test1HTML = '%s_test1_output.html' % self.toolname + + def makeXML(self): + """ + Create a Galaxy xml tool wrapper for the new script as a string to write out + fixme - use templating or something less fugly than this example of what we produce + + + a tabular file + + reverse.py --script_path "$runMe" --interpreter "python" + --tool_name "reverse" --input_tab "$input1" --output_tab "$tab_file" + + + + + + + + + + + +**What it Does** + +Reverse the columns in a tabular file + + + + + +# reverse order of columns in a tabular file +import sys +inp = sys.argv[1] +outp = sys.argv[2] +i = open(inp,'r') +o = open(outp,'w') +for row in i: + rs = row.rstrip().split('\t') + rs.reverse() + o.write('\t'.join(rs)) + o.write('\n') +i.close() +o.close() + + + + + + + """ + newXML=""" + %(tooldesc)s + %(command)s + + %(inputs)s + + + %(outputs)s + + + + %(script)s + + + %(tooltests)s + + %(help)s + + """ # needs a dict with toolname, toolid, interpreter, scriptname, command, inputs as a multi line string ready to write, outputs ditto, help ditto + + newCommand=""" + %(toolname)s.py --script_path "$runMe" --interpreter "%(interpreter)s" + --tool_name "%(toolname)s" %(command_inputs)s %(command_outputs)s + """ # may NOT be an input or htmlout + tooltestsTabOnly = """ + + + + + """ + tooltestsHTMLOnly = """ + + + + + """ + tooltestsBoth = """ + + + + + + """ + xdict = {} + xdict['tool_version'] = self.opts.tool_version + xdict['test1Input'] = self.test1Input + xdict['test1HTML'] = self.test1HTML + xdict['test1Output'] = self.test1Output + if self.opts.make_HTML and self.opts.output_tab <> 'None': + xdict['tooltests'] = tooltestsBoth % xdict + elif self.opts.make_HTML: + xdict['tooltests'] = tooltestsHTMLOnly % xdict + else: + xdict['tooltests'] = tooltestsTabOnly % xdict + xdict['script'] = self.escapedScript + # configfile is least painful way to embed script to avoid external dependencies + # but requires escaping of <, > and $ to avoid Mako parsing + if self.opts.help_text: + xdict['help'] = open(self.opts.help_text,'r').read() + else: + xdict['help'] = 'Please ask the tool author for help as none was supplied at tool generation' + coda = ['**Script**','Pressing execute will run the following code over your input file and generate some outputs in your history::'] + coda.append(self.indentedScript) + coda.append('**Attribution** This Galaxy tool was created by %s at %s\nusing the Galaxy Tool Factory.' % (self.opts.user_email,timenow())) + coda.append('See %s for details of that project' % (toolFactoryURL)) + coda.append('Please cite: Creating re-usable tools from scripts: The Galaxy Tool Factory. Ross Lazarus; Antony Kaspi; Mark Ziemann; The Galaxy Team. ') + coda.append('Bioinformatics 2012; doi: 10.1093/bioinformatics/bts573') + xdict['help'] = '%s\n%s' % (xdict['help'],'\n'.join(coda)) + if self.opts.tool_desc: + xdict['tooldesc'] = '%s' % self.opts.tool_desc + else: + xdict['tooldesc'] = '' + xdict['command_outputs'] = '' + xdict['outputs'] = '' + if self.opts.input_tab <> 'None': + xdict['command_inputs'] = '--input_tab "$input1" ' # the space may matter a lot if we append something + xdict['inputs'] = ' \n' % self.inputFormats + else: + xdict['command_inputs'] = '' # assume no input - eg a random data generator + xdict['inputs'] = '' + xdict['inputs'] += ' \n' % self.toolname + xdict['toolname'] = self.toolname + xdict['toolid'] = self.toolid + xdict['interpreter'] = self.opts.interpreter + xdict['scriptname'] = self.sfile + if self.opts.make_HTML: + xdict['command_outputs'] += ' --output_dir "$html_file.files_path" --output_html "$html_file" --make_HTML "yes" ' + xdict['outputs'] += ' \n' + if self.opts.output_tab <> 'None': + xdict['command_outputs'] += ' --output_tab "$tab_file"' + xdict['outputs'] += ' \n' % self.outFormats + xdict['command'] = newCommand % xdict + xmls = newXML % xdict + xf = open(self.xmlfile,'w') + xf.write(xmls) + xf.write('\n') + xf.close() + # ready for the tarball + + + def makeTooltar(self): + """ + a tool is a gz tarball with eg + /toolname/tool.xml /toolname/tool.py /toolname/test-data/test1_in.foo ... + """ + retval = self.run() + if retval: + print >> sys.stderr,'## Run failed. Cannot build yet. Please fix and retry' + sys.exit(1) + self.makeXML() + tdir = self.toolname + os.mkdir(tdir) + if self.opts.input_tab <> 'None': # no reproducible test otherwise? TODO: maybe.. + testdir = os.path.join(tdir,'test-data') + os.mkdir(testdir) # make tests directory + shutil.copyfile(self.opts.input_tab,os.path.join(testdir,self.test1Input)) + if self.opts.output_tab <> 'None': + shutil.copyfile(self.opts.output_tab,os.path.join(testdir,self.test1Output)) + if self.opts.make_HTML: + shutil.copyfile(self.opts.output_html,os.path.join(testdir,self.test1HTML)) + if self.opts.output_dir: + shutil.copyfile(self.tlog,os.path.join(testdir,'test1_out.log')) + op = '%s.py' % self.toolname # new name + outpiname = os.path.join(tdir,op) # path for the tool tarball + pyin = os.path.basename(self.pyfile) # our name - we rewrite ourselves (TM) + notes = ['# %s - a self annotated version of %s generated by running %s\n' % (op,pyin,pyin),] + notes.append('# to make a new Galaxy tool called %s\n' % self.toolname) + notes.append('# User %s at %s\n' % (self.opts.user_email,timenow())) + pi = open(self.pyfile,'r').readlines() # our code becomes new tool wrapper (!) - first Galaxy worm + notes += pi + outpi = open(outpiname,'w') + outpi.write(''.join(notes)) + outpi.write('\n') + outpi.close() + stname = os.path.join(tdir,self.sfile) + if not os.path.exists(stname): + shutil.copyfile(self.sfile, stname) + xtname = os.path.join(tdir,self.xmlfile) + if not os.path.exists(xtname): + shutil.copyfile(self.xmlfile,xtname) + tarpath = "%s.gz" % self.toolname + tar = tarfile.open(tarpath, "w:gz") + tar.add(tdir,arcname=self.toolname) + tar.close() + shutil.copyfile(tarpath,self.opts.new_tool) + shutil.rmtree(tdir) + ## TODO: replace with optional direct upload to local toolshed? + return retval + + + def compressPDF(self,inpdf=None,thumbformat='png'): + """need absolute path to pdf + """ + assert os.path.isfile(inpdf), "## Input %s supplied to %s compressPDF not found" % (inpdf,self.myName) + hf,hlog = tempfile.mkstemp(suffix="%s.log" % self.toolname) + sto = open(hlog,'w') + outpdf = '%s_compressed' % inpdf + cl = ["gs", "-sDEVICE=pdfwrite", "-dNOPAUSE", "-dBATCH","-dPDFSETTINGS=/printer", "-sOutputFile=%s" % outpdf,inpdf] + x = subprocess.Popen(cl,stdout=sto,stderr=sto,cwd=self.opts.output_dir) + retval1 = x.wait() + if retval1 == 0: + os.unlink(inpdf) + shutil.move(outpdf,inpdf) + outpng = '%s.%s' % (os.path.splitext(inpdf)[0],thumbformat) + if self.useGM: + cl2 = ['gm convert', inpdf, outpng] + else: # assume imagemagick + cl2 = ['convert', inpdf, outpng] + x = subprocess.Popen(cl2,stdout=sto,stderr=sto,cwd=self.opts.output_dir) + retval2 = x.wait() + sto.close() + retval = retval1 or retval2 + return retval + + + def getfSize(self,fpath,outpath): + """ + format a nice file size string + """ + size = '' + fp = os.path.join(outpath,fpath) + if os.path.isfile(fp): + size = '0 B' + n = float(os.path.getsize(fp)) + if n > 2**20: + size = '%1.1f MB' % (n/2**20) + elif n > 2**10: + size = '%1.1f KB' % (n/2**10) + elif n > 0: + size = '%d B' % (int(n)) + return size + + def makeHtml(self): + """ Create an HTML file content to list all the artifacts found in the output_dir + """ + + galhtmlprefix = """ + + + + + + + +
+ """ + galhtmlattr = """
This tool (%s) was generated by the Galaxy Tool Factory

""" + galhtmlpostfix = """
\n""" + + flist = os.listdir(self.opts.output_dir) + flist = [x for x in flist if x <> 'Rplots.pdf'] + flist.sort() + html = [] + html.append(galhtmlprefix % progname) + html.append('
Galaxy Tool "%s" run at %s

' % (self.toolname,timenow())) + fhtml = [] + if len(flist) > 0: + logfiles = [x for x in flist if x.lower().endswith('.log')] # log file names determine sections + logfiles.sort() + logfiles = [x for x in logfiles if os.path.abspath(x) <> os.path.abspath(self.tlog)] + logfiles.append(os.path.abspath(self.tlog)) # make it the last one + pdflist = [] + npdf = len([x for x in flist if os.path.splitext(x)[-1].lower() == '.pdf']) + for rownum,fname in enumerate(flist): + dname,e = os.path.splitext(fname) + sfsize = self.getfSize(fname,self.opts.output_dir) + if e.lower() == '.pdf' : # compress and make a thumbnail + thumb = '%s.%s' % (dname,self.thumbformat) + pdff = os.path.join(self.opts.output_dir,fname) + retval = self.compressPDF(inpdf=pdff,thumbformat=self.thumbformat) + if retval == 0: + pdflist.append((fname,thumb)) + if (rownum+1) % 2 == 0: + fhtml.append('%s%s' % (fname,fname,sfsize)) + else: + fhtml.append('%s%s' % (fname,fname,sfsize)) + for logfname in logfiles: # expect at least tlog - if more + if os.path.abspath(logfname) == os.path.abspath(self.tlog): # handled later + sectionname = 'All tool run' + if (len(logfiles) > 1): + sectionname = 'Other' + ourpdfs = pdflist + else: + realname = os.path.basename(logfname) + sectionname = os.path.splitext(realname)[0].split('_')[0] # break in case _ added to log + ourpdfs = [x for x in pdflist if os.path.basename(x[0]).split('_')[0] == sectionname] + pdflist = [x for x in pdflist if os.path.basename(x[0]).split('_')[0] <> sectionname] # remove + nacross = 1 + npdf = len(ourpdfs) + + if npdf > 0: + nacross = math.sqrt(npdf) ## int(round(math.log(npdf,2))) + if int(nacross)**2 != npdf: + nacross += 1 + nacross = int(nacross) + width = min(400,int(1200/nacross)) + html.append('
%s images and outputs
' % sectionname) + html.append('(Click on a thumbnail image to download the corresponding original PDF image)
') + ntogo = nacross # counter for table row padding with empty cells + html.append('
\n') + for i,paths in enumerate(ourpdfs): + fname,thumb = paths + s= """\n""" % (fname,thumb,fname,width,fname) + if ((i+1) % nacross == 0): + s += '\n' + ntogo = 0 + if i < (npdf - 1): # more to come + s += '' + ntogo = nacross + else: + ntogo -= 1 + html.append(s) + if html[-1].strip().endswith(''): + html.append('
Image called %s
\n') + else: + if ntogo > 0: # pad + html.append(' '*ntogo) + html.append('\n') + logt = open(logfname,'r').readlines() + logtext = [x for x in logt if x.strip() > ''] + html.append('
%s log output
' % sectionname) + if len(logtext) > 1: + html.append('\n
\n')
+                    html += logtext
+                    html.append('\n
\n') + else: + html.append('%s is empty
' % logfname) + if len(fhtml) > 0: + fhtml.insert(0,'
\n') + fhtml.append('
Output File Name (click to view)Size

') + html.append('
All output files available for downloading
\n') + html += fhtml # add all non-pdf files to the end of the display + else: + html.append('
### Error - %s returned no files - please confirm that parameters are sane
' % self.opts.interpreter) + html.append(galhtmlpostfix) + htmlf = file(self.opts.output_html,'w') + htmlf.write('\n'.join(html)) + htmlf.write('\n') + htmlf.close() + self.html = html + + + def run(self): + """ + scripts must be small enough not to fill the pipe! + """ + if self.treatbashSpecial and self.opts.interpreter in ['bash','sh']: + retval = self.runBash() + else: + if self.opts.output_dir: + sto = open(self.tlog,'w') + sto.write('## Toolfactory generated command line = %s\n' % ' '.join(self.cl)) + sto.flush() + p = subprocess.Popen(self.cl,shell=False,stdout=sto,stderr=sto,stdin=subprocess.PIPE,cwd=self.opts.output_dir) + else: + p = subprocess.Popen(self.cl,shell=False,stdin=subprocess.PIPE) + p.stdin.write(self.script) + p.stdin.close() + retval = p.wait() + if self.opts.output_dir: + sto.close() + if self.opts.make_HTML: + self.makeHtml() + return retval + + def runBash(self): + """ + cannot use - for bash so use self.sfile + """ + if self.opts.output_dir: + s = '## Toolfactory generated command line = %s\n' % ' '.join(self.cl) + sto = open(self.tlog,'w') + sto.write(s) + sto.flush() + p = subprocess.Popen(self.cl,shell=False,stdout=sto,stderr=sto,cwd=self.opts.output_dir) + else: + p = subprocess.Popen(self.cl,shell=False) + retval = p.wait() + if self.opts.output_dir: + sto.close() + if self.opts.make_HTML: + self.makeHtml() + return retval + + +def main(): + u = """ + This is a Galaxy wrapper. It expects to be called by a special purpose tool.xml as: + rgBaseScriptWrapper.py --script_path "$scriptPath" --tool_name "foo" --interpreter "Rscript" + + """ + op = optparse.OptionParser() + a = op.add_option + a('--script_path',default=None) + a('--tool_name',default=None) + a('--interpreter',default=None) + a('--output_dir',default=None) + a('--output_html',default=None) + a('--input_tab',default="None") + a('--output_tab',default="None") + a('--user_email',default='Unknown') + a('--bad_user',default=None) + a('--make_Tool',default=None) + a('--make_HTML',default=None) + a('--help_text',default=None) + a('--tool_desc',default=None) + a('--new_tool',default=None) + a('--tool_version',default=None) + opts, args = op.parse_args() + assert not opts.bad_user,'UNAUTHORISED: %s is NOT authorized to use this tool until Galaxy admin adds %s to admin_users in universe_wsgi.ini' % (opts.bad_user,opts.bad_user) + assert opts.tool_name,'## Tool Factory expects a tool name - eg --tool_name=DESeq' + assert opts.interpreter,'## Tool Factory wrapper expects an interpreter - eg --interpreter=Rscript' + assert os.path.isfile(opts.script_path),'## Tool Factory wrapper expects a script path - eg --script_path=foo.R' + if opts.output_dir: + try: + os.makedirs(opts.output_dir) + except: + pass + r = ScriptRunner(opts) + if opts.make_Tool: + retcode = r.makeTooltar() + else: + retcode = r.run() + os.unlink(r.sfile) + if retcode: + sys.exit(retcode) # indicate failure to job runner + + +if __name__ == "__main__": + main() + + diff -r 643164729057 -r 91e77df18a7f rgedgeR/rgedgeRpaired.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/rgedgeR/rgedgeRpaired.xml Fri Jul 26 22:04:58 2013 -0400 @@ -0,0 +1,918 @@ + + models using BioConductor packages + + biocbasics + package_r3 + + + + rgToolFactory.py --script_path "$runme" --interpreter "Rscript" --tool_name "DifferentialCounts" + --output_dir "$html_file.files_path" --output_html "$html_file" --output_tab "$outtab" --make_HTML "yes" + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + nsamp) { + dm =dm[1:nsamp,] + #sub = paste('Showing',nsamp,'contigs ranked for evidence for differential abundance out of',nprobes,'total') + } + newcolnames = substr(colnames(dm),1,20) + colnames(dm) = newcolnames + pdf(outpdfname) + heatmap.2(dm,main=myTitle,ColSideColors=pcols,col=topo.colors(100),dendrogram="col",key=T,density.info='none', + Rowv=F,scale='row',trace='none',margins=c(8,8),cexRow=0.4,cexCol=0.5) + dev.off() +} + +hmap = function(cmat,nmeans=4,outpdfname="heatMap.pdf",nsamp=250,TName='Treatment',group=NA,myTitle="Title goes here") +{ + # for 2 groups only was + #col.map = function(g) {if (g==TName) "#FF0000" else "#0000FF"} + #pcols = unlist(lapply(group,col.map)) + gu = unique(group) + colours = rainbow(length(gu),start=0.3,end=0.6) + pcols = colours[match(group,gu)] + nrows = nrow(cmat) + mtitle = paste(myTitle,'Heatmap: n contigs =',nrows) + if (nrows > nsamp) { + cmat = cmat[c(1:nsamp),] + mtitle = paste('Heatmap: Top ',nsamp,' DE contigs (of ',nrows,')',sep='') + } + newcolnames = substr(colnames(cmat),1,20) + colnames(cmat) = newcolnames + pdf(outpdfname) + heatmap(cmat,scale='row',main=mtitle,cexRow=0.3,cexCol=0.4,Rowv=NA,ColSideColors=pcols) + dev.off() +} + +qqPlot = function(descr='qqplot',pvector, outpdf='qqplot.pdf',...) +# stolen from https://gist.github.com/703512 +{ + o = -log10(sort(pvector,decreasing=F)) + e = -log10( 1:length(o)/length(o) ) + o[o==-Inf] = reallysmall + o[o==Inf] = reallybig + maint = descr + pdf(outpdf) + plot(e,o,pch=19,cex=1, main=maint, ..., + xlab=expression(Expected~~-log[10](italic(p))), + ylab=expression(Observed~~-log[10](italic(p))), + xlim=c(0,max(e)), ylim=c(0,max(o))) + lines(e,e,col="red") + grid(col = "lightgray", lty = "dotted") + dev.off() +} + +smearPlot = function(DGEList,deTags, outSmear, outMain) + { + pdf(outSmear) + plotSmear(DGEList,de.tags=deTags,main=outMain) + grid(col="lightgray", lty="dotted") + dev.off() + } + +boxPlot = function(rawrs,cleanrs,maint,myTitle,pdfname) +{ # + nc = ncol(rawrs) + for (i in c(1:nc)) {rawrs[(rawrs[,i] < 0),i] = NA} + fullnames = colnames(rawrs) + newcolnames = substr(colnames(rawrs),1,20) + colnames(rawrs) = newcolnames + newcolnames = substr(colnames(cleanrs),1,20) + colnames(cleanrs) = newcolnames + defpar = par(no.readonly=T) + print.noquote('raw contig counts by sample:') + print.noquote(summary(rawrs)) + print.noquote('normalised contig counts by sample:') + print.noquote(summary(cleanrs)) + pdf(pdfname) + par(mfrow=c(1,2)) + boxplot(rawrs,varwidth=T,notch=T,ylab='log contig count',col="maroon",las=3,cex.axis=0.35,main=paste('Raw:',maint)) + grid(col="lightgray",lty="dotted") + boxplot(cleanrs,varwidth=T,notch=T,ylab='log contig count',col="maroon",las=3,cex.axis=0.35,main=paste('After ',maint)) + grid(col="lightgray",lty="dotted") + dev.off() + pdfname = "sample_counts_histogram.pdf" + nc = ncol(rawrs) + print.noquote(paste('Using ncol rawrs=',nc)) + ncroot = round(sqrt(nc)) + if (ncroot*ncroot < nc) { ncroot = ncroot + 1 } + m = c() + for (i in c(1:nc)) { + rhist = hist(rawrs[,i],breaks=100,plot=F) + m = append(m,max(rhist\$counts)) + } + ymax = max(m) + pdf(pdfname) + par(mfrow=c(ncroot,ncroot)) + for (i in c(1:nc)) { + hist(rawrs[,i], main=paste("Contig logcount",i), xlab='log raw count', col="maroon", + breaks=100,sub=fullnames[i],cex=0.8,ylim=c(0,ymax)) + } + dev.off() + par(defpar) + +} + +cumPlot = function(rawrs,cleanrs,maint,myTitle) +{ # updated to use ecdf + pdfname = "Filtering_rowsum_bar_charts.pdf" + defpar = par(no.readonly=T) + lrs = log(rawrs,10) + lim = max(lrs) + pdf(pdfname) + par(mfrow=c(2,1)) + hist(lrs,breaks=100,main=paste('Before:',maint),xlab="# Reads (log)", + ylab="Count",col="maroon",sub=myTitle, xlim=c(0,lim),las=1) + grid(col="lightgray", lty="dotted") + lrs = log(cleanrs,10) + hist(lrs,breaks=100,main=paste('After:',maint),xlab="# Reads (log)", + ylab="Count",col="maroon",sub=myTitle,xlim=c(0,lim),las=1) + grid(col="lightgray", lty="dotted") + dev.off() + par(defpar) +} + +cumPlot1 = function(rawrs,cleanrs,maint,myTitle) +{ # updated to use ecdf + pdfname = paste(gsub(" ","", myTitle , fixed=TRUE),"RowsumCum.pdf",sep='_') + pdf(pdfname) + par(mfrow=c(2,1)) + lastx = max(rawrs) + rawe = knots(ecdf(rawrs)) + cleane = knots(ecdf(cleanrs)) + cy = 1:length(cleane)/length(cleane) + ry = 1:length(rawe)/length(rawe) + plot(rawe,ry,type='l',main=paste('Before',maint),xlab="Log Contig Total Reads", + ylab="Cumulative proportion",col="maroon",log='x',xlim=c(1,lastx),sub=myTitle) + grid(col="blue") + plot(cleane,cy,type='l',main=paste('After',maint),xlab="Log Contig Total Reads", + ylab="Cumulative proportion",col="maroon",log='x',xlim=c(1,lastx),sub=myTitle) + grid(col="blue") + dev.off() +} + + + +doGSEA = function(y=NULL,design=NULL,histgmt="", + bigmt="/data/genomes/gsea/3.1/Abetterchoice_nocgp_c2_c3_c5_symbols_all.gmt", + ntest=0, myTitle="myTitle", outfname="GSEA.xls", minnin=5, maxnin=2000,fdrthresh=0.05,fdrtype="BH") +{ + sink('Camera.log') + genesets = c() + if (bigmt > "") + { + bigenesets = readLines(bigmt) + genesets = bigenesets + } + if (histgmt > "") + { + hgenesets = readLines(histgmt) + if (bigmt > "") { + genesets = rbind(genesets,hgenesets) + } else { + genesets = hgenesets + } # use only history if no bi + } + print.noquote(paste("@@@read",length(genesets), 'genesets from',histgmt,bigmt)) + genesets = strsplit(genesets,'\t') # tabular. genesetid\tURLorwhatever\tgene_1\t..\tgene_n + outf = outfname + head=paste(myTitle,'edgeR GSEA') + write(head,file=outfname,append=F) + ntest=length(genesets) + urownames = toupper(rownames(y)) + upcam = c() + downcam = c() + for (i in 1:ntest) { + gs = unlist(genesets[i]) + g = gs[1] # geneset_id + u = gs[2] + if (u > "") { u = paste("",u,"",sep="") } + glist = gs[3:length(gs)] # member gene symbols + glist = toupper(glist) + inglist = urownames %in% glist + nin = sum(inglist) + if ((nin > minnin) && (nin < maxnin)) { + ### print(paste('@@found',sum(inglist),'genes in glist')) + camres = camera(y=y,index=inglist,design=design) + if (! is.null(camres)) { + rownames(camres) = g # gene set name + camres = cbind(GeneSet=g,URL=u,camres) + if (camres\$Direction == "Up") + { + upcam = rbind(upcam,camres) } else { + downcam = rbind(downcam,camres) + } + } + } + } + uscam = upcam[order(upcam\$PValue),] + unadjp = uscam\$PValue + uscam\$adjPValue = p.adjust(unadjp,method=fdrtype) + nup = max(10,sum((uscam\$adjPValue < fdrthresh))) + dscam = downcam[order(downcam\$PValue),] + unadjp = dscam\$PValue + dscam\$adjPValue = p.adjust(unadjp,method=fdrtype) + ndown = max(10,sum((dscam\$adjPValue < fdrthresh))) + write.table(uscam,file=paste('camera_up',outfname,sep='_'),quote=F,sep='\t',row.names=F) + write.table(dscam,file=paste('camera_down',outfname,sep='_'),quote=F,sep='\t',row.names=F) + print.noquote(paste('@@@@@ Camera up top',nup,'gene sets:')) + write.table(head(uscam,nup),file="",quote=F,sep='\t',row.names=F) + print.noquote(paste('@@@@@ Camera down top',ndown,'gene sets:')) + write.table(head(dscam,ndown),file="",quote=F,sep='\t',row.names=F) + sink() +} + + + +edgeIt = function (Count_Matrix,group,outputfilename,fdrtype='fdr',priordf=5, + fdrthresh=0.05,outputdir='.', myTitle='Differential Counts',libSize=c(),useNDF=F, + filterquantile=0.2, subjects=c(),mydesign=NULL, + doDESeq2=T,doVoom=T,doCamera=T,doedgeR=T,org='hg19', + histgmt="", bigmt="/data/genomes/gsea/3.1/Abetterchoice_nocgp_c2_c3_c5_symbols_all.gmt", + doCook=F,DESeq_fitType="parameteric") +{ + # Error handling + if (length(unique(group))!=2){ + print("Number of conditions identified in experiment does not equal 2") + q() + } + require(edgeR) + options(width = 512) + mt = paste(unlist(strsplit(myTitle,'_')),collapse=" ") + allN = nrow(Count_Matrix) + nscut = round(ncol(Count_Matrix)/2) + colTotmillionreads = colSums(Count_Matrix)/1e6 + counts.dataframe = as.data.frame(c()) + rawrs = rowSums(Count_Matrix) + nonzerod = Count_Matrix[(rawrs > 0),] # remove all zero count genes + nzN = nrow(nonzerod) + nzrs = rowSums(nonzerod) + zN = allN - nzN + print('# Quantiles for non-zero row counts:',quote=F) + print(quantile(nzrs,probs=seq(0,1,0.1)),quote=F) + if (useNDF == "T") + { + gt1rpin3 = rowSums(Count_Matrix/expandAsMatrix(colTotmillionreads,dim(Count_Matrix)) >= 1) >= nscut + lo = colSums(Count_Matrix[!gt1rpin3,]) + workCM = Count_Matrix[gt1rpin3,] + cleanrs = rowSums(workCM) + cleanN = length(cleanrs) + meth = paste( "After removing",length(lo),"contigs with fewer than ",nscut," sample read counts >= 1 per million, there are",sep="") + print(paste("Read",allN,"contigs. Removed",zN,"contigs with no reads.",meth,cleanN,"contigs"),quote=F) + maint = paste('Filter >=1/million reads in >=',nscut,'samples') + } else { + useme = (nzrs > quantile(nzrs,filterquantile)) + workCM = nonzerod[useme,] + lo = colSums(nonzerod[!useme,]) + cleanrs = rowSums(workCM) + cleanN = length(cleanrs) + meth = paste("After filtering at count quantile =",filterquantile,", there are",sep="") + print(paste('Read',allN,"contigs. Removed",zN,"with no reads.",meth,cleanN,"contigs"),quote=F) + maint = paste('Filter below',filterquantile,'quantile') + } + cumPlot(rawrs=rawrs,cleanrs=cleanrs,maint=maint,myTitle=myTitle) + allgenes = rownames(workCM) + reg = "^chr([0-9]+):([0-9]+)-([0-9]+)" + genecards=" 0.8) # is ucsc style string + { + print("@@ using ucsc substitution for urls") + contigurls = paste0(ucsc,"&position=chr",testreg[,2],":",testreg[,3],"-",testreg[,4],"\'>",allgenes,"") + } else { + print("@@ using genecards substitution for urls") + contigurls = paste0(genecards,allgenes,"\'>",allgenes,"") + } + print.noquote("# urls") + print.noquote(head(contigurls)) + print(paste("# Total low count contigs per sample = ",paste(lo,collapse=',')),quote=F) + cmrowsums = rowSums(workCM) + TName=unique(group)[1] + CName=unique(group)[2] + if (is.null(mydesign)) { + if (length(subjects) == 0) + { + mydesign = model.matrix(~group) + } + else { + subjf = factor(subjects) + mydesign = model.matrix(~subjf+group) # we block on subject so make group last to simplify finding it + } + } + print.noquote(paste('Using samples:',paste(colnames(workCM),collapse=','))) + print.noquote('Using design matrix:') + print.noquote(mydesign) + if (doedgeR) { + sink('edgeR.log') + #### Setup DGEList object + DGEList = DGEList(counts=workCM, group = group) + DGEList = calcNormFactors(DGEList) + + DGEList = estimateGLMCommonDisp(DGEList,mydesign) + comdisp = DGEList\$common.dispersion + DGEList = estimateGLMTrendedDisp(DGEList,mydesign) + if (edgeR_priordf > 0) { + print.noquote(paste("prior.df =",edgeR_priordf)) + DGEList = estimateGLMTagwiseDisp(DGEList,mydesign,prior.df = edgeR_priordf) + } else { + DGEList = estimateGLMTagwiseDisp(DGEList,mydesign) + } + DGLM = glmFit(DGEList,design=mydesign) + DE = glmLRT(DGLM,coef=ncol(DGLM\$design)) # always last one - subject is first if needed + efflib = DGEList\$samples\$lib.size*DGEList\$samples\$norm.factors + normData = (1e+06*DGEList\$counts/efflib) + uoutput = cbind( + Name=as.character(rownames(DGEList\$counts)), + DE\$table, + adj.p.value=p.adjust(DE\$table\$PValue, method=fdrtype), + Dispersion=DGEList\$tagwise.dispersion,totreads=cmrowsums,normData, + DGEList\$counts + ) + soutput = uoutput[order(DE\$table\$PValue),] # sorted into p value order - for quick toptable + goodness = gof(DGLM, pcutoff=fdrthresh) + if (sum(goodness\$outlier) > 0) { + print.noquote('GLM outliers:') + print(paste(rownames(DGLM)[(goodness\$outlier)],collapse=','),quote=F) + } else { + print('No GLM fit outlier genes found\n') + } + z = limma::zscoreGamma(goodness\$gof.statistic, shape=goodness\$df/2, scale=2) + pdf("edgeR_GoodnessofFit.pdf") + qq = qqnorm(z, panel.first=grid(), main="tagwise dispersion") + abline(0,1,lwd=3) + points(qq\$x[goodness\$outlier],qq\$y[goodness\$outlier], pch=16, col="maroon") + dev.off() + estpriorn = getPriorN(DGEList) + print(paste("Common Dispersion =",comdisp,"CV = ",sqrt(comdisp),"getPriorN = ",estpriorn),quote=F) + efflib = DGEList\$samples\$lib.size*DGEList\$samples\$norm.factors + normData = (1e+06*DGEList\$counts/efflib) + uniqueg = unique(group) + #### Plot MDS + sample_colors = match(group,levels(group)) + sampleTypes = levels(factor(group)) + print.noquote(sampleTypes) + pdf("edgeR_MDSplot.pdf") + plotMDS.DGEList(DGEList,main=paste("edgeR MDS for",myTitle),cex=0.5,col=sample_colors,pch=sample_colors) + legend(x="topleft", legend = sampleTypes,col=c(1:length(sampleTypes)), pch=19) + grid(col="blue") + dev.off() + colnames(normData) = paste( colnames(normData),'N',sep="_") + print(paste('Raw sample read totals',paste(colSums(nonzerod,na.rm=T),collapse=','))) + nzd = data.frame(log(nonzerod + 1e-2,10)) + boxPlot(rawrs=nzd,cleanrs=log(normData,10),maint='TMM Normalisation',myTitle=myTitle,pdfname="edgeR_raw_norm_counts_box.pdf") + write.table(soutput,outputfilename, quote=FALSE, sep="\t",row.names=F) + tt = cbind( + Name=as.character(rownames(DGEList\$counts)), + DE\$table, + adj.p.value=p.adjust(DE\$table\$PValue, method=fdrtype), + Dispersion=DGEList\$tagwise.dispersion,totreads=cmrowsums + ) + print.noquote("# edgeR Top tags\n") + tt = cbind(tt,URL=contigurls) # add to end so table isn't laid out strangely + tt = tt[order(DE\$table\$PValue),] + print.noquote(tt[1:50,]) + deTags = rownames(uoutput[uoutput\$adj.p.value < fdrthresh,]) + nsig = length(deTags) + print(paste('#',nsig,'tags significant at adj p=',fdrthresh),quote=F) + deColours = ifelse(deTags,'red','black') + pdf("edgeR_BCV_vs_abundance.pdf") + plotBCV(DGEList, cex=0.3, main="Biological CV vs abundance") + dev.off() + dg = DGEList[order(DE\$table\$PValue),] + #normData = (1e+06 * dg\$counts/expandAsMatrix(dg\$samples\$lib.size, dim(dg))) + efflib = dg\$samples\$lib.size*dg\$samples\$norm.factors + normData = (1e+06*dg\$counts/efflib) + outpdfname="edgeR_heatmap.pdf" + hmap2(normData,nsamp=100,TName=TName,group=group,outpdfname=outpdfname,myTitle=myTitle) + outSmear = "edgeR_smearplot.pdf" + outMain = paste("Smear Plot for ",TName,' Vs ',CName,' (FDR@',fdrthresh,' N = ',nsig,')',sep='') + smearPlot(DGEList=DGEList,deTags=deTags, outSmear=outSmear, outMain = outMain) + qqPlot(descr=paste(myTitle,'edgeR QQ plot'),pvector=DE\$table\$PValue,outpdf='edgeR_qqplot.pdf') + norm.factor = DGEList\$samples\$norm.factors + topresults.edgeR = soutput[which(soutput\$adj.p.value < fdrthresh), ] + edgeRcountsindex = which(allgenes %in% rownames(topresults.edgeR)) + edgeRcounts = rep(0, length(allgenes)) + edgeRcounts[edgeRcountsindex] = 1 # Create venn diagram of hits + sink() + } ### doedgeR + if (doDESeq2 == T) + { + sink("DESeq2.log") + # DESeq2 + require('DESeq2') + library('RColorBrewer') + pdata = data.frame(Name=colnames(workCM),Rx=group,row.names=colnames(workCM)) + deSEQds = DESeqDataSetFromMatrix(countData = workCM, colData = pdata, design = formula(~ Rx)) + #DESeq2 = DESeq(deSEQds,fitType='local',pAdjustMethod=fdrtype) + #rDESeq = results(DESeq2) + #newCountDataSet(workCM, group) + deSeqDatsizefac = estimateSizeFactors(deSEQds) + deSeqDatdisp = estimateDispersions(deSeqDatsizefac,fitType=DESeq_fitType) + resDESeq = nbinomWaldTest(deSeqDatdisp, pAdjustMethod=fdrtype) + rDESeq = as.data.frame(results(resDESeq)) + rDESeq = cbind(Contig=rownames(workCM),rDESeq,NReads=cmrowsums,URL=contigurls) + srDESeq = rDESeq[order(rDESeq\$pvalue),] + qqPlot(descr=paste(myTitle,'DESeq2 qqplot'),pvector=rDESeq\$pvalue,outpdf='DESeq2_qqplot.pdf') + cat("# DESeq top 50\n") + print.noquote(srDESeq[1:50,]) + write.table(srDESeq,paste(mt,'DESeq2_TopTable.xls',sep='_'), quote=FALSE, sep="\t",row.names=F) + topresults.DESeq = rDESeq[which(rDESeq\$padj < fdrthresh), ] + DESeqcountsindex = which(allgenes %in% rownames(topresults.DESeq)) + DESeqcounts = rep(0, length(allgenes)) + DESeqcounts[DESeqcountsindex] = 1 + pdf("DESeq2_dispersion_estimates.pdf") + plotDispEsts(resDESeq) + dev.off() + ysmall = abs(min(rDESeq\$log2FoldChange)) + ybig = abs(max(rDESeq\$log2FoldChange)) + ylimit = min(4,ysmall,ybig) + pdf("DESeq2_MA_plot.pdf") + plotMA(resDESeq,main=paste(myTitle,"DESeq2 MA plot"),ylim=c(-ylimit,ylimit)) + dev.off() + rlogres = rlogTransformation(resDESeq) + sampledists = dist( t( assay(rlogres) ) ) + sdmat = as.matrix(sampledists) + pdf("DESeq2_sample_distance_plot.pdf") + heatmap.2(sdmat,trace="none",main=paste(myTitle,"DESeq2 sample distances"), + col = colorRampPalette( rev(brewer.pal(9, "RdBu")) )(255)) + dev.off() + sink() + result = try( (ppca = plotPCA( varianceStabilizingTransformation(deSeqDatdisp,blind=T), intgroup=c("Rx","Name")) ) ) + if ("try-error" %in% class(result)) { + print.noquote('DESeq2 plotPCA failed.') + } else { + pdf("DESeq2_PCA_plot.pdf") + #### wtf - print? Seems needed to get this to work + print(ppca) + dev.off() + } + } + + if (doVoom == T) { + sink('VOOM.log') + pdf("VOOM_mean_variance_plot.pdf") + dat.voomed = voom(DGEList, mydesign, plot = TRUE, lib.size = colSums(workCM) * norm.factor) + dev.off() + # Use limma to fit data + fit = lmFit(dat.voomed, mydesign) + fit = eBayes(fit) + rvoom = topTable(fit, coef = length(colnames(mydesign)), adj = fdrtype, n = Inf, sort="none") + qqPlot(descr=paste(myTitle,'VOOM-limma QQ plot'),pvector=rvoom\$P.Value,outpdf='VOOM_qqplot.pdf') + rownames(rvoom) = rownames(workCM) + rvoom = cbind(rvoom,NReads=cmrowsums,URL=contigurls) + srvoom = rvoom[order(rvoom\$P.Value),] + write.table(srvoom,paste(mt,'VOOM_topTable.xls',sep='_'), quote=FALSE, sep="\t",row.names=F) + # Use an FDR cutoff to find interesting samples for edgeR, DESeq and voom/limma + topresults.voom = srvoom[which(rvoom\$adj.P.Val < fdrthresh), ] + voomcountsindex = which(allgenes %in% topresults.voom\$ID) + voomcounts = rep(0, length(allgenes)) + voomcounts[voomcountsindex] = 1 + cat("# VOOM top 50\n") + print(srvoom[1:50,]) + sink() + } + + if (doCamera) { + doGSEA(y=DGEList,design=mydesign,histgmt=histgmt,bigmt=bigmt,ntest=20,myTitle=myTitle, + outfname=paste(mt,"GSEA.xls",sep="_"),fdrthresh=fdrthresh,fdrtype=fdrtype) + } + + if ((doDESeq2==T) || (doVoom==T) || (doedgeR==T)) { + if ((doVoom==T) && (doDESeq2==T) && (doedgeR==T)) { + vennmain = paste(mt,'Voom,edgeR and DESeq2 overlap at FDR=',fdrthresh) + counts.dataframe = data.frame(edgeR = edgeRcounts, DESeq2 = DESeqcounts, + VOOM_limma = voomcounts, row.names = allgenes) + } else if ((doDESeq2==T) && (doedgeR==T)) { + vennmain = paste(mt,'DESeq2 and edgeR overlap at FDR=',fdrthresh) + counts.dataframe = data.frame(edgeR = edgeRcounts, DESeq2 = DESeqcounts, row.names = allgenes) + } else if ((doVoom==T) && (doedgeR==T)) { + vennmain = paste(mt,'Voom and edgeR overlap at FDR=',fdrthresh) + counts.dataframe = data.frame(edgeR = edgeRcounts, VOOM_limma = voomcounts, row.names = allgenes) + } + + if (nrow(counts.dataframe > 1)) { + counts.venn = vennCounts(counts.dataframe) + vennf = "Venn_significant_genes_overlap.pdf" + pdf(vennf) + vennDiagram(counts.venn,main=vennmain,col="maroon") + dev.off() + } + } #### doDESeq2 or doVoom + #Return our main table + uoutput + +} +#### Done + +###sink(stdout(),append=T,type="message") +builtin_gmt="" +history_gmt="" +doDESeq2 = $DESeq2.doDESeq2 # make these T or F +doVoom = $doVoom +doCamera = $camera.doCamera +doedgeR = $edgeR.doedgeR +edgeR_priordf = 0 + +#if $DESeq2.doDESeq2 == "T" + DESeq_fitType = "$DESeq2.DESeq_fitType" +#end if +#if $edgeR.doedgeR == "T" +edgeR_priordf = $edgeR.edgeR_priordf +#end if + +#if $camera.doCamera == 'T' +#if $camera.gmtSource.refgmtSource == "indexed" or $camera.gmtSource.refgmtSource == "both": + builtin_gmt = "${camera.gmtSource.builtinGMT.fields.path}" +#end if +#if $camera.gmtSource.refgmtSource == "history" or $camera.gmtSource.refgmtSource == "both": + history_gmt = "${camera.gmtSource.ownGMT}" + history_gmt_name = "${camera.gmtSource.ownGMT.name}" + #end if +#end if + + + +Out_Dir = "$html_file.files_path" +Input = "$input1" +TreatmentName = "$treatment_name" +TreatmentCols = "$Treat_cols" +ControlName = "$control_name" +ControlCols= "$Control_cols" +outputfilename = "$outtab" +org = "$input1.dbkey" +if (org == "") { org = "hg19"} +fdrtype = "$fdrtype" +fdrthresh = $fdrthresh +useNDF = "$useNDF" +fQ = $fQ # non-differential centile cutoff +myTitle = "$title" +subjects = c($subjectids) +nsubj = length(subjects) +if (nsubj > 0) { +if (doDESeq2) { + print('WARNING - cannot yet use DESeq2 for 2 way anova - see the docs') + doDESeq2 = F + } +} +TCols = as.numeric(strsplit(TreatmentCols,",")[[1]])-1 +CCols = as.numeric(strsplit(ControlCols,",")[[1]])-1 +cat('Got TCols=') +cat(TCols) +cat('; CCols=') +cat(CCols) +cat('\n') +useCols = c(TCols,CCols) +if (file.exists(Out_Dir) == F) dir.create(Out_Dir) +Count_Matrix = read.table(Input,header=T,row.names=1,sep='\t') #Load tab file assume header +snames = colnames(Count_Matrix) +nsamples = length(snames) +if (nsubj > 0 & nsubj != nsamples) { +options("show.error.messages"=T) +mess = paste('Fatal error: Supplied subject id list',paste(subjects,collapse=','), + 'has length',nsubj,'but there are',nsamples,'samples',paste(snames,collapse=',')) +write(mess, stderr()) +quit(save="no",status=4) +} + +Count_Matrix = Count_Matrix[,useCols] ### reorder columns +if (length(subjects) != 0) {subjects = subjects[useCols]} +rn = rownames(Count_Matrix) +islib = rn %in% c('librarySize','NotInBedRegions') +LibSizes = Count_Matrix[subset(rn,islib),][1] # take first +Count_Matrix = Count_Matrix[subset(rn,! islib),] +group = c(rep(TreatmentName,length(TCols)), rep(ControlName,length(CCols)) ) #Build a group descriptor +group = factor(group, levels=c(ControlName,TreatmentName)) +colnames(Count_Matrix) = paste(group,colnames(Count_Matrix),sep="_") #Relable columns +results = edgeIt(Count_Matrix=Count_Matrix,group=group,outputfilename=outputfilename, + fdrtype='BH',priordf=edgeR_priordf,fdrthresh=0.05,outputdir='.', + myTitle=myTitle,useNDF=F,libSize=c(),filterquantile=fQ,subjects=c(), + doDESeq2=doDESeq2,doVoom=doVoom,doCamera=doCamera,doedgeR=doedgeR,org=org, + histgmt=history_gmt,bigmt=builtin_gmt,DESeq_fitType=DESeq_fitType) +sessionInfo() +]]> + + + + +---- + +**What it does** + +Allows short read sequence counts from controlled experiments to be analysed for differentially expressed genes. +Optionally adds a term for subject if not all samples are independent or if some other factor needs to be blocked in the design. + +**Input** + +Requires a count matrix as a tabular file. These are best made using the companion HTSeq_ based counter Galaxy wrapper +and your fave gene model to generate inputs. Each row is a genomic feature (gene or exon eg) and each column the +non-negative integer count of reads from one sample overlapping the feature. +The matrix must have a header row uniquely identifying the source samples, and unique row names in +the first column. Typically the row names are gene symbols or probe ids for downstream use in GSEA and other methods. + +**Specifying comparisons** + +This is basically dumbed down for two factors - case vs control. + +More complex interfaces are possible but painful at present. +Probably need to specify a phenotype file to do this better. +Work in progress. Send code. + +If you have (eg) paired samples and wish to include a term in the GLM to account for some other factor (subject in the case of paired samples), +put a comma separated list of indicators for every sample (whether modelled or not!) indicating (eg) the subject number or +A list of integers, one for each subject or an empty string if samples are all independent. +If not empty, there must be exactly as many integers in the supplied integer list as there are columns (samples) in the count matrix. +Integers for samples that are not in the analysis *must* be present in the string as filler even if not used. + +So if you have 2 pairs out of 6 samples, you need to put in unique integers for the unpaired ones +eg if you had 6 samples with the first two independent but the second and third pairs each being from independent subjects. you might use +8,9,1,1,2,2 +as subject IDs to indicate two paired samples from the same subject in columns 3/4 and 5/6 + +**Methods available** + +You can run 3 popular Bioconductor packages available for count data. + +edgeR - see edgeR_ for details + +VOOM/limma - see limma_VOOM_ for details + +DESeq2 - see DESeq2_ for details + +and optionally camera in edgeR which works better if MSigDB is installed. + +**Outputs** + +Some helpful plots and analysis results. Note that most of these are produced using R code +suggested by the excellent documentation and vignettes for the Bioconductor +packages invoked. The Tool Factory is used to automatically lay these out for you to enjoy. + +***old rant on changes to Bioconductor package variable names between versions*** + +The edgeR authors made a small cosmetic change in the name of one important variable (from p.value to PValue) +breaking this and all other code that assumed the old name for this variable, +between edgeR2.4.4 and 2.4.6 (the version for R 2.14 as at the time of writing). +This means that all code using edgeR is sensitive to the version. I think this was a very unwise thing +to do because it wasted hours of my time to track down and will similarly cost other edgeR users dearly +when their old scripts break. This tool currently now works with 2.4.6. + +**Note on prior.N** + +http://seqanswers.com/forums/showthread.php?t=5591 says: + +*prior.n* + +The value for prior.n determines the amount of smoothing of tagwise dispersions towards the common dispersion. +You can think of it as like a "weight" for the common value. (It is actually the weight for the common likelihood +in the weighted likelihood equation). The larger the value for prior.n, the more smoothing, i.e. the closer your +tagwise dispersion estimates will be to the common dispersion. If you use a prior.n of 1, then that gives the +common likelihood the weight of one observation. + +In answer to your question, it is a good thing to squeeze the tagwise dispersions towards a common value, +or else you will be using very unreliable estimates of the dispersion. I would not recommend using the value that +you obtained from estimateSmoothing()---this is far too small and would result in virtually no moderation +(squeezing) of the tagwise dispersions. How many samples do you have in your experiment? +What is the experimental design? If you have few samples (less than 6) then I would suggest a prior.n of at least 10. +If you have more samples, then the tagwise dispersion estimates will be more reliable, +so you could consider using a smaller prior.n, although I would hesitate to use a prior.n less than 5. + + +From Bioconductor Digest, Vol 118, Issue 5, Gordon writes: + +Dear Dorota, + +The important settings are prior.df and trend. + +prior.n and prior.df are related through prior.df = prior.n * residual.df, +and your experiment has residual.df = 36 - 12 = 24. So the old setting of +prior.n=10 is equivalent for your data to prior.df = 240, a very large +value. Going the other way, the new setting of prior.df=10 is equivalent +to prior.n=10/24. + +To recover old results with the current software you would use + + estimateTagwiseDisp(object, prior.df=240, trend="none") + +To get the new default from old software you would use + + estimateTagwiseDisp(object, prior.n=10/24, trend=TRUE) + +Actually the old trend method is equivalent to trend="loess" in the new +software. You should use plotBCV(object) to see whether a trend is +required. + +Note you could also use + + prior.n = getPriorN(object, prior.df=10) + +to map between prior.df and prior.n. + +---- + +**Attributions** + +edgeR - edgeR_ + +VOOM/limma - limma_VOOM_ + +DESeq2 - DESeq2_ for details + +See above for Bioconductor package documentation for packages exposed in Galaxy by this tool and app store package. + +Galaxy_ (that's what you are using right now!) for gluing everything together + +Otherwise, all code and documentation comprising this tool was written by Ross Lazarus and is +licensed to you under the LGPL_ like other rgenetics artefacts + +.. _LGPL: http://www.gnu.org/copyleft/lesser.html +.. _HTSeq: http://www-huber.embl.de/users/anders/HTSeq/doc/index.html +.. _edgeR: http://www.bioconductor.org/packages/release/bioc/html/edgeR.html +.. _DESeq2: http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html +.. _limma_VOOM: http://www.bioconductor.org/packages/release/bioc/html/limma.html +.. _Galaxy: http://getgalaxy.org + + + + + diff -r 643164729057 -r 91e77df18a7f rgedgeR/test-data/edgeRtest1out.html --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/rgedgeR/test-data/edgeRtest1out.html Fri Jul 26 22:04:58 2013 -0400 @@ -0,0 +1,813 @@ + + + + + + + + +
+ +
Galaxy Tool "edgeR" run at 12/06/2013 16:42:51

+
+ + + + + + + + + + + + + + + + + + + + + + + + + +
Image called edgeRtest_BCV_vs_abundance.pdfImage called edgeRtest_MDSplot.pdfImage called edgeRtest_RowsumCum.pdf
Image called edgeRtest_Smearplot.pdfImage called edgeRtest_heatmap.pdfImage called edgeRtest_pval_qq.pdf
Image called edgeRtest_sampleBoxplot.pdfImage called edgeRtest_samplehistplot.pdfImage called edgeRtest_venn.pdf
Image called edgeRtest_voomplot.pdf  
+ +
+ + + + + + + + + + + + + + + +
Output File Name (click to view)Size
edgeR.Rscript19.7 KB)
edgeR_runner.log79.2 KB)
edgeRtest_BCV_vs_abundance.pdf16.4 KB)
edgeRtest_DESeq_TopTable.xls135.1 KB)
edgeRtest_MDSplot.pdf4.9 KB)
edgeRtest_RowsumCum.pdf6.3 KB)
edgeRtest_Smearplot.pdf16.7 KB)
edgeRtest_VOOM_topTable.xls131.4 KB)
edgeRtest_heatmap.pdf11.3 KB)
edgeRtest_pval_qq.pdf15.9 KB)
edgeRtest_sampleBoxplot.pdf7.8 KB)
edgeRtest_samplehistplot.pdf10.9 KB)
edgeRtest_venn.pdf9.7 KB)
edgeRtest_voomplot.pdf16.7 KB)

+
Rscript log
+
+## Toolfactory generated command line = Rscript - None /data/home/rlazarus/galaxy-central/database/files/000/dataset_2.dat
+
+Loading required package: gtools
+
+Loading required package: gdata
+
+gdata: read.xls support for 'XLS' (Excel 97-2004) files ENABLED.
+
+gdata: read.xls support for 'XLSX' (Excel 2007+) files ENABLED.
+
+Attaching package: ‘gdata’
+
+The following object(s) are masked from ‘package:stats’:
+
+    nobs
+
+The following object(s) are masked from ‘package:utils’:
+
+    object.size
+
+Loading required package: caTools
+
+Loading required package: grid
+
+Loading required package: KernSmooth
+
+KernSmooth 2.23 loaded
+
+Copyright M. P. Wand 1997-2009
+
+Loading required package: MASS
+
+Attaching package: ‘gplots’
+
+The following object(s) are masked from ‘package:stats’:
+
+    lowess
+
+Loading required package: Biobase
+
+Loading required package: BiocGenerics
+
+Loading required package: methods
+
+Attaching package: ‘BiocGenerics’
+
+The following object(s) are masked from ‘package:gdata’:
+
+    combine
+
+The following object(s) are masked from ‘package:stats’:
+
+    xtabs
+
+The following object(s) are masked from ‘package:base’:
+
+    anyDuplicated, cbind, colnames, duplicated, eval, Filter, Find,
+
+    get, intersect, lapply, Map, mapply, mget, order, paste, pmax,
+
+    pmax.int, pmin, pmin.int, Position, rbind, Reduce, rep.int,
+
+    rownames, sapply, setdiff, table, tapply, union, unique
+
+Welcome to Bioconductor
+
+    Vignettes contain introductory material; view with
+
+    'browseVignettes()'. To cite Bioconductor, see
+
+    'citation("Biobase")', and for packages 'citation("pkgname")'.
+
+Loading required package: locfit
+
+locfit 1.5-9.1 	 2013-03-22
+
+Loading required package: lattice
+
+Warning message:
+
+found methods to import for function ‘eapply’ but not the generic itself 
+
+Loading required package: limma
+
+Attaching package: ‘limma’
+
+The following object(s) are masked from ‘package:DESeq’:
+
+    plotMA
+
+# got TCols=2 3 4 8; CCols=1 5 6 7
+
+[1] # Quantiles for non-zero row counts:
+
+       0%       10%       20%       30%       40%       50%       60%       70%       80%       90%      100% 
+
+      1.0       1.0       2.0       3.0       4.0       8.0      13.0      24.0      86.6     753.0 2325567.0 
+
+[1] Read 3242 contigs. Removed 1494 with no reads. After filtering at count quantile =0.3, there are 1141 contigs
+
+[1] # Total low count contigs per sample =  145,111,155,120,170,67,203,86
+
+Calculating library sizes from column totals.
+
+[1] Using samples: case_X11706He_AGTTCC_L001_R1_001_trimmed.fastq_bwa.sam.bam,case_X11699He_GGCTAC_L001_R1_001_trimmed.fastq_bwa.sam.bam,case_X11698He_TAGCTT_L001_R1_001_trimmed.fastq_bwa.sam.bam,case_X11700He_CTTGTA_L001_R1_001_trimmed.fastq_bwa.sam.bam,control_X11706Liv_CAAAAG_L003_R1_001_trimmed.fastq_bwa.sam.bam,control_X11700Liv_ATTCCT_L003_R1_001_trimmed.fastq_bwa.sam.bam,control_X11698Liv_ACTGAT_L003_R1_001_trimmed.fastq_bwa.sam.bam,control_X11699Liv_ATGAGC_L003_R1_001_trimmed.fastq_bwa.sam.bam
+
+[1] Using design matrix:
+
+  (Intercept) groupcase
+
+1           1         1
+
+2           1         1
+
+3           1         1
+
+4           1         1
+
+5           1         0
+
+6           1         0
+
+7           1         0
+
+8           1         0
+
+attr(,"assign")
+
+[1] 0 1
+
+attr(,"contrasts")
+
+attr(,"contrasts")$group
+
+[1] contr.treatment
+
+[1] "No GLM fit outlier genes found\n"
+
+[1] Common Dispersion = 0.24088423096811 CV =  0.490799583300669 getPriorN =  3.33333333333333
+
+[1] "Raw sample read totals 1440960,1341813,2888924,1428365,2443751,1644652,1682104,1806045"
+
+[1] raw contig counts by sample:
+
+ case_X11706He_AGTTCC case_X11699He_GGCTAC case_X11698He_TAGCTT case_X11700He_CTTGTA control_X11706Liv_CA control_X11700Liv_AT control_X11698Liv_AC control_X11699Liv_AT
+
+ Min.   :0.0043       Min.   :0.0043       Min.   :0.0043       Min.   :0.0043       Min.   :0.0043       Min.   :0.0043       Min.   :0.0043       Min.   :0.0043      
+
+ 1st Qu.:0.0043       1st Qu.:0.0043       1st Qu.:0.0043       1st Qu.:0.0043       1st Qu.:0.3032       1st Qu.:0.0043       1st Qu.:0.3032       1st Qu.:0.0043      
+
+ Median :0.4786       Median :0.4786       Median :0.4786       Median :0.4786       Median :0.7789       Median :0.6031       Median :0.6998       Median :0.6031      
+
+ Mean   :0.9210       Mean   :0.9428       Mean   :1.0205       Mean   :0.9753       Mean   :1.0519       Mean   :1.0343       Mean   :0.9855       Mean   :0.9966      
+
+ 3rd Qu.:1.5770       3rd Qu.:1.5855       3rd Qu.:1.7161       3rd Qu.:1.6022       3rd Qu.:1.5410       3rd Qu.:1.6335       3rd Qu.:1.4473       3rd Qu.:1.5534      
+
+ Max.   :5.3609       Max.   :5.3589       Max.   :5.6967       Max.   :5.3702       Max.   :5.8209       Max.   :5.6905       Max.   :5.7999       Max.   :5.7215      
+
+ NA's   :902          NA's   :957          NA's   :821          NA's   :950          NA's   :650          NA's   :969          NA's   :664          NA's   :886         
+
+[1] normalised contig counts by sample:
+
+ case_X11706He_AGTTCC case_X11699He_GGCTAC case_X11698He_TAGCTT case_X11700He_CTTGTA control_X11706Liv_CA control_X11700Liv_AT control_X11698Liv_AC control_X11699Liv_AT
+
+ Min.   :-Inf         Min.   :-Inf         Min.   :-Inf         Min.   :-Inf         Min.   :-Inf         Min.   :-Inf         Min.   :-Inf         Min.   :-Inf        
+
+ 1st Qu.:-Inf         1st Qu.:-Inf         1st Qu.:-Inf         1st Qu.:-Inf         1st Qu.:   0         1st Qu.:-Inf         1st Qu.:   0         1st Qu.:-Inf        
+
+ Median :   0         Median :   0         Median :   0         Median :   0         Median :   0         Median :   0         Median :   0         Median :   0        
+
+ Mean   :-Inf         Mean   :-Inf         Mean   :-Inf         Mean   :-Inf         Mean   :-Inf         Mean   :-Inf         Mean   :-Inf         Mean   :-Inf        
+
+ 3rd Qu.:   1         3rd Qu.:   1         3rd Qu.:   1         3rd Qu.:   1         3rd Qu.:   1         3rd Qu.:   1         3rd Qu.:   1         3rd Qu.:   1        
+
+ Max.   :   5         Max.   :   5         Max.   :   6         Max.   :   5         Max.   :   6         Max.   :   6         Max.   :   6         Max.   :   5        
+
+[1] Using ncol rawrs= 8
+
+[1] # writing output
+
+[1] # urls
+
+0610005C13Rik 0610007N19Rik 0610008F07Rik 0610009L18Rik 0610012G03Rik 0610031O16Rik# Top tags
+
+                       Name      logFC    logCPM        LR       PValue  adj.p.value Dispersion totreads case_X11706He_AGTTCC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11699He_GGCTAC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11698He_TAGCTT_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11700He_CTTGTA_L001_R1_001_trimmed.fastq_bwa.sam.bam control_X11706Liv_CAAAAG_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11700Liv_ATTCCT_L003_R1_001_trimmed.fastq_bwa.sam.bam
+
+Mir122a             Mir122a -10.661348 12.537949 445.91203 5.594743e-99 6.383602e-96 0.10205510    90428                                                   6.644669                                               2.864648e+00                                               2.012369e+01                                               4.756523e+00                                                   2.084166e+04                                                   1.681895e+04
+
+Mir192               Mir192  -7.109019 17.247415 366.13166 1.301558e-81 7.425390e-79 0.07765487  2325567                                                1779.663879                                               1.294412e+03                                               4.493546e+03                                               1.933526e+03                                                   4.635990e+05                                                   3.403123e+05
+
+Mir208b             Mir208b  13.423720  9.822338 355.41782 2.801296e-79 1.065426e-76 0.12444059    14756                                                1245.907022                                               5.701390e+02                                               4.930344e+03                                               8.819024e+02                                                   7.453220e-01                                                   0.000000e+00
+
+Mir208a             Mir208a  11.977586  8.388290 348.81540 7.675451e-78 2.189422e-75 0.09936798     4638                                                 433.789590                                               7.125278e+02                                               1.006599e+03                                               6.497485e+02                                                   0.000000e+00                                                   0.000000e+00
+
+Mir499               Mir499  14.488216  8.612770 291.12794 2.823507e-65 6.443244e-63 0.13481620     6527                                                 608.439028                                               5.066577e+02                                               2.521609e+03                                               2.703574e+02                                                   0.000000e+00                                                   0.000000e+00
+
+Mir149               Mir149   6.869731  8.785665 253.01986 5.702930e-57 1.084507e-54 0.09834684     6164                                                 774.657675                                               4.595714e+02                                               1.710514e+03                                               7.663947e+02                                                   1.750400e+01                                                   6.246465e+00
+
+Mir490               Mir490   8.453930  6.909596 251.05610 1.528281e-56 2.491098e-54 0.09745005     1741                                                 209.881557                                               2.177498e+02                                               5.205387e+02                                               1.957941e+02                                                   1.038505e+00                                                   5.537224e-01
+
+Mir802               Mir802 -12.499678  6.629391 237.56161 1.337812e-53 1.908055e-51 0.10939981     1514                                                   0.000000                                               0.000000e+00                                               0.000000e+00                                               0.000000e+00                                                   3.056548e+02                                                   1.641034e+02
+
+Mir204               Mir204   7.371179  7.558996 237.12302 1.667365e-53 2.113848e-51 0.10292005     2601                                                 425.578854                                               3.264164e+02                                               6.462477e+02                                               3.754819e+02                                                   3.040281e+00                                                   6.923364e-01
+
+Mir1948             Mir1948  -7.583326  7.293456 204.79641 1.875897e-46 2.140399e-44 0.11833999     2404                                                   1.216112                                               6.923364e-01                                               1.661167e+00                                               1.636942e+00                                                   6.476848e+02                                                   3.852783e+02
+
+Mir3073             Mir3073 -11.748556  5.881462 200.03350 2.053625e-45 2.130170e-43 0.11068562      904                                                   0.000000                                               0.000000e+00                                               0.000000e+00                                               0.000000e+00                                                   2.073471e+02                                                   7.546467e+01
+
+Mir194-2           Mir194-2  -5.439827  7.860048 165.57239 6.859199e-38 6.521955e-36 0.10671020     3570                                                  10.520726                                               6.138532e+00                                               1.490644e+01                                               9.513045e+00                                                   9.214105e+02                                                   6.003547e+02
+
+Mir10b               Mir10b   5.032898 13.880770 160.06580 1.094640e-36 9.607570e-35 0.10795434   197340                                               25838.822630                                               3.216361e+04                                               3.631730e+04                                               3.446522e+04                                                   1.825385e+03                                                   3.738617e+02
+
+Mir101b             Mir101b  -3.925287 11.936090 120.62427 4.618143e-28 3.763787e-26 0.09663075    59019                                                 219.816815                                               3.012250e+02                                               8.119232e+02                                               4.270695e+02                                                   1.014507e+04                                                   7.747970e+03
+
+Mir182               Mir182  -5.244384  8.942708 118.92815 1.085926e-27 8.260276e-26 0.14993443     7189                                                  16.962242                                               1.439678e+01                                               2.209871e+01                                               3.130352e+01                                                   9.840056e+02                                                   6.154406e+02
+
+Mir200b             Mir200b  -6.725348  5.765187 115.73189 5.440989e-27 3.880105e-25 0.16712490      888                                                   2.432225                                               0.000000e+00                                               1.661167e+00                                               0.000000e+00                                                   3.696797e+02                                                   9.750871e+01
+
+Mir181c             Mir181c   3.611867 10.569815 107.82011 2.943153e-25 1.975375e-23 0.09385270    23605                                                2420.852087                                               1.892879e+03                                               4.093093e+03                                               1.902590e+03                                                   2.320365e+02                                                   2.727878e+02
+
+Mir181d             Mir181d   3.489976  7.232064 102.18661 5.053019e-24 3.203053e-22 0.08738498     2139                                                 310.053946                                               2.021522e+02                                               5.398233e+02                                               2.956660e+02                                                   3.648337e+01                                                   1.453906e+01
+
+Mir21                 Mir21  -3.077771 13.881500  91.37324 1.189745e-21 7.144733e-20 0.08494272   229120                                                3207.212585                                               2.190826e+03                                               4.348911e+03                                               2.010566e+03                                                   2.715400e+04                                                   3.322794e+04
+
+Mir199b             Mir199b   5.411476  4.695869  86.70372 1.260596e-20 7.191701e-19 0.15656183      370                                                  43.403268                                               4.481024e+01                                               9.924938e+01                                               4.925255e+01                                                   1.384673e+00                                                   5.537224e-01
+
+Mir193               Mir193  -5.360157  4.775477  84.66127 3.541205e-20 1.924055e-18 0.15546497      421                                                   1.490644                                               1.189131e+00                                               2.800640e+00                                               6.940516e-01                                                   1.015454e+02                                                   3.496299e+01
+
+Mir23a               Mir23a   3.003071  9.467420  82.28684 1.177029e-19 6.104501e-18 0.08920345    10118                                                1149.889322                                               1.127958e+03                                               2.471518e+03                                               1.096325e+03                                                   1.505832e+02                                                   1.268024e+02
+
+Mir195               Mir195   3.060546  8.152797  81.39727 1.846126e-19 9.158392e-18 0.09133016     3962                                                 429.134881                                               2.831909e+02                                               9.227086e+02                                               4.708957e+02                                                   1.106253e+02                                                   7.773378e+01
+
+Ahsg                   Ahsg -10.101343  6.638589  79.82133 4.098447e-19 1.923759e-17 0.42092199     1524                                                   0.000000                                               5.537224e-01                                               0.000000e+00                                               0.000000e+00                                                   3.484153e+02                                                   1.260288e+01
+
+Mir148a             Mir148a  -2.789375 16.047169  79.76589 4.215073e-19 1.923759e-17 0.08196054  1002397                                               15925.598251                                               8.528200e+03                                               2.207082e+04                                               1.059020e+04                                                   1.927239e+05                                                   1.054456e+05
+
+Dnm3os               Dnm3os   3.189443  6.618834  79.19614 5.623979e-19 2.468062e-17 0.09496531     1401                                                 134.000827                                               8.716715e+01                                               3.652078e+02                                               1.825316e+02                                                   3.710848e+01                                                   2.498586e+01
+
+Mir27a               Mir27a   2.921772 10.581277  77.26988 1.491231e-18 6.301832e-17 0.09125059    21886                                                2383.612353                                               2.512174e+03                                               5.321988e+03                                               2.361690e+03                                                   3.285136e+02                                                   3.477377e+02
+
+Mir429               Mir429  -5.341934  4.951103  76.38758 2.331128e-18 9.499346e-17 0.18324117      499                                                   1.783696                                               7.001600e-01                                               2.082155e+00                                               1.824168e+00                                                   9.173458e+01                                                   4.540524e+01
+
+Mir214               Mir214   3.125906  6.202347  74.28213 6.771387e-18 2.664191e-16 0.09532762     1048                                                 110.058161                                               5.642542e+01                                               1.982326e+02                                               9.371492e+01                                                   3.353949e+01                                                   1.545870e+01
+
+Mir200a             Mir200a  -7.592263  4.059943  73.71426 9.028450e-18 3.433821e-16 0.22417820      264                                                   0.000000                                               0.000000e+00                                               5.945653e-01                                               0.000000e+00                                                   9.022671e+01                                                   3.222698e+01
+
+Mir194-1           Mir194-1  -3.970219  5.393899  73.22380 1.157517e-17 4.260408e-16 0.13095886      635                                                   3.567392                                               9.102080e+00                                               5.552413e+00                                               3.648337e+00                                                   7.615701e+01                                                   8.859559e+01
+
+Mir378               Mir378   2.954320  8.053368  73.09647 1.234655e-17 4.402315e-16 0.09566655     4075                                                 425.697272                                               3.921392e+02                                               1.155915e+03                                               1.883155e+02                                                   7.641369e+01                                                   3.683119e+01
+
+Mir322               Mir322   3.058569  8.948242  72.83721 1.407969e-17 4.868160e-16 0.10365695     7074                                                1012.892580                                               6.718588e+02                                               1.784708e+03                                               8.550716e+02                                                   1.587027e+02                                                   7.892635e+01
+
+Mir215               Mir215   2.908934  6.381924  70.31105 5.065317e-17 1.699861e-15 0.09002036     1182                                                 123.075020                                               1.610368e+02                                               2.727623e+02                                               1.222193e+02                                                   1.938542e+01                                                   1.716540e+01
+
+Cyp3a25             Cyp3a25  -9.778602  3.913257  70.02642 5.851533e-17 1.907600e-15 0.26445237      226                                                   0.000000                                               0.000000e+00                                               0.000000e+00                                               0.000000e+00                                                   5.664447e+01                                                   1.783696e+01
+
+Mir183               Mir183  -4.214462  5.247929  66.31101 3.851010e-16 1.220556e-14 0.15955164      563                                                   8.401920                                               1.388103e+00                                               2.432225e+00                                               2.423177e+00                                                   8.638070e+01                                                   2.905572e+01
+
+Mir181a-2         Mir181a-2   2.837713  7.573798  65.52900 5.726500e-16 1.765929e-14 0.09834030     2817                                                 288.826664                                               1.377749e+02                                               6.395494e+02                                               1.821098e+02                                                   5.813511e+01                                                   4.102501e+01
+
+Mir155               Mir155   3.734310  4.975364  65.07600 7.206449e-16 2.163831e-14 0.13295455      463                                                  33.966543                                               5.888044e+01                                               1.141565e+02                                               5.251200e+01                                                   9.716723e+00                                                   1.824168e+00
+
+Snord91a           Snord91a  -2.818342  6.592251  64.29345 1.072014e-15 3.136329e-14 0.09422446     1437                                                  13.846728                                               1.605795e+01                                               3.314807e+01                                               1.863305e+01                                                   2.158272e+02                                                   1.792410e+02
+
+Mir125b-2         Mir125b-2   2.852233  8.081093  64.20822 1.119409e-15 3.193115e-14 0.10276578     3837                                                 315.520541                                               4.404853e+02                                               7.634219e+02                                               4.852109e+02                                                   1.082721e+02                                                   9.060036e+01
+
+Serpina4-ps1   Serpina4-ps1 -10.090564  4.224051  64.02094 1.231040e-15 3.425894e-14 0.34406452      295                                                   0.000000                                               0.000000e+00                                               0.000000e+00                                               0.000000e+00                                                   1.080717e+02                                                   1.129674e+01
+
+Mir133b             Mir133b   9.293392  3.404704  63.59576 1.527577e-15 4.149919e-14 0.26404392      159                                                  17.719118                                               1.064012e+01                                               5.738979e+01                                               1.426957e+01                                                   0.000000e+00                                                   0.000000e+00
+
+Gm10069             Gm10069  -7.475459  6.400326  63.15445 1.911172e-15 5.071271e-14 0.41541432     1309                                                   1.189131                                               1.400320e+00                                               6.940516e-01                                               6.080561e-01                                                   1.914310e+02                                                   6.644669e+00
+
+Mir871               Mir871  -5.633588  4.283233  62.67513 2.437746e-15 6.321517e-14 0.21907826      290                                                   1.227706                                               7.453220e-01                                               5.945653e-01                                               0.000000e+00                                                   5.691223e+01                                                   4.925255e+01
+
+Mir184               Mir184   5.002968  4.123691  61.97642 3.475940e-15 8.813439e-14 0.19289992      247                                                  33.443087                                               1.419290e+01                                               4.983502e+01                                               2.168948e+01                                                   2.981288e+00                                                   0.000000e+00
+
+Mir132               Mir132   2.702863  5.866134  58.80773 1.738573e-14 4.312416e-13 0.09148478      857                                                  82.695634                                               4.327103e+01                                               1.821747e+02                                               6.097608e+01                                                   2.832224e+01                                                   1.308044e+01
+
+Snord104           Snord104  -2.492063 11.108871  58.55547 1.976394e-14 4.798012e-13 0.09093023    33458                                                 614.929745                                               4.378004e+02                                               8.629974e+02                                               5.144081e+02                                                   3.826761e+03                                                   4.181256e+03
+
+Mir3074-2         Mir3074-2   2.635505  7.871042  57.72938 3.007773e-14 7.149726e-13 0.09900198     3470                                                 450.903031                                               3.206519e+02                                               8.354691e+02                                               1.716994e+02                                                   8.970303e+01                                                   5.197290e+01
+
+Mir24-2             Mir24-2   2.635505  7.871042  57.68174 3.081509e-14 7.175515e-13 0.09908656     3470                                                 479.987359                                               2.746892e+02                                               9.620198e+02                                               3.442496e+02                                                   9.850509e+01                                                   4.396336e+01
+
+5430416N02Rik 5430416N02Rik  -3.267228  5.202367  56.75137 4.945455e-14 1.128553e-12 0.12456687      564                                                   6.080561                                               1.730841e+00                                               1.273562e+01                                               4.910826e+00                                                   1.453378e+02                                                   5.886197e+01
+
+              control_X11698Liv_ACTGAT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11699Liv_ATGAGC_L003_R1_001_trimmed.fastq_bwa.sam.bam case_X11706He_AGTTCC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11699He_GGCTAC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11698He_TAGCTT_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11700He_CTTGTA_L001_R1_001_trimmed.fastq_bwa.sam.bam control_X11706Liv_CAAAAG_L003_R1_001_trimmed.fastq_bwa.sam.bam
+
+Mir122a                                                         7.242557e+03                                                   8.468313e+03                                                         12                                                          7                                                         27                                                          8                                                          29767
+
+Mir192                                                          3.835916e+05                                                   1.822922e+05                                                       3214                                                       3163                                                       6029                                                       3252                                                         662133
+
+Mir208b                                                         0.000000e+00                                                   0.000000e+00                                                       2049                                                       1647                                                       8904                                                       2155                                                              1
+
+Mir208a                                                         0.000000e+00                                                   5.537224e-01                                                       1060                                                        956                                                       1693                                                        928                                                              0
+
+Mir499                                                          0.000000e+00                                                   0.000000e+00                                                        869                                                        730                                                       4147                                                        781                                                              0
+
+Mir149                                                          4.864449e+00                                                   5.538691e+00                                                       1399                                                       1123                                                       2295                                                       1289                                                             25
+
+Mir490                                                          0.000000e+00                                                   7.453220e-01                                                        353                                                        311                                                        750                                                        322                                                              3
+
+Mir802                                                          1.557723e+02                                                   2.092870e+02                                                          0                                                          0                                                          0                                                          0                                                            552
+
+Mir204                                                          5.537224e-01                                                   3.683119e+00                                                        571                                                        549                                                        923                                                        541                                                              5
+
+Mir1948                                                         1.813414e+02                                                   4.282299e+02                                                          2                                                          2                                                          3                                                          4                                                            869
+
+Mir3073                                                         7.253764e+01                                                   8.757639e+01                                                          0                                                          0                                                          0                                                          0                                                            341
+
+Mir194-2                                                        2.669366e+02                                                   3.046280e+02                                                         19                                                         15                                                         20                                                         16                                                           1316
+
+Mir10b                                                          6.583760e+02                                                   7.272114e+02                                                      34668                                                      54096                                                      51870                                                      49658                                                           3002
+
+Mir101b                                                         1.128042e+04                                                   6.628420e+03                                                        635                                                        544                                                       1984                                                        573                                                          17063
+
+Mir182                                                          2.366022e+03                                                   6.536603e+02                                                         49                                                         26                                                         54                                                         42                                                           1655
+
+Mir200b                                                         4.621056e+01                                                   1.075780e+02                                                          4                                                          0                                                          3                                                          0                                                            496
+
+Mir181c                                                         2.283131e+02                                                   2.989683e+02                                                       3488                                                       3113                                                      11824                                                       3436                                                            567
+
+Mir181d                                                         2.547123e+01                                                   1.555095e+01                                                        416                                                        340                                                        771                                                        426                                                             60
+
+Mir21                                                           2.570722e+04                                                   3.537908e+04                                                       4621                                                       3603                                                      12563                                                       3631                                                          66353
+
+Mir199b                                                         0.000000e+00                                                   2.981288e+00                                                         73                                                         64                                                        143                                                         81                                                              4
+
+Mir193                                                          3.377707e+01                                                   3.396654e+01                                                          2                                                          2                                                          4                                                          1                                                            167
+
+Mir23a                                                          8.921333e+01                                                   2.437203e+02                                                       1934                                                       1611                                                       3561                                                       1803                                                            435
+
+Mir195                                                          4.560421e+01                                                   4.154019e+01                                                        775                                                        692                                                       1238                                                        792                                                            158
+
+Ahsg                                                            6.100714e+02                                                   2.432225e+01                                                          0                                                          1                                                          0                                                          0                                                            586
+
+Mir148a                                                         1.795497e+05                                                   1.342747e+05                                                      26191                                                      24636                                                      39859                                                      25878                                                         258578
+
+Dnm3os                                                          1.216112e+01                                                   1.384673e+01                                                        242                                                        213                                                        490                                                        307                                                             53
+
+Mir27a                                                          1.636942e+02                                                   5.664447e+02                                                       4009                                                       3588                                                       7668                                                       3884                                                            949
+
+Mir429                                                          1.677865e+01                                                   7.527752e+01                                                          3                                                          1                                                          3                                                          3                                                            265
+
+Mir214                                                          1.190272e+01                                                   2.012750e+01                                                        181                                                        163                                                        358                                                        229                                                             45
+
+Mir200a                                                         1.142355e+01                                                   2.602495e+01                                                          0                                                          0                                                          1                                                          0                                                            130
+
+Mir194-1                                                        3.314807e+01                                                   1.050904e+02                                                          6                                                         13                                                          8                                                          6                                                            220
+
+Mir378                                                          9.167460e+01                                                   6.302392e+01                                                        608                                                        565                                                       1901                                                        544                                                            138
+
+Mir322                                                          5.038874e+01                                                   9.166874e+01                                                       1359                                                       1130                                                       2549                                                       1232                                                            261
+
+Mir215                                                          1.145859e+01                                                   2.683159e+01                                                        207                                                        230                                                        393                                                        201                                                             56
+
+Cyp3a25                                                         6.511488e+01                                                   1.873939e+01                                                          0                                                          0                                                          0                                                          0                                                             76
+
+Mir183                                                          1.349033e+02                                                   7.729349e+01                                                         12                                                          2                                                          4                                                          7                                                            156
+
+Mir181a-2                                                       8.051840e+01                                                   5.691223e+01                                                        475                                                        398                                                       1155                                                        445                                                             78
+
+Mir155                                                          2.769346e+00                                                   4.983502e+00                                                         83                                                         79                                                        192                                                         75                                                             14
+
+Snord91a                                                        2.484705e+02                                                   1.732960e+02                                                         40                                                         29                                                         81                                                         25                                                            363
+
+Mir125b-2                                                       2.077009e+01                                                   7.364508e+01                                                        771                                                        591                                                       1284                                                        693                                                            156
+
+Serpina4-ps1                                                    8.191872e+01                                                   9.716723e+00                                                          0                                                          0                                                          0                                                          0                                                            145
+
+Mir133b                                                         0.000000e+00                                                   0.000000e+00                                                         32                                                         26                                                         77                                                         24                                                              0
+
+Gm10069                                                         2.868741e+02                                                   2.757691e+01                                                          2                                                          2                                                          1                                                          1                                                            553
+
+Mir871                                                          1.003888e+01                                                   5.149619e+01                                                          3                                                          1                                                          1                                                          0                                                             82
+
+Mir184                                                          1.400320e+00                                                   1.388103e+00                                                         55                                                         41                                                         90                                                         53                                                              4
+
+Mir132                                                          2.100480e+01                                                   1.943345e+01                                                        136                                                        125                                                        329                                                        149                                                             38
+
+Snord104                                                        4.430701e+03                                                   4.212863e+03                                                        886                                                        720                                                       2493                                                        929                                                           9351
+
+Mir3074-2                                                       4.844593e+01                                                   8.323914e+01                                                        644                                                        462                                                       1374                                                        496                                                            162
+
+Mir24-2                                                         3.599196e+01                                                   5.729296e+01                                                        644                                                        462                                                       1374                                                        496                                                            162
+
+5430416N02Rik                                                   5.531264e+01                                                   9.786128e+01                                                         10                                                          5                                                         23                                                         12                                                            195
+
+              control_X11700Liv_ATTCCT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11698Liv_ACTGAT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11699Liv_ATGAGC_L003_R1_001_trimmed.fastq_bwa.sam.bam ntotreads                                                                                                   URL
+
+Mir122a                                                                24233                                                          11911                                                          24463     90428             Mir122a
+
+Mir192                                                                490327                                                         630849                                                         526600   2325567               Mir192
+
+Mir208b                                                                    0                                                              0                                                              0     14756             Mir208b
+
+Mir208a                                                                    0                                                              0                                                              1      4638             Mir208a
+
+Mir499                                                                     0                                                              0                                                              0      6527               Mir499
+
+Mir149                                                                     9                                                              8                                                             16      6164               Mir149
+
+Mir490                                                                     1                                                              0                                                              1      1741               Mir490
+
+Mir802                                                                   401                                                            209                                                            352      1514               Mir802
+
+Mir204                                                                     2                                                              1                                                              9      2601               Mir204
+
+Mir1948                                                                  648                                                            259                                                            617      2404             Mir1948
+
+Mir3073                                                                  218                                                            131                                                            214       904             Mir3073
+
+Mir194-2                                                                 865                                                            439                                                            880      3570           Mir194-2
+
+Mir10b                                                                  1080                                                           1189                                                           1777    197340               Mir10b
+
+Mir101b                                                                11066                                                          16253                                                          10901     59019             Mir101b
+
+Mir182                                                                   879                                                           3409                                                           1075      7189               Mir182
+
+Mir200b                                                                  164                                                             66                                                            155       888             Mir200b
+
+Mir181c                                                                  366                                                            384                                                            427     23605             Mir181c
+
+Mir181d                                                                   42                                                             46                                                             38      2139             Mir181d
+
+Mir21                                                                  44582                                                          43237                                                          50530    229120                 Mir21
+
+Mir199b                                                                    1                                                              0                                                              4       370             Mir199b
+
+Mir193                                                                   101                                                             61                                                             83       421               Mir193
+
+Mir23a                                                                   229                                                            218                                                            327     10118               Mir23a
+
+Mir195                                                                   112                                                             75                                                            120      3962               Mir195
+
+Ahsg                                                                      18                                                            879                                                             40      1524                   Ahsg
+
+Mir148a                                                               177349                                                         256441                                                         193465   1002397             Mir148a
+
+Dnm3os                                                                    36                                                             20                                                             40      1401               Dnm3os
+
+Mir27a                                                                   628                                                            400                                                            760     21886               Mir27a
+
+Mir429                                                                    82                                                             41                                                            101       499               Mir429
+
+Mir214                                                                    26                                                             17                                                             29      1048               Mir214
+
+Mir200a                                                                   53                                                             33                                                             47       264             Mir200a
+
+Mir194-1                                                                 160                                                             81                                                            141       635           Mir194-1
+
+Mir378                                                                    90                                                            123                                                            106      4075               Mir378
+
+Mir322                                                                   228                                                             91                                                            224      7074               Mir322
+
+Mir215                                                                    31                                                             28                                                             36      1182               Mir215
+
+Cyp3a25                                                                   30                                                             93                                                             27       226             Cyp3a25
+
+Mir183                                                                    71                                                            181                                                            130       563               Mir183
+
+Mir181a-2                                                                 69                                                            115                                                             82      2817         Mir181a-2
+
+Mir155                                                                     3                                                              8                                                              9       463               Mir155
+
+Snord91a                                                                 256                                                            358                                                            285      1437           Snord91a
+
+Mir125b-2                                                                149                                                             60                                                            133      3837         Mir125b-2
+
+Serpina4-ps1                                                              19                                                            117                                                             14       295   Serpina4-ps1
+
+Mir133b                                                                    0                                                              0                                                              0       159             Mir133b
+
+Gm10069                                                                   12                                                            701                                                             37      1309             Gm10069
+
+Mir871                                                                    81                                                             29                                                             93       290               Mir871
+
+Mir184                                                                     0                                                              2                                                              2       247               Mir184
+
+Mir132                                                                    22                                                             30                                                             28       857               Mir132
+
+Snord104                                                                5610                                                           7452                                                           6017     33458           Snord104
+
+Mir3074-2                                                                127                                                             65                                                            140      3470         Mir3074-2
+
+Mir24-2                                                                  127                                                             65                                                            140      3470             Mir24-2
+
+5430416N02Rik                                                             99                                                             79                                                            141       564 5430416N02Rik
+
+[1] # 445 tags significant at adj p= 0.05
+
+[1] # deTags
+
+[1] "0610005C13Rik" "0610007N19Rik" "0610008F07Rik" "0610012G03Rik" "0610031O16Rik" "1110038B12Rik"
+
+# DESeq top 50
+
+               id     baseMean    baseMeanA    baseMeanB   foldChange log2FoldChange         pval         padj
+
+536       Mir122a  10112.31117 2.021162e+04 1.300527e+01 6.434550e-04     -10.601873 5.488369e-57 6.262229e-54
+
+645       Mir208a    616.76981 2.430915e-01 1.233297e+03 5.073385e+03      12.308733 6.046036e-53 3.449263e-50
+
+600        Mir192 271352.97636 5.385927e+05 4.113233e+03 7.637001e-03      -7.032778 1.311798e-38 4.989204e-36
+
+568        Mir149    810.35429 1.224267e+01 1.608466e+03 1.313819e+02       7.037623 7.073079e-37 2.017596e-34
+
+791        Mir490    220.99790 9.450198e-01 4.410508e+02 4.667107e+02       8.866385 1.354865e-36 3.091803e-34
+
+642        Mir204    347.57397 3.737736e+00 6.914102e+02 1.849810e+02       7.531234 8.058436e-36 1.532446e-33
+
+844        Mir802    166.83414 3.336683e+02 0.000000e+00 0.000000e+00           -Inf 3.255469e-35 5.306414e-33
+
+698       Mir3073     98.93199 1.978640e+02 0.000000e+00 0.000000e+00           -Inf 1.111351e-28 1.585065e-26
+
+646       Mir208b   1649.77924 1.445675e-01 3.299414e+03 2.282265e+04      14.478178 7.505934e-28 9.515857e-26
+
+614       Mir1948    263.89527 5.246983e+02 3.092210e+00 5.893310e-03      -7.406706 1.534188e-26 1.750509e-24
+
+608      Mir194-2    391.65678 7.637703e+02 1.954329e+01 2.558791e-02      -5.288394 1.622682e-24 1.683163e-22
+
+796        Mir499    712.45950 0.000000e+00 1.424919e+03          Inf            Inf 2.577318e-19 2.450600e-17
+
+585       Mir181c   2765.96510 3.955399e+02 5.136390e+03 1.298577e+01       3.698860 1.942959e-17 1.705320e-15
+
+631       Mir199b     47.84725 1.818862e+00 9.387565e+01 5.161231e+01       5.689643 4.012622e-15 3.270287e-13
+
+530        Mir10b  27820.40551 1.501200e+03 5.413961e+04 3.606421e+01       5.172496 3.069912e-14 2.335180e-12
+
+586       Mir181d    275.22797 4.254569e+01 5.079103e+02 1.193800e+01       3.577489 5.204519e-14 3.711473e-12
+
+602        Mir193     45.70861 8.913819e+01 2.279021e+00 2.556728e-02      -5.289558 1.657854e-13 1.112713e-11
+
+637       Mir200a     27.09009 5.402561e+01 1.545578e-01 2.860824e-03      -8.449354 2.466964e-13 1.563781e-11
+
+722        Mir322    899.53469 1.797868e+02 1.619283e+03 9.006682e+00       3.170996 1.359804e-12 8.165978e-11
+
+756        Mir378    483.21548 1.056050e+02 8.608260e+02 8.151374e+00       3.027043 2.046396e-12 1.160965e-10
+
+523       Mir101b   6846.19683 1.280838e+04 8.840090e+02 6.901800e-02      -3.856884 2.136745e-12 1.160965e-10
+
+552       Mir133b     19.46050 0.000000e+00 3.892101e+01          Inf            Inf 2.388227e-12 1.238621e-10
+
+663        Mir23a   1289.18043 2.671574e+02 2.311203e+03 8.651093e+00       3.112882 3.093049e-12 1.534421e-10
+
+671        Mir27a   2788.72629 6.066392e+02 4.970813e+03 8.194019e+00       3.034571 5.970859e-12 2.838646e-10
+
+616        Mir195    519.86200 1.038507e+02 9.358733e+02 9.011721e+00       3.171803 6.245320e-12 2.850364e-10
+
+607      Mir194-1     71.10636 1.325374e+02 9.675354e+00 7.300095e-02      -3.775941 1.405195e-11 6.166645e-10
+
+344        Dnm3os    179.61643 3.285433e+01 3.263785e+02 9.934110e+00       3.312391 1.503324e-11 6.352936e-10
+
+590        Mir184     32.23796 1.645700e+00 6.283021e+01 3.817841e+01       5.254685 1.644760e-11 6.702396e-10
+
+582     Mir181a-2    347.82506 8.313904e+01 6.125111e+02 7.367310e+00       2.881138 2.735424e-11 1.076248e-09
+
+541     Mir125b-2    493.08516 1.122867e+02 8.738836e+02 7.782608e+00       2.960254 5.662328e-11 2.153572e-09
+
+573        Mir155     57.66182 7.341434e+00 1.079822e+02 1.470860e+01       3.878588 6.173765e-11 2.272344e-09
+
+464        Gm5441     20.24317 2.430915e-01 4.024325e+01 1.655478e+02       7.371104 6.503611e-11 2.318944e-09
+
+571        Mir153     19.90411 2.430915e-01 3.956513e+01 1.627582e+02       7.346586 8.675688e-11 2.999685e-09
+
+649         Mir21  26121.31011 4.639963e+04 5.842995e+03 1.259276e-01      -2.989333 1.048681e-10 3.519252e-09
+
+654        Mir214    134.69038 2.546966e+01 2.439111e+02 9.576536e+00       3.259504 1.338023e-10 4.361954e-09
+
+638       Mir200b     87.13638 1.725548e+02 1.717931e+00 9.955853e-03      -6.650239 2.397950e-10 7.600169e-09
+
+666       Mir24-2    424.48288 1.104079e+02 7.385578e+02 6.689355e+00       2.741867 4.542021e-10 1.363802e-08
+
+700     Mir3074-2    424.48288 1.104079e+02 7.385578e+02 6.689355e+00       2.741867 4.542021e-10 1.363802e-08
+
+845        Mir871     33.03360 6.461640e+01 1.450790e+00 2.245235e-02      -5.476990 5.839129e-10 1.708320e-08
+
+767        Mir429     49.75018 9.677270e+01 2.727653e+00 2.818618e-02      -5.148868 6.903556e-10 1.969239e-08
+
+655        Mir215    152.78082 3.329953e+01 2.722621e+02 8.176156e+00       3.031423 8.994273e-10 2.503040e-08
+
+566       Mir148a 118994.46955 2.065090e+05 3.147993e+04 1.524386e-01      -2.713700 1.022762e-09 2.772155e-08
+
+569        Mir150    553.20599 1.318948e+02 9.745171e+02 7.388592e+00       2.885299 1.044721e-09 2.772155e-08
+
+628       Mir1983     14.04997 0.000000e+00 2.809994e+01          Inf            Inf 2.859722e-09 7.415778e-08
+
+581     Mir181a-1     59.53826 1.179305e+01 1.072835e+02 9.097179e+00       3.185419 3.537731e-09 8.970115e-08
+
+784        Mir470     18.37716 3.608521e+01 6.691037e-01 1.854232e-02      -5.753034 7.467127e-09 1.852172e-07
+
+630     Mir199a-2     44.84878 7.384673e+00 8.231288e+01 1.114645e+01       3.478512 8.433815e-09 2.047443e-07
+
+550        Mir132    106.46062 2.691980e+01 1.860014e+02 6.909467e+00       2.788574 1.329950e-08 3.161402e-07
+
+76  2610203C20Rik     79.17666 1.649993e+01 1.418534e+02 8.597210e+00       3.103869 1.594438e-08 3.712764e-07
+
+540     Mir125b-1     79.01988 1.649993e+01 1.415398e+02 8.578206e+00       3.100676 1.650666e-08 3.766821e-07
+
+# VOOM top 50
+
+                ID      logFC    AveExpr          t      P.Value    adj.P.Val         B
+
+600         Mir192  -6.948883 14.6763803 -42.722954 2.301199e-16 2.625668e-13 27.266471
+
+536        Mir122a -10.426125  8.1698641 -21.722912 2.859682e-12 1.087633e-09 17.760171
+
+568         Mir149   7.030463  6.3160807  20.883835 4.915491e-12 1.402144e-09 17.277609
+
+645        Mir208a  11.015018  3.9395538  23.252407 1.118938e-12 6.383542e-10 17.208662
+
+530         Mir10b   5.130915 12.2628672  18.242023 3.124991e-11 4.963978e-09 16.221503
+
+561       Mir143hg   2.249221 16.2444825  18.082481 3.521739e-11 4.963978e-09 16.026695
+
+560         Mir143   2.251067 16.2358599  18.081481 3.524391e-11 4.963978e-09 16.026084
+
+646        Mir208b  12.433228  4.6076218  19.592458 1.179199e-11 2.690931e-09 15.683666
+
+523        Mir101b  -3.765722 10.8508440 -16.538566 1.180419e-10 1.036045e-08 14.900027
+
+796         Mir499  11.567529  3.7874598  17.942086 3.915496e-11 4.963978e-09 14.821741
+
+566        Mir148a  -2.638213 15.4435820 -16.548188 1.171186e-10 1.036045e-08 14.814792
+
+844         Mir802  -9.158434  2.9157675 -17.316522 6.338616e-11 7.232360e-09 14.381577
+
+698        Mir3073  -8.420542  2.5457189 -16.702657 1.033066e-10 1.036045e-08 13.985845
+
+649          Mir21  -2.938537 13.1642917 -15.375404 3.148335e-10 2.394833e-08 13.867698
+
+585        Mir181c   3.742560  9.6295577  15.242361 3.537063e-10 2.522368e-08 13.810405
+
+791         Mir490   8.474378  3.7506957  16.259650 1.484816e-10 1.210125e-08 13.424617
+
+663         Mir23a   3.165768  8.7896592  14.631179 6.110682e-10 3.873493e-08 13.269174
+
+586        Mir181d   3.636211  6.3713218  14.317073 8.157508e-10 4.898798e-08 12.956333
+
+642         Mir204   7.684425  4.7751735  15.033484 4.254268e-10 2.855364e-08 12.822427
+
+671         Mir27a   3.106935  9.9255796  13.838251 1.281011e-09 6.960160e-08 12.513086
+
+552        Mir133b   6.493549  1.2544862  13.969968 1.129934e-09 6.446275e-08 11.982684
+
+608       Mir194-2  -5.264136  6.0897615 -13.044012 2.792884e-09 1.448491e-07 11.715753
+
+616         Mir195   3.216595  7.4509350  12.869478 3.332788e-09 1.653353e-07 11.587523
+
+756         Mir378   3.097393  7.3832049  11.684163 1.171371e-08 5.346138e-07 10.329692
+
+672         Mir27b   1.976376 15.0957731  11.756036 1.082197e-08 5.144946e-07 10.127719
+
+1017      Snord104  -2.337374 10.6109024 -11.495675 1.444482e-08 6.339052e-07 10.023395
+
+722         Mir322   3.296618  8.2153415  11.415362 1.580762e-08 6.441605e-07 10.008472
+
+655         Mir215   3.045873  5.7544234  11.148134 2.141822e-08 8.082459e-07  9.753268
+
+628        Mir1983   5.895500  0.9931851  11.445812 1.527548e-08 6.441605e-07  9.749260
+
+344         Dnm3os   3.363344  5.8607432  11.092261 2.283960e-08 8.082459e-07  9.689496
+
+637        Mir200a  -6.191561  1.7981309 -11.322172 1.756229e-08 6.909853e-07  9.662295
+
+587         Mir182  -4.903995  7.1511683 -11.074468 2.331304e-08 8.082459e-07  9.658842
+
+582      Mir181a-2   3.048298  6.9414651  11.072128 2.337609e-08 8.082459e-07  9.644017
+
+614        Mir1948  -7.195525  4.5513493 -11.005492 2.524936e-08 8.473388e-07  9.341794
+
+654         Mir214   3.280874  5.4784451  10.768257 3.332555e-08 1.086413e-06  9.318504
+
+571         Mir153   5.963803  1.4386315  10.727082 3.498742e-08 1.093990e-06  9.035569
+
+318        Cyp3a25  -6.318200  1.4888933 -10.698226 3.620443e-08 1.093990e-06  9.024973
+
+464         Gm5441   5.982176  1.4484953  10.692891 3.643436e-08 1.093990e-06  9.000362
+
+541      Mir125b-2   3.077678  7.4316058  10.446668 4.893073e-08 1.431538e-06  8.884250
+
+551      Mir133a-1   5.144671  0.5903264  10.358205 5.447229e-08 1.553822e-06  8.575535
+
+550         Mir132   2.847559  5.3211839  10.110952 7.380297e-08 2.004981e-06  8.531491
+
+13   1110038B12Rik  -2.226702 10.8487089 -10.194609 6.655312e-08 1.852125e-06  8.439308
+
+922        Rabggtb  -1.935779  9.9874171  -9.928995 9.262821e-08 2.457879e-06  8.133384
+
+569         Mir150   2.938531  7.6297870   9.842102 1.033602e-07 2.620755e-06  8.116464
+
+800         Mir504   5.256127  0.6221088   9.892894 9.693595e-08 2.513725e-06  8.068853
+
+573         Mir155   3.906600  3.9899000   9.732173 1.188627e-07 2.712448e-06  8.046518
+
+666        Mir24-2   2.833979  7.3083691   9.767192 1.136724e-07 2.646944e-06  8.030550
+
+700      Mir3074-2   2.833979  7.3083691   9.767192 1.136724e-07 2.646944e-06  8.030550
+
+664         Mir23b   2.124129  9.8141190   9.806316 1.081569e-07 2.625681e-06  7.979464
+
+631        Mir199b   5.752816  2.8805143   9.823920 1.057683e-07 2.623514e-06  7.979387
+
+Warning messages:
+
+1: In bplt(at[i], wid = width[i], stats = z$stats[, i], out = z$out[z$group ==  :
+
+  Outlier (-Inf) in boxplot 5 is not drawn
+
+2: In bplt(at[i], wid = width[i], stats = z$stats[, i], out = z$out[z$group ==  :
+
+  Outlier (-Inf) in boxplot 7 is not drawn
+
+3: In bxp(list(stats = c(-Inf, -Inf, -0.158608221176912, 0.826586442285183,  :
+
+  some notches went outside hinges ('box'): maybe set notch=FALSE
+
+4: In par(defpar) : calling par(new=TRUE) with no plot
+
+R version 2.15.1 (2012-06-22)
+
+Platform: x86_64-unknown-linux-gnu (64-bit)
+
+locale:
+
+ [1] LC_CTYPE=en_AU.UTF-8       LC_NUMERIC=C               LC_TIME=en_AU.UTF-8        LC_COLLATE=en_AU.UTF-8     LC_MONETARY=en_AU.UTF-8    LC_MESSAGES=en_AU.UTF-8    LC_PAPER=C                 LC_NAME=C                  LC_ADDRESS=C               LC_TELEPHONE=C             LC_MEASUREMENT=en_AU.UTF-8 LC_IDENTIFICATION=C       
+
+attached base packages:
+
+[1] methods   grid      stats     graphics  grDevices utils     datasets  base     
+
+other attached packages:
+
+ [1] edgeR_3.0.8        limma_3.14.4       DESeq_1.10.1       lattice_0.20-15    locfit_1.5-9.1     Biobase_2.18.0     BiocGenerics_0.4.0 gplots_2.11.0      MASS_7.3-23        KernSmooth_2.23-10 caTools_1.14       gdata_2.12.0.2     gtools_2.7.1       stringr_0.6.2     
+
+loaded via a namespace (and not attached):
+
+ [1] annotate_1.36.0      AnnotationDbi_1.20.7 bitops_1.0-5         DBI_0.2-7            genefilter_1.40.0    geneplotter_1.36.0   IRanges_1.16.6       parallel_2.15.1      RColorBrewer_1.0-5   RSQLite_0.11.3       splines_2.15.1       stats4_2.15.1        survival_2.37-4      XML_3.96-1.1         xtable_1.7-1        
+
+
+ +
This tool (edgeR) was generated by the Galaxy Tool Factory

+
+ diff -r 643164729057 -r 91e77df18a7f rgedgeR/test-data/edgeRtest1out.xls --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/rgedgeR/test-data/edgeRtest1out.xls Fri Jul 26 22:04:58 2013 -0400 @@ -0,0 +1,1142 @@ +Name logFC logCPM LR PValue adj.p.value Dispersion totreads case_X11706He_AGTTCC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11699He_GGCTAC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11698He_TAGCTT_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11700He_CTTGTA_L001_R1_001_trimmed.fastq_bwa.sam.bam control_X11706Liv_CAAAAG_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11700Liv_ATTCCT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11698Liv_ACTGAT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11699Liv_ATGAGC_L003_R1_001_trimmed.fastq_bwa.sam.bam case_X11706He_AGTTCC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11699He_GGCTAC_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11698He_TAGCTT_L001_R1_001_trimmed.fastq_bwa.sam.bam case_X11700He_CTTGTA_L001_R1_001_trimmed.fastq_bwa.sam.bam control_X11706Liv_CAAAAG_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11700Liv_ATTCCT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11698Liv_ACTGAT_L003_R1_001_trimmed.fastq_bwa.sam.bam control_X11699Liv_ATGAGC_L003_R1_001_trimmed.fastq_bwa.sam.bam +Mir122a -10.6613482198735 12.5379487184339 445.912028851371 5.59474310450852e-99 6.38360188224422e-96 0.102055096028295 90428 6.64466912039531 2.86464823551992 20.1236936368881 4.75652253016081 20841.6623198401 16818.9531619257 7242.55663282834 8468.31297344994 12 7 27 8 29767 24233 11911 24463 +Mir192 -7.10901851688288 17.2474145508888 366.131659138235 1.3015583263717e-81 7.42539025195057e-79 0.0776548700185199 2325567 1779.66387941254 1294.41176699279 4493.54625691845 1933.52640851037 463599.032378899 340312.253828562 383591.60517699 182292.180510107 3214 3163 6029 3252 662133 490327 630849 526600 +Mir208b 13.4237202575152 9.82233816515324 355.417815392181 2.80129611458365e-79 1.06542628891331e-76 0.124440587141633 14756 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0.998669131632702 0.592374698826143 42 1.49064397310282 2.37826126508041 7.70175985212621 2.08215489150238 9.72889817187923 1.73084106067325 0.553722426699609 0 2 4 11 3 16 5 1 0 diff -r 643164729057 -r 91e77df18a7f rgedgeR/test-data/gentestdata.sh --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/rgedgeR/test-data/gentestdata.sh Fri Jul 26 22:04:58 2013 -0400 @@ -0,0 +1,9 @@ +#!/bin/bash +# generate test data for rgGSEA +# ross lazarus June 2013 +# adjust gseajar_path ! +GSEAJAR_PATH=/home/rlazarus/galaxy-central/tool_dependency_dir/gsea_jar/2.0.12/fubar/rg_gsea_test/8e291f464aa0/jars/gsea2-2.0.12.jar +python ../rgGSEA.py --input_tab "gsea_test_DGE.xls" --adjpvalcol "5" --signcol "2" --idcol "1" --outhtml "gseatestout.html" --input_name "gsea_test" --setMax "500" --setMin "15" --nPerm "10" --plotTop "20" --gsea_jar "$GSEAJAR_PATH" --output_dir "gseatestout" --mode "Max_probe" +--title "GSEA test" --builtin_gmt "gseatestdata.gmt" + + diff -r 643164729057 -r 91e77df18a7f 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1 +Ufd1l 0 0 1 0 0 6 0 0 +Uqcc 5 0 2 1 0 8 1 1 +Vash2 0 0 0 0 0 0 0 0 +Vaultrc5 243 258 161 383 159 262 286 270 +Vax2os 0 0 0 0 0 0 0 0 +Vmn1r-ps79 0 0 0 0 0 0 0 0 +Vmn2r-ps11 0 0 0 1 0 0 0 0 +Vmn2r-ps129 0 0 0 0 0 0 0 0 +Vmn2r-ps159 0 0 0 0 0 0 0 0 +Vmn2r-ps54 0 0 1 0 0 0 0 0 +Vmn2r-ps60 0 0 0 0 0 0 0 0 +Vmn2r29 2 0 0 0 0 0 0 0 +Vps39 3 1 0 0 0 8 0 0 +Vsig8 194 50 35 100 99 74 109 50 +Wac 15 0 0 0 2 15 2 2 +Wbscr25 0 2 1 0 0 1 0 0 +Wdr13 2 2 1 3 0 1 0 3 +Wdr73 4 0 0 0 0 2 1 0 +Wiz 10 0 1 6 1 3 3 2 +Wls 1 1 0 3 2 0 0 1 +Wwp1 40 0 2 2 4 56 10 4 +Xist 0 0 0 0 0 8 0 0 +Yaf2 2 0 0 0 0 6 0 2 +Yars2 1 0 0 0 1 1 0 0 +Yipf2 1 0 0 1 0 10 0 0 +Ythdf3 16 4 1 4 3 19 3 2 +Zbtb24 1 0 0 1 0 0 0 0 +Zc3h14 11 1 0 0 1 2 2 2 +Zc3h7a 336 148 60 20 17 99 14 60 +Zf12 0 0 0 0 0 0 0 0 +Zfa-ps 0 0 0 0 0 0 0 0 +Zfhx2as 0 0 0 0 0 1 0 0 +Zfp133-ps 0 0 0 0 0 0 0 0 +Zfp207 5 0 2 0 0 9 1 2 +Zfp326 7 2 1 1 0 4 4 0 +Zfp389 0 0 0 0 0 0 0 0 +Zfp410 10 0 2 0 0 6 2 0 +Zfp414 1 0 0 0 0 4 0 0 +Zfp57 0 0 0 0 0 0 0 0 +Zfp572 0 0 0 0 0 0 0 0 +Zfp672 0 0 0 0 0 2 0 0 +Zfp783 0 0 0 0 0 0 0 0 +Zfp809 7 1 0 0 0 4 2 0 +Zfp821 0 0 0 2 2 0 0 0 +Zfp862 4 0 0 0 0 10 2 0 +Zim3 0 0 0 0 0 0 0 0 +Zmynd8 3 5 4 4 3 8 2 1 +Znf41-ps 0 0 0 0 0 0 0 0 +Zp4-ps 0 0 0 0 0 0 0 0 +Zscan4a 0 0 0 0 0 0 0 0 +Zxda 0 0 0 0 0 0 0 0 diff -r 643164729057 -r 91e77df18a7f rgedgeR/tool_dependencies.xml --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/rgedgeR/tool_dependencies.xml Fri Jul 26 22:04:58 2013 -0400 @@ -0,0 +1,27 @@ + + + + + + + + + + + + + + $INSTALL_DIR + echo "source('http://bioconductor.org/biocLite.R')" > $INSTALL_DIR/runme.R + echo "installme=c('edgeR','limma','DESeq','DESeq2','gplots')" >> $INSTALL_DIR/runme.R + echo "biocLite()" >> $INSTALL_DIR/runme.R + echo "biocLite(installme)" >> $INSTALL_DIR/runme.R + echo "quit(save='no')" >> $INSTALL_DIR/runme.R + export PATH=$PATH && export R_HOME=$R_HOME && export R_LIBS=$R_LIBS && R CMD BATCH $INSTALL_DIR/runme.R + + + Installs some basic bioc packages for the edgeR wrapper + It's clunky but this is the only way I could get anything installed into the package_r3 R + + +