changeset 0:61dffb20b6f9 draft

planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/ruvseq commit b95582cea8320d5488056a9576474f79cec53be8
author iuc
date Wed, 05 Sep 2018 15:54:16 -0400
parents
children c24765926774
files get_deseq_dataset.R ruvseq.R ruvseq.xml test-data/GSM461176_untreat_single.counts test-data/GSM461176_untreat_single.counts.noheader test-data/GSM461177_untreat_paired.counts test-data/GSM461177_untreat_paired.counts.noheader test-data/GSM461178_untreat_paired.counts test-data/GSM461178_untreat_paired.counts.noheader test-data/GSM461179_treat_single.counts test-data/GSM461179_treat_single.counts.noheader test-data/GSM461180_treat_paired.counts test-data/GSM461180_treat_paired.counts.noheader test-data/GSM461181_treat_paired.counts test-data/GSM461181_treat_paired.counts.noheader test-data/GSM461182_untreat_single.counts test-data/GSM461182_untreat_single.counts.noheader test-data/ruvseq_diag.pdf test-data/ruvseq_diag_sailfish.pdf test-data/sailfish/sailfish_quant.sf1.tab test-data/sailfish/sailfish_quant.sf2.tab test-data/sailfish/sailfish_quant.sf3.tab test-data/sailfish/sailfish_quant.sf4.tab test-data/sailfish/sailfish_quant.sf5.tab test-data/sailfish/sailfish_quant.sf6.tab test-data/tx2gene.tab
diffstat 26 files changed, 56749 insertions(+), 0 deletions(-) [+]
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/get_deseq_dataset.R	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,69 @@
+get_deseq_dataset <- function(sampleTable, header, designFormula, tximport, txtype, tx2gene) {
+
+  dir <- ""
+
+  if (!is.null(header)) {
+    hasHeader <- TRUE
+  } else {
+    hasHeader <- FALSE
+  }
+
+  if (!is.null(tximport)) {
+    if (is.null(tx2gene)) stop("A transcript-to-gene map or a GTF file is required for tximport")
+    if (tolower(file_ext(opt$tx2gene)) == "gtf") {
+      gtfFile <-tx2gene
+    } else {
+      gtfFile <- NULL
+      tx2gene <- read.table(tx2gene, header=FALSE)
+    }
+    useTXI <- TRUE
+  } else {
+    useTXI <- FALSE
+  }
+
+  if (!useTXI & hasHeader) {
+      countfiles <- lapply(as.character(sampleTable$filename), function(x){read.delim(x, row.names=1)})
+      tbl <- do.call("cbind", countfiles)
+      colnames(tbl) <- rownames(sampleTable) # take sample ids from header
+
+      # check for htseq report lines (from DESeqDataSetFromHTSeqCount function)
+      oldSpecialNames <- c("no_feature", "ambiguous", "too_low_aQual",
+          "not_aligned", "alignment_not_unique")
+      specialRows <- (substr(rownames(tbl), 1, 1) == "_") | rownames(tbl) %in% oldSpecialNames
+      tbl <- tbl[!specialRows, , drop = FALSE]
+
+      dds <- DESeqDataSetFromMatrix(countData = tbl,
+                                    colData = subset(sampleTable, select=-(filename)),
+                                    design = designFormula)
+  } else if (!useTXI & !hasHeader) {
+
+    # construct the object from HTSeq files
+    dds <- DESeqDataSetFromHTSeqCount(sampleTable = sampleTable,
+                                      directory = dir,
+                                      design =  designFormula)
+    colnames(dds) <- row.names(sampleTable)
+
+  } else {
+      # construct the object using tximport
+      # first need to make the tx2gene table
+      # this takes ~2-3 minutes using Bioconductor functions
+      if (!is.null(gtfFile)) {
+        suppressPackageStartupMessages({
+          library("GenomicFeatures")
+        })
+        txdb <- makeTxDbFromGFF(gtfFile, format="gtf")
+        k <- keys(txdb, keytype = "GENEID")
+        df <- select(txdb, keys = k, keytype = "GENEID", columns = "TXNAME")
+        tx2gene <- df[, 2:1]  # tx ID, then gene ID
+      }
+      library("tximport")
+      txiFiles <- as.character(sampleTable$filename)
+      labs <- row.names(sampleTable)
+      names(txiFiles) <- labs
+      txi <- tximport(txiFiles, type=txtype, tx2gene=tx2gene)
+      dds <- DESeqDataSetFromTximport(txi,
+                                      subset(sampleTable, select=-c(filename)),
+                                      designFormula)
+  }
+  return(dds)
+}
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/ruvseq.R	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,198 @@
+# setup R error handling to go to stderr
+library("getopt")
+options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } )
+options(stringAsFactors = FALSE, useFancyQuotes = FALSE)
+
+setup_cmdline_options <- function() {
+  args <- commandArgs(trailingOnly = TRUE)
+  spec <- matrix(c(
+    "help", "h", 0, "logical",
+    "alpha", "a", 1, "double",
+    "min_mean_count", "min_c", 1, "double",
+    "min_k", "min_k", 1, "double",
+    "max_k", "max_k", 1, "double",
+    "sample_json", "s", 1, "character",
+    "plots" , "p", 1, "character",
+    "header", "H", 0, "logical",
+    "txtype", "y", 1, "character",
+    "tx2gene", "x", 1, "character"), # a space-sep tx-to-gene map or GTF file (auto detect .gtf/.GTF)
+    byrow=TRUE, ncol=4)
+
+  opt <- getopt(spec)
+  # if help was asked for print a friendly message
+  # and exit with a non-zero error code
+  if (!is.null(opt$help)) {
+    cat(getopt(spec, usage=TRUE))
+    q(status=1)
+  } else {
+    load_libraries()
+  }
+  return(opt)
+}
+
+load_libraries <- function() {
+  # Allows displaying help without waiting for libraries to load
+  library("tools")
+  library("jsonlite")
+  library("reshape2")
+  library("RUVSeq")
+  library("RColorBrewer")
+  library("tximport")
+  library("DESeq2")
+  library("ggrepel")
+}
+
+source_local <- function(fname){
+    argv <- commandArgs(trailingOnly = FALSE)
+    base_dir <- dirname(substring(argv[grep("--file=", argv)], 8))
+    source(paste(base_dir, fname, sep="/"))
+}
+
+# Source get_deseq_dataset.R for getting deseq dataset from htseq/featurecounts/tximport
+source_local('get_deseq_dataset.R')
+
+# RUVseq function definitions
+
+plot_pca_rle <- function (set, title) {
+  x <- pData(set)[,1]
+  colors <- brewer.pal(3, "Set2")
+  label <- paste0(' for ', title)
+  plotRLE(set, outline=FALSE, ylim=c(-4, 4), col=colors[x])
+  title(main=paste0("RLE", label))
+  plotPCA(set, col=colors[x], cex=1.2)
+  title(main=paste0("PCA", label))
+}
+
+plot_factors_of_unwanted_variation <- function(set, method, k){
+  pd <- pData(set)
+  pd['sample'] <- row.names(pd)
+  colnames(pd)[1] <- 'condition'
+  d = melt(pd, id.vars = c('sample', 'condition'))
+  d['x'] <- 1  # There is no information on the X, so we just fake it to be able to do a scatterplot
+  print(ggplot(d, aes(x=x, y=value, color=condition, label=sample)) +
+  geom_point() +
+  ggtitle(paste0('Factors of unwanted variation for method: ', method, ", k=", k)) +
+  facet_wrap( ~ variable, scales = "free_x") +
+  geom_text_repel() +
+  theme(axis.title.x=element_blank(),
+        axis.text.x=element_blank(),
+        axis.ticks.x=element_blank(),
+        plot.title = element_text(hjust = 0.5))
+  )
+}
+
+create_seq_expression_set <- function (dds, min_mean_count) {
+  count_values <- counts(dds)
+  filter <- apply(count_values, 1, function(x) mean(x) > min_mean_count)
+  filtered <- count_values[filter,]
+  set = newSeqExpressionSet(as.matrix(count_values),
+                            phenoData = data.frame(colData(dds)$condition, row.names=colnames(filtered)))
+  plot_pca_rle(set = set, title = 'raw data')
+  set <- betweenLaneNormalization(set, which="upper")
+  plot_pca_rle(set = set, title = 'upper quartile normalized')
+  return(set)
+}
+
+get_empirical_control_genes <- function(set, cutoff_p) {
+  x <- pData(set)[,1]
+  design <- model.matrix(~x, data=pData(set))
+  y <- DGEList(counts=counts(set), group=x)
+  y <- calcNormFactors(y, method="upperquartile")
+  y <- estimateGLMCommonDisp(y, design)
+  y <- estimateGLMTagwiseDisp(y, design)
+  fit <- glmFit(y, design)
+  lrt <- glmLRT(fit, coef=2)
+  top <- topTags(lrt, n=nrow(set))$table
+  top_rows <- rownames(top)[which(top$PValue > cutoff_p)]
+  empirical <- rownames(set)[which(!(rownames(set) %in% top_rows))]
+  return(empirical)
+}
+
+ruv_control_gene_method <- function(set, k, control_genes='empirical', cutoff_p=0.2) {
+  if (control_genes == 'empirical') {
+    control_genes = get_empirical_control_genes(set, cutoff_p=cutoff_p)
+  }
+  set <- RUVg(set, control_genes, k=k)
+  plot_pca_rle(set, paste0("RUVg with empirical control genes, k=", k))
+  plot_factors_of_unwanted_variation(set, method="RUVg with empirical control genes", k=k)
+  return(set)
+}
+
+ruv_residual_method <- function(set, k) {
+  genes <- rownames(counts(set))
+  x <- pData(set)[,1]
+  # Initial edger residuals
+  design <- model.matrix(~x, data=pData(set))
+  y <- DGEList(counts=counts(set), group=x)
+  y <- calcNormFactors(y, method="upperquartile")
+  y <- estimateGLMCommonDisp(y, design)
+  y <- estimateGLMTagwiseDisp(y, design)
+  fit <- glmFit(y, design)
+  res <- residuals(fit, type="deviance")
+  set <- RUVr(set, genes, k=k, res)
+  plot_pca_rle(set = set, title = paste0('RUVr using residuals, k=', k))
+  plot_factors_of_unwanted_variation(set, method="RUVr using residuals", k=k)
+  return(set)
+}
+
+ruv_replicate_method <- function (set, k) {
+  genes <- rownames(counts(set))
+  x <- pData(set)[,1]
+  differences <- makeGroups(x)
+  set <- RUVs(set, genes, k=k, differences)
+  plot_pca_rle(set, paste0('RUVs with replicate samples, k=', k))
+  plot_factors_of_unwanted_variation(set, method="RUVs using replicates", k=k)
+  return(set)
+}
+
+get_differentially_expressed_genes <- function(dds, contrast, alpha=0.01) {
+  r <- results(dds, contrast=contrast, alpha=alpha)
+  return(rownames(r[which(r$padj < alpha),]))
+}
+
+opt <- setup_cmdline_options()
+alpha <- opt$alpha
+min_k <- opt$min_k
+max_k <- opt$max_k
+sample_json <- fromJSON(opt$sample_json)
+sample_paths <- sample_json$path
+sample_names <- sample_json$label
+condition <- as.factor(sample_json$condition)
+sampleTable <- data.frame(samplename=sample_names,
+                          filename = sample_paths,
+                          condition=condition)
+rownames(sampleTable) <- sample_names
+
+dds <- get_deseq_dataset(sampleTable, header=opt$header, designFormula= ~ condition, tximport=opt$txtype, txtype=opt$txtype, tx2gene=opt$tx2gene)
+if (!is.null(opt$plots)) {
+  pdf(opt$plots)
+}
+
+# Run through the ruvseq variants
+set <- create_seq_expression_set(dds, min_mean_count = opt$min_mean_count)
+result <- list(no_correction = set)
+for (k in seq(min_k, max_k)) {
+  result[[paste0('residual_method_k', k)]] <- ruv_residual_method(set, k=k)
+  result[[paste0('replicate_method_k', k)]] <- ruv_replicate_method(set, k=k)
+  result[[paste0('control_method_k', k)]] <- ruv_control_gene_method(set, k=k, cutoff_p=0.5)
+}
+
+for (name in names(result)) {
+  if (!startsWith(name, "no_correction")) {
+    set <- result[[name]]
+    unwanted_variation <- pData(set)
+    df <- data.frame(identifier = rownames(unwanted_variation))
+    df <- cbind(df, unwanted_variation)
+    colnames(df)[2] <- 'condition'
+    write.table(df, file=paste0("batch_effects_", name, ".tabular"),  sep="\t", quote=F, row.names=F)
+  }
+}
+
+# close the plot device
+if (!is.null(opt$plots)) {
+  cat("closing plot device\n")
+  dev.off()
+}
+
+cat("Session information:\n\n")
+sessionInfo()
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/ruvseq.xml	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,307 @@
+<tool id="ruvseq" name="Remove Unwanted Variation" version="1.12.0">
+    <description>from RNA-seq data</description>
+    <requirements>
+        <requirement type="package" version="1.12.0">bioconductor-ruvseq</requirement>
+        <requirement type="package" version="1.18.1">bioconductor-deseq2</requirement>
+        <requirement type="package" version="1.6.0">bioconductor-tximport</requirement>
+        <requirement type="package" version="1.30.0">bioconductor-genomicfeatures</requirement>
+        <requirement type="package" version="0.6.5">r-ggrepel</requirement>
+    </requirements>
+    <stdio>
+        <regex match="Execution halted"
+           source="both"
+           level="fatal"
+           description="Execution halted." />
+        <regex match="Error in"
+           source="both"
+           level="fatal"
+           description="An undefined error occurred, please check your input carefully and contact your administrator." />
+        <regex match="Fatal error"
+           source="both"
+           level="fatal"
+           description="An undefined error occurred, please check your input carefully and contact your administrator." />
+    </stdio>
+    <version_command><![CDATA[
+echo $(R --version | grep version | grep -v GNU)", RUVSeq version" $(R --vanilla --slave -e "library(RUVSeq); cat(sessionInfo()\$otherPkgs\$RUVSeq\$Version)" 2> /dev/null | grep -v -i "WARNING: ")
+    ]]></version_command>
+    <command><![CDATA[
+#if $tximport.tximport_selector == 'tximport':
+    #if $tximport.mapping_format.mapping_format_selector == 'gtf':
+        ln -s '$tximport.mapping_format.gtf_file' mapping.gtf &&
+    #else:
+        ln -s '$tximport.mapping_format.tabular_file' mapping.txt &&
+    #end if
+#end if
+
+Rscript '${__tool_directory__}/ruvseq.R'
+#if $pdf:
+    -p '$plots'
+#end if
+
+--sample_json '$sampleTable'
+
+$header
+
+--min_k $min_k
+--max_k $max_k
+
+#if $tximport.tximport_selector == 'tximport':
+    --txtype $tximport.txtype
+    #if $tximport.mapping_format.mapping_format_selector == 'gtf':
+        --tx2gene mapping.gtf
+    #else:
+        --tx2gene mapping.txt
+    #end if
+#end if
+]]></command>
+    <configfiles>
+        <configfile name="sampleTable">
+#import json
+#set sample_table = []
+#for $level in $rep_factorLevel:
+    #for $file in $level.countsFile:
+        #silent $sample_table.append({"path": str($file), "label": str($file.element_identifier), "condition": str($level.factorLevel)})
+    #end for
+#end for
+#echo json.dumps($sample_table)
+        </configfile>
+    </configfiles>
+    <inputs>
+        <repeat name="rep_factorLevel" title="Factor level" min="2" default="2">
+            <param name="factorLevel" type="text" value="FactorLevel" label="Specify a factor level, typical values could be 'tumor', 'normal', 'treated' or 'control'"
+                help="Only letters, numbers and underscores will be retained in this field">
+                <sanitizer>
+                    <valid initial="string.letters,string.digits"><add value="_" /></valid>
+                </sanitizer>
+            </param>
+            <param name="countsFile" type="data" format="tabular" multiple="true" label="Counts file(s)"/>
+        </repeat>
+        <param name="min_k" type="integer" value="1" min="1" label="Try to find at least this many factors of unwanted variation" />
+        <param name="max_k" type="integer" value="1" min="1" label="Try to find at most this many factors of unwanted variation" />
+        <param name="min_mean_count" type="integer" value="5" min="0" label="Ignore genes with fewer than this many counts on average" />
+        <param name="header" type="boolean" truevalue="-H" falsevalue="" checked="true" label="Files have header?" help="If this option is set to Yes, the tool will assume that the count files have column headers in the first row. Default: Yes" />
+
+        <conditional name="tximport">
+            <param name="tximport_selector" type="select" label="Choice of Input data">
+                <option value="count" selected="True">Count data (e.g. from HTSeq-count, featureCounts or StringTie)</option>
+                <option value="tximport">TPM values (e.g. from kallisto, sailfish or salmon)</option>
+            </param>
+            <when value="tximport">
+                <param name="txtype" type="select" label="Program used to generate TPMs">
+                    <option value="kallisto">kallisto</option>
+                    <option value="sailfish">Sailfish</option>
+                    <option value="salmon">Salmon</option>
+                </param>
+                <conditional name="mapping_format">
+                    <param name="mapping_format_selector" type="select" label="Gene mapping format">
+                        <option value="gtf" selected="True">GTF</option>
+                        <option value="tabular">Transcript-ID and Gene-ID mapping file</option>
+                    </param>
+                    <when value="gtf">
+                        <param name="gtf_file" type="data" format="gtf,gff3" label="GTF/GFF3 file with Transcript - Gene mapping"/>
+                    </when>
+                    <when value="tabular">
+                        <param name="tabular_file" type="data" format="tabular" label="Tabular file with Transcript - Gene mapping"/>
+                    </when>
+                </conditional>
+            </when>
+            <when value="count" />
+        </conditional>
+        <param name="pdf" type="boolean" truevalue="1" falsevalue="0" checked="true"
+            label="Visualising the analysis results"
+            help="output an additional PDF files" />
+    </inputs>
+    <outputs>
+        <collection name="unwanted_variation" type="list" label="RUVSeq covariate files on ${on_string}">
+            <discover_datasets pattern="(?P&lt;designation&gt;.+)\.tabular" format="tabular" directory="." visible="false"/>
+        </collection>
+        <data format="pdf" name="plots" label="RUVSeq diagonstic plots on ${on_string}">
+            <filter>pdf == True</filter>
+        </data>
+    </outputs>
+    <tests>
+        <!--Ensure counts files with header works -->
+        <test>
+            <repeat name="rep_factorLevel">
+                <param name="factorLevel" value="Treated"/>
+                <param name="countsFile" value="GSM461179_treat_single.counts,GSM461180_treat_paired.counts,GSM461181_treat_paired.counts"/>
+            </repeat>
+            <repeat name="rep_factorLevel">
+                <param name="factorLevel" value="Untreated"/>
+                <param name="countsFile" value="GSM461176_untreat_single.counts,GSM461177_untreat_paired.counts,GSM461178_untreat_paired.counts,GSM461182_untreat_single.counts"/>
+            </repeat>
+            <param name="pdf" value="true"/>
+            <output name="plots" file="ruvseq_diag.pdf" ftype="pdf" compare="sim_size"/>
+            <output_collection name="unwanted_variation" type="list">
+                <element name="batch_effects_control_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="GSM461179.*\tTreated\t-0.49.*"/>
+                    </assert_contents>
+                </element>
+                <element name="batch_effects_replicate_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="GSM461179.*\tTreated\t-0.25.*"/>
+                    </assert_contents>
+                </element>
+                <element name="batch_effects_residual_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="GSM461179.*\tTreated\t-0.60.*"/>
+                    </assert_contents>
+                </element>
+            </output_collection>
+        </test>
+        <!--Ensure counts files without header works -->
+        <test>
+            <repeat name="rep_factorLevel">
+                <param name="factorLevel" value="Treated"/>
+                <param name="countsFile" value="GSM461179_treat_single.counts.noheader,GSM461180_treat_paired.counts.noheader,GSM461181_treat_paired.counts.noheader"/>
+            </repeat>
+            <repeat name="rep_factorLevel">
+                <param name="factorLevel" value="Untreated"/>
+                <param name="countsFile" value="GSM461176_untreat_single.counts.noheader,GSM461177_untreat_paired.counts.noheader,GSM461178_untreat_paired.counts.noheader,GSM461182_untreat_single.counts.noheader"/>
+            </repeat>
+            <param name="pdf" value="true"/>
+            <param name="header" value="false"/>
+            <output name="plots" file="ruvseq_diag.pdf" ftype="pdf" compare="sim_size"/>
+            <output_collection name="unwanted_variation" type="list">
+                <element name="batch_effects_control_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="GSM461179.*\tTreated\t-0.49.*"/>
+                    </assert_contents>
+                </element>
+                <element name="batch_effects_replicate_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="GSM461179.*\tTreated\t-0.25.*"/>
+                    </assert_contents>
+                </element>
+                <element name="batch_effects_residual_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="GSM461179.*\tTreated\t-0.60.*"/>
+                    </assert_contents>
+                </element>
+            </output_collection>
+        </test>
+        <!--Ensure sailfish files work -->
+        <test>
+            <repeat name="rep_factorLevel">
+                <param name="factorLevel" value="Treated"/>
+                <param name="countsFile" value="sailfish/sailfish_quant.sf1.tab,sailfish/sailfish_quant.sf2.tab,sailfish/sailfish_quant.sf3.tab"/>
+            </repeat>
+            <repeat name="rep_factorLevel">
+                <param name="factorLevel" value="Untreated"/>
+                    <param name="countsFile" value="sailfish/sailfish_quant.sf4.tab,sailfish/sailfish_quant.sf5.tab,sailfish/sailfish_quant.sf6.tab"/>
+            </repeat>
+            <param name="pdf" value="true"/>
+            <param name="tximport_selector" value="tximport"/>
+            <param name="txtype" value="sailfish"/>
+            <param name="mapping_format_selector" value="tabular"/>
+            <param name="tabular_file" value="tx2gene.tab"/>
+            <output name="plots" file="ruvseq_diag_sailfish.pdf" ftype="pdf" compare="sim_size"/>
+            <output_collection name="unwanted_variation" type="list">
+                <element name="batch_effects_control_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="sailfish_quant.sf1.tab\tTreated\t-0.28.*"/>
+                    </assert_contents>
+                </element>
+                <element name="batch_effects_replicate_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="sailfish_quant.sf1.tab\tTreated\t-0.44.*"/>
+                    </assert_contents>
+                </element>
+                <element name="batch_effects_residual_method_k1">
+                    <assert_contents>
+                        <has_text_matching expression="identifier\tcondition\tW_1"/>
+                        <has_text_matching expression="sailfish_quant.sf1.tab\tTreated\t-0.22.*"/>
+                    </assert_contents>
+                </element>
+            </output_collection>
+        </test>
+    </tests>
+    <help><![CDATA[
+.. class:: infomark
+
+**What it does**
+
+RUVSeq normalizes RNA-seq data using factor analysis of control genes or samples. RUVSeq has been designed for detecting unwanted variation using replicate sample information. The current RUVSeq Galaxy tool only implements estimating unwanted variation for primary factors.
+RUVSeq implements 3 different methods for the estimation of unwanted variation:
+
+RUVg estimates the factors of unwanted variation using control genes
+
+RUVs estimates the factors of unwanted variation using replicate samples
+
+RUVr estimating the factors of unwanted variation using residuals.
+
+This tool runs all RUV methods and outputs diagnostic plots and tables with covariates that
+may be used for differential expression analsys.
+
+-----
+
+**Inputs**
+
+**Count Files**
+
+RUVSeq_ takes count tables generated from **featureCounts**, **HTSeq-count** or **StringTie** as input. Count tables must be generated for each sample individually. One header row is assumed, but files with no header (e.g from HTSeq) can be input with the *Files have header?* option set to No.
+
+RUVSeq_ can also take transcript-level counts from quantification tools such as, **kallisto**, **Salmon** and **Sailfish**, and this Galaxy wrapper incorporates the Bioconductor tximport_ package to process the transcript counts for DESeq2.
+
+**Salmon or Sailfish Files**
+
+Salmon or Sailfish ``quant.sf`` files can be imported by setting type to *Salmon* or *Sailfish* respectively above. Note: for previous version of Salmon or Sailfish, in which the quant.sf files start with comment lines you will need to remove the comment lines before inputting here. An example of the format is shown below.
+
+Example:
+
+============ ========== =============== =========== ===========
+Name         Length     EffectiveLength TPM         NumReads
+------------ ---------- --------------- ----------- -----------
+NR_001526    164        20.4518         0           0
+NR_001526_1  164        20.4518         0           0
+NR_001526_2  164        20.4518         0           0
+NM_130786    1764       1956.04         2.47415     109.165
+NR_015380    2129       2139.53         1.77331     85.5821
+NM_001198818 9360       7796.58         2.38616e-07 4.19648e-05
+NM_001198819 9527       7964.62         0           0
+NM_001198820 9410       7855.78         0           0
+NM_014576    9267       7714.88         0.0481114   8.37255
+============ ========== =============== =========== ===========
+
+**kallisto Files**
+
+kallisto ``abundance.tsv`` files can be imported by setting type to *kallisto* above. An example of the format is shown below.
+
+Example:
+
+============ ========== =============== =========== ===========
+target_id    length     eff_length      est_counts  tpm
+------------ ---------- --------------- ----------- -----------
+NR_001526    164        20.4518         0           0
+NR_001526_1  164        20.4518         0           0
+NR_001526_2  164        20.4518         0           0
+NM_130786    1764       1956.04         109.165     2.47415
+NR_015380    2129       2139.53         85.5821     1.77331
+NM_001198818 9360       7796.58         4.19648e-05 2.38616e-07
+NM_001198819 9527       7964.62         0           0
+NM_001198820 9410       7855.78         0           0
+NM_014576    9267       7714.88         8.37255     0.0481114
+============ ========== =============== =========== ===========
+
+-----
+
+**Output**
+
+RUVSeq_ generates a tabular file for each method and each k of variation as well as a summary PDF.
+
+.. _RUVSeq: http://master.bioconductor.org/packages/release/bioc/html/RUVSeq.html
+.. _tximport: https://bioconductor.org/packages/devel/bioc/vignettes/tximport/inst/doc/tximport.html
+    ]]></help>
+    <citations>
+        <citation type="doi">10.1038/nbt.2931</citation>
+    </citations>
+</tool>
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461176_untreat_single.counts	Wed Sep 05 15:54:16 2018 -0400
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461176_untreat_single.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461177_untreat_paired.counts	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4005 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461177_untreat_paired.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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+no_feature	349912
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+alignment_not_unique	13300038
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461178_untreat_paired.counts	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4005 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461178_untreat_paired.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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+too_low_aQual	0
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+alignment_not_unique	11365569
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461179_treat_single.counts	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4005 @@
+GeneID	treat1
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+alignment_not_unique	14421559
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461179_treat_single.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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+alignment_not_unique	14421559
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461180_treat_paired.counts	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4005 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461180_treat_paired.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461181_treat_paired.counts	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4005 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461181_treat_paired.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461182_untreat_single.counts	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4005 @@
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/GSM461182_untreat_single.counts.noheader	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,4004 @@
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+no_feature	165407
+ambiguous	48373
+too_low_aQual	0
+not_aligned	0
+alignment_not_unique	6896814
Binary file test-data/ruvseq_diag.pdf has changed
Binary file test-data/ruvseq_diag_sailfish.pdf has changed
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/sailfish/sailfish_quant.sf1.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+Name	Length	EffectiveLength	TPM	NumReads
+NM_017409	1957	1759.2	0	0
+NM_014212	2035	1837.2	0	0
+NM_173860	847	649.348	0	0
+NM_017410	2395	2555.05	0.0173509	1
+NM_014620	2305	2261.56	0.114436	5.83783
+NM_153633	1665	1561.63	3.55316	125.162
+NM_018953	1610	1412.2	0	0
+NR_003084	1644	1446.2	0	0
+NM_004503	1680	1482.2	0	0
+NM_153693	2077	1879.2	0	0
+NM_022658	2286	2088.2	0	0
+NM_006897	1539	1341.2	0	0
+NM_001168316	2281	2071	0.085625	4
+NM_174914	2383	2153.11	0	0
+NR_031764	1851	1659.15	0	0
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/sailfish/sailfish_quant.sf2.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+Name	Length	EffectiveLength	TPM	NumReads
+NM_017409	1957	1764.64	0	0
+NM_014212	2035	2171.69	0.0161522	1
+NM_173860	847	655.215	0	0
+NM_017410	2395	2567.91	0.01366	1
+NM_014620	2305	2235.74	0	0
+NM_153633	1665	1576.16	1.7359	78
+NM_018953	1610	1726.1	1.31761e-06	6.48367e-05
+NR_003084	1644	1704.36	0.0411608	1.99994
+NM_004503	1680	1679.47	0.10443	5
+NM_153693	2077	2069.83	0	0
+NM_022658	2286	2093.64	0	0
+NM_006897	1539	1557.46	0.0675669	3
+NM_001168316	2281	2088.64	0	0
+NM_174914	2383	2190.64	0	0
+NR_031764	1851	1658.64	0	0
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/sailfish/sailfish_quant.sf3.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+Name	Length	EffectiveLength	TPM	NumReads
+NM_017409	1957	1760.44	0	0
+NM_014212	2035	2127.85	0.0280759	2
+NM_173860	847	650.573	0	0
+NM_017410	2395	2198.44	0	0
+NM_014620	2305	2223.92	0.0661957	4.92838
+NM_153633	1665	1536.71	2.08127	107.072
+NM_018953	1610	1685.74	1.23894e-08	6.99193e-07
+NR_003084	1644	1658.72	0.0720329	4
+NM_004503	1680	1638.79	0.0546819	3
+NM_153693	2077	2026.99	0	0
+NM_022658	2286	2089.44	0	0
+NM_006897	1539	1342.44	0	0
+NM_001168316	2281	2047.58	0	0
+NM_174914	2383	2143.36	0	0
+NR_031764	1851	1659.97	0.0359895	2
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/sailfish/sailfish_quant.sf4.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+Name	Length	EffectiveLength	TPM	NumReads
+NM_017409	1957	1738.38	0	0
+NM_014212	2035	1816.38	0	0
+NM_173860	847	628.444	0	0
+NM_017410	2395	2176.38	0	0
+NM_014620	2305	2086.38	0	0
+NM_153633	1665	1510.43	2.31417	89.0334
+NM_018953	1610	1391.38	0	0
+NR_003084	1644	1425.38	0	0
+NM_004503	1680	1612.63	0.0730348	3
+NM_153693	2077	1998.27	0	0
+NM_022658	2286	2067.38	0	0
+NM_006897	1539	1320.38	0	0
+NM_001168316	2281	2033.58	0	0
+NM_174914	2383	2129.01	0	0
+NR_031764	1851	1639.11	0.071855	3
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/sailfish/sailfish_quant.sf5.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+Name	Length	EffectiveLength	TPM	NumReads
+NM_017409	1957	1781.23	0	0
+NM_014212	2035	1859.23	0	0
+NM_173860	847	671.305	0	0
+NM_017410	2395	2219.23	0	0
+NM_014620	2305	2195.37	0	0
+NM_153633	1665	1545.8	3.39643	119.002
+NM_018953	1610	1715.91	0.128558	5
+NR_003084	1644	1468.23	0	0
+NM_004503	1680	1654.16	0.0800141	3
+NM_153693	2077	2034.63	0	0
+NM_022658	2286	2148.18	0.0205378	1
+NM_006897	1539	1363.23	0	0
+NM_001168316	2281	2061.52	0	0
+NM_174914	2383	2156.64	0	0
+NR_031764	1851	1671.47	0.0527904	2
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/sailfish/sailfish_quant.sf6.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+Name	Length	EffectiveLength	TPM	NumReads
+NM_017409	1957	1747.9	0	0
+NM_014212	2035	1825.9	0	0
+NM_173860	847	638.159	0	0
+NM_017410	2395	2562.99	0.0171188	1
+NM_014620	2305	2272.12	0.152979	7.92213
+NM_153633	1665	1572.52	3.54659	127.112
+NM_018953	1610	1731.28	0.0760279	3
+NR_003084	1644	1434.9	0	0
+NM_004503	1680	1673.79	0.104853	4
+NM_153693	2077	2057.73	0	0
+NM_022658	2286	2185.1	0.0200793	1
+NM_006897	1539	1329.9	0	0
+NM_001168316	2281	2047.55	0	0
+NM_174914	2383	2143.62	0	0
+NR_031764	1851	1663.05	0.0263825	1
--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/test-data/tx2gene.tab	Wed Sep 05 15:54:16 2018 -0400
@@ -0,0 +1,16 @@
+TXNAME	GENEID
+NM_001168316	DDX11L1
+NM_174914	DDX11L1
+NR_031764	DDX11L1
+NM_004503	WASH7P
+NM_006897	WASH7P
+NM_014212	WASH7P
+NM_014620	WASH7P
+NM_017409	WASH7P
+NM_017410	WASH7P
+NM_018953	MIR6859-2
+NM_022658	MIR6859-1
+NM_153633	WASH7P
+NM_153693	WASH7P
+NM_173860	WASH7P
+NR_003084	WASH7P