# HG changeset patch # User iuc # Date 1536177256 14400 # Node ID 61dffb20b6f90cc707bd8a6951efbe41db19ecea planemo upload for repository https://github.com/galaxyproject/tools-iuc/tree/master/tools/ruvseq commit b95582cea8320d5488056a9576474f79cec53be8 diff -r 000000000000 -r 61dffb20b6f9 get_deseq_dataset.R --- /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$filename) + labs <- row.names(sampleTable) + names(txiFiles) <- labs + txi <- tximport(txiFiles, type=txtype, tx2gene=tx2gene) + dds <- DESeqDataSetFromTximport(txi, + subset(sampleTable, select=-c(filename)), + designFormula) + } + return(dds) +} diff -r 000000000000 -r 61dffb20b6f9 ruvseq.R --- /dev/null Thu Jan 01 00:00:00 1970 +0000 +++ b/ruvseq.R Wed Sep 05 15:54:16 2018 -0400 @@ -0,0 +1,198 @@ +# setup R error handling to go to stderr +library("getopt") +options( show.error.messages=F, error = function () { cat( geterrmessage(), file=stderr() ); q( "no", 1, F ) } ) +options(stringAsFactors = FALSE, useFancyQuotes = FALSE) + +setup_cmdline_options <- function() { + args <- commandArgs(trailingOnly = TRUE) + spec <- matrix(c( + "help", "h", 0, "logical", + "alpha", "a", 1, "double", + "min_mean_count", "min_c", 1, "double", + "min_k", "min_k", 1, "double", + "max_k", "max_k", 1, "double", + "sample_json", "s", 1, "character", + "plots" , "p", 1, "character", + "header", "H", 0, "logical", + "txtype", "y", 1, "character", + "tx2gene", "x", 1, "character"), # a space-sep tx-to-gene map or GTF file (auto detect .gtf/.GTF) + byrow=TRUE, ncol=4) + + opt <- getopt(spec) + # if help was asked for print a friendly message + # and exit with a non-zero error code + if (!is.null(opt$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() diff -r 000000000000 -r 61dffb20b6f9 ruvseq.xml --- /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 @@ + + from RNA-seq data + + bioconductor-ruvseq + bioconductor-deseq2 + bioconductor-tximport + bioconductor-genomicfeatures + r-ggrepel + + + + + + + /dev/null | grep -v -i "WARNING: ") + ]]> + + + +#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) + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + pdf == True + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + 10.1038/nbt.2931 + + diff -r 000000000000 -r 61dffb20b6f9 test-data/GSM461176_untreat_single.counts --- /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 @@ -0,0 +1,4005 @@ +GeneID untreat1 +FBgn0000003 0 +FBgn0000008 15 +FBgn0000014 0 +FBgn0000015 0 +FBgn0000017 1227 +FBgn0000018 92 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test-data/sailfish/sailfish_quant.sf1.tab --- /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 diff -r 000000000000 -r 61dffb20b6f9 test-data/sailfish/sailfish_quant.sf2.tab --- /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 diff -r 000000000000 -r 61dffb20b6f9 test-data/sailfish/sailfish_quant.sf3.tab --- /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 diff -r 000000000000 -r 61dffb20b6f9 test-data/sailfish/sailfish_quant.sf4.tab --- /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 diff -r 000000000000 -r 61dffb20b6f9 test-data/sailfish/sailfish_quant.sf5.tab --- /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 diff -r 000000000000 -r 61dffb20b6f9 test-data/sailfish/sailfish_quant.sf6.tab --- /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 diff -r 000000000000 -r 61dffb20b6f9 test-data/tx2gene.tab --- /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