annotate edgeR_repenrich2.R @ 2:cfb06f8e8f52 draft

planemo upload for repository https://github.com/ARTbio/tools-artbio/tree/main/tools/repenrich2 commit 4ac07201d6267f5efd5c2af20db7f53fce5af8af

# setup R error handling to go to stderr
options(show.error.messages = FALSE, error = function() {
    cat(geterrmessage(), file = stderr())
    q("no", 1, FALSE)
})

# To not crash galaxy with an UTF8 error with not-US LC settings.
loc <- Sys.setlocale("LC_MESSAGES", "en_US.UTF-8")

# load libraries
library("getopt")
library("tools")
library("rjson")
suppressPackageStartupMessages({
    library("edgeR")
    library("limma")
})

options(stringAsFactors = FALSE, useFancyQuotes = FALSE)

# get options, using the spec as defined by the enclosed list.
spec <- matrix(
    c(
        "quiet", "q", 0, "logical",
        "outfile", "o", 1, "character",
        "countsfile", "n", 1, "character",
        "factorName", "N", 1, "character",
        "levelNameA", "A", 1, "character",
        "levelNameB", "B", 1, "character",
        "levelAfiles", "a", 1, "character",
        "levelBfiles", "b", 1, "character",
        "plots", "p", 1, "character"
    ),
    byrow = TRUE, ncol = 4
)
opt <- getopt(spec)

# build levels A and B file lists
filesA <- fromJSON(opt$levelAfiles, method = "C", unexpected.escape = "error")
filesB <- fromJSON(opt$levelBfiles, method = "C", unexpected.escape = "error")
listA <- list()
indice <- 0
listA[["level"]] <- opt$levelNameA
for (file in filesA) {
    indice <- indice + 1
    listA[[paste0(opt$levelNameA, "_", indice)]] <- read.delim(file, header = FALSE)
}
listB <- list()
indice <- 0
listB[["level"]] <- opt$levelNameB
for (file in filesB) {
    indice <- indice + 1
    listB[[paste0(opt$levelNameB, "_", indice)]] <- read.delim(file, header = FALSE)
}

# build a counts table
counts <- data.frame(row.names = listA[[2]][, 1])
for (element in names(listA[-1])) {
    counts <- cbind(counts, listA[[element]][, 4])
}
for (element in names(listB[-1])) {
    counts <- cbind(counts, listB[[element]][, 4])
}
colnames(counts) <- c(names(listA[-1]), names(listB[-1]))
sizes <- colSums(counts)

# build a meta data object
meta <- data.frame(
    row.names = colnames(counts),
    condition = c(rep(opt$levelNameA, length(filesA)), rep(opt$levelNameB, length(filesB))),
    libsize = sizes
)


# Define the library size and conditions for the GLM
libsize <- meta$libsize
condition <- factor(meta$condition)
design <- model.matrix(~ 0 + condition)
colnames(design) <- levels(condition)

# Build a DGE object for the GLM
y <- DGEList(counts = counts, lib.size = libsize)

# Normalize the data
y <- calcNormFactors(y)

# Estimate the variance
y <- estimateGLMCommonDisp(y, design)
y <- estimateGLMTrendedDisp(y, design)
y <- estimateGLMTagwiseDisp(y, design)

# Builds and outputs an object to contain the normalized read abundance in counts per million of reads
cpm <- cpm(y, log = FALSE, lib.size = libsize)
cpm <- as.data.frame(cpm)
colnames(cpm) <- colnames(counts)
if (!is.null(opt$countsfile)) {
    normalizedAbundance <- data.frame(Tag = rownames(cpm))
    normalizedAbundance <- cbind(normalizedAbundance, cpm)
    write.table(normalizedAbundance, file = opt$countsfile, sep = "\t", col.names = TRUE, row.names = FALSE, quote = FALSE)
}

# Conduct fitting of the GLM
yfit <- glmFit(y, design)

# Initialize result matrices to contain the results of the GLM
results <- matrix(nrow = dim(counts)[1], ncol = 0)
logfc <- matrix(nrow = dim(counts)[1], ncol = 0)

# Make the comparisons for the GLM
my.contrasts <- makeContrasts(
    paste0(opt$levelNameA, "_", opt$levelNameB, " = ", opt$levelNameA, " - ", opt$levelNameB),
    levels = design
)

# Define the contrasts used in the comparisons
allcontrasts <- paste0(opt$levelNameA, " vs ", opt$levelNameB)

# Conduct a for loop that will do the fitting of the GLM for each comparison
# Put the results into the results objects
lrt <- glmLRT(yfit, contrast = my.contrasts[, 1])
res <- topTags(lrt, n = dim(c)[1], sort.by = "none")$table
results <- cbind(results, res[, c(1, 5)])
logfc <- cbind(logfc, res[c(1)])

# Add the repeat types back into the results.
# We should still have the same order as the input data
results$class <- listA[[2]][, 2]
results$type <- listA[[2]][, 3]
# Sort the results table by the FDR
results <- results[with(results, order(FDR)), ]

# Plot Fold Changes for repeat classes and types

# open the device and plots
if (!is.null(opt$plots)) {
    pdf(opt$plots)
    plotMDS(y, main = "Multidimensional Scaling Plot Of Distances Between Samples")
    plotBCV(y, xlab = "Gene abundance (Average log CPM)", main = "Biological Coefficient of Variation Plot")
    logFC <- results[, "logFC"]
    # Plot the repeat classes
    classes <- with(results, reorder(class, -logFC, median))
    classes
    par(mar = c(6, 10, 4, 1))
    boxplot(logFC ~ classes,
        data = results, outline = FALSE, horizontal = TRUE,
        las = 2, xlab = "log2(Fold Change)", ylab = "", cex.axis = 0.7, main = paste0(allcontrasts, ", by Class")
    )
    abline(v = 0)
    # Plot the repeat types
    types <- with(results, reorder(type, -logFC, median))
    boxplot(logFC ~ types,
        data = results, outline = FALSE, horizontal = TRUE,
        las = 2, xlab = "log2(Fold Change)", ylab = "", cex.axis = 0.7, main = paste0(allcontrasts, ", by Type")
    )
    abline(v = 0)
    # volcano plot
    TEdata <- cbind(rownames(results), as.data.frame(results), score = -log(results$FDR, 10))
    colnames(TEdata) <- c("Tag", "log2FC", "FDR", "Class", "Type", "score")
    color <- ifelse(TEdata$FDR < 0.05, "red", "black")
    s <- subset(TEdata, FDR < 0.01)
    with(TEdata, plot(log2FC, score, pch = 20, col = color, main = "Volcano plot (all tag types)", ylab = "-log10(FDR)"))
    text(s[, 2], s[, 6], labels = s[, 5], pos = seq(from = 1, to = 3), cex = 0.5)
}

# close the plot device
if (!is.null(opt$plots)) {
    cat("closing plot device\n")
    dev.off()
}

# Save the results
results <- cbind(TE_item = rownames(results), results)
colnames(results) <- c("TE_item", "log2FC", "FDR", "Class", "Type")
results$log2FC <- format(results$log2FC, digits = 5)
results$FDR <- format(results$FDR, digits = 5)
write.table(results, opt$outfile, quote = FALSE, sep = "\t", col.names = TRUE, row.names = FALSE)