R/ViewControls.R

In OscarBrock/gCrisprTools: Suite of Functions for Pooled Crispr Screen QC and Analysis

##' @title View nontargeting guides within an experiment
##' @description This function tries to identify, and then plot the abundance of, the full set of non-targeting controls from an ExpressionSet
##' object. Ideally, the user will supply a geneSymbol present in the appropriate annotation file that uniquely identifies the nontargeting gRNAs.
##' Absent this, the the function will search for common identifier used by nontargeting controls (geneID 'no_gid', or geneSymbol NA).
##' @param eset An ExpressionSet  object containing, at minimum, a matrix of gRNA abundances extractable with the \code{exprs} function.
##' @param annotation An annotation data.frame for the experiment. gRNAs are annotated by
##' row, and must minimally contain columns \code{geneSymbol} and \code{geneID}.
##' @param sampleKey A sample key, supplied as an ordered factor linking the samples to experimental
##' variables. The \code{names} attribute should exactly match those present in \code{eset}, and the control condition is assumed to be
##' the first \code{level}.
##' @param geneSymb The \code{geneSymbol} identifier in \code{annotation} that corresponds to nontargeting gRNAs. If absent, \code{ct.ViewControls} will
##' attempt to infer nontargeting guides by searching for \code{'no_gid'} or \code{NA} in the appropriate columns.
##' @param normalize Logical indicating whether to attempt to normalize the data in the \code{eset} by DESeq size factors present in the metadata. If \code{TRUE},
##' then the metadata must contain a column containing these factors, named \code{sizeFactor.crispr-gRNA}.
##' @param lib.size An optional vector of voom-appropriate library size adjustment factors, usually calculated with \code{\link[edgeR]{calcNormFactors}} 
##' and transformed to reflect the appropriate library size. These adjustment factors are interpreted as the total library sizes for each sample, 
##' and if absent will be extrapolated from the columnwise count sums of the \code{exprs} slot of the \code{eset}.
##' @return An image of nontargeting control gRNA abundances on the default device.
##' @author Russell Bainer
##' @examples 
##' data('es')
##' data('ann')
##' 
##' #Build the sample key
##' library(Biobase)
##' sk <- ordered(relevel(as.factor(pData(es)$TREATMENT_NAME), 'ControlReference'))
##' names(sk) <- row.names(pData(es))
##' 
##' ct.viewControls(es, ann, sk, geneSymb = NULL, normalize = FALSE)
##' ct.viewControls(es, ann, sk, geneSymb = NULL, normalize = TRUE)
##' @export

ct.viewControls <- function(eset, annotation, sampleKey, geneSymb = NULL, normalize = TRUE, lib.size = NULL) {
    # current.graphic.params <- par(no.readonly = TRUE) on.exit(suppressWarnings(par(current.graphic.params)))

    annotation <- ct.prepareAnnotation(annotation, object = eset, throw.error = FALSE)
    sampleKey <- ct.keyCheck(sampleKey, eset)

    if (is.null(lib.size)) {
        lib.size <- colSums(exprs(eset))
    } else if (!is.numeric(lib.size) | (length(lib.size) != ncol(eset))) {
        stop("If specified, lib.size must be a numeric vector of length equal to the number of samples.")
    }

    # Find the row.names that correspond to the guides.
    if (!is.null(geneSymb)) {
        if (!(geneSymb %in% annotation$geneSymbol)) {
            geneSymb <- NULL
        }
    }

    if (is.null(geneSymb)) {
        message("No control geneSymbol supplied, so I'll use the default of 'NoTarget'.")
        ntc <- row.names(annotation)[annotation$geneSymbol == "NoTarget"]
    } else {
        ntc <- row.names(annotation)[annotation$geneSymbol == geneSymb]
    }

    if (length(ntc) < 1) {
        stop("No controls detected.")
    }

    colorSpace <- colorRampPalette(c("lightblue", "darkred"))(length(ntc))
    plottitle <- paste0(geneSymb, " Guide Abundance (Raw Reads)")

    if (normalize) {
        message("Normalizing gRNAs by median scaling.")
        if (is.null(annotation)) {
            eset <- ct.normalizeGuides(eset, "scale", sampleKey = sampleKey, lib.size = lib.size, plot.it = FALSE)
        } else {
            eset <- ct.normalizeGuides(eset, "scale", annotation = annotation, sampleKey = sampleKey, lib.size = lib.size, plot.it = FALSE)
        }
        plottitle <- paste0(geneSymb, " Guide Abundance (Median Scaled)")
    }

    counts <- exprs(eset)[ntc, names(sampleKey)[order(sampleKey)]]
    counts <- (log2(t(counts + 0.5)/(lib.size + 1) * 1e+06))
    counts <- counts[, order(colMeans(counts))]

    # Set up and draw the plot
    ylimit <- range(counts) + c(-1, 1)
    xlimit <- nrow(counts)

    par(mar = c(10, 4, 4, 6), xpd = TRUE)
    plot(seq_len(xlimit), counts[, 1], main = plottitle, ylab = "Log2 Normalized Counts", xlab = "", xaxt = "n", xlim = c(1, xlimit), ylim = ylimit, type = "l", lwd = 2, 
        col = colorSpace[1])
    for (q in 2:ncol(counts)) {
        lines(seq_len(xlimit), counts[, q], lwd = 2, col = colorSpace[q])
    }
    axis(side = 1, labels = sampleKey[order(sampleKey)], at = seq_len(xlimit), las = 3)
    legend(xlimit + 1, ylimit[2], legend = colnames(counts), fill = colorSpace, cex = 0.5)
}