R/StackedGuides.R

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

##' @title View a stacked representation of the most variable targets or individual guides within an experiment, 
##' as a percentage of the total aligned reads
##' @description This function identifies the gRNAs or targets that change the most from sample to sample within an experiment as a percentage of 
##' the entire library. It then plots the abundance of the top \code{nguides} as a stacked barplot for all samples in the experiment. The purpose of this 
##' algorithm is to detect potential distortions in the library composition that might not be properly controlled by sample normalization, and so 
##' the most variable entites are defined by calculating the percent of aligned reads that they contribute to each sample, and then ranking each entity
##' by the range of these percentages across all samples. Consequently, gRNAs or Targets that are highly abundant in at least one condition will be 
##' are more likely to be identified. 
##' @param eset An ExpressionSet  object containing, at minimum, a matrix of gRNA abundances extractable with the exprs() function, and a metadata 
##' object containing a column named \code{SAMPLE_LABEL} containing unique identifers for each sample. The \code{colnames} should be syntactically 
##' @param sampleKey An optional 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 set is assumed to be 
##' the first \code{level}.
##' @param nguides The number of guides (or targets) to display.    
##' @param plotType A string indicating whether the individual guides should be displayed ('\code{gRNA}'), or if they should be aggregated into target-level
##' estimates ('\code{Target}') according to the \code{geneSymbol} column in the \code{annotation} object.  
##' @param annotation An optional data.frame containing an annotation object to be used to aggregate the guides into targets. gRNAs are annotated by row, 
##' and must minimally contain a column \code{geneSymbol} indicating the target elements.
##' @param ylimit An optional numeric vector of length 2 specifying the y limits for the plot, useful in comparin across studies. 
##' @param subset An optional character vector containing the sample labels to be used in the analysis; all elements must be contained in the \code{colnames} of the specified \code{eset}. 
##' @return A stacked barplot displaying the appropriate entities on the default device. 
##' @author Russell Bainer
##' @import ggplot2
##' @examples 
##' data('es')
##' data('ann')
##' ct.stackGuides(es, nguides = 20, plotType = 'Target', annotation = ann, ylimit = NULL, subset = NULL)
##' @export

ct.stackGuides <- function(eset, sampleKey = NULL, nguides = 20, plotType = "gRNA", annotation = NULL, ylimit = NULL, subset = NULL) {
    current.graphic.params <- par(no.readonly = TRUE)
    on.exit(suppressWarnings(par(current.graphic.params)))

    if (!requireNamespace("ggplot2")) {
        stop("The ggplot2 package is required")
    }
    if (!is.numeric(nguides)) {
        stop("Please specify a numeric number of guides to display.")
    }
    if (!methods::is(eset, "ExpressionSet")) {
        stop("eset must be an expressionset object.")
    }
    if (!(plotType %in% c("gRNA", "Target"))) {
        stop("Please specify \"gRNA\" or \"Target\" to be displayed.")
    }

    # Check eset colnames
    d <- exprs(eset)

    if (is.null(sampleKey)) {
        sampleKey <- ordered(rep("", ncol(d)))
        names(sampleKey) <- colnames(d)
    }

    if (any(make.names(colnames(d)) != colnames(d))) {
        warning("Some of the sample names are not syntactically valid. Coercing.")
        sampleKey <- sampleKey[colnames(d)]
        names(sampleKey) <- make.names(colnames(d))
        colnames(d) <- make.names(colnames(d))
    }

    # If a fit is included, check to see if it's valid and then subset the expression and pheno data appropriately.
    if (!is.null(subset)) {
        if (!is.character(subset)) {
            stop("subset should be a character vector containing the labels of the samples that you wish to analyze.")
        }
        subset <- make.names(subset)

        if (length(setdiff(subset, colnames(d))) != 0) {
            stop("Not all of the samples in subset are present in the specified eset.")
        }

        d <- d[, subset]
        sampleKey <- sampleKey[subset]
    }
    
    sampleKey <- ct.keyCheck(sampleKey, d)
    
    # idiosyncracies of ggplot forces rearrangement of factor labels for proper plotting.
    sampleKey <- sampleKey[order(sampleKey)]



    # Everything ok, moving on.
    plottitle <- paste0("Top ", nguides, " Most Variable ", plotType, "s Across Experimental Condition")

    if (plotType == "Target") {
        if (is.null(annotation)) {
            stop("An annotation object containing a \"geneSymbol\" column must be supplied to 
           display target-level representation.")
        }

        annotation <- ct.prepareAnnotation(annotation, throw.error = FALSE)

        if (sum(is.na(annotation$geneSymbol)) > 0) {
            message("Converting missing values in the annotation file to \"NoTarget\".")
            annotation$geneSymbol[is.na(annotation$geneSymbol)] <- "NoTarget"
        }

        # Convert all gRNA abundance to % representation
        message("Summarizing gRNA counts into targets.")
        d <- t(vapply(levels(annotation$geneSymbol), function(x) {
            if (sum(annotation$geneSymbol %in% x) > 1) {
                colSums(d[row.names(annotation)[annotation$geneSymbol %in% x], ])
            } else {
                d[row.names(annotation)[annotation$geneSymbol %in% x], ]
            }
        }, numeric(ncol(d))))

    }

    d <- apply(d, 2, function(x) {
        x/sum(x, na.rm = TRUE)
    })
    d <- d[order(apply(d, 1, function(x) {
        range(x, na.rm = TRUE)[2] - range(x, na.rm = TRUE)[1]
    }), decreasing = TRUE), names(sampleKey)[order(sampleKey)]]
    d <- d[seq_len(nguides), ]

    plotframe <- data.frame(gRNA = rep(row.names(d), ncol(d)), Condition = rep(paste0(colnames(d), "_", sampleKey[colnames(d)]), each = nrow(d)), ReadProportion = as.numeric(d))

    colorScale <- colorRampPalette(c("white", "red", "blue", "black"))(nguides)

    # as requested by Sarah, highlight the NTCs if they are present.
    if (is.element("NoTarget", levels(plotframe$gRNA))) {
        colorScale[is.element(levels(plotframe$gRNA), "NoTarget")] <- "forestgreen"
    }

    # also scale the legend as appropriate for the number of guides:
    legend.scale.factor <- 15/nguides

    if (!is.null(ylimit)) {
        if (!is.numeric(ylimit) | (length(ylimit) != 2)) {
            stop("The ylimit variable must be NULL, or a numeric vector of length 2.")
        }
        ggplot(plotframe, aes_string(x = "Condition", y = "ReadProportion", fill = "gRNA")) + geom_bar(stat = "identity", position = "stack") + theme(axis.text.x = element_text(angle = 90, 
            hjust = 1)) + scale_fill_manual(values = colorScale) + ggtitle(plottitle) + coord_cartesian(ylim = c(ylimit[1], ylimit[2])) + ylab("Proportion of Total Reads") + 
            theme(legend.key.size = unit(legend.scale.factor, "cm"), legend.title = element_blank())

    } else {
        ggplot(plotframe, aes_string(x = "Condition", y = "ReadProportion", fill = "gRNA")) + geom_bar(stat = "identity", position = "stack") + theme(axis.text.x = element_text(angle = 90, 
            hjust = 1)) + scale_fill_manual(values = colorScale) + ggtitle(plottitle) + ylab("Proportion of Total Reads") + theme(legend.key.size = unit(legend.scale.factor, 
            "cm"), legend.title = element_blank())
    }
}