R/voomWeightsFromCPM.R

In steveped/extraChIPs: Additional functions for working with ChIP-Seq data

#' @title Estimate voom precision weights directly From CPM values
#'
#' @description Estimate voom precision weights directly From CPM values
#'
#' @details
#' This function takes CPM or logCPM values and estimates the precision weights
#' as would be done by providing counts directly to \link[limma]{voom}.
#' Using this function enables the use of logCPM values which have been
#' normalised using other methods such as Conditional-Quantile or
#' Smooth-Quantile Normalisation.
#'
#' The precision weights are returned as part of the \code{EList} output, and
#' these are automatically passed to the function \link[limma]{lmFit}
#' during model fitting.
#' This will ensure that the mean-variance relationship is appropriate for
#' the linear modelling steps as performed by limma.
#'
#' Initial sample weights can be passed to the function, and should be
#' calculated using \link[limma]{arrayWeights} called on the normalised logCPM
#' values.
#' The returned sample weights will be different to these, given that the
#' function \link[limma]{voomWithQualityWeights} performs two rounds of
#' estimation.
#' The first is on the initial data, with the inappropriate mean-variance
#' relationship, whilst the second round is after incorporation of the precision
#' weights.
#'
#' @param cpm Matrix of CPM or logCPM values
#' @param design The design matrix for the experiment
#' @param w0 Initial vector of sample weights. Should be calculated using
#' \link[limma]{arrayWeights}
#' @param lib.size Initial library sizes. Must be provided as these are no
#' estimable from CPM values
#' @param isLogCPM logical(1). Indicates whether the data is log2 transformed
#' already. Most commonly (e.g. if using the output of cqn) it will be,
#' @param span Width of the smoothing window used for the lowess mean-variance
#' trend. Expressed as a proportion between 0 and 1.
#' @param ... Passed to lmFit internally
#'
#' @return
#' An object of class \code{EList} as would be output by voom.
#' Importantly, there will be no \code{genes} element, although this can be
#' added later.
#' Similarly, the returned \code{targets} element will only contain sample
#' names and library sizes.
#' This can be incorporated with any other metadata as required.
#'
#' Plotting data is always returned, noting the the value \code{sx} has
#' been offset by the library sizes and will be simple logCPM values.
#' As such, the fitted \code{Amean} is also returned in this list element.
#'
#' If initial sample weights were provided, modified weights will also be
#' returned, as the initial function \link[limma]{voomWithQualityWeights}
#' performs two rounds of estimation of sample weights.
#' Here we would simply provide the initial weights a priori, with the
#' second round performed within the function.
#' Importantly, this second round of sample weight estimation uses the precision
#' weights ensuring the correct mean-variance relationship is used for the final
#' estimation of sample weights
#'
#' @examples
#' bamFiles <- system.file("exdata", c("rep1.bam", "rep2.bam"), package="csaw")
#' wc <- csaw::windowCounts(bamFiles, filter=1)
#' cpm <- edgeR::cpm(wc, log = TRUE)
#' el <- voomWeightsFromCPM(cpm, lib.size = wc$totals)
#'
#' @importFrom methods new
#' @export
voomWeightsFromCPM <- function(
        cpm, design = NULL, w0 = NULL, lib.size = NULL, isLogCPM = TRUE,
        span = 0.5, ...
){

    if (!requireNamespace('limma', quietly = TRUE))
        stop("Please install 'limma' to use this function.")

    ## Most checks taken from voom internals
    stopifnot(.voomChecks(cpm, w0, lib.size, isLogCPM))

    ## Make sure we are on the log scale
    if (!isLogCPM) cpm <- log2(cpm)

    ## Sort out the design matrix
    i <- ncol(cpm)
    nm <- colnames(cpm)
    if (is.null(nm)) nm <- seq_len(i)
    if (is.null(design))
        design <- matrix(1, i, 1, dimnames = list(nm, "GrandMean"))

    ## Run the voom steps
    v <- .calculateVoomWeights(cpm, design, w0, lib.size, span, ...)

    ## Initialise output
    targets <- data.frame(sample = nm, lib.size = lib.size)
    if (!is.null(v$aw)) targets$sample.weights <- v$aw
    out <- list(E = cpm, weights = v$w, design = design, targets = targets)
    out$voom.xy <- list(
        x = v$sx, y = v$sy, Amean = v$fit$Amean,
        xlab = "log2( count size + 0.5 )", ylab = "Sqrt ( standard deviation )"
    )
    out$voom.line <- v$l

    ## Return an EList
    new("EList", out)

}

#' @importFrom stats approxfun lowess
#' @keywords internal
.calculateVoomWeights <- function(cpm, design, w0, lib.size, span, ...) {
    ## Taken from the main body of voom
    fit <- limma::lmFit(cpm, design, weights = w0, ...)
    if (is.null(fit$Amean)) fit$Amean <- rowMeans(cpm, na.rm = TRUE)
    sx <- fit$Amean + mean(log2(lib.size + 1)) - log2(1e+06)
    sy <- sqrt(fit$sigma)
    l <- lowess(sx, sy, f = span)
    f <- approxfun(l, rule = 2, ties = list("ordered", mean))
    if (fit$rank < ncol(design)) {
        j <- fit$pivot[seq_len(fit$rank)]
        fitted.values <- fit$coefficients[, j, drop = FALSE] %*%
            t(fit$design[, j, drop = FALSE])
    }
    else {
        fitted.values <- fit$coefficients %*% t(fit$design)
    }
    fitted.cpm <- 2^fitted.values
    fitted.count <- 1e-06 * t(t(fitted.cpm) * (lib.size + 1))
    fitted.logcount <- log2(fitted.count)
    w <- 1/f(fitted.logcount)^4
    dim(w) <- dim(fitted.logcount)

    ## If array weights are provided, the values for `w` would then be used for
    ## a second round of estimation, and scaled by these weights. Again, taken
    ## from limma::voomWithArrayWeights()
    aw <- c()
    if (!is.null(w0)) {
        ## Use all defaults from this function, providing the precision weights
        aw <- limma::arrayWeights(
            object = cpm, design = design, weights = w, var.design = NULL,
            var.group = NULL, prior.n = 10, method = "auto", maxiter = 50,
            tol = 1e-5, trace = FALSE
        )
        w <- t(aw * t(w))
    }
    v <- list(fit = fit, w = w, sx = sx, sy = sy, l = l)
    if (!is.null(aw)) v$aw <- aw
    return(v)
}

.voomChecks <- function(cpm, w0, lib.size, isLogCPM) {
    i <- ncol(cpm)
    n <- nrow(cpm)
    m <- min(cpm)
    if (n < 2L) stop("Need at least two genes to fit a mean-variance trend")
    if (is.na(m)) stop("NA values not allowed")
    if (m < 0 & !isLogCPM) stop("Negative CPM values not allowed")
    if (m == 0 & !isLogCPM)
        stop("Please ensure an offset is used for estimation of CPM values.")
    ## Check the initial weights
    if (!is.null(w0)) {
        stopifnot(is.numeric(w0))
        if (length(w0) != i) stop("Supplied weights do not match the data")
    }
    ## Library sizes must be supplied & valid
    if (is.null(lib.size))
        stop("Library sizes must be provded as these cannot estimated from CPM")
    if (length(lib.size) != i) stop("Library sizes do not match the data")
    stopifnot(is.numeric(lib.size))
    if (!all(lib.size > 0)) stop("Library sizes must be > 0")
    TRUE
}