.pvalueAdjustment_kvdb <- function(baseMean, filter, pValue,
theta, alpha=0.05, pAdjustMethod="BH") {
## this function has been adapted from the pValueAdjustment function from the DESeq2 Bioconductor package and was originally written by Mike Love.
require(genefilter)
# perform independent filtering
if (missing(filter)) {
filter <- baseMean
}
if (missing(theta)) {
lowerQuantile <- mean(filter == 0)
if (lowerQuantile < .95) upperQuantile <- .95 else upperQuantile <- 1
theta <- seq(lowerQuantile, upperQuantile, length=50)
}
# do filtering using genefilter
stopifnot(length(theta) > 1)
filtPadj <- filtered_p(filter=filter, test=pValue,
theta=theta, method=pAdjustMethod)
numRej <- colSums(filtPadj < alpha, na.rm = TRUE)
# prevent over-aggressive filtering when all genes are null,
# by requiring the max number of rejections is above a fitted curve.
# If the max number of rejection is not greater than 10, then don't
# perform independent filtering at all.
lo.fit <- lowess(numRej ~ theta, f=1/5)
if (max(numRej) <= 10) {
j <- 1
} else {
residual <- if (all(numRej==0)) {
0
} else {
numRej[numRej > 0] - lo.fit$y[numRej > 0]
}
thresh <- max(lo.fit$y) - sqrt(mean(residual^2))
j <- if (any(numRej > thresh)) {
which(numRej > thresh)[1]
} else {
1
}
}
padj <- filtPadj[, j, drop=TRUE]
cutoffs <- quantile(filter, theta)
filterThreshold <- cutoffs[j]
filterNumRej <- data.frame(theta=theta, numRej=numRej)
filterTheta <- theta[j]
return(list(padj=padj, filterThreshold=filterThreshold, filterTheta=filterTheta, filterNumRej = filterNumRej, lo.fit=lo.fit, alpha=alpha))
}
#' Estimate ZINB count component posterior probabilities
#'
#' Estimate posterior probabilities to belong to the count component according to a zero-inflated negative binomial (ZINB) model. Internally, edgeR is used for the estimation of the NB component.
#'
#' @param counts A count matrix with feature-wise expression values. Values in this matrix must be integers.
#' @param design Design matrix specifying the experimental design.
#' @param designZI The design for the zero-excess model. If \code{NULL}, the effective library size (defined as the sequencing depth multiplied by the normalization factors) is used by default.
#' @param normalization The normalization method to use. Can be one of \code{"TMM"}, \code{"DESeq2"} or \code{"phyloseq"}. If none of the methods are of interest, global normalization factors can also be given as input in the \code{normFactors} argument.
#' If \code{"TMM"}, the trimmed mean of M-values (Robinson & Oshlack, 2010) normalization is used as implemented in \code{edgeR}.
#' If \code{"DESeq2"}, the default median-of-ratios method from the \code{DESeq2} package (Love et al., 2014) is used for normalization.
#' If \code{"DESeq2_poscounts"}, an adapted median-of-ratios method now implemented in \code{DESeq2}. This method was originally first implemented in the \code{phyloseq} package (McMurdie & Holmes, 2013). The adaptation ensures that genes with zero counts can be used for the purpose of normalization.
#' @param normFactors A vector of user-supplied global normalization factors for every sample. The normalization factors should be sorted according to the samples in the count matrix.
#' @param colData Only applicable if \code{normalization="DESeq2"} or \code{normalization="phyloseq"}. The \code{colData} with pheno data for constructing a \code{\link[DESeq2]{DESeqDataSet-class}} object.
#' @param designFormula Only applicable if \code{normalization="DESeq2"} or \code{normalization="phyloseq"}. The design formula required for constructing a \code{\link[DESeq2]{DESeqDataSet-class}} object.
#' @param maxit The number of iterations for the EM-algorithm. 200 by default, but larger may be useful for large datasets (many samples). Convergence of the posterior probabilities can be checked by following the distribution of posterior probabilities over iterations with \code{plotW}. The EM-algorithm will automatically stop if convergence is achieved before the maximum number of iterations.
#' @param llOffset Offset added to likelihood to avoid taking the log of 0. Defaults to $1e-6$.
#' @param plot Logical. Should the BCV plot be plotted in every iteration?
#' @param plotW Logical. Should the distribution of posterior probabilities for all zeros in the count matrix be plotted in every iteration?
#' @name zeroWeightsLS
#' @rdname zeroWeightsLS
#' @references
#'
#' Robinson MD and Oshlack A (2010). "A scaling normalization method for differential expression analysis of RNA-seq data." Genome Biology, 11, pp. 25.
#'
#' Love MI, Huber W and Anders S (2014). "Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2." Genome Biology, 15, pp. 550.
#'
#' McMurdie PJ and Holmes S (2013). "phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data." PLoS ONE, 8(4), pp. e61217.
#'
#' @examples
#' data(islamEset,package="zingeR")
#' islam=exprs(islamEset)[1:2000,]
#' design=model.matrix(~pData(islamEset)[,1])
#' zeroWeights=zeroWeightsLS(counts=islam, design=design, maxit=200)
#' @export
zeroWeightsLS <- function(counts, design, maxit=200, normalization="TMM", colData, designFormula, normFactors=NULL, plot=FALSE, plotW=FALSE, verbose=TRUE, designZI=NULL, llTol=1e-4, llOffset=1e-6){
if (!is.integer(counts)) {
if (any(round(counts) != counts)) {
stop("some values in assay are not integers")
}
message("converting counts to integer mode")
mode(counts) <- "integer"
}
#### normalization
require(edgeR)
if(plot | plotW) par(mfrow=c(1,plot+plotW))
counts <- DGEList(counts)
if(normalization=="TMM"){
counts <- suppressWarnings(edgeR::calcNormFactors(counts))
} else if(normalization=="DESeq2"){
dse = DESeqDataSetFromMatrix(counts$counts, colData=colData, design=designFormula)
dse = DESeq2::estimateSizeFactors(dse)
counts$samples$norm.factors = 1/dse$sizeFactor
} else if(normalization=="DESeq2_poscounts"){
dse = DESeqDataSetFromMatrix(counts$counts, colData=colData, design=designFormula)
dse = DESeq2::estimateSizeFactors(dse, type = "poscounts")
counts$samples$norm.factors = 1/dse$sizeFactor
}
# user input normalization factors
if(!is.null(normFactors)) counts$samples$norm.factors = normFactors
effLibSize <- counts$samples$lib.size*counts$samples$norm.factors
logEffLibSize <- log(effLibSize)
## initialize EM
zeroId <- counts$counts==0
w <- matrix(1,nrow=nrow(counts),ncol=ncol(counts), dimnames=list(c(1:nrow(counts)), NULL))
## starting values based on P(zero) in the library
for(k in 1:ncol(w)) w[counts$counts[,k]==0,k] <- 1-mean(counts$counts[,k]==0)
llOld <- matrix(-1e4,nrow=nrow(counts),ncol=ncol(counts))
likCOld <- matrix(0,nrow=nrow(counts),ncol=ncol(counts))
converged=FALSE
j=0
for(i in 1:maxit){
j=j+1
zeroId <- counts$counts==0
counts$weights <- w
### M-step counts
#only estimate dispersions after weight convergence
if(i==1 | converged){
#estimateDisp faster starting from edgeR v3.19
#counts <- estimateGLMCommonDisp(counts, design, interval=c(0,10))
#counts <- estimateGLMTagwiseDisp(counts, design, prior.df=0, min.row.sum=1)
counts <- estimateDisp(counts, design, prior.df=0, min.row.sum=1)
}
if(plot) plotBCV(counts)
fit <- glmFit(counts, design)
likC <- dnbinom(counts$counts, mu=fit$fitted.values, size=1/counts$tagwise.dispersion)
### M-step mixture parameter: model zero probability
successes <- colSums(1-w) #P(zero)
failures <- colSums(w) #1-P(zero)
if(is.null(designZI)){
zeroFit <- suppressWarnings(glm(cbind(successes,failures) ~ logEffLibSize, family="binomial"))} else{
zeroFit <- suppressWarnings(glm(cbind(successes,failures) ~-1+designZI, family="binomial"))}
pi0Hat <- predict(zeroFit,type="response")
## E-step: Given estimated parameters, calculate expected value of weights
pi0HatMat <- expandAsMatrix(pi0Hat,dim=dim(counts),byrow=TRUE)
w <- 1-pi0HatMat*zeroId/(pi0HatMat*zeroId+(1-pi0HatMat)*likC*zeroId+1e-15)
## data log-likelihood
if(i>1) llOld=ll
ll <- log(pi0HatMat*zeroId + (1-pi0HatMat)*likC + llOffset)
delta <- (rowSums(ll)-rowSums(llOld))/(rowSums(llOld)+llTol)
if(mean(abs(delta) < llTol)>.999){ #if 99.9% has converged
if(j==1 & mean(abs(delta) < llTol)>.999){ #final convergence?
cat(paste0("converged. \n")) ; return(w)}
j=0
converged=TRUE} else {converged=FALSE}
if(verbose) cat(paste0("iteration: ",i,". mean conv.: ",round(mean(abs(delta) < llTol),5),"\n"))
if(plotW) hist(w[zeroId],main=paste0("iteration: ",i,". mean conv.: ",mean(abs(delta) < llTol)))
## if maximum iterations reached but convergence low, keep on iterating
if(i==maxit & mean(abs(delta) < llTol)<0.9) maxit=maxit+1
}
return(w)
}
#' Zero-inflation adjusted statistical tests for assessing differential expression.
#'
#' This function recycles an old version of the \code{\link[edgeR]{glmLRT}} method that allows an F-test with adjusted denominator degrees of freedom to account for the downweighting in the zero-inflation model.
#'
#' @param fit a \code{\link[edgeR]{DGEGLM-class}} object, usually output from \code{\link[edgeR]{glmFit}}.
#' @param coef integer or character vector indicating which coefficients of the linear model are to be tested equal to zero. Values must be columns or column names of design. Defaults to the last coefficient. Ignored if \code{contrast} is specified.
#' @param contrast numeric vector or matrix specifying one or more contrasts of the linear model coefficients to be tested equal to zero. Number of rows must equal to the number of columns of \code{design}. If specified, then takes precedence over \code{coef}.
#' @param ZI Logical, specifying whether the degrees of freedom in the statistical test should be adjusted according to the weights in the \code{fit} object to account for the downweighting. Defaults to TRUE and this option is highly recommended.
#' @references
#' McCarthy, DJ, Chen, Y, Smyth, GK (2012). Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Research 40, 4288-4297.
#' @seealso \code{\link[edgeR]{glmLRT}}
#' @examples
#' library(edgeR)
#' data(islamEset,package="zingeR")
#' islam=exprs(islamEset)[1:2000,]
#' design=model.matrix(~pData(islamEset)[,1])
#' d=DGEList(islam)
#' d=calcNormFactors(d)
#' d=estimateWeightedDispersions(d,design, maxit=200)
#' fit=glmFit(d,design)
#' lrt=glmWeightedF(fit,coef=2)
#' @name glmWeightedF
#' @rdname glmWeightedF
#' @export
glmWeightedF <- function(glmfit, coef=ncol(glmfit$design), contrast=NULL, test="F", ZI=TRUE, independentFiltering=TRUE, filter=NULL)
## original function obtained from https://github.com/Bioconductor-mirror/edgeR/blob/release-3.0/R/glmfit.R
# Tagwise likelihood ratio tests for DGEGLM
# Gordon Smyth, Davis McCarthy and Yunshun Chen.
# Created 1 July 2010. Last modified 22 Nov 2013.
{
# Check glmfit
if(!is(glmfit,"DGEGLM")) {
if(is(glmfit,"DGEList") && is(coef,"DGEGLM")) {
stop("First argument is no longer required. Rerun with just the glmfit and coef/contrast arguments.")
}
stop("glmfit must be an DGEGLM object (usually produced by glmFit).")
}
if(is.null(glmfit$AveLogCPM)) glmfit$AveLogCPM <- aveLogCPM(glmfit)
nlibs <- ncol(glmfit)
# Check test
test <- match.arg(test,c("F","f","chisq"))
if(test=="f") test <- "F"
# Check design matrix
design <- as.matrix(glmfit$design)
nbeta <- ncol(design)
if(nbeta < 2) stop("Need at least two columns for design, usually the first is the intercept column")
coef.names <- colnames(design)
# Evaluate logFC for coef to be tested
# Note that contrast takes precedence over coef: if contrast is given
# then reform design matrix so that contrast of interest is last column.
if(is.null(contrast)) {
if(length(coef) > 1) coef <- unique(coef)
if(is.character(coef)) {
check.coef <- coef %in% colnames(design)
if(any(!check.coef)) stop("One or more named coef arguments do not match a column of the design matrix.")
coef.name <- coef
coef <- match(coef, colnames(design))
}
else
coef.name <- coef.names[coef]
logFC <- glmfit$coefficients[,coef,drop=FALSE]/log(2)
} else {
contrast <- as.matrix(contrast)
qrc <- qr(contrast)
ncontrasts <- qrc$rank
if(ncontrasts==0) stop("contrasts are all zero")
coef <- 1:ncontrasts
if(ncontrasts < ncol(contrast)) contrast <- contrast[,qrc$pivot[coef]]
logFC <- drop((glmfit$coefficients %*% contrast)/log(2))
if(ncontrasts>1) {
coef.name <- paste("LR test of",ncontrasts,"contrasts")
} else {
contrast <- drop(contrast)
i <- contrast!=0
coef.name <- paste(paste(contrast[i],coef.names[i],sep="*"),collapse=" ")
}
Dvec <- rep.int(1,nlibs)
Dvec[coef] <- diag(qrc$qr)[coef]
Q <- qr.Q(qrc,complete=TRUE,Dvec=Dvec)
design <- design %*% Q
}
if(length(coef)==1) logFC <- as.vector(logFC)
# Null design matrix
design0 <- design[,-coef,drop=FALSE]
# Null fit
fit.null <- glmFit(glmfit$counts,design=design0,offset=glmfit$offset,weights=glmfit$weights,dispersion=glmfit$dispersion,prior.count=0)
# Likelihood ratio statistic
LR <- fit.null$deviance - glmfit$deviance
### ADDED
if(ZI) fit.null$df.residual <- rowSums(fit.null$weights)-ncol(design0)
if(ZI) glmfit$df.residual <- rowSums(glmfit$weights)-ncol(design)
## END ADDED
df.test <- fit.null$df.residual - glmfit$df.residual ## okay
# Chisquare or F-test
# LRT.pvalue <- switch(test,
# "F" = {
# phi <- quantile(glmfit$dispersion,p=0.5)
# mu <- quantile(glmfit$fitted.values,p=0.5)
# gamma.prop <- (phi*mu/(1 + phi*mu))^2
# prior.df <- glmfit$prior.df
# if(is.null(prior.df)) prior.df <- 20
# glmfit$df.total <- glmfit$df.residual + prior.df/gamma.prop
# pf(LR/df.test, df1=df.test, df2=glmfit$df.total, lower.tail = FALSE, log.p = FALSE)
# },
# "chisq" = pchisq(LR, df=df.test, lower.tail = FALSE, log.p = FALSE)
# )
LRT.pvalue <- {
phi <- quantile(glmfit$dispersion,p=0.5)
mu <- quantile(glmfit$fitted.values,p=0.5)
gamma.prop <- (phi*mu/(1 + phi*mu))^2
prior.df <- glmfit$prior.df
if(is.null(prior.df)) prior.df <- 20
glmfit$df.total <- glmfit$df.residual + prior.df/gamma.prop
pf(LR/df.test, df1=df.test, df2=glmfit$df.total, lower.tail = FALSE, log.p = FALSE)
}
rn <- rownames(glmfit)
if(is.null(rn))
rn <- 1:nrow(glmfit)
else
rn <- make.unique(rn)
tab <- data.frame(
logFC=logFC,
logCPM=glmfit$AveLogCPM,
LR=LR,
PValue=LRT.pvalue,
row.names=rn
)
glmfit$counts <- NULL
glmfit$table <- tab
glmfit$comparison <- coef.name
glmfit$df.test <- df.test
res <- new("DGELRT",unclass(glmfit))
if(independentFiltering){
if(is.null(filter)) filter=rowMeans(glmfit$fitted.values) #aprrox. linear w\ basemean
res <- independentFiltering(res,filter=filter, objectType="edgeR")
} else return(res)
}
#' Perform independent filtering in differential expression analysis.
#'
#' This function uses the \code{DESeq2} independent filtering method to increase detection power in high throughput gene expression studies.
#'
#' @param object Either a \code{\link[edgeR]{DGELRT-class}} object or a \code{\link{data.frame}} with differential expression results.
#' @param filter The characteristic to use for filtering, usually a measure of normalized mean expression for the features.
#' @param objectType Either \code{"edgeR"} or \code{"limma"}. If \code{"edgeR"}, it is assumed that \code{object} is of class \code{\link[edgeR]{DGELRT-class}}, the output of \code{\link[edgeR]{glmLRT}}. If \code{"limma"}, it is assumed that \code{object} is a \code{\link{data.frame}} and the output of a limma-voom analysis.
#' @seealso \code{\link[DESeq2]{results}}
#' @references
#' Michael I Love, Wolfgang Huber, and Simon Anders. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biology, 15(12):550, dec 2014.
#' @name independentFiltering
#' @rdname independentFiltering
#' @examples
#' library(limma)
#' data(islamEset,package="zingeR")
#' islam=exprs(islamEset)[1:2000,]
#' design=model.matrix(~pData(islamEset)[,1])
#' d=DGEList(islam)
#' nf=calcNormFactors(islam)
#' y=zeroWeightedVoom(d,design,nf=nf,maxit=200)
#' fit=lmWeightedFit(y,design)
#' fit=eBayes(fit)
#' tt=topTable(fit,coef=2,sort.by="none",number=nrow(fit))
#' baseMean=unname(rowMeans(sweep(d$counts,2,nf,FUN="*")))
#' ttFiltered=independentFiltering(tt,filter=baseMean, objectType="limma")
#' @export
independentFiltering <- function(object, filter, objectType=c("edgeR","limma")){
if(objectType=="edgeR"){
hlp <- .pvalueAdjustment_kvdb(filter=filter, pValue=object$table$PValue)
object$table$padjFilter <- hlp$padj
return(object)
} else if(objectType=="limma"){
hlp <- .pvalueAdjustment_kvdb(filter=filter, pValue=object$P.Value)
object$padjFilter <- hlp$padj
return(object)
} else stop("objectType must be either one of 'edgeR' or 'limma'.")
}
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