diffSplice <- function(fit,...) UseMethod("diffSplice") diffSplice.MArrayLM <- function(fit,geneid,exonid=NULL,robust=FALSE,legacy=FALSE,verbose=TRUE,...) # Test for differential exon usage between conditions # using linear model fit of exon data. # Gordon Smyth and Charity Law # Created 13 Dec 2013. Last modified 4 Apr 2025. { # Make sure there is always an annotation frame exon.genes <- fit$genes if(is.null(exon.genes)) exon.genes <- data.frame(ExonID=1:nrow(fit)) # Get ID columns for genes and exons if(length(geneid)==1) { genecolname <- as.character(geneid) geneid <- exon.genes[[genecolname]] } else { exon.genes$GeneID <- geneid genecolname <- "GeneID" } if(is.null(exonid)) { exoncolname <- NULL } else { if(length(exonid)==1) { exoncolname <- as.character(exonid) exonid <- exon.genes[[exoncolname]] } else { exon.genes$ExonID <- exonid exoncolname <- "ExonID" } } # Treat NA geneids as genes with one exon if(anyNA(geneid)) { isna <- which(is.na(geneid)) geneid[isna] <- paste0("NA",1:length(isna)) } # Sort by geneid if(is.null(exonid)) o <- order(geneid) else o <- order(geneid,exonid) geneid <- geneid[o] exon.genes <- exon.genes[o,,drop=FALSE] exon.coefficients <- fit$coefficients[o,,drop=FALSE] exon.stdev.unscaled <- fit$stdev.unscaled[o,,drop=FALSE] exon.df.residual <- fit$df.residual[o] exon.s2 <- fit$sigma[o]^2 if(min(exon.df.residual) < 1e-6) exon.s2[exon.df.residual < 1e-6] <- 0 # Count exons by gene and get genewise variances exon.stat <- cbind(1,exon.df.residual,exon.df.residual*exon.s2) gene.sum <- rowsum(exon.stat,geneid,reorder=FALSE) gene.nexons <- gene.sum[,1] gene.df.residual <- gene.sum[,2] gene.s2 <- gene.sum[,3] / gene.sum[,2] if(verbose) { cat("Total number of exons: ", length(geneid), "\n") cat("Total number of genes: ", length(gene.nexons), "\n") cat("Number of genes with 1 exon: ", sum(gene.nexons==1), "\n") cat("Mean number of exons in a gene: ", round(mean(gene.nexons),0), "\n") cat("Max number of exons in a gene: ", max(gene.nexons), "\n") } # Posterior genewise variances squeeze <- squeezeVar(var=gene.s2, df=gene.df.residual, robust=robust, legacy=legacy) # Remove genes with only 1 exon gene.keep <- gene.nexons>1 ngenes <- sum(gene.keep) if(ngenes==0) stop("No genes with more than one exon") exon.keep <- rep(gene.keep,gene.nexons) geneid <- geneid[exon.keep] exon.genes <- exon.genes[exon.keep,,drop=FALSE] exon.coefficients <- exon.coefficients[exon.keep,,drop=FALSE] exon.stdev.unscaled <- exon.stdev.unscaled[exon.keep,,drop=FALSE] exon.df.residual <- exon.df.residual[exon.keep] gene.nexons <- gene.nexons[gene.keep] gene.df.test <- gene.nexons-1 gene.df.residual <- gene.df.residual[gene.keep] if(length(squeeze$df.prior) > 1L) squeeze$df.prior <- squeeze$df.prior[gene.keep] gene.df.total <- gene.df.residual+squeeze$df.prior gene.df.total <- pmin(gene.df.total,sum(gene.df.residual)) gene.s2.post <- squeeze$var.post[gene.keep] # Genewise betas u2 <- 1/exon.stdev.unscaled^2 u2.rowsum <- rowsum(u2,geneid,reorder=FALSE) gene.betabar <- rowsum(exon.coefficients*u2,geneid,reorder=FALSE) / u2.rowsum # T-statistics for exon-level tests g <- rep(1:ngenes,times=gene.nexons) exon.coefficients <- exon.coefficients-gene.betabar[g,,drop=FALSE] exon.t <- exon.coefficients / exon.stdev.unscaled / sqrt(gene.s2.post[g]) gene.F <- rowsum(exon.t^2,geneid,reorder=FALSE) / gene.df.test exon.1mleverage <- 1 - (u2 / u2.rowsum[g,,drop=FALSE]) exon.coefficients <- exon.coefficients / exon.1mleverage exon.t <- exon.t / sqrt(exon.1mleverage) exon.p.value <- 2 * pt(abs(exon.t), df=gene.df.total[g], lower.tail=FALSE) gene.F.p.value <- pf(gene.F, df1=gene.df.test, df2=gene.df.total, lower.tail=FALSE) # Exon level output out <- new("MArrayLM",list()) out$genes <- exon.genes out$genecolname <- genecolname out$exoncolname <- exoncolname out$coefficients <- exon.coefficients out$t <- exon.t out$p.value <- exon.p.value # Gene level output out$gene.df.prior <- squeeze$df.prior out$gene.df.residual <- gene.df.residual out$gene.df.total <- gene.df.total out$gene.s2 <- gene.s2[gene.keep] out$gene.s2.post <- gene.s2.post out$gene.F <- gene.F out$gene.F.p.value <- gene.F.p.value # Which columns of exon.genes contain gene level annotation? gene.lastexon <- cumsum(gene.nexons) gene.firstexon <- gene.lastexon-gene.nexons+1 no <- logical(nrow(exon.genes)) isdup <- vapply(exon.genes,duplicated,no)[-gene.firstexon,,drop=FALSE] isgenelevel <- apply(isdup,2,all) out$gene.genes <- exon.genes[gene.lastexon,isgenelevel, drop=FALSE] row.names(out$gene.genes) <- out$gene.genes[[genecolname]] out$gene.genes$NExons <- gene.nexons out$gene.firstexon <- gene.firstexon out$gene.lastexon <- gene.lastexon # Simes adjustment of exon level p-values # Full Simes adjustment implemented 2 March 2025. Previously a # modified version on the top nexons-1 exons for each gene was used. penalty <- rep_len(1L,length(g)) penalty[gene.firstexon[-1]] <- 1L-gene.nexons[-ngenes] penalty <- cumsum(penalty) penalty <- rep(gene.nexons,gene.nexons) / penalty out$gene.simes.p.value <- gene.F.p.value for (j in 1:ncol(fit)) { o <- order(g,exon.p.value[,j]) p.adj <- exon.p.value[o,j] * penalty o <- order(g,p.adj) out$gene.simes.p.value[,j] <- p.adj[o][gene.firstexon] } # Bonferroni adjustment of exon level p-values out$gene.bonferroni.p.value <- gene.F.p.value for (j in 1:ncol(fit)) { o <- order(g,exon.p.value[,j]) p.adj <- pmin(exon.p.value[o,j][gene.firstexon]*(gene.nexons),1) out$gene.bonferroni.p.value[,j] <- p.adj } out }