From 7a786e2954ff74384633bcd3280abb31316f633e Mon Sep 17 00:00:00 2001 From: stepanv1 Date: Tue, 29 Sep 2015 15:16:28 -0400 Subject: [PATCH] Major update to comply with Bioconductor requirements. --- DESCRIPTION | 2 +- NEWS | 4 +- R/funtoonorm.R | 747 +++++++++++++++------------ man/agreement.Rd | 10 +- man/funtooNorm-package.Rd | 7 +- man/funtoonorm.Rd | 73 +-- vignettes/funtooNorm.Rnw | 70 ++- vignettes/valid.jpg | Bin 212764 -> 0 bytes vignettes/validationcurvesPCRlow.jpg | Bin 0 -> 84320 bytes vignettes/validationcurvesPLSlow.jpg | Bin 0 -> 85780 bytes 10 files changed, 523 insertions(+), 390 deletions(-) delete mode 100644 vignettes/valid.jpg create mode 100644 vignettes/validationcurvesPCRlow.jpg create mode 100644 vignettes/validationcurvesPLSlow.jpg diff --git a/DESCRIPTION b/DESCRIPTION index 6de53a6..b1d9a95 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -8,7 +8,7 @@ Maintainer: Stepan Grinek Description: Provides a function to normalize Illumina Infinium Human Methylation 450 BeadChip (Illumina 450K), correcting for tissue and/or cell type. License: GPL-3 Imports: pls -Suggests: BiocStyle, illuminaio +Suggests: BiocStyle, illuminaio, minfi Depends: R(>= 2.10.0) LazyData: true biocViews: DNAMethylation, Preprocessing, Normalization \ No newline at end of file diff --git a/NEWS b/NEWS index b8df44c..45be3cb 100644 --- a/NEWS +++ b/NEWS @@ -12,6 +12,6 @@ \section{Version 0.99.1}{ \itemize{ \item Few corrections - } -} \ No newline at end of file +} + diff --git a/R/funtoonorm.R b/R/funtoonorm.R index ea19f92..9f68f28 100644 --- a/R/funtoonorm.R +++ b/R/funtoonorm.R @@ -1,355 +1,456 @@ # Function to normalize Illumina Infinium Human Methylation 450 BeadChip (Illumina 450K) with multiple tissues or cell types. #Main function of the package.s funtoonorm <- function(sigA, sigB, Annot=NULL, - controlred, controlgrn, cp.types=NULL, cell_type, ncmp=4, - save.quant=TRUE, save.loess=TRUE, apply.loess=TRUE, validate=FALSE) + controlred, controlgrn, cp.types=NULL, cell_type, ncmp=4, ncv.fold=10, + logged.data = FALSE, save.quant=TRUE, type.fits="PCR", apply.fits=TRUE, validate=FALSE) { - #################################################################################### - # functions - logitfn <- function(x) { log(x/(1-x)) } - ## - mult1 <- function(x, vec1) return(x*vec1) - ## - sum1 <- function(x, v2) { return(x + v2) } - ## - extractqnt <- function(x, i, AB, ncmp) { - return(x$fitted.values[i,AB,ncmp]) } - ## - ## just the usual lm functgion - lmfunction1<- function(yvec, xmat) { - if (sum(is.na(yvec))==0) { - fit1 <- lm(yvec ~ cell_type/Disease + Sex + age_quant, data = xmat) - cfit1 <- summary(fit1)$coefficients - fit2 <- anova(fit1) - outp <- c(cfit1[2:nrow(cfit1),4],fit2[4,5]) - names(outp) <- c(rownames(cfit1[2:nrow(cfit1)]),rownames(fit2)[4]) - return(outp) - }} - ## - qqfn <- function(vect1) { - nnonmiss <- sum(!is.na(vect1)) - xaxs <- sort(-log10( (1:nnonmiss - 0.5) / nnonmiss)) - plot(c(0, max(xaxs)), c(0, max(-log10(vect1), na.rm=TRUE)), type='n', - xlab='Expected', ylab='Observed') - abline(0,1) - points(xaxs, sort(-log10(vect1[!is.na(vect1)])), pch=16, cex=0.4) + #################################################################################### + # functions + logitfn <- function(x) { log(x/(1-x)) } + ## + mult1 <- function(x, vec1) return(x*vec1) + ## + sum1 <- function(x, v2) { return(x + v2) } + ## + calcbeta <- function(A,B,offset) { + return((exp(B)-1)/(exp(A) + exp(B)-2 + offset)) + } + extractqnt <- function(x, i, AB, ncmp) { + return(x$fitted.values[i,AB,ncmp]) } + ## + NotNumeric<-function(a){ + return(any(!is.finite(as.matrix(a)))) + } + ## + is.wholenumber <- function(x){abs(x - round(x)) == 0} + ## + denom.offset <- 100 # to avoid dividing by zero Illumina uses 100! + + #Function adjusts beta values with results of loess regression + applyfuntoonorm <- function(lfits, sigA, sigB, Annot, qntllist) { + + wh.red <- which(Annot$Type=='I' & Annot$Color=="Red") + wh.grn <- which(Annot$Type=='I' & Annot$Color=="Grn") + wh.II <- which(Annot$Type=='II') + + rankmatA.red <- (apply(sigA[wh.red,],2, rank) - 0.5)/length(wh.red) + rankmatA.grn <- (apply(sigA[wh.grn,],2, rank) - 0.5)/length(wh.grn) + rankmatA.II <- (apply(sigA[wh.II,],2, rank) - 0.5)/length(wh.II) + + rankmatB.red <- (apply(sigB[wh.red,],2, rank) - 0.5)/length(wh.red) + rankmatB.grn <- (apply(sigB[wh.grn,],2, rank) - 0.5)/length(wh.grn) + rankmatB.II <- (apply(sigB[wh.II,],2, rank) - 0.5)/length(wh.II) + + predmatA.red <- matrix(NA, nrow=nrow(rankmatA.red), ncol = ncol(rankmatA.red)) + predmatA.grn <- matrix(NA, nrow=nrow(rankmatA.grn), ncol = ncol(rankmatA.grn)) + predmatA.II <- matrix(NA, nrow=nrow(rankmatA.II), ncol = ncol(rankmatA.II)) + predmatB.red <- matrix(NA, nrow=nrow(rankmatB.red), ncol = ncol(rankmatB.red)) + predmatB.grn <- matrix(NA, nrow=nrow(rankmatB.grn), ncol = ncol(rankmatB.grn)) + predmatB.II <- matrix(NA, nrow=nrow(rankmatB.II), ncol = ncol(rankmatB.II)) + for (i in (1:ncol(predmatA.red))) { + predmatA.red[,i]<-approx(qntllist,lfits[[1]][i,],xout=rankmatA.red[,i])$y + predmatA.grn[,i]<-approx(qntllist,lfits[[2]][i,],xout=rankmatA.grn[,i])$y + predmatA.II[,i]<-approx(qntllist,lfits[[3]][i,],xout=rankmatA.II[,i])$y + predmatB.red[,i]<-approx(qntllist,lfits[[4]][i,],xout=rankmatB.red[,i])$y + predmatB.grn[,i]<-approx(qntllist,lfits[[5]][i,],xout=rankmatB.grn[,i])$y + predmatB.II[,i]<-approx(qntllist,lfits[[6]][i,],xout=rankmatB.II[,i])$y } - ## - qqfn2 <- function(vect1, vect2) { - nnonmiss <- sum(!is.na(vect1)) - xaxs <- sort(-log10( (1:nnonmiss - 0.5) / nnonmiss)) - plot(c(0, max(xaxs)), c(0, max(-log10(vect1), na.rm=TRUE)), type='n', - xlab='Expected', ylab='Observed') - abline(0,1) - points(xaxs, sort(-log10(vect1[!is.na(vect1)])), pch=16, cex=0.4, col=1) - nnonmiss <- sum(!is.na(vect2)) - xaxs <- sort(-log10( (1:nnonmiss - 0.5) / nnonmiss)) - points(xaxs, sort(-log10(vect2[!is.na(vect2)])), pch=16, cex=0.4, col=2) + predmatA <- matrix(NA, nrow(sigA), ncol(predmatA.red)) + predmatB <- predmatA + predmatA[wh.red,] <- predmatA.red + predmatA[wh.grn,] <- predmatA.grn + predmatA[wh.II,] <- predmatA.II + predmatB[wh.red,] <- predmatB.red + predmatB[wh.grn,] <- predmatB.grn + predmatB[wh.II,] <- predmatB.II + + newBeta <- calcbeta(predmatA, predmatB, denom.offset) + origBeta <- as.matrix(calcbeta(sigA, sigB, denom.offset)) + rownames(newBeta) <- rownames(origBeta) + colnames(newBeta) <- colnames(origBeta) + return(list(origBeta=origBeta, newBeta=newBeta)) + } + ############################################################################### + + + if (is.null(Annot)) { + cat("Since Annot is NULL using default annotation.", '\n') + data('Annot', package='funtooNorm', envir = environment()) + } + + # obtain names of types of control probes if not provided + if (!is.null(cp.types)) { cp.types.tab <- table(cp.types) } + if (is.null(cp.types)) { + cp.types.tab <- table(rownames(controlgrn)) + if (any(names(cp.types.tab)=="NEGATIVE")) { + cp.types <- rownames(controlgrn) + } + if (!(any(names(cp.types.tab)=="NEGATIVE"))) { + cat("Since cp.types is NULL and rownames of control probe matrices do not contain,", "\n", + "this information, analysis will use default cp.types.", '\n') + data('cp.types', package='funtooNorm', envir = environment()) + cp.types.tab <- table(cp.types) + } + } + + #checking sanity of the data + cat("Checking sanity of the data...", '\n') + if (NotNumeric(sigA)){stop("There are non-numeric values in the matrix", '\n')} + if (NotNumeric(sigB)){stop("There are non-numeric values in the matrix", '\n')} + if (NotNumeric(controlred)){stop("There are non-numeric values in the matrix", '\n')} + if (NotNumeric(controlgrn)){stop("There are non-numeric values in the matrix", '\n')} + if (any(cell_type == '' | typeof(cell_type) != 'character' | is.na(cell_type))) {{stop("There are non-character values in cell_type", '\n')}} + if (length(unique(cell_type))<2) {{stop("There should be more that one tissue or cell type in cell_type variable", '\n')}} + if (any(cp.types == '' | typeof(cp.types) != 'character' | is.na(cp.types))) {{stop("There are non-character values in cp.types", '\n')}} + if (type.fits !="PCR" & type.fits !="PLS") {{stop("type.fits must be either PCR or PLS","\n")}} + + #check that ID's match between sigA, sigB and control probe data + if (!(identical(colnames(controlgrn), colnames(sigA)) & + identical(colnames(controlred), colnames(sigA)) & + identical(colnames(sigB), colnames(sigA)))) { + if ((ncol(controlgrn) == ncol(sigA)) & (ncol(controlred) == ncol(sigA)) + & (ncol(controlred) == ncol(sigB)) & (length(cp.types) == ncol(sigB)) & (length(cell_type) == ncol(sigB))){ + ix <- sort(colnames(controlred), index.return = TRUE)$ix + controlgrn<-controlgrn[,ix]; sigA<-sigA[,ix]; sigB<-sigB[,ix]; + if (!(identical(colnames(controlgrn), colnames(sigA)) & + identical(colnames(controlred), colnames(sigA)) & + identical(colnames(sigB), colnames(sigA)))){ + stop("Sample names do not match.", '\n')} + + } else{stop("Data dimensions or samples names do not match.", '\n')} + } + if (type.fits!="PCR" & type.fits!="PLS") {stop("type.fits must be either PCR or PLS", "\n")} + + if ((max(controlred)<20 & max(sigA)>25) | (max(controlred)>25 & max(sigA)<20)) { + stop("apparent inconsistency w.r.t. log transformation of sigA/B data and control data \n") + } + if (max(sigA, na.rm=TRUE)>25) { + cat("Assuming data have not been previously log transformed, and applying a log transformation, \n") + logged.data <- FALSE + } + + + #check if annotation is ok, and subset annotation on the probes from Sig files + if(any(!(rownames(sigA) %in% rownames(Annot)))){stop("Probe names do not match annotation entries", '\n')} + cpg<-intersect(rownames(sigA), rownames(Annot)) + Annot=Annot[cpg,] + + cat("Data is ok.", '\n') + + if (!logged.data) { # log transformation if data are not already logged at input + sigA <- log2(1 + sigA) + sigB <- log2(1 + sigB) + } + + wh.red <- which(Annot$Type=='I' & Annot$Color=="Red") + wh.grn <- which(Annot$Type=='I' & Annot$Color=="Grn") + wh.II <- which(Annot$Type=='II') + + nr <- nrow(sigA) + qntllist <- c(seq((0.5)/nr, 0.0009, 0.0001), seq(0.001, 0.009, 0.001), seq(0.01,0.99,0.002), + seq(0.991,0.999,0.001), seq(0.999 ,(nr-0.5)/nr,0.0001 )) + qntllist <- c(qntllist, (nr - 0.5)/nr) # add one more at the top end! + nqnt <- length(qntllist) # number of desired quantiles + + if (type.fits=="PCR") method.mvr <- "svdpc" + if (type.fits=="PLS") method.mvr <- "kernelpls" + + ####! TO DO good to add special treatment of Y chromosome. See Fortin et al. particularly for Y + ## columns of quantilesA,B are the desired quantiles. Rows are samples + if (save.quant) { + quantilesA.red <- matrix(NA, ncol(sigA), nqnt) + quantilesA.grn <- quantilesA.red; quantilesA.II <- quantilesA.red + quantilesB.red <- matrix(NA, ncol(sigA), nqnt) + quantilesB.grn <- quantilesA.red; quantilesB.II <- quantilesA.red + for (i in (1:ncol(sigA))) { + quantilesA.red[i,] <- quantile(sigA[wh.red,i],qntllist) + quantilesA.grn[i,] <- quantile(sigA[wh.grn,i],qntllist) + quantilesA.II[i,] <- quantile(sigA[wh.II,i],qntllist) + quantilesB.red[i,] <- quantile(sigB[wh.red,i],qntllist) + quantilesB.grn[i,] <- quantile(sigB[wh.grn,i],qntllist) + quantilesB.II[i,] <- quantile(sigB[wh.II,i],qntllist) } + save(quantilesA.red, file="quantilesA.red.RData") + save(quantilesA.grn, file="quantilesA.grn.RData") + save(quantilesA.II, file="quantilesA.II.RData") + save(quantilesB.red, file="quantilesB.red.RData") + save(quantilesB.grn, file="quantilesB.grn.RData") + save(quantilesB.II, file="quantilesB.II.RData") + } # if save.quant + if (!save.quant) { + load("quantilesA.red.RData") + load("quantilesA.grn.RData") + load("quantilesA.II.RData") + load("quantilesB.red.RData") + load("quantilesB.grn.RData") + load("quantilesB.II.RData") + } + + if (!logged.data) { # same assumption as for signalA, signalB w.r.t. log transformation + controlgrn <- log2(1 + controlgrn) + controlred <- log2(1 + controlred) + } + + # construct control probe summaries, averages by type of control probe + # then specifically create columns by cell type + for (k in (1:length(cp.types.tab))) { + temp1 <- apply(controlred[cp.types==names(cp.types.tab[k]),],2,mean) + temp2 <- apply(controlgrn[cp.types==names(cp.types.tab[k]),],2,mean) + if (k==1) { + mat.by.ct <- cbind(temp1,temp2) } + if (k>1) { mat.by.ct <- cbind(mat.by.ct, cbind(temp1,temp2)) } + } # end for mat.by.ct is 15 columns - means for each type of control probe + + ind=matrix(FALSE,ncol=length(cell_type),nrow=length(unique(cell_type))) + for(j in 1:length(unique(cell_type))){ + ind[j,]<- (cell_type==names(table(cell_type))[j]) + } + # now add additional sets of 15 columns, each specific to one cell type + ctl.covmat <- cbind(mat.by.ct, apply(mat.by.ct, 2, mult1, ind[1,])) + for(j in 2:length(unique(cell_type))){ + ctl.covmat <- cbind(ctl.covmat, apply(mat.by.ct, 2, mult1, ind[j,])) + } + + svd.ctlcovmat <- svd(ctl.covmat) + numcomp <- min(c(which(cumsum(svd.ctlcovmat$d)/sum(svd.ctlcovmat$d)>=0.98),8)) # set max to 8 + # validation graphs look better with no more than 8 + pcs.ctlcovmat <- svd.ctlcovmat$u[,1:numcomp] + + if (save.quant) { + save(svd.ctlcovmat, file="svd.ctlcovmat.RData") + } + if (!save.quant) { + load("svd.ctlcovmat.RData") + } + + ###################################################################################### + #validation chunk + if (validate){ - NotNumeric<-function(a){ - return(any(!is.finite(as.matrix(a)))) - } + if (NotNumeric(numcomp) | numcomp>20) { + stop('There may be a problem with the SVD decomposition of the control matrix: number of components needed is either>20 or missing', "\n") + } - is.wholenumber <- function(x){abs(x - round(x)) == 0} + cat('\n', 'Starting validation with ', numcomp , ' components...', '\n') - #Function adjusts beta values with results of loess regression - applyfuntoonorm <- function(loessfits, sigA, sigB, Annot) { - - wh.red <- which(Annot$Type=='I' & Annot$Color=="Red") - wh.grn <- which(Annot$Type=='I' & Annot$Color=="Grn") - wh.II <- which(Annot$Type=='II') - - rankmatA.red <- (apply(sigA[wh.red,],2, rank) - 0.5)/length(wh.red) - rankmatA.grn <- (apply(sigA[wh.grn,],2, rank) - 0.5)/length(wh.grn) - rankmatA.II <- (apply(sigA[wh.II,],2, rank) - 0.5)/length(wh.II) - - rankmatB.red <- (apply(sigB[wh.red,],2, rank) - 0.5)/length(wh.red) - rankmatB.grn <- (apply(sigB[wh.grn,],2, rank) - 0.5)/length(wh.grn) - rankmatB.II <- (apply(sigB[wh.II,],2, rank) - 0.5)/length(wh.II) - - predmatA.red <- matrix(NA, nrow=nrow(rankmatA.red), ncol = ncol(rankmatA.red)) - predmatA.grn <- matrix(NA, nrow=nrow(rankmatA.grn), ncol = ncol(rankmatA.grn)) - predmatA.II <- matrix(NA, nrow=nrow(rankmatA.II), ncol = ncol(rankmatA.II)) - predmatB.red <- matrix(NA, nrow=nrow(rankmatB.red), ncol = ncol(rankmatB.red)) - predmatB.grn <- matrix(NA, nrow=nrow(rankmatB.grn), ncol = ncol(rankmatB.grn)) - predmatB.II <- matrix(NA, nrow=nrow(rankmatB.II), ncol = ncol(rankmatB.II)) - for (i in (1:ncol(predmatA.red))) { - predmatA.red[,i] <- predict(loessfits[[1]][[i]], newdata = logitfn(rankmatA.red[,i])) - predmatA.grn[,i] <- predict(loessfits[[2]][[i]], newdata = logitfn(rankmatA.grn[,i])) - predmatA.II[,i] <- predict(loessfits[[3]][[i]], newdata = logitfn(rankmatA.II[,i])) - predmatB.red[,i] <- predict(loessfits[[4]][[i]], newdata = logitfn(rankmatB.red[,i])) - predmatB.grn[,i] <- predict(loessfits[[5]][[i]], newdata = logitfn(rankmatB.grn[,i])) - predmatB.II[,i] <- predict(loessfits[[6]][[i]], newdata = logitfn(rankmatB.II[,i])) - } - predmatA <- matrix(NA, nrow(sigA), ncol(predmatA.red)) - predmatB <- predmatA - predmatA[wh.red,] <- predmatA.red - predmatA[wh.grn,] <- predmatA.grn - predmatA[wh.II,] <- predmatA.II - predmatB[wh.red,] <- predmatB.red - predmatB[wh.grn,] <- predmatB.grn - predmatB[wh.II,] <- predmatB.II + cores=1 #probably does not make sense to use parallelisation + pls.options(parallel = cores) + + ns <- nrow(quantilesA.red) + mat1P.Ared <- array(NA, dim=c(ns, ncol=nqnt, numcomp)) + mat1P.Agrn <- mat1P.Ared; mat1P.AII <- mat1P.Ared; mat1P.Bred <- mat1P.Ared + mat1P.Bgrn <- mat1P.Ared; mat1P.BII <- mat1P.Ared; mat2P.Ared <- mat1P.Ared; + mat2P.Agrn <- mat1P.Ared; mat2P.AII <- mat1P.Ared; mat2P.Bred <- mat1P.Ared; + mat2P.Bgrn <- mat1P.Ared; mat2P.BII <- mat1P.Ared; + + # potential for speedup + # by using an apply function here somehow? + for (j in (1:nqnt)) { + cvindex <- sample(ceiling((1:ns)/(ns/ncv.fold))) # cross validation groups + for (k in (1:10)) { + wh.cv <- which(cvindex==k) + tempfit <- mvr(quantilesA.red[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "svdpc") + mat1P.Ared[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesA.grn[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "svdpc") + mat1P.Agrn[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesA.II[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "svdpc") + mat1P.AII[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesB.red[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "svdpc") + mat1P.Bred[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesB.grn[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "svdpc") + mat1P.Bgrn[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesB.II[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "svdpc") + mat1P.BII[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) - newBeta <- (exp(predmatB)-1)/(exp(predmatA) + exp(predmatB)-2) - - origBeta <- (exp(sigB)-1)/(exp(sigA) + exp(sigB) - 2 + 0.1) - origBeta <- (origBeta * 999 + 0.5)/1000 - rownames(newBeta) <- rownames(origBeta) - colnames(newBeta) <- colnames(origBeta) - return(list(origBeta=origBeta, newBeta=newBeta)) + tempfit <- mvr(quantilesA.red[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "kernelpls") + mat2P.Ared[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesA.grn[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "kernelpls") + mat2P.Agrn[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesA.II[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "kernelpls") + mat2P.AII[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesB.red[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "kernelpls") + mat2P.Bred[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesB.grn[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "kernelpls") + mat2P.Bgrn[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + tempfit <- mvr(quantilesB.II[-wh.cv,j] ~ ctl.covmat[-wh.cv,],ncomp=numcomp, method = "kernelpls") + mat2P.BII[wh.cv, j, ] <- predict(tempfit, newdat = ctl.covmat[wh.cv,]) + } # for k + } # for j + + rmse1.Ared <- matrix(NA, nrow = numcomp, ncol=nqnt) + rmse1.Agrn <- rmse1.Ared; rmse1.AII <- rmse1.Ared + rmse2.Ared <- rmse1.Ared; + rmse2.Agrn <- rmse1.Ared; rmse2.AII <- rmse1.Ared + rmse1.Bred <- rmse1.Ared + rmse1.Bgrn <- rmse1.Ared; rmse1.BII <- rmse1.Ared + rmse2.Bred <- rmse1.Ared; + rmse2.Bgrn <- rmse1.Ared; rmse2.BII <- rmse1.Ared + + for (i.n in (1:numcomp)) { + rmse1.Ared[i.n,] <- sqrt(apply((mat1P.Ared[,,i.n]-quantilesA.red)^2, 2, sum)) + rmse1.Agrn[i.n,] <- sqrt(apply((mat1P.Agrn[,,i.n]-quantilesA.grn)^2, 2, sum)) + rmse1.AII[i.n,] <- sqrt(apply((mat1P.AII[,,i.n]-quantilesA.II)^2, 2, sum)) + rmse1.Bred[i.n,] <- sqrt(apply((mat1P.Bred[,,i.n]-quantilesB.red)^2, 2, sum)) + rmse1.Bgrn[i.n,] <- sqrt(apply((mat1P.Bgrn[,,i.n]-quantilesB.grn)^2, 2, sum)) + rmse1.BII[i.n,] <- sqrt(apply((mat1P.BII[,,i.n]-quantilesB.II)^2, 2, sum)) + + rmse2.Ared[i.n,] <- sqrt(apply((mat2P.Ared[,,i.n]-quantilesA.red)^2, 2, sum)) + rmse2.Agrn[i.n,] <- sqrt(apply((mat2P.Agrn[,,i.n]-quantilesA.grn)^2, 2, sum)) + rmse2.AII[i.n,] <- sqrt(apply((mat2P.AII[,,i.n]-quantilesA.II)^2, 2, sum)) + rmse2.Bred[i.n,] <- sqrt(apply((mat2P.Bred[,,i.n]-quantilesB.red)^2, 2, sum)) + rmse2.Bgrn[i.n,] <- sqrt(apply((mat2P.Bgrn[,,i.n]-quantilesB.grn)^2, 2, sum)) + rmse2.BII[i.n,] <- sqrt(apply((mat2P.BII[,,i.n]-quantilesB.II)^2, 2, sum)) } - ############################################################################### + pdf(file = 'validationcurves.PCR.pdf', height=7, width=7) + par(omi=c(0,0,0,0)) + m <- matrix(c(1,2,3,4,5,6,7,7,7), nrow = 3, ncol = 3, byrow = TRUE) + layout(mat = m, heights = c(0.3,0.3,0.15)) + par(mar = c(2,2,0.2, 0.2), mgp = c(1,0,0)) + sq <- 2; while (max(rmse1.Ared[,sq])>1.3*max(rmse1.Ared[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse1.Ared)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse1.Ared[,sq:nqnt])), "PCR, A I red", cex=1.0) - if (is.null(Annot)) - {cat("Since Annot is NULL using default annotation.", '\n') - data('Annot', package='funtooNorm', envir = environment()) - } - if (is.null(cp.types)) - {cat("Since cp.types is NULL using default cp.types.", '\n') - data('cp.types', package='funtooNorm', envir = environment()) - } + sq <- 2; while (max(rmse1.Agrn[,sq])>1.3*max(rmse1.Agrn[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse1.Agrn)[sq:nqnt,]), type='l', lty=1,col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse1.Agrn[,sq:nqnt])), "PCR, A I green", cex=1.0) - #checking sanity of the data - cat("Checking sanity of the data...", '\n') - if (NotNumeric(sigA)){stop("There are non-numeric values in the matrix", '\n')} - if (NotNumeric(sigB)){stop("There are non-numeric values in the matrix", '\n')} - if (NotNumeric(controlred)){stop("There are non-numeric values in the matrix", '\n')} - if (NotNumeric(controlgrn)){stop("There are non-numeric values in the matrix", '\n')} - if (any(cell_type == '' | typeof(cell_type) != 'character' | is.na(cell_type))) {{stop("There are non-character values in cell_type", '\n')}} - if (length(unique(cell_type))<2) {{stop("There should be more that one tissue or cell type in cell_type variable", '\n')}} - if (any(cp.types == '' | typeof(cp.types) != 'character' | is.na(cp.types))) {{stop("There are non-character values in cp.types", '\n')}} - - #check that ID's match between sigA, sigB and control probe data - if (!(identical(colnames(controlgrn), colnames(sigA)) & - identical(colnames(controlred), colnames(sigA)) & - identical(colnames(sigB), colnames(sigA)))) { - if ((ncol(controlgrn) == ncol(sigA)) & (ncol(controlred) == ncol(sigA)) - & (ncol(controlred) == ncol(sigB)) & (length(cp.types) == ncol(sigB)) & (length(cell_type) == ncol(sigB))){ - ix <- sort(colnames(controlred), index.return = T)$ix - controlgrn<-controlgrn[,ix]; sigA<-sigA[,ix]; sigB<-sigB[,ix]; - if (!(identical(colnames(controlgrn), colnames(sigA)) & - identical(colnames(controlred), colnames(sigA)) & - identical(colnames(sigB), colnames(sigA)))){ - stop("Sample names do not match.", '\n')} - - } else{stop("Data dimensions or samples names do not match.", '\n')} - } + sq <- 2; while (max(rmse1.AII[,sq])>1.3*max(rmse1.AII[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse1.AII)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse1.AII[,sq:nqnt])), "PCR, A II", cex=1.0) - #check if annotation is ok, and subset annotation on the probes from Sig files - if(any(!(rownames(sigA) %in% rownames(Annot)))){stop("Probe names do not match annotation entries", '\n')} - cpg<-intersect(rownames(sigA), rownames(Annot)) - Annot=Annot[cpg,] - - cat("Data is ok.", '\n') + sq <- 2; while (max(rmse1.Bred[,sq])>1.3*max(rmse1.Bred[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse1.Bred)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse1.Bred[,sq:nqnt])), "PCR, B I red", cex=1.0) - sigA <- log2(1 + sigA) - sigB <- log2(1 + sigB) - - nr <- nrow(sigA) - #qntllist <- c((0.5)/nr, seq(0.001, 0.009, 0.001), seq(0.01,0.05,0.01), - # seq(0.07,0.93,by=0.02), seq(0.95,0.99,0.01), seq(0.991,0.999,0.001), - # (nr-0.5)/nr) - qntllist <- c(seq((0.5)/nr, 0.0009, 0.0001), seq(0.001, 0.009, 0.001), seq(0.01,0.05,0.01), - seq(0.07,0.93,by=0.02), seq(0.95,0.99,0.01), seq(0.991,0.999,0.001), - seq(0.999 ,(nr-0.5)/nr,0.0001 )) - nqnt <- length(qntllist) # number of desired quantiles - - ####! TODO good to add special treatment of X and Y chromosomes. See Fortin et al. particularly for Y - if (save.quant) { - quantilesA.red <- matrix(NA, ncol(sigA), nqnt) - quantilesA.grn <- quantilesA.red; quantilesA.II <- quantilesA.red - quantilesB.red <- matrix(NA, ncol(sigA), nqnt) - quantilesB.grn <- quantilesA.red; quantilesB.II <- quantilesA.red - # need separate estimation for X but the X chromosome is currently removed - #quantiles.Xred <- quantiles.red - #quantiles.Xgrn <- quantiles.red - #quantiles.XII <- quantiles.red - for (j in (1:nqnt)) { - quantilesA.red[,j] <- apply(sigA[Annot$Type=="I" & Annot$Color=='Red',], - 2, quantile, qntllist[j]) - quantilesA.grn[,j] <- apply(sigA[Annot$Type=="I" & Annot$Color=='Grn',], - 2, quantile, qntllist[j]) - quantilesA.II[,j] <- apply(sigA[Annot$Type=="II",], - 2, quantile, qntllist[j]) - quantilesB.red[,j] <- apply(sigB[Annot$Type=="I" & Annot$Color=='Red',], - 2, quantile, qntllist[j]) - quantilesB.grn[,j] <- apply(sigB[Annot$Type=="I" & Annot$Color=='Grn',], - 2, quantile, qntllist[j]) - quantilesB.II[,j] <- apply(sigB[Annot$Type=="II",], - 2, quantile, qntllist[j]) - # quantiles.Xred[,j] <- apply(JPNorm_ABeta[Annot$Type=="I" & Annot$Color=='Red' & - # Annot$chr=="X",], 2, quantile, j/nqnt) - # quantiles.Xgrn[,j] <- apply(JPNorm_ABeta[Annot$Type=="I" & Annot$Color=='Grn' & - # Annot$chr=="X",], 2, quantile, j/nqnt) - # quantiles.XII[,j] <- apply(JPNorm_ABeta[Annot$Type=="II" & Annot$chr=="X",], - # 2, quantile, j/nqnt) - } # for j - save(quantilesA.red, file="quantilesA.red.RData") - save(quantilesA.grn, file="quantilesA.grn.RData") - save(quantilesA.II, file="quantilesA.II.RData") - save(quantilesB.red, file="quantilesB.red.RData") - save(quantilesB.grn, file="quantilesB.grn.RData") - save(quantilesB.II, file="quantilesB.II.RData") - } # if save.quant - if (!save.quant) { - load("quantilesA.red.RData") - load("quantilesA.grn.RData") - load("quantilesA.II.RData") - load("quantilesB.red.RData") - load("quantilesB.grn.RData") - load("quantilesB.II.RData") - } + sq <- 2; while (max(rmse1.Bgrn[,sq])>1.3*max(rmse1.Bgrn[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse1.Bgrn)[sq:nqnt,]), type='l', lty=1,col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse1.Bgrn[,sq:nqnt])), "PCR, B I green", cex=1.0) - - controlgrn <- log2(1 + controlgrn) - controlred <- log2(1 + controlred) + sq <- 2; while (max(rmse1.BII[,sq])>1.3*max(rmse1.BII[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse1.BII)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse1.BII[,sq:nqnt])), "PCR, B II", cex=1.0) - # construct control probe summaries, averages by type of control probe - # then specifically create columns by cell type - cp.type.tab <- table(cp.types) - for (k in (1:length(cp.type.tab))) { - temp1 <- apply(controlred[cp.types==names(cp.type.tab[k]),],2,mean) - temp2 <- apply(controlgrn[cp.types==names(cp.type.tab[k]),],2,mean) - if (k==1) { - mat.by.ct <- cbind(temp1,temp2) } - if (k>1) { mat.by.ct <- cbind(mat.by.ct, cbind(temp1,temp2)) } - } # end for mat.by.ct is 15 columns - means for each type of control probe - - ind=matrix(FALSE,ncol=length(cell_type),nrow=length(unique(cell_type))) - for(j in 1:length(unique(cell_type))){ - ind[j,]<- (cell_type==names(table(cell_type))[j]) - } - # now add additional sets of 15 columns, each specific to one cell type - ctl.covmat <- cbind(mat.by.ct, apply(mat.by.ct, 2, mult1, ind[1,])) - for(j in 2:length(unique(cell_type))){ - ctl.covmat <- cbind(ctl.covmat, apply(mat.by.ct, 2, mult1, ind[j,])) - } + par(mar=c(0, 0, 1, 0)) + plot.new() + ltylist <- rep(1, min(numcomp,8)) + if (numcomp>8) ltylist <- c(ltylist, rep(2, numcomp-8)) + legend(title='Number of components:', x = "top",inset = 0, + legend = 1:numcomp, col=1:numcomp, lty=ltylist, cex=1.1, horiz = TRUE) + dev.off() - if (save.quant) { - save(ctl.covmat, file="ctl.covmat.RData") - } - if (!save.quant) { - load("ctl.covmat.RData") - } + pdf(file = 'validationcurves.PLS.pdf', height=7, width=7) + par(omi=c(0,0,0,0)) + m <- matrix(c(1,2,3,4,5,6,7,7,7), nrow = 3, ncol = 3, byrow = TRUE) + layout(mat = m, heights = c(0.3,0.3,0.15)) - ###################################################################################### - #validation chunck - if (validate){ - if (NotNumeric(validate)){stop('Validate parameter should be integer bigger than 1')} - else{if(!is.wholenumber(validate) | (validate<=1)){ - stop('Validate parameter should be integer bigger than 1')} - } - - cat('\n', 'Starting validation with ', validate , ' PLS components...', '\n') + par(mar = c(2,2,0.2, 0.2), mgp = c(1,0,0)) + sq <- 2; while (max(rmse2.Ared[,sq])>1.3*max(rmse2.Ared[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse2.Ared)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse2.Ared[,sq:nqnt])), "PLS, A I red", cex=1.0) - cores=1 #probably does not make sense to use parallelisation - pls.options(parallel = cores) - - fit2cvA.red <- plsr(quantilesA.red ~ ctl.covmat, ncomp=validate, validation="CV") - fit2cvB.red <- plsr(quantilesB.red ~ ctl.covmat, ncomp=validate, validation="CV") - fit2cvA.grn <- plsr(quantilesA.grn ~ ctl.covmat, ncomp=validate, validation="CV") - fit2cvB.grn <- plsr(quantilesB.grn ~ ctl.covmat, ncomp=validate, validation="CV") - fit2cvA.II <- plsr(quantilesA.II ~ ctl.covmat, ncomp=validate, validation="CV") - fit2cvB.II <- plsr(quantilesB.II ~ ctl.covmat, ncomp=validate, validation="CV") - - pdf(file = 'validationCurves.pdf') - par(mar=c(5.1, 4.1, 5.1, 2.1)) - m <- matrix(c(1,1,1,2,3,4,5,6,7,8,8,8), nrow = 4, ncol = 3, byrow = TRUE) - #mtext("Root mean square error of prediction ", line=2, font=2, cex=1.2) - layout(mat = m, heights = c(0.2,0.35,0.35,0.2)) - - par(mar=c(0, 0, 0, 0)) - plot.new() - text(0.5, 0.4,"Root mean square error of prediction",cex=2,font=1.5) - par(mar=c(3, 3, 3, 3), mgp = c(2.0, 0.5, 0)) - matplot(t(apply(RMSEP(fit2cvA.red, estimate='adjCV', intercept=F)$val, 2, function(x) x)), ylab="Error", main= 'A red', type = "l", col=1:validate, lty=1) - matplot(t(apply(RMSEP(fit2cvA.grn, estimate='adjCV', intercept=F)$val, 2, function(x) x)), ylab="", main= 'A grn', type = "l" , col=1:validate, lty=1) - matplot(t(apply(RMSEP(fit2cvA.II, estimate='adjCV', intercept=F)$val, 2, function(x) x)), ylab="", main= 'A II', type = "l" , col=1:validate, lty=1) - matplot(t(apply(RMSEP(fit2cvB.red, estimate='adjCV', intercept=F)$val, 2, function(x) x)), ylab="Error", xlab="Quantiles", main= 'B red', type = "l" , col=1:validate, lty=1) - matplot(t(apply(RMSEP(fit2cvB.grn, estimate='adjCV', intercept=F)$val, 2, function(x) x)), xlab="Quantiles", ylab="", main= 'B grn', type = "l" , col=1:validate, lty=1) - matplot(t(apply(RMSEP(fit2cvB.II, estimate='adjCV', intercept=F)$val, 2, function(x) x)), xlab="Quantiles", ylab="", main= 'B II', type = "l" , col=1:validate, lty=1) - - par(mar=c(0, 0, 1, 0)) - # c(bottom, left, top, right) - plot.new() - #plot.new() - #plot(1, type = "n", axes=FALSE, xlab="", ylab="") - #plot_colors <- c("blue","black", "green", "orange", "pink") - legend(title='Number of PLS components:', x = "top",inset = 0, - legend = 1:validate, - col=1:validate, lty=1, cex=1, horiz = TRUE) - #title("My 'Title' in a strange place", side = 3, line = -21, outer = TRUE) - dev.off() - - - - cat('Done. Check your working directory for the file "validationCurves.pdf"', '\n') - return() - } + sq <- 2; while (max(rmse2.Agrn[,sq])>1.3*max(rmse2.Agrn[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse2.Agrn)[sq:nqnt,]), type='l', lty=1,col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse2.Agrn[,sq:nqnt])), "PLS, A I green", cex=1.0) + sq <- 2; while (max(rmse2.AII[,sq])>1.3*max(rmse2.AII[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse2.AII)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse2.AII[,sq:nqnt])), "PLS, A II", cex=1.0) + sq <- 2; while (max(rmse2.Bred[,sq])>1.3*max(rmse2.Bred[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse2.Bred)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse2.Bred[,sq:nqnt])), "PLS, B I red", cex=1.0) + sq <- 2; while (max(rmse2.Bgrn[,sq])>1.3*max(rmse2.Bgrn[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse2.Bgrn)[sq:nqnt,]), type='l', lty=1,col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse2.Bgrn[,sq:nqnt])), "PLS, B I green", cex=1.0) - # here are the actual fits, one for each quantile in qntllist - fit2.red <- list() ; fit2.grn <- list(); fit2.II <- list() - for (j in (1:nqnt)) { - fit2.red <- c(fit2.red, list( - plsr(cbind(quantilesA.red[,j], quantilesB.red[,j]) ~ ctl.covmat, ncomp=ncmp))) - fit2.grn <- c(fit2.grn, list( - plsr(cbind(quantilesA.grn[,j], quantilesB.grn[,j]) ~ ctl.covmat, ncomp=ncmp))) - fit2.II <- c(fit2.II, list( - plsr(cbind(quantilesA.II[,j], quantilesB.II[,j]) ~ ctl.covmat, ncomp=ncmp))) - } + sq <- 2; while (max(rmse2.BII[,sq])>1.3*max(rmse2.BII[,0.4*nqnt])) sq<-sq+1 + matplot(qntllist[sq:nqnt], sqrt(t(rmse2.BII)[sq:nqnt,]), type='l', lty=1, col=1:numcomp, xlab = 'Percentiles', ylab='RMSE') + text(0.6, max(sqrt(rmse2.BII[,sq:nqnt])), "PLS, B II", cex=1.0) - # smooth the predictions so can get a value for any quantile - loessfitsA.red <- list() - loessfitsA.grn <- list() - loessfitsA.II <- list() - loessfitsB.red <- list() - loessfitsB.grn <- list() - loessfitsB.II <- list() - xq <- logitfn(qntllist) - for (i in (1:nrow(quantilesA.red))) { # i: over samples - # assemble the fitted values into a vector - fittedvals <- unlist(lapply(fit2.red, extractqnt, i=i, AB=1, ncmp=ncmp)) - coef.fit <- loess(fittedvals ~ xq, span = 0.15) - loessfitsA.red <- c(loessfitsA.red, list(coef.fit)) - fittedvals <- unlist(lapply(fit2.grn, extractqnt, i=i, AB=1, ncmp=ncmp)) - coef.fit <- loess(fittedvals ~ xq, span = 0.15) - loessfitsA.grn <- c(loessfitsA.grn, list(coef.fit)) - fittedvals <- unlist(lapply(fit2.II, extractqnt, i=i, AB=1, ncmp=ncmp)) - coef.fit <- loess(fittedvals ~ xq, span = 0.15) - loessfitsA.II <- c(loessfitsA.II, list(coef.fit)) - - fittedvals <- unlist(lapply(fit2.red, extractqnt, i=i, AB=2, ncmp=ncmp)) - coef.fit <- loess(fittedvals ~ xq, span = 0.15) - loessfitsB.red <- c(loessfitsB.red, list(coef.fit)) - fittedvals <- unlist(lapply(fit2.grn, extractqnt, i=i, AB=2, ncmp=ncmp)) - coef.fit <- loess(fittedvals ~ xq, span = 0.15) - loessfitsB.grn <- c(loessfitsB.grn, list(coef.fit)) - fittedvals <- unlist(lapply(fit2.II, extractqnt, i=i, AB=2, ncmp=ncmp)) - coef.fit <- loess(fittedvals ~ xq, span = 0.15) - loessfitsB.II <- c(loessfitsB.II, list(coef.fit)) - } - if (save.loess) { - save(loessfitsA.red, file="loessfitsA.red") - save(loessfitsA.grn, file="loessfitsA.grn") - save(loessfitsA.II, file="loessfitsA.II") - save(loessfitsB.red, file="loessfitsB.red") - save(loessfitsB.grn, file="loessfitsB.grn") - save(loessfitsB.II, file="loessfitsB.II") - } - loessfits <- list(loessfitsA.red, loessfitsA.grn, loessfitsA.II, - loessfitsB.red, loessfitsB.grn, loessfitsB.II) - if (apply.loess) { - betamatrices <- applyfuntoonorm(loessfits, sigA, sigB, Annot) - return(betamatrices) - } else { return(loessfits) } + par(mar=c(0, 0, 1, 0)) + plot.new() + ltylist <- rep(1, min(numcomp,8)) + if (numcomp>8) ltylist <- c(ltylist, rep(2, numcomp-8)) + legend(title='Number of components:', x = "top",inset = 0, + legend = 1:numcomp, col=1:numcomp, lty=ltylist, cex=1.1, horiz = TRUE) + dev.off() + + selquant <- sort(sample((1:nqnt), 10)) + rmse.red <- array(NA, dim = c(length(selquant), length(selquant), numcomp)) + betaP.red <- rmse.red + rmse.grn <- rmse.red; betaP.grn <- betaP.red + rmse.II <- rmse.red; betaP.II <- betaP.red + for (i.n in (1:numcomp)) { + for (i.s1 in (1:length(selquant))) { + for (i.s2 in (1:length(selquant))) { + betaP <- calcbeta(mat1P.Ared[,selquant[i.s1], i.n], mat1P.Bred[,selquant[i.s2],i.n],offset=0.0001) + betaO <- calcbeta(quantilesA.red[,selquant[i.s1]], quantilesB.red[,selquant[i.s2]], offset=0.0001) + rmse.red[i.s1,i.s2, i.n] <- sqrt(sum((betaP-betaO)^2)) + betaP.red[i.s1,i.s2, i.n] <- mean(betaP) + betaP <- calcbeta(mat1P.Agrn[,selquant[i.s1], i.n], mat1P.Bgrn[,selquant[i.s2],i.n],offset=0.0001) + betaO <- calcbeta(quantilesA.grn[,selquant[i.s1]], quantilesB.grn[,selquant[i.s2]], offset=0.0001) + rmse.grn[i.s1,i.s2, i.n] <- sqrt(sum((betaP-betaO)^2)) + betaP.grn[i.s1,i.s2, i.n] <- mean(betaP) + betaP <- calcbeta(mat1P.AII[,selquant[i.s1], i.n], mat1P.BII[,selquant[i.s2],i.n],offset=0.0001) + betaO <- calcbeta(quantilesA.II[,selquant[i.s1]], quantilesB.II[,selquant[i.s2]], offset=0.0001) + rmse.II[i.s1,i.s2, i.n] <- sqrt(sum((betaP-betaO)^2)) + betaP.II[i.s1,i.s2, i.n] <- mean(betaP) + }} # i.s1, i.s2 + } # i.n + + #pdf(file = 'validationcurves.beta.PCR.pdf', height=7, width=7) + #par(mfrow = c(2,2), omi=c(0,0,0,0), mar = rep(0,4), mgp = c(-1,0,0)) + + #plot(c(0,1), c(min(rmse.red), max(rmse.red)), type='n', xlab='', ylab='RMSE') + #for (i.n in (1:8)) { + # points(betaP.red[,,i.n], rmse.red[,,i.n], col=i.n, pch=16, cex=0.7) } + + #plot(c(0,1), c(min(rmse.grn), max(rmse.grn)), type='n', xlab='', ylab='RMSE') + #for (i.n in (1:8)) { + # points(betaP.grn[,,i.n], rmse.grn[,,i.n], col=i.n, pch=16, cex=0.7) } + + #plot(c(0,1), c(min(rmse.II), max(rmse.II)), type='n', xlab='', ylab='RMSE') + #for (i.n in (1:8)) { + # points(betaP.II[,,i.n], rmse.II[,,i.n], col=i.n, pch=16, cex=0.7) } + + #plot.new() + #legend(title='Number of components:', x = "top", + # legend = 1:numcomp, col=1:numcomp, cex=1.1, lty=1, horiz = FALSE) + + #dev.off() + + cat('Done. Check your working directory for the file "validationcurves.PCR.pdf" and validationcurves.PLS.pdf"', '\n') + return() + } # end if validate + ######################### + + # could add in here different values of ncmp for different probe types + predA.red <- matrix(NA, nrow = ncol(sigA), ncol = nqnt) + predA.grn <- matrix(NA, nrow = ncol(sigA), ncol = nqnt) + predA.II <- matrix(NA, nrow = ncol(sigA), ncol = nqnt) + predB.red <- matrix(NA, nrow = ncol(sigA), ncol = nqnt) + predB.grn <- matrix(NA, nrow = ncol(sigA), ncol = nqnt) + predB.II <- matrix(NA, nrow = ncol(sigA), ncol = nqnt) + for (j in (1:nqnt)) { + predA.red[,j] <- mvr(quantilesA.red[,j] ~ ctl.covmat, ncomp=ncmp, method=method.mvr)$fitted.values[,1,ncmp] + predB.red[,j] <- mvr(quantilesB.red[,j] ~ ctl.covmat, ncomp=ncmp, method=method.mvr)$fitted.values[,1,ncmp] + predA.grn[,j] <- mvr(quantilesA.grn[,j] ~ ctl.covmat, ncomp=ncmp, method=method.mvr)$fitted.values[,1,ncmp] + predB.grn[,j] <- mvr(quantilesB.grn[,j] ~ ctl.covmat, ncomp=ncmp, method=method.mvr)$fitted.values[,1,ncmp] + predA.II[,j] <- mvr(quantilesA.II[,j] ~ ctl.covmat, ncomp=ncmp, method=method.mvr)$fitted.values[,1,ncmp] + predB.II[,j] <- mvr(quantilesB.II[,j] ~ ctl.covmat, ncomp=ncmp, method=method.mvr)$fitted.values[,1,ncmp] + } # for j + + lfits <- list(predA.red, predA.grn, predA.II, + predB.red, predB.grn, predB.II) + if (apply.fits) { + betamatrices <- applyfuntoonorm(lfits, sigA, sigB, Annot, qntllist) + return(betamatrices) + } else { return(lfits) } } # end function diff --git a/man/agreement.Rd b/man/agreement.Rd index 25785e0..dd5e25c 100644 --- a/man/agreement.Rd +++ b/man/agreement.Rd @@ -38,11 +38,11 @@ #This call will perform normalization of the data: ncmp <- 4 - funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, Annot=NULL, - controlred=matred, controlgrn=matgrn, - cp.types=NULL, cell_type = cell_type, - ncmp=4, save.quant=TRUE, save.loess=TRUE, apply.loess=TRUE, - validate=FALSE) + funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, Annot=Annotsample, + controlred=matred, controlgrn=matgrn, + cp.types=NULL, cell_type = cell_type, + logged.data=FALSE, save.quant=TRUE, ncmp=ncmp, apply.fits=TRUE, + validate=FALSE) #To calculate measures of agreement before and after normalization agreement(funtoonormout[[1]], individualID) diff --git a/man/funtooNorm-package.Rd b/man/funtooNorm-package.Rd index 5862bd6..6e96c55 100644 --- a/man/funtooNorm-package.Rd +++ b/man/funtooNorm-package.Rd @@ -27,8 +27,9 @@ License: \tab GPL-3\cr %% data("data") %% ncmp <- 4 %% funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, Annot=Annotsample, -%% controlred=matred, controlgrn=matgrn, -%% cp.types=cp.types, cell_type = cell_type, -%% ncmp=ncmp, save.quant=TRUE, save.loess=TRUE, apply.loess=TRUE, logit.quant=TRUE, validate=FALSE) +%% controlred=matred, controlgrn=matgrn, +%% cp.types=cp.types, cell_type = cell_type, +%% logged.data=FALSE, save.quant=FALSE, ncmp=ncmp, apply.fits=TRUE, +%% validate=FALSE) %%} diff --git a/man/funtoonorm.Rd b/man/funtoonorm.Rd index 13b7941..48a08b1 100644 --- a/man/funtoonorm.Rd +++ b/man/funtoonorm.Rd @@ -9,73 +9,82 @@ This function performs normalization of Illumina Infinium Human Methylation 450 } \usage{ funtoonorm(sigA, sigB, Annot = NULL, controlred, controlgrn, -cp.types = NULL, cell_type, ncmp = 4, save.quant = TRUE, save.loess = TRUE, -apply.loess = TRUE, validate = FALSE) +cp.types = NULL, cell_type, ncmp = 4, +ncv.fold = 10, logged.data=FALSE, save.quant=TRUE, +type.fits="PCR", apply.fits = TRUE, validate = FALSE) } \arguments{ \item{sigA, sigB}{ -Matrices containing the signal A and signal B results extracted from the IDAT files. -} -\item{controlred, controlgrn}{ -Matrices containing control probe data. +Matrices containing the signal A and signal B results extracted from the IDAT files. (The user should know whether or not these data have undergone a log transformation prior to being sent into funtooNorm.) } \item{Annot}{ Annotation matrix. Supplied by default. } - +\item{controlred, controlgrn}{ +Matrices containing control probe data. (Again, the user should know whether the data have been log transformed prior to calling funtooNorm). +} \item{cp.types}{ -Vector of types of control probes. +Vector of types of control probes. (Often available as the row names from the control probe information). } \item{cell_type}{ Vector of cell type (or tissue type) information. } \item{ncmp}{ -Number of partial least squares components used in the model fitting. +Number of components, from either principal component regression or partial least squares regression, used in the model fitting when \verb{validate=FALSE}. } +\item{ncv.fold}{ +Number of cross-validation partitions. +} +\item{logged.data}{ +Logical, \verb{TRUE} if data have been previously log transformed (sigA, sigB, controlred, controlgrn), and \verb{FALSE} if not. +} + \item{save.quant}{ Logical, whether to save calculated quantiles. } -\item{save.loess}{ -Logical, whether to save calculated results of loess regression. +\item{type.fits}{ +Values are either \verb{"PLS"}, or \verb{"PCR"} for partial least squares or principal component regression, respectively. The default is \verb{"PCR"}. } -\item{apply.loess}{ -Logical, whether to apply results of loess regression. If TRUE, two matrices are returned, one the data before normalization and one after normalization. -normalised beta values is returned. +\item{apply.fits}{ +Logical, whether to apply results of the PCR/PLS fits to normalize the data. If \verb{TRUE}, two matrices are returned, one the data before normalization and one after normalization. +This would normally be set to \verb{FALSE} when \verb{validate=TRUE}. } \item{validate}{ -Either FALSE, or the maximum number of PLS components to be explored in cross-validation. -If FALSE, the normalization corrections are calculated using \verb{ncmp} partial least squares (PLS) components. if not FALSE, then a number must be supplied. This number will be the maximum number of PLS components used when exploring model fit performance across a range of \verb{ncmp} values ranging from 1 to the supplied number. +Logical. If \verb{FALSE}, the normalization corrections are calculated using \verb{ncmp} components. +If \verb{TRUE}, then a range of numbers of components are explored graphically. } } \details{ -The funtooNorm function operates in one of two modes. If validate=FALSE, then the normalization corrections are calculated using the supplied value of \verb{ncmp} to fix the number of partial least squares (PLS) components. If validate is an integer, K>1, (e.g. K=5), then cross-validation is performed exploring performance across a range of values for \verb{ncmp} ranging from 1 to K. +The funtooNorm function operates in one of two modes. If \verb{validate=FALSE}, then the normalization corrections are calculated using the supplied value of \verb{ncmp} to specify the number of components. +If \verb{validate=TRUE}, then cross-validation is performed exploring performance across a range of values. The maximum value is set to 8 to facilite the visualization of results. } \value{The values returned depend on the parameters chosen. \itemize{ -\item If validate is not FALSE (i.e. validate=K), the function creates a pdf file containing a series of plots showing residual error curves across percentiles of the signal distributions, to facilitate the choice of an appropriate value for \verb{ncmp}. No object is returned by the function. +\item If validate is TRUE the function creates two pdf files, each containing a series of plots showing root mean squared error curves across percentiles of the signal distributions, obtained from cross-validation, to facilitate the choice of an appropriate value for \verb{ncmp}. No object is returned by the function. -\item If validate = FALSE, then funtoonorm has the following behaviour: +\item If validate is FALSE, then \verb{funtoonorm} has the following behaviour: \itemize{ - \item If apply.loess = FALSE the function will not return any object. - However, if save.loess=TRUE or if save.quant=TRUE then RData objects will be saved to disk for future use. - \item If apply.less= TRUE, then the function returns a list of 2 objects. The first, 'origBeta', is the matrix of Beta avalues before normalization, and the second, 'newBeta' is the Beta values after normalization. + \item If apply.fits = FALSE the function will not return any object. + However, if save.quant=TRUE then RData objects will be saved to disk for future use. + \item If apply.fits= TRUE, then the function returns a list of 2 objects. The first, 'origBeta', is the matrix of Beta values before normalization, and the second, 'newBeta' is the Beta values after normalization. } } } \examples{ -%% to normalize methylation data: -ncmp <- 4 -funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, Annot=Annotsample, - controlred=matred, controlgrn=matgrn, - cp.types=cp.types, cell_type = cell_type, - ncmp=ncmp, save.quant=TRUE, save.loess=TRUE, apply.loess=TRUE, - validate=FALSE) %%to choose the number of components: funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, controlred=matred, controlgrn=matgrn, - cp.types=cp.types, cell_type = cell_type, - ncmp=4, save.quant=TRUE, save.loess=TRUE, - apply.loess=FALSE, validate=5) + cp.types=NULL, cell_type = cell_type, + logged.data=FALSE, save.quant=TRUE, apply.fits=FALSE, + validate=TRUE) +%% to normalize methylation data, assuming save.quant was set to TRUE in the previous call: +ncmp <- 4 +funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, Annot=Annotsample, + controlred=matred, controlgrn=matgrn, + cp.types=NULL, cell_type = cell_type, + logged.data=FALSE, save.quant=FALSE, ncmp=ncmp, apply.fits=TRUE, + validate=FALSE) + } diff --git a/vignettes/funtooNorm.Rnw b/vignettes/funtooNorm.Rnw index 2073720..dbcd641 100644 --- a/vignettes/funtooNorm.Rnw +++ b/vignettes/funtooNorm.Rnw @@ -49,8 +49,6 @@ Percentiles of methylation levels may vary across cell types and hence the origi Normalization separately for each cell type may introduce unwanted variability if sample sizes are small. Therefore, this algorithm provides flexibility while optimizing the sample size used to estimate the corrections. -A partial least squares (PLS) fit is included as the core of the algorithm, followed by smoothing across percentiles. - Note that the current version of the package does not do a good job of normalizing the Y chromosome probes; the funNorm method performs better. In a subsequent version of the package we will address this issue. @@ -75,7 +73,7 @@ The program also requires the following information, but default matrices are pr \item \texttt{Annot:} An annotation table, containing information on probe names, probe type, and color (for probes of type I). This can be extracted from the Illumina annotation information for the Infinium BeadChip. A default annotation table is provided if not supplied including all probes on the 450K array. -\item \texttt{cp.types:} A list of the types of control probes to be used in the normalization. +\item \texttt{cp.types:} A list of the types of control probes to be used in the normalization. This may be obtainable from the row names of the control probe matrices (controlred, controlgrn). \end{itemize} Finally, a number of parameters control whether intermediate calculations should be stored, simply so that the analysis can be performed in stages if desired. @@ -88,51 +86,76 @@ Here is a basic call to normalize this sample data set: %\begin{Verbatim} <<>>= library(funtooNorm) - ncmp <- 4 + ncmp <-4 funtoonormout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, Annot=NULL, controlred=matred, controlgrn=matgrn, cp.types=NULL, cell_type = cell_type, - ncmp=4, save.quant=TRUE, save.loess=TRUE, - apply.loess=TRUE, validate=FALSE) + ncmp=4, ncv.fold=10, logged.data=FALSE, save.quant=TRUE, + type.fits="PCR", apply.fits=TRUE, validate=FALSE) @ %\end{Verbatim} -\texttt{save.quant} implies that the quantiles should be saved; \texttt{save.loess} means that loess fits to the curves should be saved; \texttt{apply.loess} means that the normalization itself should be applied to all the data based on the loess fits; \texttt{logit.quant} asks whether the quantiles should be logit-transformed prior to fitting PLS models. +FuntooNorm will fit either principal component regression (PCR) or partial least squares regression (PLS) by specifying \texttt{type.fits="PCR"} or \texttt{type.fits="PLS"}. The default is set to "PCR" to match funNorm. +An important user-chosen parameter is \texttt{ncmp}, the number of components to be included in either of these two models; these components are calculated from the control probe data and cell type data. + +Choice of the number of components can be facilitated by examining a series of fits with different numbers of components. +When \texttt{validate=TRUE}, funtooNorm produces two files showing the root mean squared errors from cross-validated fits, for different numbers of components. Results are displayed across the quantiles of the signal distributions, separately for A and B signals, and for type I red, type I green, and type II probes. +By default, funtooNorm will perform 10-fold cross-validation, but this can be changed with the parameter \texttt{ncv.fold}. -An important user-chosen parameter is \texttt{ncmp}, the number of PLS components to be included in the model. -This can be chosen after examining a series of fits with different numbers of PLS components, together with a cross-validation procedure to assess how well the quantiles are modelled by the control probe data. -By setting $validate=N$, for a maximum of $N$ components, the algorithm will graph the cross-validated errors across the percentiles for -models with $1, 2, ... , N$ PLS components. -Examination of these graphs will enable choice of the best value for \texttt{ncmp} for each data set. +Other parameters include: \\ +\texttt{save.quant:} When \texttt{TRUE}, the quantiles should be saved. When \texttt{FALSE}, saved quantiles from a previous run will be loaded and used. \\ +\texttt{apply.fits:} When \texttt{TRUE}, the results of the model fitting process should be used - i.e. the original data should be normalized. This parameter can be set fo \texttt{FALSE} when exploring the desired number of components. \\ +\texttt{logged.data:} If \texttt{TRUE}, the \texttt{sigA} and \texttt{sigB} matrices, as well as the control probe data matrices (controlred, controlgrn) are assumed to have been previously log transformed prior to sending the data +into the algorithm. If \texttt{logged.data=FALSE}, then these data will be log transformed (log(1+x)) inside the algorithm. \\ -The following call performs cross-validation to assess the performance of the model fitting for \texttt{ncmp} between 1 and 5. -Note that here the \texttt{ncmp} parameter is not specified. +The following call performs cross-validation to assess the performance of the model fitting. +Note that here the \texttt{ncmp} parameter does not need to be specified. %\begin{Verbatim} <<>>= #This call will perform cross validation to find optimal value #of parameter ncmp for PLS regression: funtoonormmout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, - controlred=matred, controlgrn=matgrn, - cp.types=cp.types, cell_type = cell_type, - save.quant=TRUE, save.loess=TRUE, - apply.loess=FALSE, validate=5) + controlred=matred, controlgrn=matgrn, cp.type = cp.types, cell_type = cell_type, + ncv.fold=10, logged.data=FALSE, save.quant=TRUE, + type.fits="PCR", apply.fits=FALSE, validate = TRUE) + @ %\end{Verbatim} -Calling the funtoonorm function with \texttt{validate = N} produce set of plots, one for each type of probe and colour (probe type I red, signal A, ... probe type II, signal B). By looking at figure \ref{val}, the goal is to choose the smallest value of \texttt{ncmp} where the cross-validated error is small. +Calling the funtoonorm function with \texttt{validate = TRUE} produces two files, corresponding to PCR and PLS regressions, where each file contains a set of plots, one for each type of probe and colour (probe type I red, signal A, ... probe type II, signal B). By looking at figures \ref{val1} and \ref{val2} the goal is to choose the smallest value of \texttt{ncmp} where the cross-validated root mean squared error is fairly small across the quantiles. We have set 4 as the default value for \texttt{ncmp}. %%Add graph and explanations of its meaning. +\begin{figure}[val1] +\centering +\includegraphics[width=10cm,height=10cm]{validationcurvesPCRlow.jpg} +\caption{Cross-validated root mean squared errors across percentiles of the signal distributions for different numbers of PCR components. +Top: signal A; Bottom: signal B; Left: probe type I red; Middle: probe type I green; Right: probe type II.} \label{val1} +\end{figure} -\begin{figure}[val] +\begin{figure}[val2] \centering -\includegraphics[width=10cm,height=10cm]{valid.jpg} -\caption{Cross-validated errors across percentiles of the signal distributions for different numbers of PLS components. Top: signal A; Bottom: signal B; Left: probe type I red; Middle: probe type I green; Right: probe type II.} \label{val} +\includegraphics[width=10cm,height=10cm]{validationcurvesPLSlow.jpg} +\caption{Cross-validated root mean squared errors across percentiles of the signal distributions for different numbers of PLS components. +Top: signal A; Bottom: signal B; Left: probe type I red; Middle: probe type I green; Right: probe type II.} \label{val2} \end{figure} +After choosing a desired number of components, in order to run the program to normalize the data, +%\begin{Verbatim} +<<>>= + #This call will normalize the data, using parameter ncmp for the number of components + # and PLR regresion + funtoonormmout <- funtoonorm(sigA=sigAsample, sigB=sigBsample, + controlred=matred, controlgrn=matgrn, cp.type = cp.types, cell_type = cell_type, + logged.data=FALSE, save.quant=FALSE, ncmp=4, + type.fits="PLS", apply.fits=TRUE, validate = FALSE) + +@ +%\end{Verbatim} + To access the performance of normalization function one can use a measure of intra-replicate differences \texttt{M}, described in \cite{funtooNorm}. We provide a function \texttt{agreement} implementing this measure. It takes as arguments a matrix of beta values and a vector of individual ID's. For the function to work some elements of individual's vector, obviously, should be identical. The returned value of \texttt{M} is expected to be similar for the data before and after normalization: %\begin{Verbatim} <<>>= @@ -143,11 +166,10 @@ agreement(funtoonormout$newBeta, individualID) # M for data after normalization \begin{thebibliography}{} -\bibitem{deLeeuw1976} de Leeuw, J., Young, F. W., and Takane, Y. (1976). Additive structure in qualitative data: An alternating least squares method with optimal scaling features. \emph{Psychometrika}, 41, 471-503. \bibitem{Fortin2014} Fortin, J.-P., et al. (2014). Functional normalization of 450K methylation array data improves replication in large cancer studies. \emph{Genome Biology}, 15: p. 503. \bibitem{Aryee2014} Aryee, M.J., et al. (2014). Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. \emph{Bioinformatics}, 30(10): p. 1363-9. \bibitem{Smith2013} Smith M., et al. (2013). illuminaio: An open source IDAT parsing tool for Illumina microarrays. \emph{F1000Research}, 2:264, 2013. -\bibitem{funtooNorm} Kathleen Oros Klein et al. (2015). \emph{funtooNorm:} An improvement of the funNorm normalization method for methylation data from multiple cell or tissue types. Manuscript in preparation. +\bibitem{funtooNorm} Kathleen Oros Klein et al. (2015). \emph{funtooNorm:} An improvement of the funNorm normalization method for methylation data from multiple cell or tissue types. Manuscript submitted. \end{thebibliography} \end{document} diff --git a/vignettes/valid.jpg b/vignettes/valid.jpg deleted file mode 100644 index 526f775b98fa215a7292eec7e93f5e81682589ef..0000000000000000000000000000000000000000 GIT binary patch literal 0 HcmV?d00001 literal 212764 zcmeFZbyOYAwlCUCSh&N&!@}L&7Y+e}1`oj{xI-YgyCgUXmH@$Bg1bWq!6CRLK#&9p zcKP-`=k7D!x#PZl-Z_80v8zXS&s9Bp_N?lvS+jpNYu(S^uK{?E6_gbK5C{aMJ#4`J z1_nY|R`$7umb!wns{F$R0KmWl9uoNB=;-9(rllx@&^Itd{FV3b$;`stMOs5c_1}O0 z=kxhj;6V}qd}063*ZyB~Ff6UyEgraT9(I2V7dH<80Qo%7%-$X@f6;jlG?BUe-{|_k zXtxIiAM%I(qHX?8FaM3_@AUECcyzR6A4&xU05mL{|3I7n2m0Th`@jy+O1pX3SzGxa zw4JRzyv^LK5bl4~#6w*nG~Ar6?HsKB;`+DrUt<3EoBkhX4=bOCvH^gsvx~2rosF#r z;y-D?%gw_B{Nr7mY5uK_LGZDE!dk!PL>08QZlK)U#kJo-EU zzzqX{#z_lLH?O}-dZ;b{KmjlST!08510Df%05iY=@B%`B7=Q%ifyaP4paU2HCV&NC z3pfI9fH&X|1OpL3ERY1e0kVL1KoL+1R06d?6VL{90|US)Fa>-CmVh;28~6bn1Lwf? zLxV&GVS@-kWFTq~1Bea83lauNg5*J}AT5vq$P{D)asqjR{6V3hSWpTm3zQEk1=WCB zK;58W&}YyBXdSc*IA}|%05zGY^21|ogz&hY(U>mS2_$4?DoB+-M7l6yb 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