From ba75252485c5e98ea50a6e98f3ddbf8f3807debe Mon Sep 17 00:00:00 2001 From: "m.jiang" Date: Tue, 24 Dec 2013 00:13:06 +0000 Subject: [PATCH] merge from git git-svn-id: https://hedgehog.fhcrc.org/bioconductor/trunk/madman/Rpacks/flowStats@85007 bc3139a8-67e5-0310-9ffc-ced21a209358 --- DESCRIPTION | 34 + NAMESPACE | 94 ++ NEWS | 17 + R/autoGate.R | 153 +++ R/curvPeaks.R | 53 + R/density1d.R | 424 +++++++ R/gaussNorm.R | 838 +++++++++++++ R/getPeakRegions.R | 38 + R/gpaSet.R | 398 +++++++ R/iProcrustes.R | 86 ++ R/idFeatures.R | 314 +++++ R/landmarkMatrix.R | 111 ++ R/landmarkMatrixWithoutFilterResult.R | 80 ++ R/normalize-methods.R | 258 ++++ R/pbin.R | 216 ++++ R/peakSeparator.R | 309 +++++ R/quadrantGate.R | 59 + R/singletGate.R | 107 ++ R/warpSet.R | 1295 +++++++++++++++++++++ data/BackGating.rda | Bin 0 -> 95584 bytes data/ITN.rda | Bin 0 -> 1356124 bytes man/BackGating.Rd | 22 + man/ITN.Rd | 18 + man/autoGate.Rd | 65 ++ man/binByRef.Rd | 45 + man/calcPBChiSquare.Rd | 54 + man/calcPearsonChi.Rd | 49 + man/curvPeaks.Rd | 72 ++ man/density1d.Rd | 151 +++ man/flowStats-package.Rd | 45 + man/gaussNorm.Rd | 97 ++ man/gpaSet.Rd | 143 +++ man/iProcrustes.Rd | 127 ++ man/idFeaturesByBackgating.Rd | 79 ++ man/landmarkMatrix.Rd | 88 ++ man/lymphGate.Rd | 199 ++++ man/normQA.Rd | 60 + man/normalize.Rd | 92 ++ man/plotBins.Rd | 66 ++ man/proBin.Rd | 106 ++ man/quadrantGate.Rd | 99 ++ man/rangeGate.Rd | 164 +++ man/singletGate.Rd | 85 ++ man/warpSet.Rd | 140 +++ vignettes/GettingStartedWithFlowStats.Rnw | 348 ++++++ 45 files changed, 7298 insertions(+) create mode 100644 DESCRIPTION create mode 100644 NAMESPACE create mode 100644 NEWS create mode 100644 R/autoGate.R create mode 100644 R/curvPeaks.R create mode 100644 R/density1d.R create mode 100644 R/gaussNorm.R create mode 100644 R/getPeakRegions.R create mode 100644 R/gpaSet.R create mode 100644 R/iProcrustes.R create mode 100644 R/idFeatures.R create mode 100644 R/landmarkMatrix.R create mode 100644 R/landmarkMatrixWithoutFilterResult.R create mode 100644 R/normalize-methods.R create mode 100644 R/pbin.R create mode 100644 R/peakSeparator.R create mode 100644 R/quadrantGate.R create mode 100644 R/singletGate.R create mode 100644 R/warpSet.R create mode 100644 data/BackGating.rda create mode 100644 data/ITN.rda create mode 100644 man/BackGating.Rd create mode 100644 man/ITN.Rd create mode 100644 man/autoGate.Rd create mode 100644 man/binByRef.Rd create mode 100644 man/calcPBChiSquare.Rd create mode 100644 man/calcPearsonChi.Rd create mode 100644 man/curvPeaks.Rd create mode 100644 man/density1d.Rd create mode 100644 man/flowStats-package.Rd create mode 100644 man/gaussNorm.Rd create mode 100644 man/gpaSet.Rd create mode 100644 man/iProcrustes.Rd create mode 100644 man/idFeaturesByBackgating.Rd create mode 100644 man/landmarkMatrix.Rd create mode 100644 man/lymphGate.Rd create mode 100644 man/normQA.Rd create mode 100644 man/normalize.Rd create mode 100644 man/plotBins.Rd create mode 100644 man/proBin.Rd create mode 100644 man/quadrantGate.Rd create mode 100644 man/rangeGate.Rd create mode 100644 man/singletGate.Rd create mode 100644 man/warpSet.Rd create mode 100644 vignettes/GettingStartedWithFlowStats.Rnw diff --git a/DESCRIPTION b/DESCRIPTION new file mode 100644 index 0000000..6ee3ff3 --- /dev/null +++ b/DESCRIPTION @@ -0,0 +1,34 @@ +Package: flowStats +Type: Package +Title: Statistical methods for the analysis of flow cytometry data +Version: 3.21.2 +Author: Florian Hahne, Nishant Gopalakrishnan, Alireza Hadj Khodabakhshi, + Chao-Jen Wong, Kyongryun Lee +Maintainer: Greg Finak and Mike Jiang +Description: Methods and functionality to analyse flow data that is beyond the + basic infrastructure provided by the flowCore package. +Depends: R (>= 2.10), flowCore (>= 1.10.0), fda (>= 2.2.6), mvoutlier, cluster, flowWorkspace (>= 3.7.29) +Suggests: flowViz, xtable +Imports: BiocGenerics, MASS, flowViz, flowCore, fda (>= 2.2.6), Biobase, methods, grDevices, + graphics, stats, utils, KernSmooth, lattice,compositions +Enhances: RBGL,ncdfFlow,graph +License: Artistic-2.0 +biocViews: FlowCytometry, CellBasedAssays, Bioinformatics +Lazyload: yes +Collate: autoGate.R + density1d.R + quadrantGate.R + getPeakRegions.R + singletGate.R + curvPeaks.R + gaussNorm.R + landmarkMatrix.R + landmarkMatrixWithoutFilterResult.R + normalize-methods.R + peakSeparator.R + warpSet.R + pbin.R + gpaSet.R + iProcrustes.R + idFeatures.R + diff --git a/NAMESPACE b/NAMESPACE new file mode 100644 index 0000000..61de8ce --- /dev/null +++ b/NAMESPACE @@ -0,0 +1,94 @@ +#Generated by codetoolsBioC version 0.0.16 +#Timestamp: Thu Feb 23 13:56:56 2012 + +#Imports: Biobase, BiocGenerics, KernSmooth, MASS, compositions, fda, +# flowCore, flowViz, flowWorkspace, grDevices, graphics, +# lattice, methods, mvoutlier, stats, utils + +importClassesFrom(flowCore, flowFrame, parameterFilter, parameters) + +importClassesFrom(methods, character, list, numeric) + +importMethodsFrom(Biobase, channel) + +importMethodsFrom(BiocGenerics, cbind, colnames, lapply, mapply, order, + paste, pmax, pmin, rbind, rownames, sapply, setdiff, + table, unique) + +importMethodsFrom(flowCore, "%in%", "%subset%", Subset, assign, + "colnames<-", description, "description<-", exprs, + "exprs<-", filter, filterDetails, fsApply, head, + "identifier<-", keyword, ncol, nrow, pData, + "pData<-", parameters, "parameters<-", phenoData, + "phenoData<-", print, sampleNames, sort, split, + summary, tail,colnames,normalize) + +importMethodsFrom(flowViz, densityplot, glpolygon, xyplot) + +importMethodsFrom(flowWorkspace, getData) + +importFrom(Biobase, listLen) + +importFrom(KernSmooth, bkde2D) + +importFrom(MASS, huber, hubers, rlm) + +importFrom(compositions, dist) + +importFrom(fda, Data2fd, create.bspline.basis, eval.fd, fdPar, + landmarkreg) + +importFrom(flowCore, boundaryFilter, char2ExpressionFilter, + curv1Filter, curv2Filter, filterList, flowFrame, + norm2Filter, quadGate, rectangleGate, sampleFilter) + +importFrom(grDevices, chull, col2rgb, contourLines, dev.cur, dev.off, + png, rainbow, rgb, x11) + +importFrom(graphics, abline, legend, lines, par, plot, points, polygon, + rect, text) + +importFrom(lattice, make.groups, panel.abline, panel.number, + panel.points, panel.polygon, panel.segments, + panel.stripplot, panel.text, stripplot, trellis.focus, + trellis.par.get, trellis.unfocus, which.packet) + +importFrom(methods, as, is, new, selectMethod) + +importFrom(mvoutlier, pcout) + +importFrom(stats, anova, approxfun, as.formula, as.hclust, chisq.test, + density, dnorm, fitted, formula, kmeans, mad, median, + na.omit, optimize, qnorm, quantile, resid, sd, var) + +importFrom(utils, capture.output) + +export("lymphGate", + "lymphFilter", + "oneDGate", + "quadrantGate", + "rangeGate", + "rangeFilter", + "warpSet", +# "warpSetGS", +# "warpSetNCDF", +# "warpSetNCDFLowMem", + "proBin", + "binByRef", + "gaussNorm", + "plotBins", + "calcPearsonChi", + "calcPBChiSquare", + "gpaSet", + "iProcrustes", + "normQA", + "singletGate" + ) + +exportClasses("lymphFilter","rangeFilter") + +exportMethods("normalize") + + +exportMethods("%in%") + diff --git a/NEWS b/NEWS new file mode 100644 index 0000000..d71d074 --- /dev/null +++ b/NEWS @@ -0,0 +1,17 @@ +************************************************** +* 1.7 SERIES PATCHED NEWS * +************************************************** + +NEW UTILITIES + +o Added an argument 'plot' to lymphGate() to produce plots of results. + +o Added an argument 'bwFac' to warpSet() to allow finer control to the + smoothing of the density kernal estimation (in curv1Filter). This bandwidth + factor would affect the number of landmarks. + +o Added two arguments 'refLine1' and refLine2' to quandrantGate() allowing + algorithm to ignore minor, yet significant, sub-populations below 'refLine1' + and 'refLine' when calculating the separator between positive and negative. + + diff --git a/R/autoGate.R b/R/autoGate.R new file mode 100644 index 0000000..378d392 --- /dev/null +++ b/R/autoGate.R @@ -0,0 +1,153 @@ +## 2D gating with norm2Filter. One first selects a rectangle area in the +## two-dimensional space as a rough preselection and applies the norm2Filter +## only to this subset in a second step. This function is now considered +## internal, use lymphGate instead. +autoGate <- function(x, ..., scale = 2.5) +{ + ## some type-checking first + flowCore:::checkClass(x, "flowSet") + flowCore:::checkClass(scale, "numeric", 1) + stains <- names(list(...)) + if(length(stains) != 2) + stop("Only know how to deal with 2 dimensions.", call.=FALSE) + ## construct initial rectangle gate and subset + rectgate2 <- rectangleGate(...) + tmp2 <- Subset(x, filter(x, rectgate2)) + ## compute norm2Filter for the rest + bcn2g <- do.call(norm2Filter, + list(stains[1], stains[2], scale = scale)) + bcn2f <- filter(tmp2, bcn2g) + ans <- Subset(tmp2, bcn2f) + list(x = ans, n2gate = bcn2g, n2gateResults = bcn2f) +} + + +## A more versatile API for autoGate. 'preselection' can be one in +## NULL: basically a regular norm2Filter operation without any +## preselection +## a character scalar: The name of one of the channels in x used for the +## preselection. Only positive cells in this channel +## will be considered to construct the rectangle gate. +## a list: The same as for autoGate, numerics defining the initial rectangular +## selection +lymphGate <- function(x, channels, preselection=NULL, scale=2.5, + bwFac=1.3, filterId="defaultLymphGate", + evaluate=TRUE, plot=FALSE, ...) +{ + ## some type-checking first + flowCore:::checkClass(channels, "character", 2) + flowCore:::checkClass(x, c("flowSet", "flowFrame")) + flowCore:::checkClass(scale, "numeric", 1) + flowCore:::checkClass(bwFac, "numeric", 1) + flowCore:::checkClass(filterId, "character", 1) + flowCore:::checkClass(evaluate, "logical", 1) + bcn2g <- do.call(norm2Filter, list(channels, scale=scale, + filterId=filterId)) + if(!is.null(preselection)){ + if(is.character(preselection)){ + ## preselect by a single stain + flowCore:::checkClass(preselection, "character", 1) + if(!preselection %in% colnames(x)) + stop(sprintf("'%s' is not a valid flow parameter in this flowSet.", + preselection), call.=FALSE) + ## collapse to a single flowFrame and find most likely positive peak + ## (essentially the one with the highest mean) after removing margin + ## events. + xc <- as(x, "flowFrame") + xc <- Subset(xc, boundaryFilter(preselection)) + xcf <- filter(xc, curv1Filter(preselection, bwFac=1.3)) + xcS <- split(xc, xcf) + xcS <- xcS[sapply(xcS, nrow)>nrow(xc)/500] + xcMax <- Subset(tail(xcS, n=1)[[1]], boundaryFilter(channels)) + ## estimate location and variance of this subset in the two other + ## channels and construct a rectangular preselection from that + m <- apply(exprs(xcMax[,channels]), 2, median) + s <- scale*apply(exprs(xcMax[,channels]), 2, mad) + rg <- list(c(m[1]-s[1], m[1]+s[1]), c(m[2]-s[2], m[2]+s[2])) + names(rg) <- channels + bcrg <- rectangleGate(.gate=rg, filterId="Preselection") + }else if(is.list(preselection)){ + ## give the preselection as an explicit rectangle + sapply(preselection, flowCore:::checkClass, "numeric", 2) + if(is.null(names(preselection))) + names(preselection) <- channels + bcrg <- rectangleGate(preselection, filterId="Preselection") + }else stop("Invalid argument 'preselection'.", call.=FALSE) + bcn2g <- bcn2g %subset% bcrg + identifier(bcn2g) <- filterId + } + ## compute the filterResult and subset only if evaluate=TRUE + xr <- fr <- NULL + if(evaluate){ + fr <- filter(x, bcn2g) + xr <- Subset(x, fr) + } + + if (evaluate & plot) { + fm <- formula(paste(sapply(channels, function(ch) paste("`", ch, "`", sep="")), + collapse="~")) + print(xyplot(fm, x, filter=bcn2g)) + + } + + return(list(x=xr, n2gate=bcn2g, n2gateResults=fr)) +} + + + + + +## =========================================================================== +## lymphFilter +## --------------------------------------------------------------------------- +## This is basically an abstraction of the lymphGate function. It allows us +## to use it as a regular gate object. +## --------------------------------------------------------------------------- +setClass("lymphFilter", + representation=representation(preselection="character", + rectDef="list", + scale="numeric", + bwFac="numeric"), + contains="parameterFilter", + prototype=list(filterId="defaultLymphFilter")) + +## Constructor. We allow for the following inputs: +## scale and bwFac are always numerics of length 1 +## channels is a characters of length 2 +## preselection is either a character scalar or a named list +## of numerics +lymphFilter <- function(channels, preselection=as.character(NULL), + scale=2.5, bwFac=1.3, filterId="defaultLymphFilter") +{ + flowCore:::checkClass(scale, "numeric", 1) + flowCore:::checkClass(bwFac, "numeric", 1) + flowCore:::checkClass(filterId, "character", 1) + flowCore:::checkClass(channels, "character", 2) + rdef <- if(is.list(preselection)){ + tmp <- preselection + preselection <- as.character(NULL) + tmp} else list() + new("lymphFilter", parameters=channels, preselection=preselection, + scale=scale, bwFac=bwFac, filterId=filterId, rectDef=rdef) +} + + +setMethod("%in%", + signature=signature("flowFrame", + table="lymphFilter"), + definition=function(x, table) + { + pre <- if(is.null(table@preselection)) table@rectDef else table@preselection + if(length(parameters(table)) != 2) + stop("lymph filters require exactly two parameters.") + tmp <- lymphGate(x, channels=parameters(table), + preselection=pre, + scale=table@scale, + bwFac=table@bwFac, + filterId=table@filterId, + eval=TRUE, + plot=FALSE) + tmp$n2gateResults@subSet + }) + + diff --git a/R/curvPeaks.R b/R/curvPeaks.R new file mode 100644 index 0000000..9323c5a --- /dev/null +++ b/R/curvPeaks.R @@ -0,0 +1,53 @@ +## parse output of curv1Filter and find modes and midpoints of the density +## regions +curvPeaks <- function(x, dat, borderQuant=0.01, n=201, from, to, densities=NULL) +{ + ## Some type-checking first + flowCore:::checkClass(x, "multipleFilterResult") + flowCore:::checkClass(dat, c("numeric","NULL")) + flowCore:::checkClass(borderQuant, "numeric", 1) + flowCore:::checkClass(n, "numeric", 1) + if(missing(from)) + from <- min(dat) + flowCore:::checkClass(from, "numeric", 1) + if(missing(to)) + to <- max(dat) + flowCore:::checkClass(to, "numeric", 1) + ## extract boundaries + bound <- attr(x@subSet, "boundaries") + + dens <- if(!is.null(densities)) densities else + density(dat, n=n, from=from, to=to, na.rm=TRUE)$y + ## iterate over regions + regPoints <- list() + peaks <- midpoints <- regions <- densFuns <- NULL + i <- 1 + if(!all(is.na(bound[[1]]))){ + #oo <- options(warn=-1) + #on.exit(options(oo)) + for(b in bound){ + ## discard regions on the margins + if(b[2] > quantile(c(from,to), borderQuant) && + b[1] < quantile(c(from,to), 1-borderQuant)){ + ## approximate density by function + afun <- approxfun(seq(from, to, len=n), dens) + sel <- seq(b[1], b[2], len=50) + regPoints[[i]] <- cbind(x=sel, y=afun(sel)) + ## compute maximum of function + m <- optimize(afun, b, maximum=TRUE) + peaks <- rbind(peaks, cbind(x=m$maximum, y= m$objective)) + regions <- rbind(regions, cbind(left=b[1], right=b[2])) + midpoints <- c(midpoints, mean(b)) + densFuns <- c(densFuns, afun) + i <- i+1 + } + } + } + if(i==1) + return(list(peaks=cbind(x=NA, y=NA), regions=cbind(left=NA, right=NA), + midpoints=NA, regPoints=list(cbind(x=NA, y=NA)), + densFuns=NA)) + return(list(peaks=peaks, regions=regions, midpoints=midpoints, + regPoints=regPoints, densFuns=densFuns)) +} + diff --git a/R/density1d.R b/R/density1d.R new file mode 100644 index 0000000..d7befde --- /dev/null +++ b/R/density1d.R @@ -0,0 +1,424 @@ +hubers1<-function (y, k = 1.5, mu, s, initmu = median(y), tol = 1e-06) +{ + mmu <- missing(mu) + ms <- missing(s) + y <- y[!is.na(y)] + n <- length(y) + if (mmu) { + mu0 <- initmu + n1 <- n - 1 + } + else { + mu0 <- mu + mu1 <- mu + n1 <- n + } + if (ms) { + s0 <- mad(y) + if (s0 == 0) + return(list(mu = mu0, s = 0)) + } + else { + s0 <- s + s1 <- s + } + th <- 2 * pnorm(k) - 1 + beta <- th + k^2 * (1 - th) - 2 * k * dnorm(k) + for (i in 1:30) { + yy <- pmin(pmax(mu0 - k * s0, y), mu0 + k * s0) + if (mmu) + mu1 <- sum(yy)/n + if (ms) { + ss <- sum((yy - mu1)^2)/n1 + s1 <- sqrt(ss/beta) + } + if ((abs(mu0 - mu1) < tol * s0) && abs(s0 - s1) < tol * + s0) + break + mu0 <- mu1 + s0 <- s1 + } + list(mu = mu0, s = s0) +} +density1d_simple <- function(x, stain, alpha="min", sd=2, plot=FALSE, borderQuant=0.1, + absolute=TRUE, inBetween=FALSE, refLine=NULL,rare=FALSE,bwFac = 1.2 + ,sig=NULL + ,peakNr=NULL + ,cutoff=0.05 + ,valley="min" + , ...) +{ + + ## some type checking first + flowCore:::checkClass(x, c("flowFrame", "flowSet")) + flowCore:::checkClass(stain, "character", 1) + if(!stain %in% colnames(x)) + stop("'", stain,"' is not a valid parameter in this flowFrame") + + flowCore:::checkClass(alpha, c("character", "numeric"), 1) +# browser() + flowCore:::checkClass(sd, "numeric", 1) + flowCore:::checkClass(plot, "logical", 1) + flowCore:::checkClass(borderQuant, "numeric", 1) + flowCore:::checkClass(absolute, "logical", 1) + if (!is.null(refLine)) + flowCore:::checkClass(refLine, "numeric", 1) + flowCore:::checkClass(inBetween, "logical", 1) + + ## collapse to flowFrame + if(is(x, "flowSet")) + x <- as(x, "flowFrame") +# browser() + + #get density curve peaks + fres<-filter(x,curv1Filter(stain,bwFac=bwFac)) + fres<-filterDetails(fres) + fhat<-fres[[1]]$fsObj$fhat + rawSig<-fres[[1]]$fsObj$x + + #fitted curve points + curve<-data.frame(x=fhat[[1]][[1]],y=fhat$est) +# browser() + curve_filtered<-subset(curve,y>=cutoff) + #peak boudaries + boundaries<-fres[[1]]$boundaries + #filter peaks as well + isNoise<-unlist(lapply(boundaries,function(curbn){ +# browser() + subCurve<-subset(curve,x>=curbn[1]&x<=curbn[2]) + peak_mode<-subCurve[which.max(subCurve$y),] + peak_mode$y=peak_mode[,"x"],] + halfS<-rawSig[rawSig>=peak_mode[,"x"]] + }else + stop("invalid value for 'sig' !") +# plot(halfS) + }else + { + ##pick LHS of far left peak or RHS of far right peak + #when there are more than 1 peak + if(!is.null(sig)) + if(sig=="L") + { + lpeak_bnd<-boundaries[[1]] + subCurve<-subset(curve,x>=lpeak_bnd[1]&x<=lpeak_bnd[2]) + peak_mode<-subCurve[which.max(subCurve$y),] +# halfCurve<-curve[curve$x<=peak_mode$x,] + halfS<-rawSig[rawSig<=peak_mode[,"x"]] + }else if(sig=="R") + { + rpeak_bnd<-boundaries[[nPeaks]] + subCurve<-subset(curve,x>=rpeak_bnd[1]&x<=rpeak_bnd[2]) + peak_mode<-subCurve[which.max(subCurve$y),] +# halfCurve<-curve[curve$x>=peak_mode$x,] + halfS<-rawSig[rawSig>=peak_mode[,"x"]] + + }else + stop("invalid value for 'sig' !") +# plot(halfS) + + } + if(is.null(sig)) + { + #use entire signal to estimate mode and sd + est <- hubers(curve$x) + }else + { +# browser() +# sd1<-sqrt(mean((halfCurve$x-peak_mode[,"x"])^2*halfCurve$y))*2 +# sd1<-sqrt(mean((halfS-peak_mode[,"x"])^2)) + sd1<-median(abs(halfS-peak_mode[,"x"])) +# sd1<-hubers(halfS)$s + est<-list(mu=peak_mode[,"x"],s=sd1) + + + + } + + loc <- est$mu + sd * est$s + + }else + { + + #get valleys + valleys<-do.call(rbind + ,lapply(1:(nPeaks-1),function(i){ + # browser() + #get valley boundaries + valleyLbound<-max(boundaries[[i]]) + valleyRbound<-min(boundaries[[i+1]]) + + # + subCurve<-subset(curve,x>=valleyLbound&x<=valleyRbound) + # plot(subCurve) + subCurve[which.min(subCurve$y),] + + }) + ) + if(valley=="min")#pick lowest valley + loc<-valleys[which.min(valleys$y),"x"] + else + loc<-valleys[valley,"x"] + + + } + ## create output if needed +# browser() + if(plot){ + plot(curve,type="l",main=paste("breakpoint for parameter", stain), cex.main=1, ...) + abline(v = loc, col = 2, lwd = 2) + + } +# browser() + return(loc) +} + +## Find most likely separator between peaks in 1D +density1d <- function(x, stain, alpha="min", sd=2, plot=FALSE, borderQuant=0.1, + absolute=TRUE, inBetween=FALSE, refLine=NULL,rare=FALSE,bwFac = 1.2 + ,sig=NULL + ,peakNr=NULL + , ...) +{ + + ## some type checking first + flowCore:::checkClass(x, c("flowFrame", "flowSet")) + flowCore:::checkClass(stain, "character", 1) + if(!stain %in% colnames(x)) + stop("'", stain,"' is not a valid parameter in this flowFrame") + + flowCore:::checkClass(alpha, c("character", "numeric"), 1) +# browser() + flowCore:::checkClass(sd, "numeric", 1) + flowCore:::checkClass(plot, "logical", 1) + flowCore:::checkClass(borderQuant, "numeric", 1) + flowCore:::checkClass(absolute, "logical", 1) + if (!is.null(refLine)) + flowCore:::checkClass(refLine, "numeric", 1) + flowCore:::checkClass(inBetween, "logical", 1) + + ## collapse to flowFrame + if(is(x, "flowSet")) + x <- as(x, "flowFrame") +# browser() + ## clip to data range (if needed) and compute density peaks + if(absolute){ + vrange <- range(x, na.rm=TRUE)[, stain] + vrange[is.nan(vrange)] <- 0 + vrange[1] <- min(vrange[1], min(exprs(x)[,stain], na.rm=TRUE)) + } + else + vrange <- range(exprs(x)[,stain], na.rm=TRUE) + + vrange[1] <- ifelse(is.null(refLine), vrange[1], max(vrange[1], refLine)) + inc <- diff(vrange)/1e5 + exprf <- char2ExpressionFilter(sprintf("`%s` > %s & `%s` < %s", stain, + vrange[1]+inc, stain, vrange[2]-inc)) + + fres <- filter(tmp <- Subset(x, exprf), curv1Filter(stain,bwFac=bwFac)) + bnds <- flowStats:::curvPeaks(fres, exprs(tmp)[, stain], borderQuant=borderQuant) + ##when peakNr is present,drop the less significant peaks by their heights + + if(!is.null(peakNr)) + { + if(peakNr=bnds$peak[,"x"]] + } + + varS<-sqrt(mean((halfS-bnds$peak[,"x"])^2)) + + est<-list(mu=bnds$peak[,"x"],s=varS) + }else + { + est <- hubers(signals) + + } + + class <- which.min(abs(est$mu - anchors)) + }else + { + est <- hubers(signals) + if(nrow(anc)==2 && inBetween) + { + left <- bnds$regions[1,"right"] + right <- bnds$regions[2,"left"] + class <- 1:2 + } + else + { + ## multiple peaks: classify as pos or neg and set boundary between + class <- apply(anc,1, which.min) + left <- ifelse(sum(class==1)>0, max(bnds$regions[class==1, "right"]), + min(bnds$regions[class==2, "left"])) + right <- ifelse(sum(class==2)>0, min(bnds$regions[class==2, "left"]), + max(bnds$regions[class==1, "right"])) + } + } + + + + ##if rare==true then assume there is only one major peak and the rare population is at the right tail +# browser() + if(rare||all(class==1)) + { + bound <- est$mu + sd * est$s + + } + else if(all(class==2)) + bound <- est$mu - sd * est$s + else{ + if(is.character(alpha)){ + if(alpha=="min"){ + sel <- (dens$x > left & dens$x <= right) + bound <- dens$x[sel][which.min(dens$y[sel])] + }else + { + alpha<-as.numeric(alpha) + if(is.na(alpha)) + stop("unknown value for alpha") + else + bound <- (1-alpha)*left + alpha*right + } + + }else + bound <- (1-alpha)*left + alpha*right + } + ## create output if needed +# browser() + if(plot){ + plot(dens, main=paste("breakpoint for parameter", stain), cex.main=1, ...) + regs <- bnds$regions + for(i in 1:nrow(regs)){ + sel <- dens$x >= regs[i,1] & dens$x <= regs[i,2] + polygon(c(dens$x[min(which(sel))], dens$x[sel], + dens$x[max(which(sel))]), c(0, dens$y[sel], 0), + col="lightgray", border=NA) + } + lines(dens, ...) + abline(v = bound, col = 2, lwd = 2) + legend("topright", c("breakpoint", "dens region"), + fill=c("red", "lightgray"), bty="n") + } + return(bound) +} + + +## A wrapper around density1D directly creating a range gate. +rangeGate <- function(x, stain, alpha="min", sd=2, plot=FALSE, borderQuant=0.1, + absolute=TRUE, filterId="defaultRectangleGate", + positive=TRUE, refLine=NULL,simple=FALSE, ...) +{ + if(simple) + loc <- density1d_simple(x=x, stain=stain, alpha=alpha, sd=sd, plot=plot, + borderQuant=borderQuant, absolute=absolute, refLine=refLine, ...) + else + loc <- density1d(x=x, stain=stain, alpha=alpha, sd=sd, plot=plot, + borderQuant=borderQuant, absolute=absolute, refLine=refLine, ...) + bounds <- if(positive) list(c(loc, Inf)) else list(c(-Inf, loc)) + names(bounds) <- stain + rectangleGate(bounds, filterId=filterId) +} + +## An old alias, this will go away soon +oneDGate <- function(...){ + .Deprecated(new="rangeGate") + rangeGate(...) +} + +## ================================================================================= +## rangeFilter +## --------------------------------------------------------------------------------- +## This is basically an abstraction of the lymphGate function. It allows us +## to use it as a regular gate object. +## --------------------------------------------------------------------------------- +setClass("rangeFilter", + representation=representation(alpha="character", + sd="numeric", + borderQuant="numeric"), + contains="parameterFilter", + prototype=list(filterId="defaultRangeFilter", + alpha="min", + sd= 2, + borderQuant=0.1)) + +## Constructor. We allow for the following inputs: +## alpha is always a character and sd and borderQuant both are always +## numerics of length 1. +## stain is a list of characters and/or transformations, y is missing +rangeFilter <- function(stain, alpha="min", sd=2, borderQuant=0.1, + filterId="defaultRangeFilter") +{ + flowCore:::checkClass(filterId, "character", 1) + flowCore:::checkClass(alpha, "character", 1) + flowCore:::checkClass(sd, "numeric", 1) + flowCore:::checkClass(borderQuant, "numeric", 1) + new("rangeFilter", parameters=stain, alpha=alpha, + sd=sd, borderQuant=borderQuant, filterId=as.character(filterId)) +} + +setMethod("%in%", + signature=signature("flowFrame", + table="rangeFilter"), + definition=function(x, table) + { + + if(length(parameters(table)) != 1) + stop("range filters require exactly one parameters.") + tmp <- rangeGate(x, stain=parameters(table), + alpha=table@alpha, + sd=table@sd, + borderQuant=table@borderQuant, + filterId=table@filterId ) + filter(x, tmp) + }) + + diff --git a/R/gaussNorm.R b/R/gaussNorm.R new file mode 100644 index 0000000..685b743 --- /dev/null +++ b/R/gaussNorm.R @@ -0,0 +1,838 @@ +## normalizes data per-channel basis by locating high density regions +## on each channel and use these as landmarks to adjust the distance +## between populations. Inputs: -flowset: the input flowset +## containing the flowcytomery expriments. -channel.names: the list +## of channels to be normalized. -max.lms: a vector in which each +## element is the maximum number of landmarks used in the +## normalization of the corresponding channel. The default values is 2 +## for all channels. -debug: if set TRUE draws before and after +## normalization plots for each sample. The plot of the i-th sample is +## stored in "paste(fname, i)" file. -base.lms: list of base +## landmarks for each channel. i.e. base.lms[[p]] is a vector of base +## landmars for channel p. If not specified the base.lms is computed. +## The peaks with density value less than peak.density.thr*maximum.peak.density are discarded. +## and of the peaks with distance less than peak.distance.thr*range.data only one is considered. + +##Normalize a GatingSet, allowing for normalization on a subset of gates +gaussNormGS <- function(gatingset, channel.names, max.lms=2, base.lms=NULL,peak.density.thr=0.05,peak.distance.thr=0.05,debug=FALSE,fname='',gate=NULL){ + if(!inherits(gatingset,"GatingSet")) + stop("gatingset must be of class GatingSet") + if(!gatingset[[1]]@isNcdf) + stop("gatingset not gated using netcdf. Just use the regular gaussNorm function, please"); + if(is.null(gate)){ + ncflowset<-graph:::nodeData(gatingset[[1]]@tree,gatingset[[1]]@nodes[1],"data")[[1]][["data"]]$ncfs + }else{ + ncflowset<-Subset(gatingset,gate) + } + #Update the column names on the ncflowset so that they are consistent with the column names on the GatingSet + colnames(ncflowset)<-colnames(getData(gatingset[[1]])) + normed<-gaussNormNC(ncflowset=ncflowset,channel.names=channel.names,max.lms=max.lms,base.lms=base.lms,peak.density.thr=peak.density.thr,peak.distance.thr=peak.distance.thr,debug=debug,fname=fname); + return(normed); +} +## Output: -returns the normalized flowset. +gaussNormNC <- function(ncflowset, channel.names, max.lms=2, base.lms=NULL, peak.density.thr=0.05, peak.distance.thr=0.05, debug=FALSE, fname=''){ + remb.flowset=remBoundaryNC(ncflowset, channel.names) #This method needs to be rewritten to deal with large data sets. + expr.list=c(1:length( ncflowset)) + normalized<-ncdfFlow:::clone.ncdfFlowSet(ncflowset) + if(length(max.lms)==1){ + max.lms=rep(max.lms, times=length(channel.names)) + } + if(length(max.lms)!=length(channel.names)){ + cat('Error: length of max.lms and channel.names doesn\'t match\n') + return(NULL) + } + names(max.lms)=channel.names + lms=extract.landmarksNC(ncflowset,remb.flowset, expr.list, channel.names, max.lms, peak.density.thr, peak.distance.thr) + if(is.null(base.lms)) + base.lms=extract.base.landmarks(lms$filter, channel.names, max.lms) + + ## finds the best matching between landmarks and base landmarks. + ## the score of a matching is defined as a function of the distance between landmark and the base landmark and the score of the landmark itself. + matched.lms=match.all.lms(lms, base.lms, channel.names, max.lms) + confidence=compute.confidence(matched.lms, base.lms) + cat('\nAdjusting the distance between landmarks\n') + newRange=matrix(ncol=length(channel.names), nrow=2) + colnames(newRange)=channel.names + newRange[,channel.names] <- c(Inf, -Inf) + for(i in expr.list){ + cat('.') + if(fname != '') + file.name=paste(fname, as.character(i), sep='.') + else + file.name='' + ## normalizing sample i. + tmp<-flowFrame(normalize.one.exprNC(ncflowset,remb.flowset$index[[i]],i, base.lms, lms$filter[,i], lms$original[,i], matched.lms[,i], channel.names, max.lms, file.name, debug)) + parameters(tmp)<-parameters(ncflowset[[i]]) + description(tmp)<-description(ncflowset[[i]]) + normalized[[sampleNames(ncflowset)[i]]] <- tmp + for(p in channel.names){ + newRange[1,p] <- min(newRange[1,p], min(exprs(normalized[[i]])[,p], na.rm=TRUE)) + newRange[2,p] <- max(newRange[2,p], max(exprs(normalized[[i]])[,p], na.rm=TRUE)) + } + } + cat('\n') + restoreBoundaryNC(ncflowset,normalized, remb.flowset, channel.names) + #updating the new ranges. + for(i in expr.list){ + for(p in channel.names){ + ip <- match(p, pData(parameters(normalized[[i]]))$name) + tmp <- parameters(normalized[[i]]) + oldRanges <- unlist(range(ncflowset[[i]],p)) + pData(tmp)[ip, c("minRange", "maxRange")] <- c(min(oldRanges[1], newRange[1,p]), + max(oldRanges[2], newRange[2,p])) + normalized[[i]]@parameters <- tmp + + } + } + list(flowset=normalized, confidence=confidence) +} + +gaussNorm <- function(flowset, channel.names, max.lms=2, base.lms=NULL, peak.density.thr=0.05, peak.distance.thr=0.05, debug=FALSE, fname=''){ + remb.flowset=remBoundary(flowset, channel.names) + expr.list=c(1:length(flowset)) + if(length(max.lms)==1){ + max.lms=rep(max.lms, times=length(channel.names)) + } + if(length(max.lms)!=length(channel.names)){ + cat('Error: length of max.lms and channel.names doesn\'t match\n') + return(NULL) + } + names(max.lms)=channel.names + lms=extract.landmarks(remb.flowset$res, expr.list, channel.names, max.lms, peak.density.thr, peak.distance.thr) + if(is.null(base.lms)) + base.lms=extract.base.landmarks(lms$filter, channel.names, max.lms) + + ## finds the best matching between landmarks and base landmarks. + ## the score of a matching is defined as a function of the distance between landmark and the base landmark and the score of the landmark itself. + matched.lms=match.all.lms(lms, base.lms, channel.names, max.lms) + confidence=compute.confidence(matched.lms, base.lms) + cat('\nAdjusting the distance between landmarks\n') + newRange=matrix(ncol=length(channel.names), nrow=2) + colnames(newRange)=channel.names + newRange[,channel.names] <- c(Inf, -Inf) + for(i in expr.list){ + cat('.') + if(fname != '') + file.name=paste(fname, as.character(i), sep='.') + else + file.name='' + ## normalizing sample i. + exprs(remb.flowset$res[[i]])=normalize.one.expr(exprs(remb.flowset$res[[i]]), base.lms, lms$filter[,i], lms$original[,i], matched.lms[,i], channel.names, max.lms, file.name, debug) + for(p in channel.names){ + newRange[1,p] <- min(newRange[1,p], min(exprs(remb.flowset$res[[i]])[,p], na.rm=TRUE)) + newRange[2,p] <- max(newRange[2,p], max(exprs(remb.flowset$res[[i]])[,p], na.rm=TRUE)) + } + } + cat('\n') + restoreBoundary(flowset, remb.flowset, channel.names) + #updating the new ranges. + for(i in expr.list){ + for(p in channel.names){ + ip <- match(p, pData(parameters(remb.flowset$res[[i]]))$name) + tmp <- parameters(remb.flowset$res[[i]]) + oldRanges <- unlist(range(flowset[[i]],p)) + pData(tmp)[ip, c("minRange", "maxRange")] <- c(min(oldRanges[1], newRange[1,p]), + max(oldRanges[2], newRange[2,p])) + remb.flowset$res[[i]]@parameters <- tmp + + } + } + list(flowset=remb.flowset$res, confidence=confidence) +} + +## shifts the data in such a way that the peak at matched.lms[i] is moved to matched.lms[i+1] for each i. +## base.lms, lms.lis and lms.original are passed for the debug purposes only. +##Works on ncdfFlowSet. +normalize.one.exprNC <- function(data,indices,i,base.lms,lms.list,lms.original,matched.lms,channel.names,max.lms,fname='',debug=FALSE){ + norm.data=exprs(data[[i]]); + norm.data[indices,]<-NA; + if(debug==TRUE){ + if(fname=='') + x11() + else + png(filename=fname, bg="white",width=1800,height=1000) + par(mfrow=c(1, length(which(max.lms!=0)))) + } + cn=1 + i=0 + ## normalizing each channel. + for (p in channel.names){ + i=i+1 + if(max.lms[i]==0){ + norm.data[,p]=data[[i]][,p] + } + else{ + ## registering the data of channel p given the matching landmarks. + norm.data[,p]=register.channel(norm.data[,p], matched.lms[[p]]) + ## drawing the pre- and post- normalization density curves. + if(debug==TRUE){ + if(length(lms.list[[p]])==0){ + A1=density(na.omit(data[,p])) + xlim=c(min(A1$x, na.rm=T), max(A1$x, na.rm=T)) + ylim=c(min(A1$y, na.rm=T), max(A1$y, na.rm=T)) + par(mfg=c(1, cn)) + plot(A1, type="l", col= "black", xlim=xlim, ylim=ylim, xlab=p, main='No lms is found') + } + else{ + A1=density(na.omit(data[,p])) + A2=density(na.omit(norm.data[,p])) + xlim=c(min(min(A1$x, na.rm=T), min(A2$x, na.rm=T)), max(max(A1$x, na.rm=T), max(A2$x, na.rm=T))) + ylim=c(min(min(A1$y, na.rm=T), min(A2$y, na.rm=T)), max(max(A1$y, na.rm=T), max(A2$y, na.rm=T))) + par(mfg=c(1, cn)) + plot(A1, type="l", col= "blue", xlim=xlim, ylim=ylim, xlab=p, main='') + Lms=matched.lms[[p]][seq(1, length(matched.lms[[p]]), by=2)] + M.Lms=matched.lms[[p]][seq(2, length(matched.lms[[p]]), by=2)] + M.Lms.index=1 + for(k in 1:(length(M.Lms))) + M.Lms.index[k]=which(base.lms[[p]]==M.Lms[k]) + points(Lms, returny(A1, Lms), pch=19, col="red") + text(Lms, returny(A1, Lms), as.character(M.Lms.index), pos=3, col='red') + points(lms.original[[p]], returny(A1, lms.original[[p]]), pch=21, col="black") ##all the peaks + par(mfg=c(1, cn)) + plot(A2, type="l", col="red", xlim=xlim, ylim=ylim, xlab=p) + points(M.Lms, returny(A2, M.Lms), pch=15, col="blue") + text(M.Lms, returny(A2, M.Lms), as.character(M.Lms.index), pos=3, col='blue') + } + cn=cn+1 + } + } + } + if(debug){ + if(fname=='') + readline() + dev.off() + } + return (norm.data) +} + + +## shifts the data in such a way that the peak at matched.lms[i] is moved to matched.lms[i+1] for each i. +## base.lms, lms.lis and lms.original are passed for the debug purposes only. +normalize.one.expr <- function(data, base.lms, lms.list, lms.original, matched.lms, channel.names, max.lms, fname='', debug=FALSE){ + norm.data=data + if(debug==TRUE){ + if(fname=='') + x11() + else + png(filename=fname, bg="white",width=1800,height=1000) + par(mfrow=c(1, length(which(max.lms!=0)))) + } + cn=1 + i=0 + ## normalizing each channel. + for (p in channel.names){ + i=i+1 + if(max.lms[i]==0){ + norm.data[,p]=data[,p] + } + else{ + ## registering the data of channel p given the matching landmarks. + norm.data[,p]=register.channel(data[,p], matched.lms[[p]]) + ## drawing the pre- and post- normalization density curves. + if(debug==TRUE){ + if(length(lms.list[[p]])==0){ + A1=density(na.omit(data[,p])) + xlim=c(min(A1$x, na.rm=T), max(A1$x, na.rm=T)) + ylim=c(min(A1$y, na.rm=T), max(A1$y, na.rm=T)) + par(mfg=c(1, cn)) + plot(A1, type="l", col= "black", xlim=xlim, ylim=ylim, xlab=p, main='No lms is found') + } + else{ + A1=density(na.omit(data[,p])) + A2=density(na.omit(norm.data[,p])) + xlim=c(min(min(A1$x, na.rm=T), min(A2$x, na.rm=T)), max(max(A1$x, na.rm=T), max(A2$x, na.rm=T))) + ylim=c(min(min(A1$y, na.rm=T), min(A2$y, na.rm=T)), max(max(A1$y, na.rm=T), max(A2$y, na.rm=T))) + par(mfg=c(1, cn)) + plot(A1, type="l", col= "blue", xlim=xlim, ylim=ylim, xlab=p, main='') + Lms=matched.lms[[p]][seq(1, length(matched.lms[[p]]), by=2)] + M.Lms=matched.lms[[p]][seq(2, length(matched.lms[[p]]), by=2)] + M.Lms.index=1 + for(k in 1:(length(M.Lms))) + M.Lms.index[k]=which(base.lms[[p]]==M.Lms[k]) + points(Lms, returny(A1, Lms), pch=19, col="red") + text(Lms, returny(A1, Lms), as.character(M.Lms.index), pos=3, col='red') + points(lms.original[[p]], returny(A1, lms.original[[p]]), pch=21, col="black") ##all the peaks + par(mfg=c(1, cn)) + plot(A2, type="l", col="red", xlim=xlim, ylim=ylim, xlab=p) + points(M.Lms, returny(A2, M.Lms), pch=15, col="blue") + text(M.Lms, returny(A2, M.Lms), as.character(M.Lms.index), pos=3, col='blue') + } + cn=cn+1 + } + } + } + if(debug){ + if(fname=='') + readline() + dev.off() + } + return (norm.data) + +} + +#Works on ncdfFlowSet rather than flowSet +## computes the channel landmarks for each flowset experiment in expr.list. +## max.lms is the maximum number of landmarks we want to shift when normalizing the data. +## for each landmark a score is assigned which is a function of its sharpness and its density value. +## output: +## lms.list$original: list of all landmarks +## lms.list$score: the score of lms.list$original landmarks +## lms.list$filtered: max.lms top score landmarks. +extract.landmarksNC<-function(ncflowset,indices,expr.list, channel.names, max.lms, peak.density.thr, peak.distance.thr){ + ## defining output variables. + lms.list=list() + lms.list$original=matrix(vector("list"), length(channel.names), length(expr.list)) + lms.list$score=matrix(vector("list"), length(channel.names), length(expr.list)) + lms.list$filter=matrix(vector("list"), length(channel.names), length(expr.list)) + row.names(lms.list$original)=channel.names + row.names(lms.list$score)=channel.names + row.names(lms.list$filter)=channel.names + max.lms.num=0 + c=1 + ## iterating over channels. + for( p in channel.names){ + if(max.lms[c]!=0){ + j=1 + for(i in expr.list){ + data=exprs(ncflowset[[i]]) + ## finding the landmarks of channel p of sample i. + data[indices$index[[i]],]<-NA + lms=landmarker(data, p, max.lms[c]) + lms.list$original[p, j]=list(lms) + ## returns the max.lms[c] top score landmarks. + filtered=filter.lms(lms, data[,p], max.lms[c], peak.density.thr, peak.distance.thr) + lms.list$filter[p, j]=list(filtered$lms) + lms.list$score[p, j]=list(filtered$score) + j=j+1 + } + } + c=c+1 + } + return(lms.list) +} + +## computes the channel landmarks for each flowset experiment in expr.list. +## max.lms is the maximum number of landmarks we want to shift when normalizing the data. +## for each landmark a score is assigned which is a function of its sharpness and its density value. +## output: +## lms.list$original: list of all landmarks +## lms.list$score: the score of lms.list$original landmarks +## lms.list$filtered: max.lms top score landmarks. + +extract.landmarks <- function(flowset, expr.list, channel.names, max.lms, peak.density.thr, peak.distance.thr){ + ## defining output variables. + lms.list=list() + lms.list$original=matrix(vector("list"), length(channel.names), length(expr.list)) + lms.list$score=matrix(vector("list"), length(channel.names), length(expr.list)) + lms.list$filter=matrix(vector("list"), length(channel.names), length(expr.list)) + row.names(lms.list$original)=channel.names + row.names(lms.list$score)=channel.names + row.names(lms.list$filter)=channel.names + max.lms.num=0 + c=1 + ## iterating over channels. + for( p in channel.names){ + if(max.lms[c]!=0){ + j=1 + for(i in expr.list){ + data=exprs(flowset[[i]]) + ## finding the landmarks of channel p of sample i. + lms=landmarker(data, p, max.lms[c]) + lms.list$original[p, j]=list(lms) + ## returns the max.lms[c] top score landmarks. + filtered=filter.lms(lms, data[,p], max.lms[c], peak.density.thr, peak.distance.thr) + lms.list$filter[p, j]=list(filtered$lms) + lms.list$score[p, j]=list(filtered$score) + j=j+1 + } + } + c=c+1 + } + return(lms.list) +} + +## computes the base landmarks. +## output: a vector of base landmarks for each channel p. +extract.base.landmarks <- function(lms, channel.names, max.lms){ + lms.list=list() + max.lms.num=0 + for( p in channel.names){ + if(max.lms[p]!=0){ + ## if the total number of landmarks for channel p is less than max.lms[p] return them as base landmarks. + if(length(unlist(lms[p,])) <= max.lms[p]){ + lms.list[[p]]=sort(unlist(lms[p,])) + } + else{ + + if (max.lms[p]==1){ + lms.list[[p]]=median(unlist(lms[p,])) + } + else { + ## first identify samples that have exactly max.lms[p] landmarks on their channel p. These landmarks are labeled from 1 to max.lms + ## the landmarks samples with less than max.lms[p] landmarks are stored in short.lms vector. + short.lms=vector() + lms.class=list() + for(jj in 1:max.lms[p]) + lms.class[[jj]]=vector() + for (ii in 1:length(lms[p,])){ + lms.p.ii=lms[p,ii][[1]] + if(length(lms.p.ii)==max.lms[p]){ + for (jj in 1:max.lms[p]) + lms.class[[jj]]=append(lms.class[[jj]], lms.p.ii[jj]) + } + else{ + short.lms=append(short.lms, lms.p.ii) + } + } + + if(length(lms.class[[1]])==0){ + cat('No curve with ', max.lms[p], ' landmarks was found for channel ', p, '. Decrease max.lms[',p,'] and try again.\n') + stop() + } + mean.lms.class=0 + for(jj in 1:max.lms[p]){ + mean.lms.class[jj]=mean(lms.class[[jj]]) + } + + ## assigning short.lms landmarks to the class of labeled landmarks wich are closer to. + if(length(short.lms)!=0){ + for(jj in 1:length(short.lms)){ + kk=which(abs(mean.lms.class-short.lms[jj])==min(abs(mean.lms.class-short.lms[jj]))) + kk=kk[1] + lms.class[[kk]]=append(lms.class[[kk]], short.lms[jj]) + } + } + lms.class.len=0 + lms.class.med=0 + for(jj in 1:max.lms[p]){ + lms.class.len[jj]=length(lms.class[[jj]]) + lms.class.med[jj]=median(lms.class[[jj]], na.rm=T) + } + s=sd(lms.class.len, na.rm=T) + m=mean(lms.class.len, na.rm=T) + ll=which(m-lms.class.len > 2*s) + ## if a class of landmarks has too few landmarks in it just ignore this class. + if(length(ll)!=0){ + cat('warning: fewer landmark classes found in channel ', p, '\n') + tmp.lms=list() + kk=1 + for(jj in (1:max.lms[p])[-ll]){ + tmp.lms[[kk]]=lms.class[[jj]] + kk=kk+1 + } + lms.class=tmp.lms + + } + + ## returning the median of each class as the base landmark. + for(jj in 1:length(lms.class)){ + lms.class.med[jj]=median(lms.class[[jj]], na.rm=T) + } + + lms.list[[p]]=lms.class.med + + } + } + } + } + return(lms.list) +} + +## finds the best matching between landmarks (lms) and the base landmarks (base.lms). +## the score of a matching is defined as a function of the distance between the base landmark and the landmark and the score of the landmark itself. +## returns: matched.lms-for each channel p of a sample i matched.lms[p, i] is a list of pairs of the form (lms, base.lms) +match.all.lms <- function(lms, base.lms, channel.names, max.lms){ + n=length(lms$filter[channel.names[1],]) ##number of samples. + matched.lms=matrix(vector("list"), length(channel.names), n) + row.names(matched.lms)=channel.names + lms.class=list() + lms.class.median=list() + for(p in channel.names){ + lms.class[[p]]=list() + lms.class.median[[p]]=list() + } + for (p in channel.names){ + if(max.lms[[p]]==0) + next + for(i in 1:n){ + matched.lms[p, i][[1]]=best.match(lms$original[p, i][[1]], lms$score[p, i][[1]], base.lms[[p]], max.lms[[p]]) + } + } + return(matched.lms) +} + +best.match <- function(lms, lms.score, base.lms, max.lms){ + comb=choose.nk(max(length(lms), length(base.lms)), min(length(lms), length(base.lms))) + sc=list() + k=1 + max.s=-1 + if(length(lms)n){ + v=v[-length(v)] + v[length(v)]=v[length(v)]+1 + } + else if(length(v)==k){ + + res=rbind(res, v) + v[length(v)]=v[length(v)]+1 + } + else{ + v=append(v, v[length(v)]+1) + } + } + return(res) +} + +## manipulates the data in such a way that the landmark at matched.lms[i] is moved to matched.lms[i+1] for each i. +register.channel <- function(data, matched.lms){ + if(length(matched.lms)==0){ + cat('*') + return (data) + } + s=m=shift=vector() + lms=vector() + for(i in seq(1,length(matched.lms), by=2)){ + shift=append(shift, matched.lms[i+1]-matched.lms[i]) + lms=append(lms, matched.lms[i]) + s=append(s, sd(na.omit(data))) + } + r.data=register.function(data, s, lms, shift) + return(r.data) +} + +returny <- function(A, X){ + y=vector() + i=1; + for( x in X){ + y[i]=A$y[which(abs(A$x-x)==min(abs(A$x-x)))[1]] + i=i+1 + } + return (y) +} + +register.function <- function(data, s, m, shift){ + sum=0 + if(length(m)==1){ + return(data+shift) + } + if(length(m)==2){ + sh=(shift[1]-shift[2]) + data=data+gau(data, s[1], m[1])*(sh/2) + data=data-gau(data, s[2], m[2])*(sh/2) + return(data+shift[1]-sh/2) + } + max.shift=which(abs(shift)==max(abs(shift)))[1] + if(shift[max.shift]>0){ + sh=(shift[max.shift]-(shift[max.shift]-min(shift[-max.shift]))/2) + } + else{ + sh=(shift[max.shift]-(shift[max.shift]-max(shift[-max.shift]))/2) + } + data=data+sh + shift=shift-sh + + for(i in 1:length(m)) + data=data+gau(data, s[i], m[i])*shift[i] + return (data) +} + + +## gaussian function used in shifting the data. +gau <- function(d, s, m){ + return(2.7182^(-((d-m)^2)/(2*s*s))) +} + +## computes the confidence value based on the matched landmarks. +compute.confidence <- function(matched.lms, base.lms){ + confidence=rep(1, times=length(matched.lms[1,])) + # for each channel c. + for(c in 1:length(base.lms)){ + for(l in 1:length(base.lms[[c]])){ + lms.list=0 + for ( i in 1:length(matched.lms[1,])){ + ind=which(matched.lms[c,i][[1]]==base.lms[[c]][l]) + if(length(ind)==0){ + lms.list[i]=NA + } + else{ + if(ind[1] %% 2 != 0) + ind[1]=ind[1]+1 + lms.list[i]=matched.lms[c,i][[1]][ind[1]-1] + } + } + d=density(na.omit(lms.list)) + den.lms=0 + for(i in 1:length(lms.list)){ + if(is.na(lms.list[i])) + den.lms[i]=NA + else + den.lms[i]=d$y[which(abs(d$x-lms.list[i])==min(abs(d$x-lms.list[i])))] + } + m.den.lms=mean(den.lms, na.rm=T) + ind1=which(den.lms>m.den.lms/4 & den.lmslms.list[i]-bw & lms.list0) + if(length(ind)==0){ + filtered$lms=vector() + filtered$score=vector() + return(filtered) + } + lms.score.ind=lms.score$ind[ind] + if(length(lms.score.ind)score[last.lms.i]){ + last.lms=lms[i] + score[last.lms.i]=0 + last.lms.i=i + } + else{ + score[i]=0 + } + } + else{ + last.lms=lms[i] + last.lms.i=i + } + } + } + return(score) +} + +#Returns the indices of each flowFrame with boundary values that should be NA. In contrast to remBoundary, doesn't copy the data and doesn't return the NA'd flowSet +remBoundaryNC<-function(ncflowset,channel.names){ + ranges<-fsApply(ncflowset,range); + index=list() + res=list() + for(i in 1:length(ncflowset)){ + d=exprs(ncflowset[[i]]) + index[[i]]=vector() + for(p in channel.names){ + ind=which(d[,p]==ranges[[i]][1,p]) + index[[i]]=append(index[[i]],ind) + #d[ind,p]=NA + ind=which(d[,p]==ranges[[i]][2,p]) + index[[i]]=append(index[[i]],ind) + #d[ind,p]=NA + } + #res[[i]]=new('flowFrame', exprs=d) + #parameters(res[[i]])=parameters(flowset[[i]]) + } + #names(res)=sampleNames(flowset) + #res=as(res, 'flowSet') + #phenoData(res)=phenoData(flowset) + return(list(index=index)) +} + +#setting the boundary values to NA. Also returns the indices of the NA values for recovery. +remBoundary <- function(flowset, channel.names){ + ranges <- fsApply(flowset, range) + index=list() + res=list() + for(i in 1:length(flowset)){ + d=exprs(flowset[[i]]) + index[[i]]=vector() + for(p in channel.names){ + ind=which(d[,p]==ranges[[i]][1,p]) + index[[i]]=append(index[[i]],ind) + d[ind,p]=NA + ind=which(d[,p]==ranges[[i]][2,p]) + index[[i]]=append(index[[i]],ind) + d[ind,p]=NA + } + res[[i]]=new('flowFrame', exprs=d) + parameters(res[[i]])=parameters(flowset[[i]]) + } + names(res)=sampleNames(flowset) + res=as(res, 'flowSet') + phenoData(res)=phenoData(flowset) + return(list(index=index, res=res)) +} + +#Restore the boundary values that were NA in the ncdfFlowSet +restoreBoundaryNC <- function(org.flowset, normalized.flowset,remb.flowset,channel.names){ + for(i in 1:length(org.flowset)){ + for(p in channel.names){ + exprs(normalized.flowset[[i]])[remb.flowset$index[[i]], p]=exprs(org.flowset[[i]])[remb.flowset$index[[i]], p] + } + } +} + +#Restore the boundary values that were set to NA. +restoreBoundary <- function(org.flowset, remb.flowset, channel.names){ + for(i in 1:length(org.flowset)){ + for(p in channel.names){ + exprs(remb.flowset$res[[i]])[remb.flowset$index[[i]], p]=exprs(org.flowset[[i]])[remb.flowset$index[[i]], p] + } + } +} + diff --git a/R/getPeakRegions.R b/R/getPeakRegions.R new file mode 100644 index 0000000..26395a4 --- /dev/null +++ b/R/getPeakRegions.R @@ -0,0 +1,38 @@ +#should be okay as is +#Pulls out the peaks of significant regions from curv1Filter results. Can be used in code that chunks the samples. +#It will be up to the calling function to organize the results for the landmarkMatrix call +getPeakRegions <- function(data, fres, parm, border=0.05, peakNr=NULL, densities=NULL, n=201, + indices=FALSE) +{ + ## Some type-checking first + flowCore:::checkClass(data, "flowSet") + flowCore:::checkClass(fres, "list") + flowCore:::checkClass(parm, "character") + flowCore:::checkClass(border, "numeric", 1) + ## get peaks of significant regions from curv1Filter results + ranges <- fsApply(data, range) + from <- min(sapply(ranges, function(z) z[1,parm]-diff(z[,parm])*0.15), na.rm=TRUE) + to <- max(sapply(ranges, function(z) z[2,parm]+diff(z[,parm])*0.15), na.rm=TRUE) + peaks <- list() + regions <- list() + eps <- .Machine$double.eps + for(i in sampleNames(data)){ + if(is.null(densities)) + { + dens <- densities + x <- exprs(data[[i]][,parm]) + r <- ranges[[i]][,parm] + x <- x[x>r[1]+eps & x 1) { + message("idFeatures: lambda must be between 0 to 1. Defaulting to + 0.1. \n") + lambda <- 0.1 + } + + if (! reference.method %in% c("median", "mean")) + stop(paste(reference.method, "is not a valid method.")) + + ## 1. take all the centoids from all the backgating and apply 'diana' + ## to get the clusters of features + cent <- bg[bg$type=="centroid", ] + # cent.translate <- .centerFigure(cent, nDim) + + ## cluster centered centroids using 'diana' + cent.clust <- .useDivCluster(cent, nDim, thres.sigma, cut.by.height=TRUE) + + ## 2. label the outliers + filt.clust <- .filterOutlier(cent.clust, lambda=lambda, keep.outlier=TRUE, + label.outlier=".outlier") + + ## 3. get reference features, do not include outliers + ## reference <- .calcReference(filt.clust, nDim) # if keep.outlier=FALSE + reference <- .calcReference( + filt.clust[!filt.clust$cluster %in% ".outlier", ], nDim, + method=reference.method) + + ## 4. register features for each samples (with bogus features) + perSample <- split(filt.clust, factor(filt.clust$sample)) + register <- lapply(perSample, .classifyFeatures, + refF=reference, nDim=nDim) + ## 5. prepare return value register (containing the reference features) + + register$reference <- reference + + if (plot.workflow) + .plotWorkFlow(cent=cent, cent.clust=cent.clust, + filt.clust=filt.clust, register=register, ask=ask) + + return(register) +} + +############################### +## get the centered figure +############################### +.centerFigure <- function(cent, nDim) +{ + ## cent: centroids of the return values of backgating + ## return translated centroids (called centered figures) + TransMatrix <- lapply(split(cent, cent$sample), + function(x) mean(x[, 1:nDim], na.rm=TRUE)) + cF <- lapply(split(cent, cent$sample), + function(x, TransMatrix) { + I <- matrix(1, nrow=nrow(x), ncol=1) + x[, 1:nDim] <- x[, 1:nDim] - I %*% TransMatrix[[x$sample[1]]] + x + }, TransMatrix) + + return(list(centered=do.call(rbind, cF), TransMatrix=TransMatrix)) +} + +################################################# +## filter out outlier cluster and cluster members +## threshold = lambda * number of total features +################################################# +.filterOutlier <-function(center, lambda=0.1, + keep.outlier=FALSE, label.outlier=NULL) +{ + if (keep.outlier & is.null(label.outlier)) + label.outlier <- ".outlier" + + nclust <- table(center$cluster) + outlier_thres <- lambda * sum(nclust) + #outlier_thres <- median(table(center$cluster)) * lambda + outlier <- names(nclust)[nclust < outlier_thres] + if (length(outlier) == length(nclust)) + stop("idFeature: chose smaller lambda.") + + ifelse(keep.outlier, + center$cluster[center$cluster %in% outlier] <- label.outlier, + center <- center[!center$cluster %in% outlier, ]) + + attr(center, "outlier_thres") <- outlier_thres + return(center) +} + +############################## +## calculate the refenrence ## +############################## +.calcReference <- function(center, nDim=NULL, method="mean") { + + ## return value: ref is a data frame with columns of channels of interest, + ## cluster, sample, and bogus. + + if (is.null(nDim)) stop("Dimension of the variables must be provided") + + ref <- do.call(rbind, lapply(split(center, center$cluster), + function(a) { + ref <- as.data.frame(t(apply(a[, 1:nDim], 2, method))) + ref$cluster <- a$cluster[1] + return(ref) + })) + ref$sample <- "reference" + ref$bogus <- rep(FALSE, nrow(ref)) + return(ref) +} + + +########################################################### +## use divisive hierarchical clustering, diana-algorithm ## +########################################################### +.useDivCluster <- function(center, nDim, thres.sigma=2.5, + cut.by.height=TRUE, k=2) { + ## sign (or re-sign the cluster label) + clustD <- cluster::diana(center[, 1:nDim]) + + ## cut into n groups by 'heightThres' or k + if (cut.by.height) { + ## heightThres indicates where the tree should be cut + heightThres <- sd(clustD$height) * thres.sigma + center$cluster <- stats::cutree(as.hclust(clustD), h=heightThres) + attr(center, "height") <- heightThres + + } + else { + center$cluster <- stats::cutree(as.hclust(clustD), k=k) + attr(center, "k") <- k + } + + attr(center, "cluster") <- clustD + return(center) +} + +###################### +## classifyFeatures ## +###################### +.classifyFeatures <- function(eachSample, refF, nDim) +{ + ## initialize parameter: dim and dim names of reg are equeal xzto that of refF + reg <- refF + reg[, 1:nDim] <- NA + reg$sample <- rep(eachSample$sample[1], nrow(refF)) + + #reg <- data.frame(apply(refF, 2, function(x) rep(NA, length(x))), + # sample=rep(eachSample$sample[1], nrow(refF)), + # bogus=rep(TRUE, nrow(refF))) + #reg$cluster <- refF$cluster + + perClust <- split(eachSample, factor(eachSample$cluster)) + + ## ignore outliers (the label not in refF$clust + label <- unique(eachSample$cluster) + anyOutlier <- label[!label %in% refF$cluster] + if (length(anyOutlier)) + perClust <- perClust[!names(perClust) %in% anyOutlier] + + + dim <- seq_len(nDim) + + for (j in 1:length(perClust)) { + lab <- perClust[[j]]$cluster[1] + if (nrow(perClust[[j]]) == 1) + reg[reg$cluster==lab, dim] <- as.matrix(perClust[[j]][, dim]) + else { + tmp <- rbind(refF[lab, dim], perClust[[j]][,dim]) + mindist <- + which.min(as.numeric(dist(tmp))[1:nrow(perClust[[j]])]) + reg[reg$cluster==lab, dim] <- as.matrix(perClust[[j]][mindist, dim]) + } + reg$bogus[reg$cluster==lab] <- FALSE + } + + ## 2. deal with NA peaks, give the refFeatures and cluster status as bogus + naIdx <- which(is.na(reg[, 1])) + + if (!identical(naIdx, integer(0))) { + reg[naIdx, dim] <- refF[naIdx, dim] + reg[naIdx, "bogus"] <- TRUE + } + + return(reg) +} + + +################################################### +## check if the number of features for +## each sample is equal to that of reference +## if the feature is missing, replace with a bogus +################################################### +.checkFeatureNum <- function(combf, reference, nDim) { + lapply(combf, function(x) { + if (nrow(x) != nrow(reference)) { + i <- nrow(x) + x[i+1,] <- x[i,] + x[i+1, 1:(nDim+1)] <- + reference[!(reference$cluster %in% x$cluster), 1:(nDim+1)] + x$bogus[i+1] = TRUE + x <- x[sort(x$cluster), ] + } + x + }) +} + + +################################ +## plot the results of workflow +################################ + +.plotWorkFlow <- function(cent, cent.clust, filt.clust, register, ask) +{ + par(ask=ask) + on.exit(par(ask=FALSE)) + ## 1. cent + fo <- as.formula(paste(names(cent)[1], "~", names(cent)[2])) + mainTitle <- do.call(paste, as.list(unique(cent$channel))) + mainTitle <- paste("Centroids of high-density areas yielded by + backgating on channel", mainTitle) + print(lattice::xyplot(fo, group=channel, + data=cent, + auto.key=list(cex=0.8, title="channel", space="right"), + main=mainTitle, + sub="potential features")) + + col <- rainbow(length(unique(cent$sample))) + key <- list(text=list(levels(factor(cent$sample))), + points=list(col=col, pch=20, alpha=0.7), + cex=0.7, title="sample") + + print(lattice::xyplot(fo, group=sample, + data=cent, + col=col, pch=20, alpha=0.7, + key=c(key, space="right"), + main=mainTitle, + sub="potential features")) + + ## 2. cent.translate$centered + #print(lattice::xyplot(fo, group=sample, + # data=cent.translate$centered, + # auto.key=list(cex=0.8, title="sample", space="right"), + # main="Centered figure: translate the center of + # centroids to the origin")) + + ## 2. cent.clust + subTitle <- paste("Clusters containing <", + format(attr(cent.clust, "height"), digit=3), + "members are considered outliers.") + + + print(plot(attr(cent.clust, "cluster"), which.plot=2, + xlab="Potential Features", + main=paste("Cut the tree at height", + format(attr(cent.clust, "height"), digit=3)) + )) + + d <- density(attr(cent.clust, "cluster")$height) + print(plot(d, main="Distribution of the cluster (diana) height")) + abline(v=attr(cent.clust, "height"), col="grey") + text(x= attr(cent.clust, "height"), y=mean(d$y)+2*sd(d$y), + labels="cutting the\n tree here", pos=4) + + print(lattice::xyplot(fo, group=cluster, + data=cent.clust, + auto.key=list(cex=0.8,title="cluster", space="right"), + main="Clusters of potential features", + sub=subTitle)) + ## plot dendrogram of attr(cent.clust, "cluster") + + + ## 3. filt.clust + print(lattice::xyplot(fo, group=cluster, + data=filt.clust, + auto.key=list(cex=0.8, title="channel", space="right"), + sub=subTitle, + main="Clusters of features and outliers")) + + ## 4. register -- final features and reference features + fo <- as.formula(paste(names(cent)[1], "~", names(cent)[2], + " | sample")) + print(lattice::xyplot(fo , group=cluster, + data=do.call(rbind, register), + auto.key=list(cex=0.8, title="label",space="right"), + main="Labeled features")) + print(lattice::xyplot(fo, group=bogus, + data=do.call(rbind, register), + auto.key=list(cex=0.8, title="Bogus",space="right"), + main="Labeled features", + sub="Missing features are those with bogus=TURE.")) + +} diff --git a/R/landmarkMatrix.R b/R/landmarkMatrix.R new file mode 100644 index 0000000..6ed4cd2 --- /dev/null +++ b/R/landmarkMatrix.R @@ -0,0 +1,111 @@ +## create matrix of landmarks by aligning modes of the data density estiamtes +## Number of modes per sample doesn't need to be the same, the algorithm will +## try to guess wich of the modes are to be aligned. +## Currently this uses k-means clustering +landmarkMatrix <- function(data, fres, parm, border=0.05, peakNr=NULL, densities=NULL, n=201, + indices=FALSE) +{ + ## Some type-checking first + flowCore:::checkClass(data, "flowSet") + flowCore:::checkClass(fres, "list") + flowCore:::checkClass(parm, "character") + flowCore:::checkClass(border, "numeric", 1) + ## get peaks of significant regions from curv1Filter results + ranges <- fsApply(data, range) + from <- min(sapply(ranges, function(z) z[1,parm]-diff(z[,parm])*0.15), na.rm=TRUE) + to <- max(sapply(ranges, function(z) z[2,parm]+diff(z[,parm])*0.15), na.rm=TRUE) + peaks <- list() + regions <- list() + eps <- .Machine$double.eps + for(i in sampleNames(data)){ + if(is.null(densities)) + { + dens <- densities + x <- exprs(data[[i]][,parm]) + r <- ranges[[i]][,parm] + x <- x[x>r[1]+eps & x 0.1)))) + if(!is.null(peakNr)) + peakNr <- min(as.numeric(names(which.max(nrPeaks)), peakNr)) + clustCenters <- if(fnrPeaks>1 && !is.null(peakNr)){ + fnrPeaks <- peakNr + apply(matrix(unlist(peaks[(listLen(peaks)==peakNr)]), ncol=peakNr, byrow=T) ,2, mean) + }else fnrPeaks + resRegions <- list() + #Check if any peaks are detected as significant (why the 0.1 threshold above?) + if(is.na(fnrPeaks)){ + return(FALSE); + } + if(fnrPeaks==1){ + single <- which(listLen(peaks)==1) + med <- median(unlist(peaks[single]), na.rm=TRUE) + rmed <- apply(t(sapply(regions[single], c)), 2, median, na.rm=TRUE) + resPeaks <- numeric(length(peaks)) + resPeaks[single] <- unlist(peaks[single]) + resRegions[single] <- regions[single] + resPeaks[-single] <- unlist(sapply(peaks[-single], + function(x) x[which.min(abs(x-med))])) + resRegions[-single] <- mapply(function(x,y) y[which.min(abs(x-med)),,drop=FALSE], + x=peaks[-single], + y=regions[-single], SIMPLIFY=FALSE) + resPeaks[is.na(resPeaks)] <- med + resRegions[is.na(resPeaks)] <- matrix(rmed, ncol=2) + names(resRegions) <- sampleNames(data) + if(!indices){ + m <- matrix(resPeaks, ncol=1) + attr(m, "regions") <- resRegions + attr(m, "cdists") <- matrix(0, nrow=length(data), ncol=1, + dimnames=list(sampleNames(data),NULL)) + return(m) + } + else + { + return(matrix(1, ncol=1, nrow=length(resPeaks), + dimnames=list(sampleNames(data), NULL))) + } + } + ## cluster peaks in k cluster where k is max number of peaks for a sample + mat <- matD <- matrix(nrow=length(peaks), ncol=fnrPeaks) + rownames(mat) <- rownames(matD) <- sampleNames(data) + pvect <- unlist(peaks) + names(pvect) <- rep(names(peaks), listLen(peaks)) + sel <- !is.na(pvect) + km <- kmeans(pvect[sel], clustCenters) + km$cluster <- match(km$cluster, order(km$centers)) + clusterDist <- numeric(length(km$cluster)) + for(i in seq_along(km$cluster)) + clusterDist[i] <- abs(pvect[i] - km$centers[km$cluster[i]]) + cList <- split(data.frame(cluster=km$cluster, dist=clusterDist, + landmark=pvect[sel], index=unlist(sapply(peaks, seq_along))[sel]), + names(pvect[sel])) + cList <- lapply(cList, function(x) x[order(x$dist),][1:min(nrow(x), + fnrPeaks),]) + ## put peaks in matrix according to clustering where row=sample and + ## col=cluster + for(i in rownames(mat)){ + cl <- cList[[i]] + tmp <- matrix(NA, ncol=2, nrow=fnrPeaks) + tmp[cl$cluster,] <- regions[[i]][cl$index,,drop=FALSE] + resRegions[[i]] <- matrix(tmp, ncol=2) + mat[i,cl$cluster] <- if(!indices) cl$landmark else cl$index + matD[i,cl$cluster] <- cl$dist/diff(ranges[[1]][,parm]) + } + resRegions <- resRegions[rownames(mat)] + attr(mat, "regions") <- resRegions + attr(mat, "cdists") <- matD + return(mat) +} + diff --git a/R/landmarkMatrixWithoutFilterResult.R b/R/landmarkMatrixWithoutFilterResult.R new file mode 100644 index 0000000..1d2ea8d --- /dev/null +++ b/R/landmarkMatrixWithoutFilterResult.R @@ -0,0 +1,80 @@ +#TODO fix formal arguments +landmarkMatrixWithoutFilterResult <- function(data, peaksandregions, parm, border=0.05, peakNr=NULL, + indices=FALSE) +{ + ##TODO maybe code to organzie the peaksandregions variable should go here.. + peaks<-do.call(c,lapply(peaksandregions,function(x)x[[parm]][[1]])) + regions<-do.call(c,lapply(peaksandregions,function(x)x[[parm]][[2]])) + + ranges <- fsApply(data, range) + nrPeaks <- table(listLen(peaks)) + fnrPeaks <- as.numeric(max(names(which(nrPeaks/sum(nrPeaks) > 0.1)))) + if(!is.null(peakNr)) + peakNr <- min(as.numeric(names(which.max(nrPeaks)), peakNr)) + clustCenters <- if(fnrPeaks>1 && !is.null(peakNr)){ + fnrPeaks <- peakNr + apply(matrix(unlist(peaks[(listLen(peaks)==peakNr)]), ncol=peakNr, byrow=T) ,2, mean) + }else fnrPeaks + resRegions <- list() + if(is.na(fnrPeaks)) + return(FALSE) + if(fnrPeaks==1){ + single <- which(listLen(peaks)==1) + med <- median(unlist(peaks[single]), na.rm=TRUE) + rmed <- apply(t(sapply(regions[single], c)), 2, median, na.rm=TRUE) + resPeaks <- numeric(length(peaks)) + resPeaks[single] <- unlist(peaks[single]) + resRegions[single] <- regions[single] + resPeaks[-single] <- unlist(sapply(peaks[-single], + function(x) x[which.min(abs(x-med))])) + resRegions[-single] <- mapply(function(x,y) y[which.min(abs(x-med)),,drop=FALSE], + x=peaks[-single], + y=regions[-single], SIMPLIFY=FALSE) + resPeaks[is.na(resPeaks)] <- med + resRegions[is.na(resPeaks)] <- matrix(rmed, ncol=2) + names(resRegions) <- sampleNames(data) + if(!indices){ + m <- matrix(resPeaks, ncol=1) + attr(m, "regions") <- resRegions + attr(m, "cdists") <- matrix(0, nrow=length(data), ncol=1, + dimnames=list(sampleNames(data),NULL)) + return(m) + } + else + { + return(matrix(1, ncol=1, nrow=length(resPeaks), + dimnames=list(sampleNames(data), NULL))) + } + } + ## cluster peaks in k cluster where k is max number of peaks for a sample + mat <- matD <- matrix(nrow=length(peaks), ncol=fnrPeaks) + rownames(mat) <- rownames(matD) <- sampleNames(data) + pvect <- unlist(peaks) + names(pvect) <- rep(names(peaks), listLen(peaks)) + sel <- !is.na(pvect) + km <- kmeans(pvect[sel], clustCenters) + km$cluster <- match(km$cluster, order(km$centers)) + clusterDist <- numeric(length(km$cluster)) + for(i in seq_along(km$cluster)) + clusterDist[i] <- abs(pvect[i] - km$centers[km$cluster[i]]) + cList <- split(data.frame(cluster=km$cluster, dist=clusterDist, + landmark=pvect[sel], index=unlist(sapply(peaks, seq_along))[sel]), + names(pvect[sel])) + cList <- lapply(cList, function(x) x[order(x$dist),][1:min(nrow(x), + fnrPeaks),]) + ## put peaks in matrix according to clustering where row=sample and + ## col=cluster + for(i in rownames(mat)){ + cl <- cList[[i]] + tmp <- matrix(NA, ncol=2, nrow=fnrPeaks) + tmp[cl$cluster,] <- regions[[i]][cl$index,,drop=FALSE] + resRegions[[i]] <- matrix(tmp, ncol=2) + mat[i,cl$cluster] <- if(!indices) cl$landmark else cl$index + matD[i,cl$cluster] <- cl$dist/diff(ranges[[1]][,parm]) + } + resRegions <- resRegions[rownames(mat)] + attr(mat, "regions") <- resRegions + attr(mat, "cdists") <- matD + return(mat) + } + diff --git a/R/normalize-methods.R b/R/normalize-methods.R new file mode 100644 index 0000000..ce93e8e --- /dev/null +++ b/R/normalize-methods.R @@ -0,0 +1,258 @@ +#Sequentially normalize a gating set by normalizing channels at each step of gating +#return a normalized gatingset +#target is the target sample to normalize against +#skip is a list of gate indices for which you want to skip normalization (i.e. dump gates that may be difficult to normalize, or FSC/SSC) +getNormGateList<-function(x){ + flowCore:::checkClass(x,"GatingSet") + bfsgates<-lapply(x,function(y)which(sapply(RBGL::bfs(y@tree),function(x)!flowWorkspace:::.isBooleanGate.graphNEL(y,x)))) + bfsgates<-unique(do.call(rbind,bfsgates)) + return(data.frame(gate=flowWorkspace:::getNodes(x[[1]], showHidden = TRUE)[bfsgates],index=t(bfsgates))) +} +plotAllGates<-function(gh,cex=2,gsubset=NULL){ + nodes<-RBGL::bfs(gh@tree) + nodes<-nodes[!sapply(nodes,function(x)flowWorkspace:::.isBooleanGate.graphNEL(gh,x))][-1L] + if(is.null(gsubset)) + l<-length(nodes) + else{ + nodes<-nodes[gsubset] + l<-length(nodes) + } + rs<-floor(sqrt(l)) + cs<-l%/%rs + while(rs*cslength(flowWorkspace::getNodes(x[[1]], showHidden = TRUE))){ + stop("Gate index out of bounds") + } + if(s>length(x)){ + stop("Sample index out of bounds") + } + if(length(x)!=length(y)){ + stop("The two gating sets should be of the same size") + } + if(!is.null(g2)){ + G2<-flowWorkspace::getGate(y[[s]],g2)@boundaries + }else{ + G2<-NULL + } + par<-flowWorkspace::getParent(x[[1]],g) + dims<-flowWorkspace::getDimensions(x[[1]],flowWorkspace::getNodes(x[[1]], showHidden = TRUE)[g]) + form<-sapply(dims,function(f)as.formula(paste("~`",f,"`",sep=""))) + print(densityplot(form[[1]],flowWorkspace::getData(x,par),main="Raw"),split=c(1,1,3,2),more=TRUE) + print(densityplot(form[[1]],flowWorkspace::getData(y,par),main="Normalized"),split=c(1,2,3,2),more=TRUE) + + print(densityplot(form[[2]],flowWorkspace::getData(x,par),main="Raw"),split=c(2,1,3,2),more=TRUE) + print(densityplot(form[[2]],flowWorkspace::getData(y,par),main="Normalized"),split=c(2,2,3,2),more=TRUE) + + print(flowWorkspace::plotGate(x[[s]],g,main="Raw"),split=c(3,1,3,2),more=TRUE) + if(!is.null(G2)){ + trellis.focus(highlight=FALSE) + panel.polygon(G2,border="red") + trellis.unfocus() + } + print(flowWorkspace::plotGate(y[[s]],g,main="Normalized"),split=c(3,2,3,2),more=FALSE) + if(!is.null(G2)){ + trellis.focus() + panel.polygon(G2,border="red") + trellis.unfocus() + } +} + + + + +setMethod("normalize",c("GatingSet","missing"),function(data,x="missing",...){ + .normalizeGatingSet(x=data,...) + }) + + +.normalizeGatingSet <- function(x,target=NULL,skipgates=NULL,skipdims=c("FSC-A","SSC-A","FSC-H","SSC-H","Time"),subsample=NULL,chunksize=10,nPeaks=list(),bwFac=2,ncdfFile = NULL, minCountThreshold = 500, ...){ + +# browser() + samples<-sampleNames(x) + valid<-target%in%samples + if(!is.null(target)){ + if(!valid){ + stop("target ",target," not in the GatingSet") + } + } + + #Get all the non-boolean gates, breadth first traversal + #Do a breadth-first traversal + nodelist<-getNodes(x[[1]],order="bfs",prefix=TRUE, showHidden = TRUE) + + bfsgates<-unlist(lapply(nodelist,function(curNode){ +# browser() + if(curNode=="root") + return(0) + else + { + curNodeInd<-as.integer(strsplit(curNode,split="\\.")[[1]][1])+1 + if(!flowWorkspace:::.isBoolGate(x[[1]],curNodeInd)) + return(curNodeInd) + } + })) + + + np<-vector("list",length(bfsgates)) + + names(np)<-bfsgates + for(p in seq_along(nPeaks)){ + np[[p]]<-nPeaks[[p]] + } + #gate-specific channel list to track normalization + parentgates<-list(); + + #Keep a vector of normalized dimensions + unnormalized<-NULL + + #Initialize master channel list + unnormalized<-colnames(getData(x[[1]])) + + message("cloning the gatingSet...") + + x<-clone(x, ncdfFile = ncdfFile) +# browser() + #for each gate, grab the dimensions and check if they are normalized. + #Normalize what hasn't been normalized yet, then do the gating. + #Set a target sample by name + for(i in bfsgates){ + + gate<-getGate(x[[1]],i) + + #Root node + if(class(gate)=="logical"){ #implies gate is NA, since NA is class logical, otherwise some flowCore gate class. + next; + }else{ + #check which dimensions are normalized already + dims<-parameters(gate) + dims<-setdiff(dims,skipdims) + if(length(dims)>0) + { + + #Data will be subset at gate g (parent) and normalized on dims of i (child) + #keep a list of normalized and unnormalized channels for each parent gate.. + #Check the gate being normalized.. make sure 74 is done correctly + if(!i%in%skipgates){ +# browser() + #Get the PARENT gate (since we'll be gating the data using gate i) + g<-getParent(x[[1]],i) + #initialize gate-specific normalization list + if(is.null(parentgates[[as.character(g)]])){ + parentgates[[as.character(g)]]<-list(); + parentgates[[as.character(g)]]$unnormalized<-unnormalized + parentgates[[as.character(g)]]$normalized<-NULL + } + + wh.dim<-dims%in%parentgates[[as.character(g)]]$unnormalized + parentgates[[as.character(g)]]$normalized<-c(parentgates[[as.character(g)]]$normalized,dims[wh.dim]); + parentgates[[as.character(g)]]$unnormalized<-setdiff(parentgates[[as.character(g)]]$unnormalized,dims) + stains<-dims[wh.dim] +# browser() + if(length(stains)!=0&gateHasSufficientData(x,g, minCountThreshold = minCountThreshold, ...)){ + #choose the np element by name + npks<-np[[as.character(i)]] + + result<-warpSetGS(x,stains=stains,gate=g,target=target,subsample=subsample,chunksize=chunksize,peakNr=npks,bwFac=bwFac,...) + + if(flowWorkspace:::isNcdf(x)){ + sapply(sampleNames(result),function(s)ncdfFlow:::updateIndices(result,s,NA)) + flowData(x)<-result + }else{ + oldfs<-flowData(x) + for(j in sampleNames(x)){ + inds<-flowWorkspace::getIndices(x[[j]],g) + oldfs[[j]]@exprs[inds,]<-result[[j]]@exprs + } + flowData(x)<-oldfs + } + recompute(x,i); + } + + + } + } + + } + + } + x +} + +#Function to check if each flowFrame at the given gate has enough data to normalize. +#Set the threshold at 500 events for each flowFrame. +gateHasSufficientData<-function(x=NULL,g=NULL,minCountThreshold=500,...){ + #x could be flowSet or ncdfFlowSet + res<-unlist(lapply(x,function(x)nrow(getData(x,g))>=minCountThreshold)) + if(all(res)) + return(TRUE) + else + { + warning("not enough events to normalize: ",paste(names(res[!res]),collapse="\n")) + return(FALSE) + } +} +# hierarchy +#}) diff --git a/R/pbin.R b/R/pbin.R new file mode 100644 index 0000000..b7daeb4 --- /dev/null +++ b/R/pbin.R @@ -0,0 +1,216 @@ + +proBin<-function(m,minEvents=500,channels=NULL) +{ + if(!is(m,"flowFrame")) + stop("m needs to be an object of class flowFrame") + + timeCol <- flowCore:::findTimeChannel(m,strict=FALSE) + cNames <-colnames(m) + if(length(timeCol)==0){ + m <- exprs(m) + }else{ + m <-exprs(m)[, cNames[!(cNames==timeCol)]] + } + if(is.null(channels)){ + channels <- colnames(m) + } + + nodeIndx=1 + node =data.frame(dataIndx=1,visited=FALSE,parent=0,left=0,right=0,stringsAsFactors=FALSE) + data=new.env(parent=emptyenv()) + data[[as.character(1)]]=m + + axisLimits=list() + axisLimits[[1]]=data.frame(axisMin=rep(-Inf,ncol(m)),axisMax=rep(Inf,ncol(m))) + + splitPars=list() + splitPars[[1]]=data.frame(splitCol=0,splitMed=0) + + while (1) + { + if( node$left[nodeIndx] !=0 && node$visited[node$left[nodeIndx]]==FALSE) + { + nodeIndx= node$left[nodeIndx] + + } + else if( node$right[nodeIndx] !=0 && node$visited[node$right[nodeIndx]]==FALSE) + { + nodeIndx= node$right[nodeIndx] + + } + else if(node$visited[nodeIndx]==FALSE) + { + node$visited[nodeIndx]=TRUE + + if(nrow(data[[as.character(nodeIndx)]])>minEvents) + { + y<-apply(data[[as.character(nodeIndx)]][,channels],2,var) + tName <- which.max(y) + t <- which(colnames(data[[as.character(nodeIndx)]])== attr(tName,"names")) + #t<-which.max(y) + x<-median(data[[as.character(nodeIndx)]][,t]) + s1=data[[as.character(nodeIndx)]][,t] > x + if(!(all(s1) || all(!s1))) + { + + m1<-data[[as.character(nodeIndx)]][s1,,drop=FALSE] + m2<-data[[as.character(nodeIndx)]][!s1,,drop=FALSE] + + temp=nrow(node)+1; + node$left[nodeIndx]=temp; + node$right[nodeIndx]=temp+1; + data[[as.character(temp)]]=m1; + data[[as.character(temp+1)]]=m2; + splitPars[[temp]]=data.frame(splitCol=t,splitMed=x) + splitPars[[temp+1]]=data.frame(splitCol=t,splitMed=x) + + axisLimits[[temp]]=axisLimits[[nodeIndx]] + axisLimits[[temp+1]]=axisLimits[[nodeIndx]] + axisLimits[[temp]]$axisMin[t]=x + axisLimits[[temp+1]]$axisMax[t]=x + + node[temp,]=list(dataIndx=as.numeric(temp),visited=FALSE,parent=nodeIndx,left=0,right=0) + node[temp+1,]=list(dataIndx=as.numeric(temp+1),visited=FALSE,parent=nodeIndx,left=0,right=0) + } + } + } + else + { + nodeIndx=node$parent[nodeIndx] + if(node$parent[nodeIndx]==0) + { + if(node$visited[node$right[nodeIndx]]==TRUE) + { + break; + } + + } + } + + } + return(list(table=node, data=data,limits=axisLimits,splitPars=splitPars)) +} + + +binByRef<-function(binRes,data) #output of proBin function, flowFrame +{ + data<-exprs(data) + lmts=binRes$table$dataIndx[binRes$table$left==0] + storeIndx=1; + binned=new.env(parent=emptyenv()) + for (i in lmts) + { + cStoreIndx<-as.character(storeIndx) + len=nrow(binRes$limits[[i]]) + binned[[cStoreIndx]]<-data + for(j in seq_len(len)) + { + indx<-!is.na(cut( binned[[cStoreIndx]][,j],binRes$limits[[i]][j,],labels=FALSE)) + binned[[cStoreIndx]]<- matrix(binned[[cStoreIndx]][indx],ncol=ncol(binned[[cStoreIndx]])) + } + storeIndx<-storeIndx+1 + } + return(binned) + +} + + +plotBins<-function(binRes,data,channels=c("FSC-H","SSC-H"),title="",residuals=NULL,shadeFactor=0.6) +{ + if(!all(channels %in% colnames(data))) + stop("Invalid channel(s)") + + timeCol <- flowCore:::findTimeChannel(data,strict=FALSE) + cNames <-colnames(data) + if(length(timeCol)==0){ + data <- exprs(data) + }else{ + data<- exprs(data)[, cNames[!(cNames==timeCol)]] + } + + plotColX<-which(colnames(data)==channels[1]) + plotColY<-which(colnames(data)==channels[2]) + + plot(x=data[,plotColX], y=data[,plotColY], + xlab=channels[1], ylab=channels[2], + main=title, col="#0080ff30") + + fill<-"transparent" + if(!is.null(residuals)) + { + res <- (((residuals - min(residuals)) / max(residuals-min(residuals)))*shadeFactor) + shade <- rgb(0,0,0, res) + } + + x.limits=range(data[,plotColX]) + y.limits=range(data[,plotColY]) + limits=binRes$limits[binRes$table$left==0] + pars=binRes$splitPars[binRes$table$left==0] + + for(i in seq_along(limits)) + { + x1=limits[[i]]$axisMin[plotColX] + x2=limits[[i]]$axisMax[plotColX] + y1=limits[[i]]$axisMin[plotColY] + y2=limits[[i]]$axisMax[plotColY] + + if(pars[[i]]$splitCol==plotColX) + { + ##plot vertical lines + y1=max(limits[[i]]$axisMin[plotColY], y.limits[1]) + y2=min(limits[[i]]$axisMax[plotColY], y.limits[2]) + x1=max(limits[[i]]$axisMin[plotColX], x.limits[1]) + x2=min(limits[[i]]$axisMax[plotColX], x.limits[2]) + } + else if(pars[[i]]$splitCol==plotColY) + { + x1=max(limits[[i]]$axisMin[plotColX], x.limits[1]) + x2=min(limits[[i]]$axisMax[plotColX], x.limits[2]) + y1=max(limits[[i]]$axisMin[plotColY], y.limits[1]) + y2=min(limits[[i]]$axisMax[plotColY], y.limits[2]) + } + + if(!is.null(residuals)) + { + fill<-shade[i] + } + + rect(x1,y1,x2,y2,border="red", col=fill) + } + +} + + +calcPearsonChi<-function(ctrlRes,sampRes) +{ + binId<-ctrlRes$table$dataIndx[ctrlRes$table$left==0] + binCount<-length(binId) + input=matrix(0,2,binCount) + for( i in seq_len(binCount)) + { + input[1,i]=nrow(ctrlRes$data[[as.character(binId[i])]]) #expected + input[2,i]=nrow(sampRes[[as.character(i)]]) #observed + } + chisq.test(input) +} + +calcPBChiSquare<-function(ctrlRes,sampRes,ctrlCount,sampCount) +{ + result<-list() + binId<-ctrlRes$table$dataIndx[ctrlRes$table$left==0] + binCount<-length(binId) + for( i in seq_len(binCount)) + { + ctrl= (nrow(ctrlRes$data[[as.character(binId[i])]]))/ctrlCount + samp=(nrow(sampRes[[as.character(i)]]))/sampCount + result$chiSq[i]<-( (samp-ctrl)^2)/(ctrl+samp) + result$expected[i]<-ctrl*ctrlCount + result$observed[i]<-samp*sampCount + result$residuals[i]<-(samp-ctrl)/(sqrt(ctrl+samp)) + } + result$pbStat<-(2*ctrlCount*sampCount*(sum(result$chiSq))/(ctrlCount+sampCount) + -(binCount-1))/(sqrt(2*(binCount-1))) + + return(result) + } + diff --git a/R/peakSeparator.R b/R/peakSeparator.R new file mode 100644 index 0000000..09000fa --- /dev/null +++ b/R/peakSeparator.R @@ -0,0 +1,309 @@ +## This is the main function to perform backgating. 'data' is supposed to be a flowSet, +## 'xy' is a character vector of length 2 giving the dimensions of interest and 'channels' +## are the additional channels used for backgating. 'fres' is an optional list of filterResults +## of the initial curv1Filter foer each parameter in channels and can be supplied in order to +## speed things up. the output of the function depends on the value of 'incidence'. If this +## is TRUE, it will be a list of incidence matrices for each sample in the set, where each matrix +## indicates whether a particular event was assign to a given peak. If 'incidence' is FALSE, a +## hook function can be supplied which directly uses the subset of events under a peak +## (as a flowFrame in the first argument). The value of xy is passed in as a second argument. +## The output is supposed to be a data frame, potentially of landmark coordinates and further +## annotation. The hook function is called for each frame in the set, for each peak in each +## channel, and the respective results are concatenated using rbind. The peak, channel and +## sample information is added automatically. The final output of the backGating function is +## the concatenated data frame in this case. +backGating <- function(data, xy, channels=setdiff(colnames(data), c(xy, "time", "Time")), + fres, hookFunction="curvRegs", incidence=FALSE) +{ + if(missing(fres)) + { + fres <- lapply(channels, function(x) filter(data, curv1Filter(x))) + names(fres) <- channels + } + allres <- NULL + subsets <- vector(mode="list", length=length(data)) + names(subsets) <- sampleNames(data) + dataColl <- as(data, "flowFrame") + for(i in seq_along(channels)) + { + fresColl <- filter(dataColl, curv1Filter(channels[i]) %subset% sampleFilter(50000)) + np <- length(fresColl)-1 + lm <- landmarkMatrix(data, fres, channels[i], indices=TRUE, + peakNr=np) + if(!all(lm==0)) + { + for(j in seq_len(ncol(lm))) + { + for(k in sampleNames(data)) + { + cat("\r ") + cat("\rProcessing channel", channels[i], "peak", j, "sample", k) + subset <- split(data[[k]], fres[[channels[i]]][[k]])[[lm[k,j]+1]] + if(!is.null(subset)) + { + tfr <- fres[[channels[i]]][[k]][[lm[k,j]+1]] + subsets[[k]] <- cbind(subsets[[k]], tfr@subSet) + colnames(subsets[[k]])[ncol(subsets[[k]])] <- + paste(channels[[i]], j, sep="_") + if(!incidence) + { + res <- do.call(hookFunction, args=list(subset, xy)) + if(!is.null(res)) + allres <- rbind(allres, cbind(res, sample=k, peak=j, + channel=channels[i])) + } + } + } + } + } + } + cat("\n") + return(if(incidence) subsets else allres) +} + + + + + + + +## Compute high-density regions using a curv2Filter, try to combine +## neighboring areas if they are within a certain threshold and return +## the polygon vertices as well as theirt centroids. +curvRegs <- function(dat, p) +{ + r <- range(dat, p) + c2 <- curv2Filter(p, bwFac=1.8) + c2Res <- filter(dat, c2) + pols <- attr(c2Res@subSet, "polygons") + polM <- lapply(pols, function(x) sapply(x[2:3], c)) + if(length(polM)>1) + polM <- pruneRegions(polM, max(apply(r, 2, diff))/5, + prod(apply(r, 2, diff))/250) + res <- NULL + for(i in seq_along(polM)) + { + tmp <- polM[[i]] + colnames(tmp) <- p + cent <- apply(tmp, 2, mean) + ntmp <- data.frame(x=cent[1], y=cent[2], population=i, + type="centroid") + colnames(ntmp)[seq_along(p)] <- p + res <- rbind(res, + rbind(data.frame(tmp, population=i, + type="polygon"), + ntmp)) + } + return(res) +} + + + + + + +## Some helpers below here... + + +## Find and score separators between peaks of a flowSet. The scoring +## is based on the overlap of normal approximations for the underlying +## data of neighbouring peaks. + +peakSeparator <- function(data, parm, fres) +{ + if(missing(fres)) + { + fres <- lapply(parm, function(x) filter(data, curv1Filter(x))) + names(fres) <- parm + } + lm <- landmarkMatrix(data, fres, parm, indices=TRUE) + ## Initially we only take peaks that are present in all samples + ## lm <- lm[,!apply(lm, 2, function(x) any(is.na(x))), drop=FALSE] + ##regions <- getRegions(data, parm, fres, lm) + subs <- getSubsets(data, parm, fres, lm) + props <- getProportions(data, parm, fres, lm) + norm <- lapply(subs, normApprox) + ol <- computeOverlap(norm) + plotting(data, norm, parm, props, ol) + +} + + +getSubsets <- function(data, parm, fres, mat) +{ + res <- list() + for(i in sampleNames(data)) + res[[i]] <- split(data[[i]][,parm], fres[[parm]][[i]])[mat[i,]+1] + return(res) +} + + + +getRegions <- function(data, parm, fres, mat) +{ + res <- list() + for(i in sampleNames(data)) + res[[i]] <- curvPeaks(fres[[parm]][[i]], exprs(data[[i]])[,parm])$regions[mat[i,],] + return(res) +} + + +getProportions <- function(data, parm, fres, mat) +{ + res <- list() + for(i in sampleNames(data)) + res[[i]] <- summary(fres[[parm]][[i]], toTable=TRUE)$p[mat[i,]+1] + return(res) +} + + +normApprox <- function(x) +{ + res <- matrix(ncol=2, nrow=length(x)) + colnames(res) <- c("mu", "sd") + for(i in seq_along(x)) + { + if(!is.null(x[[i]])) + res[i,] <- c(median(exprs(x[[i]]), na.rm=TRUE), mad(exprs(x[[i]]), na.rm=TRUE)) + + } + return(res) +} + + + +plotting <- function(data, norm, parm, props, ol) +{ + opar <- par(ask=TRUE) + on.exit(par(opar)) + for(i in sampleNames(data)) + { + plot(density(exprs(data[[i]][,parm])), main=parm) + r <- range(exprs(data[[i]][,parm])) + for(j in 1:nrow(norm[[i]])) + { + sq <- seq(r[1], r[2], len=500) + tt <- dnorm(sq, mean=norm[[i]][j,1], sd=norm[[i]][j,2])*props[[i]][j] + lines(sq, tt, col=j+1) + if(!is.na(ol[[i]][j])) + { + xl <- if(nrow(norm[[i]]) >= j+1) mean(c(norm[[i]][j,1], norm[[i]][j+1,1])) + else norm[[i]][j,1] + text(xl, par("usr")[4]-(par("usr")[4]/5), signif(ol[[i]][j],2)) + } + } + } + +} + + + +computeOverlap <- function(norm) +{ + res <- list() + for(n in names(norm)) + { + nn <- norm[[n]] + lr <- min(nn[,1] - 10*nn[,2], na.rm=TRUE) + rr <- max(nn[,1] + 10*nn[,2], na.rm=TRUE) + tmp <- NULL + if(nrow(nn)>1) + { + for(j in 2:nrow(nn)) + { + sq <- seq(lr, rr, len=500) + d1 <- dnorm(sq, mean=nn[j-1,1], sd=nn[j-1,2]) + d2 <- dnorm(sq, mean=nn[j,1], sd=nn[j,2]) + tmp <- c(tmp) + } + } + res[[n]] <- tmp + } + return(res) +} + + + + + +## Compute the area within a polygon by triangular approximation +area.in.polygon <- function(x) +{ + n<-nrow(x) + D <- diff(x[c(1:n, 1), ]) + A <- x[, 1] * D[, 2] - x[, 2] * D[, 1] + abs(sum(A)/2) +} + + +## A simple Hausdorff distance algorithm for two sets of polygon +## vertices. The proper solution is to compute distances between +## edges, however this is only defined for convex polygons (which +## could be forced by first calling chull...) +## Note that this makes sense only if the two dimensions are on a +## similar scale, e.g., FSC vs SSC +simpleHd <- function (A, B) +{ + hd <- 0 + n1 <- nrow(A) + n2 <- nrow(B) + d1 <- c() + d2 <- c() + if (n1 * n2 == 0) + hd <- -1 + else { + stopifnot(is.numeric(A)) + stopifnot(is.numeric(B)) + for (i in 1:n1) d1[i] <- min(sqrt((B[,1]-A[i,1])^2 + (B[,2]-A[i,2])^2)) + for (i in 1:n2) d2[i] <- min(sqrt((A[,1]-B[i,1])^2 + (A[,2]-B[i,2])^2)) + hd <- max(max(d1), max(d2)) + } + hd +} + + +## The pariwise distance matrix for a list of polygons, based on the +## Hausdorff distance metrix. +hdMat <- function(pols) +{ + mat <- matrix(ncol=length(pols), nrow=length(pols)) + for(i in seq_along(pols)) + for(j in seq_along(pols)) + mat[i,j] <- simpleHd(pols[[i]], pols[[j]]) + return(mat) +} + + +## Collapse two polygons to their common convex hull. +combinePols <- function(p1, p2) +{ + pc <- rbind(p1, p2) + pc[chull(pc),] +} + + +## Take a list of polygons and collapse all areas that are closer than +## 'dt' in the Hausdorff distance metric. Also drop all polygons with +## areas smaller than 'st'. +pruneRegions <- function(pols, dt, st) +{ + ssel <- sapply(pols, area.in.polygon) > st + if(any(ssel)) + pols <- pols[ssel] + else + return(pols) + d <- hdMat(pols) + sel <- which(hdMat(pols) < dt, arr.ind=TRUE) + sel <- sel[apply(sel, 1, diff)!=0, ] + while(length(sel)) + { + pols[[sel[1,1]]] <- combinePols(pols[[sel[1,1]]], + pols[[sel[1,2]]]) + + pols <- pols[-sel[1,2]] + d <- hdMat(pols) + sel <- which(hdMat(pols) < dt, arr.ind=TRUE) + sel <- sel[apply(sel, 1, diff)!=0, ] + } + return(pols) +} diff --git a/R/quadrantGate.R b/R/quadrantGate.R new file mode 100644 index 0000000..644f676 --- /dev/null +++ b/R/quadrantGate.R @@ -0,0 +1,59 @@ +## Do quad gating of a flowSet or flowFrame +quadrantGate <- function(x, stains, alpha = c("min", "min"), sd=c(2,2), + plot=FALSE, filterId="defaultQuadGate", + refLine.1=NULL, refLine.2=NULL + ,rare=c(FALSE,FALSE) + ,sig=c(NULL,NULL) + ,...) +{ + ## some type checking first + flowCore:::checkClass(x, c("flowFrame", "flowSet")) + flowCore:::checkClass(stains, "character", 2) + mt <- stains %in% colnames(x) + if(!all(mt)) + stop("Invalid stain(s) not matching the flowSet:\n ", + paste(stains[!mt], collapse=", ")) + alpha <- rep(alpha, length = 2)[1:2] + flowCore:::checkClass(alpha, c("character", "numeric"), 2) + sd <- rep(sd, length=2)[1:2] + rare <- rep(rare, length=2)[1:2] + sig <- rep(sig, length=2)[1:2] + + flowCore:::checkClass(sd, "numeric", 2) + + if (!is.null(refLine.1)) + flowCore:::checkClass(refLine.1, "numeric",1) + if (!is.null(refLine.2)) + flowCore:::checkClass(refLine.2, "numeric",1) + + flowCore:::checkClass(plot, "logical", 1) + flowCore:::checkClass(filterId, "character", 1) + + ## set up plotting device (if needed) + if(plot){ + opar <- par(mfrow=c(2,2)) + on.exit(par(opar)) + } + ## collapse to flowFrame and compute gates for each of the two channels + if(is(x, "flowSet")) + x <- as(x, "flowFrame") + boundX <- density1d(x, stains[1], alpha = alpha[1], plot, sd=sd[1], + refLine = refLine.1,rare=rare[1],sig=sig[1], ...) + boundY <- density1d(x, stains[2], alpha = alpha[2], plot, sd=sd[2], + refLine = refLine.2,rare=rare[2], sig=sig[2],...) + + + ## add final plot if needed + if(plot){ + ## FIXME: Ugly hack foe name space issue + plot <- selectMethod("plot", c("flowFrame", "character")) + plot(x, stains, main=paste(basename(description(x)$FIL),"\n Quad-gate for parameters\n", + paste(stains, collapse=" and ")), cex.main=1) + abline(h = boundY, v = boundX) + } + ## create the quadGate object + gate <- c(boundX, boundY) + names(gate) <- stains + return(quadGate(.gate=gate, filterId=filterId)) +} + diff --git a/R/singletGate.R b/R/singletGate.R new file mode 100644 index 0000000..29f0008 --- /dev/null +++ b/R/singletGate.R @@ -0,0 +1,107 @@ +#' Creates a singlet polygon gate using the prediction bands from a robust linear model +#' +#' We construct a singlet gate by applying a robust linear model with +#' \code{\link[MASS]{rlm}}. By default, we model the forward-scatter height +#' (FSC-H)as a function of forward-scatter area (FSC-A). If \code{sidescatter} +#' is given, forward-scatter height is as a function of \code{area} + +#' \code{sidescatter} + \code{sidescatter / area}. +#' +#' Because \code{\link[MASS]{rlm}} relies on iteratively reweighted least +#' squares (IRLS), the runtime to construct a singlet gate is dependent in part +#' on the number of observations in \code{x}. To improve the runtime, we provide +#' an option to subsample randomly a subset of \code{x}. A percentage of +#' observations to subsample can be given in \code{subsample_pct}. By default, no +#' subsampling is applied. +#' +#' @param x a \code{\link[flowCore:flowFrame-class]{flowFrame}} object +#' @param area character giving the channel name that records the signal +#' intensity as peak area +#' @param height character giving the channel name that records the signal +#' intensity as peak heightchannel name of height +#' @param sidescatter character giving an optional channel name for the +#' sidescatter signal. By default, ignored. +#' @param prediction_level a numeric value between 0 and 1 specifying the level +#' to use for the prediction bands +#' @param subsample_pct a numeric value between 0 and 1 indicating the percentage +#' of observations that should be randomly selected from \code{x} to construct +#' the gate. By default, no subsampling is performed. +#' @param wider_gate logical value. If \code{TRUE}, the prediction bands used to +#' construct the singlet gate use the robust fitted weights, which increase +#' prediction uncertainty, especially for large FSC-A. This leads to wider gates, +#' which are sometimes desired. +#' @param filterId the name for the filter that is returned +#' @param ... additional arguments passed to \code{\link[MASS]{rlm}} +#' @return a \code{\link[flowCore]{polygonGate}} object with the singlet gate +singletGate <- function(x, area = "FSC-A", height = "FSC-H", sidescatter = NULL, + prediction_level = 0.99, subsample_pct = NULL, + wider_gate = FALSE, filterId = "singlet", ...) { + flowCore:::checkClass(x, "flowFrame") + flowCore:::checkClass(area, "character") + flowCore:::checkClass(height, "character") + if (!is.null(sidescatter)) { + flowCore:::checkClass(sidescatter, "character") + } + if (length(area) + length(height) != 2) { + stop("Each of 'area' and 'height' must be 'character' vectors of length 1.") + } + + x <- data.frame(exprs(x[, c(area, height, sidescatter)])) + + # If specified, subsample from 'x' + if (!is.null(subsample_pct)) { + subsample_pct <- as.numeric(subsample_pct) + if (subsample_pct <= 0 || subsample_pct > 1) { + warning("The subsampling percentage must be between 0 and 1. ", + "Setting percentage to default value...", call. = FALSE) + subsample_pct <- 1 + } + n <- nrow(x) + x <- x[sample(x = seq_len(nrow(x)), size = subsample_pct * n), ] + } + + channel_names <- c(area, height) + area <- make.names(area) + height <- make.names(height) + rlm_formula <- paste(make.names(height), make.names(area), sep = " ~ ") + if (!is.null(sidescatter)) { + sidescatter <- make.names(sidescatter) + ssc_ratio <- paste0("I(", sidescatter, " / ", area, ")") + rlm_formula <- paste(rlm_formula, sidescatter, ssc_ratio, sep = " + ") + } + rlm_formula <- as.formula(rlm_formula) + + rlm_fit <- rlm(rlm_formula, data = x, ...) + + if (!rlm_fit$converged) { + warning("The IRLS algorithm employed in 'rlm' did not converge.") + } + + # Creates polygon gate based on the prediction bands at the minimum and maximum + # 'area' observation using the trained robust linear model. + which_min <- which.min(x[[area]]) + which_max <- which.max(x[[area]]) + x_extrema <- x[c(which_min, which_max), ] + + # Prediction weights. By default, these are weighted equally. If 'wider_gate' + # is set, the weights from 'rlm' are used. The weight for the maximum FSC-A + # is usually much smaller and yields a more uncertain prediction interval. + # This leads to a wider gate. + prediction_weights <- c(1, 1) + if (wider_gate) { + prediction_weights <- rlm_fit$w[c(which_min, which_max)] + } + + predictions <- predict(rlm_fit, x_extrema, interval = "prediction", + level = prediction_level, weights = prediction_weights) + + # Create a matrix of the vertices using the prediction bands at the minimum + # and maximum values of x. The ordering matters. Must go clockwise. + # Otherwise, the polygon is not convex and makes an X-shape. + gate_vertices <- rbind(cbind(x_extrema[[area]][1], predictions[1, "lwr"]), + cbind(x_extrema[[area]][1], predictions[1, "upr"]), + cbind(x_extrema[[area]][2], predictions[2, "upr"]), + cbind(x_extrema[[area]][2], predictions[2, "lwr"])) + colnames(gate_vertices) <- channel_names + + polygonGate(gate_vertices, filterId = filterId) +} diff --git a/R/warpSet.R b/R/warpSet.R new file mode 100644 index 0000000..17da965 --- /dev/null +++ b/R/warpSet.R @@ -0,0 +1,1295 @@ +## Align data in a flowSet by estimating high density regions and using this +## information as landmarks. This works separately on each parameter. + + +warpSetGS <- function(x,stains, grouping=NULL, monwrd=TRUE, subsample=NULL, + peakNr=NULL, clipRange=0.01, nbreaks=11, fres, bwFac=2, + warpFuns=FALSE,gate=NULL,target=NULL,chunksize=10, + ...){ + #Perform normalization on the subset + #return the normalized data + if(!inherits(x,"GatingSet")) + stop("x must be of class GatingSet") + if(!flowWorkspace:::isNcdf(x)){ + #TODO code the regular flowSet (not ncdfFlowSet) normalization code. + message("Gating Set not gated using netcdf. We'll use the regular warpSet function"); + if(is.null(gate)){ + flowset<-getData(x); + }else{ + if(gate<=length(flowWorkspace:::getNodes(x[[1]], showHidden = TRUE))){ + flowset<-getData(x,gate); + }else{ + stop("gate ",gate," out of range"); + } + } + colnames(flowset)<-colnames(getData(x[[1]])) + warped<-warpSet(x=flowset,stains=stains,grouping=grouping,monwrd=monwrd,subsample=subsample,peakNr=peakNr,clipRange=clipRange,nbreaks=nbreaks,fres=fres,bwFac=bwFac,warpFuns=warpFuns,target=target,...); + }else{ + if(is.null(gate)){ + #ncflowset<-graph:::nodeData(x[[1]]@tree,x[[1]]@nodes[1],"data")[[1]][["data"]]$ncfs + ncflowset<-flowWorkspace:::flowData(x) + #subset for the correct samples + ncflowset<-ncflowset[flowWorkspace:::sampleNames(x)] + #ncflowset<-ncflowset[setdiff(sampleNames(ncflowset),setdiff(sampleNames(ncflowset),sampleNames(x)))] + + }else{ + #check if the gate is in range + if(gate<=length(flowWorkspace:::getNodes(x[[1]], showHidden = TRUE))){ +# browser() + ncflowset <- getData(x,gate) + #subset for the correct samples + #ncflowset<-ncflowset[setdiff(sampleNames(ncflowset),setdiff(sampleNames(ncflowset),sampleNames(x)))] + ncflowset <- ncflowset[sampleNames(x)] + }else{ + stop("gate ",gate," out of range"); + } + } + #Update the column names on the ncflowset so that they are consistent with the column names on the GatingSet + colnames(ncflowset)<-colnames(getData(x[[1]])) + warped<-warpSetNCDF(x=ncflowset,stains=stains,grouping=grouping,monwrd=monwrd,subsample=subsample,peakNr=peakNr,clipRange=clipRange,nbreaks=nbreaks,fres=fres,bwFac=bwFac,warpFuns=warpFuns,target=target,chunksize=chunksize,...); + #Should the warped ncdfflowset be assigned to the GatingSet data environment? + #Do we now need to clear the indices after calling Subset? + return(warped); + } +} +warpSetNCDFLowMem <- function(x, stains, grouping=NULL, monwrd=TRUE, subsample=NULL, + peakNr=NULL, clipRange=0.01, nbreaks=11, fres=NULL, bwFac=2, + warpFuns=FALSE,target=NULL,chunksize=10,isNew=FALSE,newNcFile=NULL, + ...) +{ + .Deprecated("warpSetNCDF") + ## Some type-checking first + flowCore:::checkClass(x, "flowSet") + flowCore:::checkClass(stains, "character") + mt <- stains %in% colnames(x) + if(!all(mt)) + stop("Invalid stain(s) not matching the flowSet:\n ", + paste(stains[!mt], collapse=", ")) + #expData <- as(x, "list") + #expData should now be x... +# browser() + if(isNew){ + expData<-ncdfFlow:::clone.ncdfFlowSet(x,isNew=TRUE,isEmpty=FALSE,ncdfFile=newNcFile) + }else{ + expData<-x; + } + samples<-sampleNames(expData) + ranges<-vector("list",length(x)); + names(ranges)<-sampleNames(x) + for(i in 1:length(x)){ranges[[i]]<-range(x[[i]])} + #ranges <- fsApply(x, range) + if(!is.null(grouping)) + flowCore:::checkClass(grouping, "character", 1) + if(!is.null(subsample)) + { + flowCore:::checkClass(subsample, "numeric", 1) + ## subsample data set before all density estimation steps + #Does Subset clobber anything in a permanent way for an ncdfFlowSet? Check this, otherwise use subsample. + x <- Subset(x, sampleFilter(size=subsample)) + } + flowCore:::checkClass(monwrd, "logical", 1) + flowCore:::checkClass(bwFac, "numeric", 1) + + ##Chunking for filterResult + chunkgroups<-(1:length(expData))%/%chunksize + chunksamples<-split(samples,chunkgroups) + chunks.size<-table(chunkgroups) + nchunks<-length(chunks.size) + densY<-vector("list",nchunks) + peaksandregions<-vector("list",nchunks) + #Goal here is to compute filterResults in chunks, then to compute and register landmarks, which take up MUCH less space. + #chunk the data and compute filterResults + ## find landmarks + for(k in 1:nchunks){ + if(is.null(formals()$fres)) + { + fres<-vector("list",nchunks) + #fres <- list() + for(p in stains){ + cat("\rEstimating landmarks for channel", p, " Sample chunk ",k,"...") + #fres[[p]] <- filter(x, curv1Filter(p, bwFac=bwFac)) + fres[[k]][[p]]<-filter(x[chunksamples[[k]]],curv1Filter(p,bwFac)) + } + cat("\n") + } + else + { + if(!is.list(fres[[k]]) || !all(stains %in% names(fres[[k]]))) + stop("The supplied list of filter results does not match ", + "the channels to warp.") + if(!all(sapply(fres, is, "filterResult"))) + stop("The argument 'fres' has to be a list of filterResultLists.") + cat("Extracting landmarks from user supplied filterResults\n") + } + + ## define some variables + nb <- 1001 + lm <- list() + z <- NULL + + ## iterate over stains + eps <- .Machine$double.eps + for(p in stains) + { + ## set up fda parameters + extend <- 0.15 + from <- min(sapply(ranges, function(z) z[1,p]-diff(z[,p])*extend), na.rm=TRUE) + to <- max(sapply(ranges, function(z) z[2,p]+diff(z[,p])*extend), na.rm=TRUE) + wbasis <- create.bspline.basis(rangeval=c(from, to), + norder=4, breaks=seq(from, to, len=nbreaks)) + + WfdPar <- fdPar(wbasis, 1, 1e-4) + ##append to the previously computed density estimates of the last chunk and now all stains + densY[[k]][[p]] <- t(fsApply(x[chunksamples[[k]]], function(z){ + r <- range(z)[,p] + z <- exprs(z) + z <- z[z[,p]>r[1]+eps & z[,p] chunkranges[[k]][[i]][2,p]-eps + rb[is.na(rb)] <- TRUE + chunkrightBoard[[k]][[i]] <- rb + #Don't exclude things beyond the range + #sel <- leftBoard[[i]] | rightBoard[[i]] + sel<-rep(FALSE,length(thisDat[[i]][,p])) + newDat[[i]] <- as.matrix(chunkfuns[[k]][[chunksamples[[k]][[i]]]](thisDat[[i]][!sel,p])) + newDat[[i]][is.na(newDat[[i]])] <- thisDat[[i]][!sel,p][is.na(newDat[[i]])] + #exprs(expData[[i]])[!sel,p] <- newDat + #ind1<-ncdfFlow:::getIndices(expData,chunksamples[[k]][i]) + #ncdfFlow::updateIndices(expData,chunksamples[[k]][i],z=NA) + #slice<-exprs(expData[[samples[i]]]) + thisDat[[i]][,p]<-newDat[[i]] + #slice[ind1,][!sel,p]<-newDat + + warpedLandmarks[chunkindices[[k]][i], ] <- chunkfuns[[k]][[chunksamples[[k]][[i]]]](landmarks[[p]][chunkindices[[k]][i],]) + newRange[1] <- chunkranges[[k]][[i]][,p][1] + newRange[2] <- chunkranges[[k]][[i]][,p][2] + #this doesn't work for ncdf flow sets in this scenario since it negatively impacts plotting + #newRange[1] <- min(newRange[1], min(exprs(expData[[samples[i]]])[,p], na.rm=TRUE)) + #newRange[2] <- max(newRange[2], max(exprs(expData[[samples[i]]])[,p], na.rm=TRUE)) + } + #Writing after normalization + cat("Writing to disk..\n"); + for(i in seq_along(chunkfuns[[k]])){ +# browser() + curSampleName<-chunksamples[[k]][i] + print(i) + print(curSampleName) + exprs(expData[[curSampleName]])<-thisDat[[i]] + gc() + } + ## make sure that edge envents are set to the extreme values + ## of the warped data range and update the parameters slot + ## accordingly + for(i in seq_along(chunkfuns[[k]])){ + #samples<-names(chunkfuns[[k]]) + minSel <- chunkleftBoard[[k]][[i]] + maxSel <- chunkrightBoard[[k]][[i]] + + ip <- match(p, pData(parameters(expData[[chunksamples[[k]][i]]]))$name) + tmp <- parameters(expData[[chunksamples[[k]][i]]]) + oldRanges <- unlist(range(expData[[chunksamples[[k]][i]]],p)) + pData(tmp)[ip, c("minRange", "maxRange")] <- c(min(oldRanges[1], newRange[1]), + max(oldRanges[2], newRange[2])) + expData[[chunksamples[[k]][i]]]@parameters <- tmp + gc() + } + + } + lm[[p]] <- list(prior=landmarks[[p]], warped=warpedLandmarks, + warpFun=funs, regions=regions, dists=dists, + hasPeak=hasPeak, revWarpFuns=funsBack) + } + } + if(warpFuns) + return(funs) + #regSet <- as(expData, "flowSet") + #regSet<-expData + phenoData(expData) <- phenoData(x) + expDat <- expData[sampleNames(x)] + attr(expData, "warping") <- lm + rm(thisDat);rm(newDat);rm(x); + gc() + expData +} + +# When isNew == FALSE, the original cdf is modified +# when isNew == TRUE, a new cdf is created +warpSetNCDF <- function(x, stains, grouping=NULL, monwrd=TRUE, subsample=NULL, + peakNr=NULL, clipRange=0.01, nbreaks=11, fres, bwFac=2, + warpFuns=FALSE,target=NULL,chunksize=10,isNew=FALSE,newNcFile=NULL, + ...) +{ + + ## Some type-checking first + flowCore:::checkClass(x, "flowSet") + flowCore:::checkClass(stains, "character") + mt <- stains %in% colnames(x) + if(!all(mt)) + stop("Invalid stain(s) not matching the flowSet:\n ", + paste(stains[!mt], collapse=", ")) + + + #expData should now be x... + if(isNew){ + expData<-ncdfFlow:::clone.ncdfFlowSet(x,isNew=TRUE,isEmpty=FALSE,ncdfFile=newNcFile) + }else{ + expData<-x; + } + samples<-sampleNames(expData) + ranges<-vector("list",length(x)); + names(ranges)<-sampleNames(x) + for(i in 1:length(x)){ranges[[i]]<-range(x[[i]])} + #ranges <- fsApply(x, range) + if(!is.null(grouping)) + flowCore:::checkClass(grouping, "character", 1) + if(!is.null(subsample)) + { + flowCore:::checkClass(subsample, "numeric", 1) + ## subsample data set before all density estimation steps + #Does Subset clobber anything in a permanent way for an ncdfFlowSet? Check this, otherwise use subsample. + x <- Subset(x, sampleFilter(size=subsample)) + } + flowCore:::checkClass(monwrd, "logical", 1) + flowCore:::checkClass(bwFac, "numeric", 1) + + ## find landmarks + if(missing(fres)) + { + fres <- list() + for(p in stains){ + cat("\rEstimating landmarks for channel", p, "...") + fres[[p]] <- filter(x[,p], curv1Filter(p, bwFac=bwFac)) + } + cat("\n") + } + else + { + if(!is.list(fres) || !all(stains %in% names(fres))) + stop("The supplied list of filter results does not match ", + "the channels to warp.") + if(!all(sapply(fres, is, "filterResult"))) + stop("The argument 'fres' has to be a list of filterResultLists.") + cat("Extracting landmarks from user supplied filterResults\n") + } + + ## define some variables + nb <- 1001 + lm <- list() + z <- NULL + + ## iterate over stains + eps <- .Machine$double.eps + for(p in stains) + { + thisX <- x[,p] + ## set up fda parameters + extend <- 0.15 + from <- min(sapply(ranges, function(z) z[1,p]-diff(z[,p])*extend), na.rm=TRUE) + to <- max(sapply(ranges, function(z) z[2,p]+diff(z[,p])*extend), na.rm=TRUE) + wbasis <- create.bspline.basis(rangeval=c(from, to), + norder=4, breaks=seq(from, to, len=nbreaks)) + WfdPar <- fdPar(wbasis, 1, 1e-4) + densY <- t(fsApply(thisX, function(z){ + r <- range(z)[,p] + z <- exprs(z) + z <- z[z[,p]>r[1]+eps & z[,p] curChunkRange[2,p]-eps + rb[is.na(rb)] <- TRUE +# curChunkrightBoard <- rb + #Don't exclude things beyond the range + #sel <- leftBoard[[i]] | rightBoard[[i]] + sel<-rep(FALSE,length(curData)) + newDat <- curChunkFun(curData[!sel]) + newDat[is.na(newDat)] <- curData[!sel][is.na(newDat)] + + warpedLandmarks[curChunkindices, ] <- curChunkFun(landmarks[curChunkindices,]) + newRange[1] <- curChunkRange[,p][1] + newRange[2] <- curChunkRange[,p][2] + + + + #Writing after normalization + exprs(curfr)[,1] <- newDat + expData[[curChunksample,only.exprs = TRUE]] <- curfr + ## make sure that edge envents are set to the extreme values + ## of the warped data range and update the parameters slot + ## accordingly + srcFr <- expData@frames[[curChunksample]] +# minSel <- curChunkleftBoard +# maxSel <- curChunkrightBoard + tmp <- parameters(srcFr) + oldRanges <- unlist(range(curfr)) + ip <- match(p, pData(tmp)[,"name"]) + pData(tmp)[ip, c("minRange", "maxRange")] <- c(min(oldRanges[1], newRange[1]) + ,max(oldRanges[2], newRange[2]) + ) + parameters(expData@frames[[curChunksample]]) <- tmp + } + + } + lm[[p]] <- list(prior=landmarks, warped=warpedLandmarks, + warpFun=funs, regions=regions, dists=dists, + hasPeak=hasPeak, revWarpFuns=funsBack) + } + } + if(warpFuns) + return(funs) + expData +} +# +warpSet <- function(x, stains, grouping=NULL, monwrd=TRUE, subsample=NULL, + peakNr=NULL, clipRange=0.01, nbreaks=11, fres, bwFac=2, + warpFuns=FALSE,target=NULL, + ...) +{ + ## Some type-checking first + flowCore:::checkClass(x, "flowSet") + flowCore:::checkClass(stains, "character") + mt <- stains %in% colnames(x) + if(!all(mt)) + stop("Invalid stain(s) not matching the flowSet:\n ", + paste(stains[!mt], collapse=", ")) + expData <- as(x, "list") #Delete for ncdfFlowSet + ranges <- fsApply(x, range) + if(!is.null(grouping)) + flowCore:::checkClass(grouping, "character", 1) + if(!is.null(subsample)) + { + flowCore:::checkClass(subsample, "numeric", 1) + ## subsample data set before all density estimation steps + x <- Subset(x, sampleFilter(size=subsample)) + } + flowCore:::checkClass(monwrd, "logical", 1) + flowCore:::checkClass(bwFac, "numeric", 1) + + ## find landmarks + if(missing(fres)) + { + fres <- list() + for(p in stains){ + cat("\rEstimating landmarks for channel", p, "...") + fres[[p]] <- filter(x, curv1Filter(p, bwFac=bwFac)) + } + cat("\n") + } + else + { + if(!is.list(fres) || !all(stains %in% names(fres))) + stop("The supplied list of filter results does not match ", + "the channels to warp.") + if(!all(sapply(fres, is, "filterResult"))) + stop("The argument 'fres' has to be a list of filterResultLists.") + cat("Extracting landmarks from user supplied filterResults\n") + } + + ## define some variables + nb <- 1001 + lm <- list() + z <- NULL + + ## iterate over stains + eps <- .Machine$double.eps + for(p in stains) + { + ## set up fda parameters + extend <- 0.15 + from <- min(sapply(ranges, function(z) z[1,p]-diff(z[,p])*extend), na.rm=TRUE) + to <- max(sapply(ranges, function(z) z[2,p]+diff(z[,p])*extend), na.rm=TRUE) +# browser() + wbasis <- create.bspline.basis(rangeval=c(from, to), + norder=4, breaks=seq(from, to, len=nbreaks)) + WfdPar <- fdPar(wbasis, 1, 1e-4) + densY <- t(fsApply(x, function(z){ + r <- range(z)[,p] + z <- exprs(z) + z <- z[z[,p]>r[1]+eps & z[,p] ranges[[i]][2,p]-eps + rb[is.na(rb)] <- TRUE + rightBoard[[i]] <- rb + #Include ALL data, none of this thresholding crap at borders. + sel <- leftBoard[[i]] | rightBoard[[i]] + #sel<-rep(FALSE,length(thisDat)) + #browser(); + newDat <- as.matrix(funs[[i]](thisDat[!sel,])) + newDat[is.na(newDat)] <- thisDat[!sel,][is.na(newDat)] + exprs(expData[[i]])[!sel,p] <- newDat + warpedLandmarks[i, ] <- funs[[i]](landmarks[i,]) + newRange[1] <- min(newRange[1], min(exprs(expData[[i]])[,p], na.rm=TRUE)) + newRange[2] <- max(newRange[2], max(exprs(expData[[i]])[,p], na.rm=TRUE)) + } + ## make sure that edge envents are set to the extreme values + ## of the warped data range and update the parameters slot + ## accordingly + for(i in seq_along(funs)){ + minSel <- leftBoard[[i]] + maxSel <- rightBoard[[i]] + exprs(expData[[i]])[minSel,p] <- as.matrix(rep(newRange[1], + sum(minSel, na.rm=TRUE)), + ncol=1) + exprs(expData[[i]])[maxSel,p] <- as.matrix(rep(newRange[2], + sum(maxSel, na.rm=TRUE)), + ncol=1) + ip <- match(p, pData(parameters(expData[[i]]))$name) + tmp <- parameters(expData[[i]]) + oldRanges <- unlist(range(expData[[i]],p)) + pData(tmp)[ip, c("minRange", "maxRange")] <- c(min(oldRanges[1], newRange[1]), + max(oldRanges[2], newRange[2])) + expData[[i]]@parameters <- tmp + } + lm[[p]] <- list(prior=landmarks, warped=warpedLandmarks, + warpFun=funs, regions=regions, dists=dists, + hasPeak=hasPeak, revWarpFuns=funsBack) + } + } + if(warpFuns) + return(funs) + regSet <- as(expData, "flowSet") + phenoData(regSet) <- phenoData(x) + regSet <- regSet[sampleNames(x)] + attr(regSet, "warping") <- lm + regSet +} + + + + + +## Some QA pots for a normalized data set +normQA <- function(data, morph=c("^fsc", "^ssc"), channels, + odat=NULL, ask=names(dev.cur())!="pdf", + grouping=NULL, tag.outliers=FALSE, + peaksOnly=TRUE) +{ + if(! "warping" %in% names(attributes(data))) + stop("This flowSet has not been normalized.") + fsc <- grep(morph[1], colnames(data), ignore.case=TRUE) + if(length(fsc)) fsc <- colnames(data)[min(fsc)] else + stop("No parameter matching regular expression '", morph[1], "'.\n", + "Please provide a valid backgating channel.") + ssc <- grep(morph[2], colnames(data), ignore.case=TRUE) + if(length(ssc)) ssc <- colnames(data)[min(ssc)] else + stop("No parameter matching regular expression '", morph[2], "'\n", + "Please provide a valid backgating channel.") + ninfo <- attr(data, "warping") + wchans <- if(missing(channels)) names(ninfo) else channels + if(!all(wchans %in% names(ninfo))) + stop("No normalization information available for one or more channels.") + if(is.null(grouping)) + { + grouping <- factor(rep(1, length(data))) + } + else + { + if(is.character(grouping)){ + if(! grouping %in% names(pData(data))) + stop("'grouping' must be a covariate in the phenoData slot of ", + "'data' or a factor of the same length as 'data'.") + grouping <- factor(pData(data)[,grouping]) + } + else + { + grouping <- factor(grouping) + } + if(length(grouping) != length(data)) + stop("'grouping' must be a covariate in the phenoData slot of ", + "'data' or a factor of the same length as 'data'.") + } + + ## Plot the amount of warping for each landmark. + if(all(c("prior", "warped", "hasPeak") %in% names(ninfo[[1]]))) + { + ## We first create a dataFrame ameanable for trellis graphics. + prior <- data.frame() + m <- lapply(ninfo, function(x) apply(x$warped, 2, mean, na.rm=TRUE)) + for(p in wchans) + { + pr <- ninfo[[p]]$prior + hp <- ninfo[[p]]$hasPeak + nc <- ncol(pr) + nr <- nrow(pr) + peak <- factor(rep(seq_len(nc), each=nr)) + present <- sapply(split(hp, peak), function(x) + sprintf("(%d/%d)", sum(x), nr)) + peakLong <- factor(paste(peak, present[peak])) + prior <- rbind(prior, + data.frame(sample=factor(sampleNames(data), + levels=sampleNames(data)), + group=factor(as.integer(grouping)), + value=as.vector(pr), + peak=peak, peakLong=peakLong, + channel=factor(p), + hasPeak=as.vector(hp), + dists=if(length(ninfo[[p]]$dists)) + 1-as.vector(ninfo[[p]]$dists) else NA)) + sel <- is.na(prior$value) + prior[sel, "value"] <- m[[p]][prior[sel, "peak"]] + } + ## A regular stripplot with dotted lines extending from the + ## adjusted landmark positions for each peak. + par(ask=ask) + on.exit(par(ask=FALSE)) + myPanelStrip <- function(x,y, groupMeans, datSet, groups, ng, ...) + { + chan <- levels(datSet$channel)[panel.number()] + thisP <- datSet[datSet$channel==chan,] + nc <- length(groupMeans[[chan]]) + nr <- nlevels(y) + col <- rep(trellis.par.get("superpose.symbol")$col[seq_len(ng)], ng) + pch <- if(ng==1) rep(c(4,20), 2) else rep(c(4, 20), each=ng) + panel.abline(v=groupMeans[[chan]], lty="dotted", col="gray") + for(i in seq_len(nc)){ + peak <- thisP[thisP$peak==i,"value"] + panel.segments(y0=seq_len(nr), y1=seq_len(nr), x0=peak, + x1=groupMeans[[chan]][i], col="gray", lty="dotted") + } + panel.stripplot(x,y, pch=pch, col=col, groups=groups, ...) + } + col <- trellis.par.get("superpose.symbol")$col[seq_len(nlevels(prior$group))] + ng <- nlevels(prior$group) + key <- list() + if(ng>1) + key <- append(key, list(points=list(col=col, pch=20), + text=list(paste("Group", levels(grouping))))) + if(!all(prior$hasPeak)) + key <- append(key, list(points=list(pch=4, col=1), + text=list("no peak detected"), + rep=FALSE)) + print(stripplot(sample~value|channel, prior, ng=ng, + groups=interaction(group, hasPeak), + panel=myPanelStrip, groupMeans=m, datSet=prior, xlab=NULL, + key=if(length(key)) c(key, cex=0.8, between=1) else NULL, + main="Amount of Landmark Adjustment\n")) + } + + ## Plot the confidence of the landmark registration step + if("dists" %in% names(ninfo[[1]])) + { + present <- lapply(split(prior, prior$channel), function(x) + sapply(split(x$hasPeak, x$peak), function(x) + sprintf("(%d/%d)", sum(x), length(data)))) + col <- trellis.par.get("superpose.symbol")$col[seq_len(nlevels(prior$group))] + key <- list() + ng <- nlevels(prior$group) + if(ng>1) + key <- append(key, list(text=list(paste("Group", levels(grouping))), + points=list(col=col, pch=1))) + print(stripplot(dists~peak |channel, prior, ylim=c(0,1.1), + ylab="Clustering Confidence", xlab="Peak", + groups=group, myLabel=present, + panel=function(myLabel, datSet, ...){ + panel.stripplot(...) + chan <- levels(datSet$channel)[panel.number()] + panel.text(seq_along(myLabel[[chan]]), 1.05, myLabel[[chan]], + cex=0.7) + panel.abline(h=1, col="gray") + }, datSet=prior, + key=if(length(key)) c(key, cex=0.8) else NULL, + main="Landmark Registration\n")) + } + + ## Plot the warping functions. + if("warpFun" %in% names(ninfo[[1]])) + { + ## We first create a dataFrame for the trellis plotting + wfRes <- data.frame() + for(p in wchans) + { + np <- 30 + xvals <- seq(range(data[[1]], p)[1,], range(data[[1]], p)[2,], len=np) + yvals <- sapply(ninfo[[p]]$warpFun, function(fun) fun(xvals)) + wfRes <- rbind(wfRes, + data.frame(sample=rep(factor(sampleNames(data), + levels=sampleNames(data)), + each=np), + group=rep(factor(as.integer(grouping)),each=np), + original=xvals, normalized=as.vector(yvals), + channel=p)) + } + col <- trellis.par.get("superpose.symbol")$col[seq_len(nlevels(wfRes$group))] + key <- list() + ng <- nlevels(wfRes$group) + if(ng>1) + key <- append(key, list(text=list(paste("Group", levels(grouping))), + lines=list(col=col, pch=1))) + print(xyplot(normalized~original|channel, wfRes, type="l", alpha=0.4, + main="Warping Functions\n", groups=group, + key=if(length(key)) c(key, cex=0.8) else NULL)) + } + + + ## Plot the projection in the FCS vs. SSC space. + if("regions" %in% names(ninfo[[1]])) + { + ## We first create rectangleGates from the peak regions and + ## compute norm2Filters in the backgating dimensions + cat("Computing the backgating information. Please wait...") + backGates <- cents <- vector(mode="list", length=length(wchans)) + names(backGates) <- names(cents) <- wchans + stand <- apply(range(data[[1]])[,c(fsc, ssc)], 2, diff) + vars <- data.frame() + noOdat <- !is.null(ninfo[[1]]$warpFun) && is.null(odat) + if(!noOdat && is.null(odat)) + stop("The un-nomalized data set needs to be supplied as argument 'odat'.") + if(noOdat) + odat <- flowCore:::copyFlowSet(data) + for(p in wchans) + { + regions <- ninfo[[p]]$regions + if(is.null(names(regions))) + names(regions) <- sampleNames(data) + wfuns <- if(noOdat) ninfo[[p]]$warpFun else { + wf <- list() + for(n in names(regions)) + wf[[n]] <- function(x) x + wf} + np <- ncol(ninfo[[p]]$warped) + bg <- cs <- vector(mode="list", length=np) + for(i in seq_len(np)) + { + g <- list() + for(j in names(regions)) + { + g[[j]] <- if(ninfo[[p]]$hasPeak[which(sampleNames(data)==j),i]) + rectangleGate(matrix(wfuns[[j]](regions[[j]][i,]), ncol=1, + dimnames=list(NULL, p))) + else rectangleGate(matrix(c(-Inf, Inf), ncol=1, + dimnames=list(NULL, p))) + } + subDat <- Subset(Subset(odat, filterList(g)), boundaryFilter(c(fsc, ssc))) + hp <- ninfo[[p]]$hasPeak[,i] + bg[[i]] <- filter(subDat, norm2Filter(c(fsc, ssc))) + bg[[i]][fsApply(subDat, nrow)<30] <- NA + cs[[i]] <- fsApply(subDat, function(x){ + if(nrow(x)>30) + apply(x[,c(fsc, ssc)], 2, mean, na.rm=TRUE) + else as.numeric(rep(NA,2))}, use.exprs=TRUE) + if(peaksOnly) + { + bg[[i]][!hp] <- NA + cs[[i]][!hp] <- rep(NA,2) + } + vars <- rbind(vars, + data.frame(value=sapply(bg[[i]], function(x) + if(is(x, "filterResult") && !is.na(filterDetails(x)[[1]]$cov) && + length(x@subSet) > 30 ) + prod(sqrt(eigen(filterDetails(x)[[1]]$cov)$values)/stand)*pi + else NA), + channel=p, peak=factor(paste("Peak", i)), + sample=factor(sampleNames(data), + levels=sampleNames(data)))) + } + backGates[[p]] <- bg + cents[[p]] <- cs + } + dummy <- data.frame(channel=factor(rep(names(backGates), + times=length(data)*listLen(backGates))), + peak=factor(unlist(sapply(listLen(backGates), + function(x) + rep(seq_len(x), + each=length(data))))), + sample=factor(sampleNames(data), levels=sampleNames(data)), + group=factor(as.integer(grouping)), + x=0, y=0) + cat("\n") + myXYPanel <- function(datSet, backGates, groups, ...) + { + wp <- which.packet() + p <- levels(datSet$channel)[wp[1]] + ng <- nlevels(groups) + bg <- backGates[[p]] + if(length(wp)==2) + bg <- bg[wp[2]] + for(i in seq_along(bg)) + { + crgb <- col2rgb(trellis.par.get("superpose.symbol")$col[as.integer(groups)]) + col <- rgb(t(crgb), alpha=255*0.12, maxColorValue=255) + colc <- rgb(t(crgb), alpha=255*0.2, maxColorValue=255) + for(f in seq_along(bg[[i]])) + if(is(bg[[i]][[f]], "filterResult") && + !is.na(filterDetails(bg[[i]][[f]])[[1]]$cov) && + length(bg[[i]][[f]]@subSet) > 30 ) + glpolygon(bg[[i]][[f]], gpar=list(gate=list(fill=col[f], + col=colc[f]))) + } + } + flowViz:::plotType("gsmooth", c(fsc, ssc)) + key <- list() + ng <- nlevels(dummy$group) + col <- trellis.par.get("superpose.symbol")$col[seq_len(ng)] + if(ng>1) + key <- append(key, list(text=list(paste("Group", levels(grouping))), + rectangles=list(col=col, pch=1))) + print(xyplot(x~y|channel+factor(paste("Peak", peak)), dummy, xlab=fsc, ylab=ssc, + xlim=range(data[[1]])[,fsc], groups=group, + ylim=range(data[[1]])[, ssc], + panel=myXYPanel, datSet=dummy, backGates=backGates, + key=if(length(key)) c(key, cex=0.8) else NULL, + main="Backgating Shape\n")) + + + ## tmp <- sapply(regions, rowMeans) + ## expval <- signif(if(!is.null(dim(tmp))) rowMeans(tmp) else mean(tmp),3) + ## legend("topleft", pch=20, col=seq_len(nc)+1, + ## legend=sprintf("Peak %d (mean %s=%g)", seq_len(nc), p, expval), + ## cex=0.8, bty="n") + + ## Now only plot the centroids of the ellipses + alldat <- Subset(as(odat, "flowFrame")[,c(fsc, ssc)], + boundaryFilter(c(fsc, ssc)) %subset% + sampleFilter(10000)) + + myXYPanelCent <- function(datSet, centroids, alldat, labels, groups, ...) + { + wp <- which.packet() + p <- levels(datSet$channel)[wp[1]] + ct <- centroids[[p]] + if(length(wp)==2) + ct <- ct[wp[2]] + exp <- exprs(alldat)[,1:2] + xr <- range(exp[,1], na.rm=TRUE) + yr <- range(exp[,2], na.rm=TRUE) + bw <- diff(apply(exp, 2, quantile, probs=c(0.05, 0.95), + na.rm=TRUE)) / 25 + range <- list(xr+c(-1,1)*bw[1]*2.5, yr+c(-1,1)*bw[2]*2.5) + z <- bkde2D(exp, bw, c(65,65), range.x=range) + ll <- contourLines(z$x1, z$x2, z$fhat, nlevels=10) + for(pg in ll) + panel.polygon(pg$x, pg$y, border="lightgray") + col <- trellis.par.get("superpose.symbol")$col[as.integer(groups)] + for(i in seq_along(ct)) + { + panel.points(ct[[i]], col=col, pch=i) + if(tag.outliers) + { + tg <- subset(datSet, channel==p & peak==i)$group + x <- split(ct[[i]][,1], tg) + y <- split(ct[[i]][,2], tg) + outliers <- unlist(mapply(function(xx, yy){ + tmp <- cbind(xx, yy) + sel <- apply(tmp, 1, function(z) any(is.na(z))) + res <- rep(FALSE, nrow(tmp)) + ol <- !pcout(tmp[!sel,], outbound=0.1)$wfinal01 + res[!sel] <- ol + return(res)}, x, y, SIMPLIFY=FALSE)) + panel.text(ct[[i]][,1][outliers], ct[[i]][,2][outliers], + labels[outliers], adj=c(1.5,1.5), cex=0.6) + } + } + } + key <- list() + np <- nlevels(dummy$peak) + if(ng>1 || np>1) + { + labs <- as.character(levels(interaction(paste("Group", levels(grouping)), + paste("Peak", levels(dummy$peak)), + sep=" "))) + key <- append(key, list(text=list(labs), + points=list(col=rep(col, nlevels(grouping)), + pch=rep(seq_len(nlevels(dummy$peak)), + each=nlevels(grouping))), + columns=ng)) + } + print(xyplot(x~y|channel, dummy, xlab=fsc, ylab=ssc, + xlim=range(data[[1]])[,fsc], groups=group, + ylim=range(data[[1]])[, ssc], + panel=myXYPanelCent, datSet=dummy, centroids=cents, + main="Backgating Location\n", alldat=alldat, labels=sampleNames(data), + key=if(length(key)) c(key, cex=0.8) else NULL)) + + + cvSum <- data.frame() + for(i in seq_along(cents)) + for(j in seq_along(cents[[i]])) + { + mc <- apply(cents[[i]][[j]], 2, median, na.rm=TRUE) + md <- t(cents[[i]][[j]]) - mc + mds <- colMeans(md/stand) + cvSum <- rbind(cvSum, data.frame(sample=factor(names(mds), + levels=unique(names(mds))), + group=factor(as.integer(grouping)), + channel=wchans[[i]], + peak=factor(paste("Peak",j)), + location=mds)) + } + cvSum <- cbind(cvSum, variation=vars[, "value"]) + myXYPanelSum <- function(x, y, labels, datSet, groups, ...) + { + wp <- which.packet() + tg <- subset(datSet, channel==levels(factor(datSet$channel))[wp[1]] & + peak==paste("Peak", wp[2]))$group + if(length(x)) + { + col <- trellis.par.get("superpose.symbol")$col[as.integer(tg)] + panel.points(x,y, col=col, pch=1) + if(tag.outliers) + { + xs <- split(x, tg) + ys <- split(y, tg) + outliers <- unlist(mapply(function(xx, yy){ + tmp <- cbind(xx, yy) + sel <- apply(tmp, 1, function(z) any(is.na(z))) + res <- rep(FALSE, nrow(tmp)) + ol <- !pcout(tmp[!sel,], outbound=0.1)$wfinal01 + res[!sel] <- ol + return(res)}, xs, ys, SIMPLIFY=FALSE)) + col <- trellis.par.get("superpose.symbol")$col[as.integer(tg)] + panel.text(x[outliers], y[outliers], labels[outliers], adj=c(1.5,1.5), + cex=0.6) + } + } + } + key <- list() + col <- trellis.par.get("superpose.symbol")$col[seq_len(ng)] + if(ng>1) + key <- append(key, list(text=list(paste("Group", levels(grouping))), + rectangles=list(col=col, pch=1))) + print(xyplot(location ~ variation|channel+peak, cvSum, panel=myXYPanelSum, + labels=sampleNames(data), main="Backgating Summary\n", + groups=group, datSet=cvSum, + key=if(length(key)) c(key, cex=0.8) else NULL)) + + } + ## Plot the relative ellipse volumes for the backgated channels + ## layout(matrix(1:length(wchans), ncol=1)) + ## for(p in wchans) + ## { + ## nc <- ncol(ninfo[[p]]$warped) + ## nr <- nrow(ninfo[[p]]$warped) + ## plot(seq_len(nc), rep(0, nc), + ## ylim=c(0,max(5, max(unlist(allV[[p]])))), type="n", + ## xlim=c(0, nc+1), xlab="peaks", ylab="variation", xaxt="n", + ## pch=pch, col=rep(seq_len(nc), each=nr), + ## main=paste("Backgating Variation", p)) + ## axis(1, at=seq_len(nc), labels=TRUE) + ## box() + ## for(i in seq_len(nc)) + ## points(rep(i, nr), allV[[p]][[i]]) + ## } + invisible() +} + + + + + + + + + + + + + + + + + + + + + + + + + + + +## An optimized object size function that is able to deal with embedded +## environments. +## setGeneric("objectSize", function(x, ...) standardGeneric("objectSize")) +## setMethod("objectSize", signature(x="ANY"), definition=function(x, ...) +## { +## realObjSize <- function(object, size=0, done=list()) +## { +## ## recursively go though environments and record the total size +## ## We also pass on pointers to all the environments that have +## ## already been counted in order to avoid infinite loops or +## ## counting things multiple times. +## objSize <- size +## if(is.environment(object) && !any(sapply(done, identical, object))) +## { +## for(i in ls(object)) +## objSize <- realObjSize(get(i,object),objSize, +## done=append(done, object)) +## } +## 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b/man/BackGating.Rd new file mode 100644 index 0000000..04c92ec --- /dev/null +++ b/man/BackGating.Rd @@ -0,0 +1,22 @@ +\name{BackGating} +\alias{BackGating} +\docType{data} +\title{Sample backgating results} +\description{ + A data frame containing the sub-populations of ITN dataset corresponding + to the high-density areas on "FSC" and "SSC" channels. This dataset is + yielded by \code{backGating} on channel CD3, CD8, and CD4 of the + \code{ITN} sample data. +} + +\usage{data(BackGating)} + +\source{ Results from executing the following code: + + data(ITN) + + flowStats:::backGating(ITN, xy=c("FSC", "SSC"), + channels=c("CD3", "CD8", "CD4")) +} + +\keyword{datasets} diff --git a/man/ITN.Rd b/man/ITN.Rd new file mode 100644 index 0000000..f494a5e --- /dev/null +++ b/man/ITN.Rd @@ -0,0 +1,18 @@ +\name{ITN} +\alias{ITN} +\docType{data} +\title{ Sample flow cytometry data } +\description{ + A \code{\link[flowCore:flowSet-class]{flowSet}} cotaining data from 15 + patients. +} +\usage{data(ITN)} +\format{ + A \code{flowSet} containing 15 + \code{\link[flowCore:flowFrame-class]{flowFrames}}. There are 3 + patient groups with 5 samples each. +} +\source{ + Immune Tolerance Network +} +\keyword{datasets} diff --git a/man/autoGate.Rd b/man/autoGate.Rd new file mode 100644 index 0000000..a9126d9 --- /dev/null +++ b/man/autoGate.Rd @@ -0,0 +1,65 @@ +\name{autoGate} +\alias{autoGate} + +\title{ Automated gating of single populations in 2D} +\description{ + This function tries to fit a single norm2Filter based on a rough + preselection of the data. This function is considered internal. Please + use the API provided by \code{\link{lymphGate}}. +} + +\usage{ +autoGate(x, \dots, scale = 2.5) +} + +\arguments{ + + \item{x}{ An object of class \code{\link[flowCore:flowSet-class]{flowSet}}} + + \item{\dots}{ Named arguments or a list of the ranges used for the + initial rough preselection. This gets passed on to + \code{\link[flowCore]{rectangleGate}}, see it's documentation for + details. } + + \item{scale}{ The \code{scale} parameter that gets passed on to + \code{\link[flowCore:norm2Filter-class]{norm2Filter}}. } +} + +\details{ + + The \code{flowSet} is first filtered using a \code{rectangleGate} and + the \code{norm2Filter} is subsequently fitted to the remaining subset. + +} + +\value{ + + A list with items: + + \item{x }{The filtered \code{flowSet}. } + + \item{n2gate }{The \code{norm2Filter} object. } + + \item{n2gateResults }{The + \code{\link[flowCore:filterResult-class]{filterResult}} after + applying the \code{norm2Filter} on the \code{flowSet}. } + +} + + +\author{ Florian Hahne } + +\seealso{ + \code{\link{lymphGate}}, + \code{\link[flowCore:norm2Filter-class]{norm2Filter}} +} + +\examples{ + +data(GvHD) +flowStats:::autoGate(GvHD[10:15], "FSC-H"=c(100,500), "SSC-H"=c(0, 400)) + +} + +\keyword{misc} + diff --git a/man/binByRef.Rd b/man/binByRef.Rd new file mode 100644 index 0000000..09584a0 --- /dev/null +++ b/man/binByRef.Rd @@ -0,0 +1,45 @@ +\name{binByRef} +\alias{binByRef} + +\title{ Bin a test data set using bins previously created by probability + binning a control dataset} + +\description{ +The bins generated by probability binning a control data set can be applied to a +test data set to perfom statistical comparisions by methods such as the +Chi-squared test or the probability binning statistic. +} + +\usage{ +binByRef(binRes, data) +} + +\arguments{ + + \item{binRes}{ The result generated by calling teh \code{probBin} + function on a control dataset.} + + \item{data}{ An object of class + \code{\link[flowCore:flowFrame-class]{flowFrame}}} + + +} + +\value{ + + An enviroment containing the matrices for each bin of the test data set +} + +\author{ Nishant Gopalakrishnan } + +\seealso{ \code{\link{plotBins}}, \code{\link{proBin}}} + +\examples{ + +data(GvHD) +resCtrl<-proBin(GvHD[[1]],200) +resSample<-binByRef(resCtrl,GvHD[[2]]) +ls(resSample) +} + +\keyword{misc} diff --git a/man/calcPBChiSquare.Rd b/man/calcPBChiSquare.Rd new file mode 100644 index 0000000..faa00fa --- /dev/null +++ b/man/calcPBChiSquare.Rd @@ -0,0 +1,54 @@ + +\name{calcPBChiSquare} +\alias{calcPBChiSquare} + +\title{ Probability binning metirc for comparing the probability binned +datasets} + +\description{ + + This function calculates the Probability binning metric proposed by + Baggerly et al. The function utilizes the data binned using the + \code{proBin} and \code{binByRef} functions. + +} + +\usage{ +calcPBChiSquare(ctrlRes,sampRes,ctrlCount,sampCount) +} + +\arguments{ + + \item{ctrlRes}{ The result generated by calling the \code{probBin} + function on a control dataset.} + + \item{sampRes}{ The result generated by calling the \code{byByRef} + function on a test sample dataset} + + \item{ctrlCount}{ The number of events in the control sample} + + \item{sampCount}{ The number of events in the test sample being compared} + +} +\value{ A list containing the statistic, p.value, observed, expected counts and +the residuals +} + +\author{Nishant Gopalakrishnan} + +\seealso{ \code{\link{proBin}}, \code{\link{calcPBChiSquare}}} + +\examples{ + +data(GvHD) +# flow frame 1 is treated as control dataset and used to generate bins +resCtrl<-proBin(GvHD[[1]][,c("FSC-H","SSC-H","Time")],200) +plotBins(resCtrl,GvHD[[1]],channels=c("FSC-H","SSC-H","Time"),title="Binned control data") +# Same bins are applied to flowFrame 16 +resSample<-binByRef(resCtrl,GvHD[[16]][,c("FSC-H","SSC-H","Time")]) +ctrlCount<-nrow(GvHD[[1]]) +sampCount<-nrow(GvHD[[16]]) +stat<-calcPBChiSquare(resCtrl,resSample,ctrlCount,sampCount) +} + +\keyword{misc} \ No newline at end of file diff --git a/man/calcPearsonChi.Rd b/man/calcPearsonChi.Rd new file mode 100644 index 0000000..080a974 --- /dev/null +++ b/man/calcPearsonChi.Rd @@ -0,0 +1,49 @@ + +\name{calcPearsonChi} +\alias{calcPearsonChi} + +\title{ Pearsons chi-square statistic for comparing the probability +binned datasets} + +\description{ +This function calculates the Pearsons chi-squared statistic for comparing +data binned using the \code{proBin} and \code{binByRef} functions.Internally, the +function utilizes the chisq.test function. +} + +\usage{ +calcPearsonChi(ctrlRes,sampRes) +} + +\arguments{ + + \item{ctrlRes}{ The result generated by calling the \code{probBin} + function on a control dataset.} + + \item{sampRes}{ The result generated by calling the \code{byByRef} + function on a sample dataset} + +} +\value{ + + A list containing the statistic, p.value, observed, expected counts + and the residuals + +} + +\author{Nishant Gopalakrishnan} + +\seealso{ \code{\link{proBin}}, \code{\link{calcPBChiSquare}}} + +\examples{ + +data(GvHD) +# flow frame 1 is treated as control dataset and used to generate bins +resCtrl<-proBin(GvHD[[1]][,c("FSC-H","SSC-H","Time")],200) +plotBins(resCtrl,GvHD[[1]],channels=c("FSC-H","SSC-H","Time"),title="Binned control data") +# Same bins are applied to flowFrame 16 +resSample<-binByRef(resCtrl,GvHD[[16]][,c("FSC-H","SSC-H","Time")]) +stat<-calcPearsonChi(resCtrl,resSample) +} + +\keyword{misc} \ No newline at end of file diff --git a/man/curvPeaks.Rd b/man/curvPeaks.Rd new file mode 100644 index 0000000..84ff9a4 --- /dev/null +++ b/man/curvPeaks.Rd @@ -0,0 +1,72 @@ +\name{curvPeaks} +\alias{curvPeaks} + + +\title{ Parse curv1Filter output } + + +\description{ + + Parse the output of + \code{\link[flowCore:curv1Filter-class]{curv1Filter}} and find modes + and midpoints of the high-density regions. This function is considered + to be internal. + +} + +\usage{ + +curvPeaks(x, dat, borderQuant = 0.01, n = 201, from, to, densities=NULL) + +} + + +\arguments{ + \item{x}{ A + \code{\link[flowCore:multipleFilterResult-class]{multipleFilterResult}} + produced by a \code{curv1Filter} operation. } + + \item{dat}{ The corresponding + \code{\link[flowCore:flowFrame-class]{flowFrame}}. } + + \item{borderQuant}{ A numeric in \code{[0,1]} giving the extreme + quantiles for which high-density regions are ignored. } + + \item{n, from, to}{ Arguments are passed on to + \code{\link[stats]{density}}. } + + \item{densities}{The optional y values of the density estimate + computed for the respective data.} + +} + + +\value{ + + A list with items + + \item{peaks }{x and y locations of the modes of the regions in the + density estimates. } + + \item{regions }{the left and right margins of the regions. } + + \item{midpoints }{the mean of \code{regions}. } + + \item{regPoints }{x and y locations of the outline of the significant + density regions. } + + \item{densFuns }{an approximation function of the density estimate } + +} + +\author{ Florian Hahne} + +\seealso{ \code{\link{landmarkMatrix}} } + +\examples{ + +data(GvHD) +tmp <- filter(GvHD[[10]], curv1Filter("FSC-H")) +res <- flowStats:::curvPeaks(tmp, exprs(GvHD[[10]])[, "FSC-H"]) + +} \ No newline at end of file diff --git a/man/density1d.Rd b/man/density1d.Rd new file mode 100644 index 0000000..23a55a7 --- /dev/null +++ b/man/density1d.Rd @@ -0,0 +1,151 @@ +\name{density1d} + +\alias{density1d} + +\title{ Find most likely separation between positive and negative + populations in 1D } + + +\description{ + + The function tries to find a reasonable split point between the two + hypothetical cell populations "positive" and "negative". This function + is considered internal, please use the API provided by + \code{\link{rangeGate}}. + +} + +\usage{ +density1d(x, stain, alpha = "min", sd = 2, plot = FALSE, borderQuant = +0.1, absolute = TRUE, inBetween = FALSE, refLine=NULL,rare=FALSE,bwFac=1.2 +,sig=NULL,peakNr=NULL, ...) +} + + + + + + +\arguments{ + + \item{x}{ A \code{\link[flowCore:flowSet-class]{flowSet}} or + \code{\link[flowCore:flowSet-class]{flowFrame}}. } + + \item{stain}{ A character scalar giving the flow parameter for which + to compute the separation. } + + \item{alpha}{ A tuning parameter that controls the location of the + split point between the two populations. This has to be a numeric in + the range \code{[0,1]}, where values closer to 0 will shift the + split point closer to the negative population and values closer to 1 + will shift towards the positive population. Additionally, the value + of \code{alpha} can be \code{"min"}, in which case the split point + will be selected as the area of lowest local density between the two + populations. } + + \item{sd}{For the case where there is only a single population, the + algorithm falls back to esitmating the mode of this population and a + robust measure of the variance of it distribution. The \code{sd} + tuning parameter controls how far away from the mode the split point + is set. } + + \item{plot}{ Create a plot of the results of the computation. } + + \item{borderQuant}{Usualy the instrument is set up in a way that the + positive population is somewhere on the high end of the measurement + range and the negative population is on the low end. This parameter + allows to disregard populations with mean values in the extreme + quantiles of the data range. It's value should be in the range + \code{[0,1]}. } + + \item{absolute}{Logical controling whether to classify a population + (positive or negative) relative to the theoretical measurment range + of the instrument or the actual range of the data. This can be set + to \code{TRUE} if the alignment of the measurment range is not + optimal and the bulk of the data is on one end of the theoretical + range.} + + \item{inBetween}{Force the algorithm to put the separator in between + two peaks. If there are more than two peaks, this argument is + ignored.} + + \item{refLine}{Either \code{NULL} or a numeric of lenth 1. If + \code{NULL}, this parameter is ignored. When it is set to a numeric, the + minor sub-population (if any) below this reference line + will be igored while determining the separator between positive and + negative.} + \item{rare}{Either \code{TRUE} or \code{FALSE}, assumes that there is one major peak, and that the rare positive population is to the right of it. Uses a robust estimate of mean and variance to gate the positive cells.} + \item{bwFac}{The bandwidth for smoothing the density estimate. User-tunable} + \item{sig}{a value of c(NULL,"L","R"),when sig is not NULL,use the half (left or right) of signal to estimate the std and mean.} + \item{peakNr}{when peakNr is not NULL,drop the less significant peaks by their heights} + + \item{\dots}{ Further arguments. } +} + +\details{ + + The algorithm first tries to identify high density regions in the + data. If the input is a \code{flowSet}, density regions will be + computed on the collapsed data, hence it should have been normalized + before (see \code{\link{warpSet}} for one possible normalization + technique). The high density regions are then clasified as positive + and negative populations, based on their mean value in the theoretical + (or absolute if argument \code{absolute=TRUE}) measurement range. In + case there are only two high-density regions the lower one is usually + clasified as the negative populations, however the heuristics in the + algorithm will force the classification towards a positive population + if the mean value is already very high. The \code{absolute} and + \code{borderQuant} arguments can be used to control this + behaviour. The split point between populations will be drawn at the + value of mimimum local density between the two populations, or, if the + \code{alpha} argument is used, somewhere between the two populations + where the value of alpha forces the point to be closer to the negative + (\code{0 - 0.5}) or closer to the positive population (\code{0.5 - + 1}). + + If there is only a single high-density region, the algorithm will fall + back to estimating the mode of the distribution + (\code{\link[MASS]{hubers}}) and a robust measure of it's variance + and, in combination with the \code{sd} argument, set the split point + somewhere in the right or left tail, depending on the classification + of the region. + + For more than two populations, the algorithm will still classify each + population into positive and negative and compute the split point + between those clusteres, similar to the two population case. + +} + + +\value{ + + + A numeric indicating the split point between positive and negative populations. +} + + +\author{ Florian Hahne } + +\seealso{ + + \code{\link{warpSet}}, \code{\link{rangeGate}} + +} + +\examples{ + +data(GvHD) +dat <- GvHD[pData(GvHD)$Patient==10] +dat <- transform(dat, "FL4-H"=asinh(`FL4-H`), "FL3-H"=asinh(`FL3-H`)) +d <- flowStats:::density1d(dat, "FL4-H", plot=TRUE) +if(require(flowViz)) +densityplot(~`FL4-H`, dat, refline=d) + +## tweaking the location +flowStats:::density1d(dat, "FL4-H", plot=TRUE, alpha=0.8) + +## only a single population +flowStats:::density1d(dat, "FL3-H", plot=TRUE) +flowStats:::density1d(dat, "FL3-H", plot=TRUE, sd=2) + +} diff --git a/man/flowStats-package.Rd b/man/flowStats-package.Rd new file mode 100644 index 0000000..39a3d5a --- /dev/null +++ b/man/flowStats-package.Rd @@ -0,0 +1,45 @@ +\name{flowStats-package} + +\alias{flowStats-package} + +\alias{flowStats} + +\docType{package} + +\title{ + +Statistical methods for flow cytometry data analysis + +} + +\description{ + +Functions, methods and classes implementing algorithmns for statistical +analysis of flow cytometry data. This involves mostly data normalization +and automated gating. + +} + +\details{ + + \tabular{ll}{ + Package: \tab flowStats\cr + Type: \tab Package\cr + Version: \tab 1.0\cr + License: \tab Artistic-2.0\cr + Lazyload: \tab yes\cr + } + +} + +\author{ + +Florian Hahne + +Maintainer: Florian Hahne + +} + + +\keyword{ package } + diff --git a/man/gaussNorm.Rd b/man/gaussNorm.Rd new file mode 100644 index 0000000..6ad67c1 --- /dev/null +++ b/man/gaussNorm.Rd @@ -0,0 +1,97 @@ +\name{gaussNorm} +\alias{gaussNorm} + +\title{Per-channel normalization based on landmark registration} + +\description{ + + This funciton normalizes a set of flow cytometry data samples by + identifying and aligning the high density regions (landmarks or peaks) for each channel. + The data of each channel is shifted in such a way that the identified high + density regions are moved to fixed locations called base landmarks. +} + +\usage{ + gaussNorm (flowset, channel.names, max.lms=2, base.lms=NULL, + peak.density.thr=0.05, peak.distance.thr=0.05, debug=FALSE, fname='') + +} + + +\arguments{ + + \item{flowset}{ A \code{\link[flowCore:flowSet-class]{flowSet}}. } + + \item{channel.names}{ A character vector of flow parameters in \code{flowset} to be + normalized. } + + \item{max.lms}{ A numeric vector of the maximum number of base + landmarks to be used for normalizing each channel. If it has only one + value that will be used as the maximum number of base landmarks for + all the channels. } + + \item{base.lms}{ A list of vector for each channel that contains the + base landmarks for normalizing that channel. If not specified the base + landmarks are computed from the set of extracted landmarks.} + + \item{peak.density.thr}{The peaks with density value less than + "peak.density.thr times maximum peak density" are discarded.} + \item{peak.distance.thr}{The sequences of peaks that are located closer than + "peak.distance.thr times range of data" are identified. Then for + each sequence only one peak (the one with the highest intensity + value) is used as a landmark. In other words no two landmarks are + located closer than "peak.distance.thr times range of data" to each other.} + \item{debug}{ Logical. Forces the function to draw before and + after normalization plots for each sample. The plot of the i-th sample is stored in \code{paste(fname, i)} file.} + + \item{fname}{ The pre- and post- normalization plots of the i-th sample is + stored in \code{paste(fname, i)} file if debug is set to + \code{TRUE}. If default value is used the plots are drawn on separate X11 windows + for each sample. In this case, the function waits for a user input to draw the plots + for the next sample.} + + +} + +\details{ + + Normalization is archived in three phases: (i) identifying high-density regions + (landmarks) for each \code{\link[flowCore:flowFrame-class]{flowFrame}} + in the \code{flowSet} for a single channel; (ii) computing the best + matching between the landmarks and a set of fixed reference + landmarks for each channel called base landmarks; (iii) manipulating + the data of each channel in such a way that each landmark is moved to + its matching base landmark. + Please note that this normalization is on a channel-by-channel + basis. Multiple channels are normalized in a loop. + + +} + + +\value{ + + A list with items \code{flowset}: normalized \code{flowSet}. + \code{confidence}: a confidence measure of the normalization procedure. + +} + + +\author{ Alireza Hadj Khodabakhshi } + +\examples{ + +data(ITN) +dat <- transform(ITN, "CD4"=asinh(CD4), "CD3"=asinh(CD3), "CD8"=asinh(CD8)) +lg <- lymphGate(dat, channels=c("CD3", "SSC"), +preselection="CD4",scale=1.5) +dat <- Subset(dat, lg$n2gate) +datr <- gaussNorm(dat, "CD8")$flowset +if(require(flowViz)){ +d1 <- densityplot(~CD8, dat, main="original", filter=curv1Filter("CD8")) +d2 <- densityplot(~CD8, datr, main="normalized", filter=curv1Filter("CD8")) +plot(d1, split=c(1,1,2,1)) +plot(d2, split=c(2,1,2,1), newpage=FALSE) +} + +} \ No newline at end of file diff --git a/man/gpaSet.Rd b/man/gpaSet.Rd new file mode 100644 index 0000000..c4e9d29 --- /dev/null +++ b/man/gpaSet.Rd @@ -0,0 +1,143 @@ +\name{gpaSet} +\alias{gpaSet} + +\title{Multi-dimensional normalization of flow cytometry data} + +\description{ + + This function performs a multi-dimensional normalization of flow cytometry + data (\code{flowSets}) using a generalized Procrustes analysis (GPA) method. + +} + +\usage{ +gpaSet(x, params, register="backgating", bgChannels=NULL, + bg=NULL, rotation.only=TRUE, + downweight.missingFeatures=FALSE, thres.sigma=2.5, + show.workflow=FALSE, + ask=names(dev.cur())!="pdf") +} + +\arguments{ + \item{x}{A \code{\link[flowCore:flowSet-class]{flowSet}}.} + + \item{params}{A character vector of length 2 describing the channels of + interest.} + + \item{register}{A character indicating the method to be used for identifying + features. Available method only includes \dQuote{backgating} at the point. + } + + \item{bgChannels}{A character vector indicating the channels used for + backgating. If NULL, \code{backGating} will find the appropriate + backgating channels.} + + \item{bg}{A data frame as the returning value of the + \code{backGating} function. If not NULL, \code{gpaSet} will skip the + \code{backGating} process and use the given data frame to extract + potential features.} + + \item{rotation.only}{Logical for coarsing a reflection matrix to a + rotation matrix.} + + \item{downweight.missingFeatures}{Logical. If TRUE, the missing + features, labeled as bogus features, are downweighted to zero. See details. } + + \item{thres.sigma}{A numerical value indicating the threshold of where + to cut the tree, e.g., as resulting from \code{diana}, into several clusters. + It is default to 2.5 sigma of the distribution of the + heights of the cluster points.} + + \item{show.workflow}{Logical. If TRUE, the workflow of + \code{gpaSet} will be displayed.} + + \item{ask}{Logical. If TRUE, the display operates in interactive + mode.} + +} + +\details{ + Normalization is achieved by first identifying features for each + \code{\link[flowCore:flowFrame-class]{flowFrame}} in the \code{flowSet} for + designated channels using backgating, subsequently labeling features, and + finally aligning the features to a reference feature in the sense of + minimizing the Frobenus norm of + \deqn{||sFQ - \bar{F}||,} + where \eqn{s} is a scalar, \eqn{Q} a rotational matrix, \eqn{F} the matrix of + features, and \eqn{\bar{F}} the reference feature. Both \eqn{s} and + \eqn{Q} are solved by using singular value decomposition (SVD). + + Note that if feature \eqn{F_{ij}} is missing, it is given a bogus + value as \eqn{\bar{F}_{ij}}. + + If \code{downweight.missingFeatures} is TRUE, the cost function + becomes + \deqn{||s W_0 FQ - W_0 \bar{F}||,} + where the weighting function \eqn{W_0} is zero if the corresponding + feature is bogus. + +} + +\value{ + The normalized \code{flowSet} with "GPA" attribute. +} + +\references{in progress} + +\author{C. J. Wong \email{cwon2@fhcrc.org}} + +\examples{ +## Example 1: calling up gpaSet directly +data(ITN) +data(BackGating) + +tl <- transformList(colnames(ITN)[3:7], asinh, transformationId="asinh") +dat <- transform(ITN, tl) + +xy = c("FSC", "SSC") +bgChannels = c("CD8", "CD4", "CD3") +## bg <- flowStats:::backGating(dat, xy=xy, channels=bgChannels) +## using pre-generated backgating results: BackGating +s <- gpaSet(dat, params=xy, bgChannels=bgChannels, bg=BackGating) + +if(require(flowViz)) { + d1 <- densityplot(~., s, channels=c("FSC", "SSC"), + layout=c(2,1), main="After GPA using bg") + d2 <- xyplot(FSC ~ SSC, as(s, "flowFrame"), + channels=c("FSC", "SSC"), main="All flowFrames") + plot(d1) + plot(d2) +} + +## view "GPA" attribute +attr(s, "GPA") + +\dontrun{ +## Example 2: using work flow and normalization objects +data(ITN) +ITN <- ITN[1:8, ] +wf <- workFlow(ITN) +tl <- transformList(colnames(ITN)[3:7], asinh, transformationId="asinh") +add(wf, tl) +x <- Data(wf[["asinh"]]) +## normalize 'FSC' and 'SSC' channels +norm <- normalization(normFun=function(x, parameters, ...) + gpaSet(x, parameters, ...), + parameters = c("FSC", "SSC"), + arguments=list(bgChannels=c("CD8", "CD3"), + register="backgating"), + normalizationId="Procrustes") + +add(wf, norm2, parent="asinh") +s <- Data(wf[["Procrustes"]]) +if(require(flowViz)) { + d1 <- densityplot(~., s, channels=c("FSC", "SSC"), + layout=c(2,1), main="After GPA using bg") + d2 <- xyplot(FSC ~ SSC, as(s, "flowFrame"), + channels=c("FSC", "SSC"), main="All flowFrames") + plot(d1) + plot(d2) +} +} ## end of dontrun + +} diff --git a/man/iProcrustes.Rd b/man/iProcrustes.Rd new file mode 100644 index 0000000..a3e8b92 --- /dev/null +++ b/man/iProcrustes.Rd @@ -0,0 +1,127 @@ +\name{iProcrustes} + +\alias{iProcrustes} + +\title{Procrustes analysis. Using singular value decomposition (SVD) to + determine a linear transformation to align the + points in X to the points in a reference matrix Y. +} + +\description{ + Based on generalized Procrustes analysis, this function determines a + linear transformation (rotation/reflection and scalling) of the points in + matrix \code{x} to align them to their reference points + in matrix \code{xbar}. The alignemnt is carried out by + minimizing the distance between the points in \code{x} and \code{xbar}. +} + +\usage{ +iProcrustes(x, xbar, rotation.only=TRUE, scalling=TRUE, translate=FALSE) +} + +\arguments{ + \item{x}{A numerical matrix to be align to points in \code{xbar}, the second + arguement. The columns represents the coordinates of the points. The + matrices \code{x} and \code{xbar} must have the same dimensions.} + + \item{xbar}{A numerical, reference matrix to which points in matrix \code{x} + are to be aligned.} + + \item{rotation.only}{Logical. When \code{rotaion.only} is TRUE, it allows the + function to lose reflection component of the linear transformation. Although + it might not give the best-fitting aligenment, when + dealing with flow cytometry data alignment, a non-reflection transformation is + prefered. When \code{rotaion.only} is FALSE, it allows the function to + retain the reflection component.} + + \item{scalling}{Logical. When \code{scalling} is FALSE, it allows the + function to calculate the linear transformation without a scalling factor. + That is, the returning scalling factor is set to \eqn{1}.} + + \item{translate}{Logical. Set \code{translate} to FALSE when the points in + matrices x and xbar are already centralized prior to applying this function. + When \code{translate} is TRUE, it allows the function to translate the + centroid the points in matrix \code{x} to that of points in \code{xbar}.} +} + +\details{ + Suppose the points in matrix \eqn{X} and \eqn{\bar{X}} are centralized + (meaning their centroids are at the origin). The + linear transformation of \eqn{X} for aligning \eqn{X} to its reference + matrix \eqn{\bar{X}}., i.e., min \eqn{||sXQ - \bar{X}||_F}, is given by: + \deqn{Q = VU^T,} + and + \deqn{s = trace(\bar{X}^TXQ) / trace(X^T X),} + where V and U are the sigular value vectors of \eqn{\bar{X}^T X} (that is, + \eqn{\bar{X}^T X = U \Sigma V^T}), and \eqn{s} is the scalling factor. + +} + +\value{ + A list of the linear tranformation with items + \item{Q}{An orthogonal, rotation/reflection matrix.} + \item{scal}{A scalling factor}. + \item{T}{(optional) A translation vector used to shift the centroid of the + points in matrix \code{x} to the origin. Returned when \code{translate} + is TRUE.} + \item{T.xbar}{(optional) Centered \code{xbar} (that is, the centroid + of the points in \code{xbar} is translated to the origin). Returned when + \code{translate} is TRUE.} + + Note that the return values of this function do not include the transformed + matrix \eqn{scal* x* Q} or \eqn{scal*(x-IT)*Q}, where \eqn{T} is the + translation vector and \eqn{I} is an \eqn{n-by-1} vector with elements + \eqn{1}. +} + +\author{C. J. Wong \email{cwon2@fhcrc.org}} + +\seealso{\code{\link{gpaSet}}} + +\examples{ +## Example 1 +x <- matrix(runif(20), nrow=10, ncol=2)+ 1.4 +s <- matrix(c(cos(60), -sin(60), sin(60), cos(60)), + nrow=2, ncol=2, byrow=TRUE) +xbar <- 2.2 *(x \%*\% s) - 0.1 + +lt <- iProcrustes(x, xbar, translate=TRUE) ## return linear transformation +lt + +## showing result +I <- matrix(1, nrow=nrow(x), ncol=1) +tx <- x - I \%*\% lt$T +## get the transformed matrix xnew +xnew <- lt$scal * (tx \%*\% lt$Q) + +if (require(lattice)) { + xyplot(V1 ~ V2, + do.call(make.groups, lapply(list(x=x, xbar=xbar, T.xbar=lt$T.xbar, + xnew=xnew),as.data.frame)), + group=which, aspect=c(0.7), pch=c(1,3,2,4), col.symbol="black", + main=("Align the points in x to xbar"), + key=list(points=list(pch=c(1,3,2,4), col="black"), space="right", + text=list(c("x", "xbar", "T.xbar", "xnew")))) +} + +## Example 2. centralized x and xbar prior to using iProcrustes +x <- matrix(runif(10), nrow=5, ncol=2) +s <- matrix(c(cos(60), -sin(60), sin(60), cos(60)), + nrow=2, ncol=2, byrow=TRUE) +xbar <- 1.2 *(x \%*\% s) - 2 +I <- matrix(1, nrow=nrow(x), ncol=1) +x <- x-(I \%*\% colMeans(x)) ## shift the centroid of points in x to the origin +xbar <- xbar - (I \%*\% colMeans(xbar)) ## shift centroid to the origin +lt <- iProcrustes(x, xbar, translate=FALSE) ## return linear transformation +## only return the rotation/reflection matrix and scalling factor +lt + +xnew=lt$scal *(x \%*\% lt$Q) ## transformed matrix aligned to centralized xbar +if (require(lattice)) { + xyplot(V1 ~ V2, + do.call(make.groups, lapply(list(x=x,xbar=xbar, + xnew=xnew), as.data.frame)), + group=which, auto.key=list(space="right")) +} + +} diff --git a/man/idFeaturesByBackgating.Rd b/man/idFeaturesByBackgating.Rd new file mode 100644 index 0000000..e83371c --- /dev/null +++ b/man/idFeaturesByBackgating.Rd @@ -0,0 +1,79 @@ +\name{idFeaturesByBackgating} +\alias{idFeatures} +\alias{idFeaturesByBackgating} + +\title{(Internal use only) Identify features of flow cytometry data using + backgating} + +\description{Identify and labeling significant features using divisive + clustering method such as \code{\link[cluster:diana]{diana}}. +} + +\usage{ +idFeaturesByBackgating(bg, nDim, thres.sigma=2.5, lambda=0.1, + reference.method="median", + plot.workflow=FALSE, ask=names(dev.cur())!="pdf") +} + +\arguments{ + \item{bg}{A data frame containing subpopulations on channels of + interests. Must be a returning result from \code{flowStats:::backGating}} + \item{nDim}{An integer indicating the length of channels of interest.} + \item{thres.sigma}{An numerical value indicating the threshold at + which to cut tree, e.g., as resulting from 'diana', into several clusters.} + \item{lambda}{A numerical value indicating the percentage of the + potential features that is used as a threshold for deciding outlier + clusters. The default value is 0.1.} + \item{reference.method}{A character vector indicating the method for + computing the reference features. If \code{median}, the reference + feature is defined by the medain of eac cluster of features. Valid + methods include \code{median} and \code{mean} only. } + \item{plot.workflow}{Logical. If TURE, display the workflow of feature + identification.} + \item{ask}{Logical. If TRUE, the display operates in interactive mode.} +} + +\details{ + Using the resulting data frame from \code{backGating} as potential + features, the algorithm follows four major steps: (i) centering + the potential features, which yields the returning value + \code{TransMatrix}, (ii) using \code{diana} to compute a clustering of + the potential features, (iii) cutting the tree into several clusters, + and (iv) accessing outliers and rendering the final registered + features with labels. + + In step three, the threshold for cutting the tree is computed by + \deqn{sd * thres.sigma,} + where \eqn{sd} is the standard deviation of the distribution of the + height between entities computed by \code{diana}. + + A cluster is determined as an outlier if the number of its members is + less than the median of the numbers of all clusters' members times 'lambda'. +} + +\value{ + \item{register}{A list containing registered features for each sample.} +} + +\author{Chao-Jen Wong} +\seealso{ + \code{\link[cluster:diana]{diana}}, + \code{\link{BackGating}}, \code{\link{gpaSet}}. +} + +\examples{ +\dontrun{ +data(ITN) +wf <- workFlow(ITN) +tl <- transformList(colnames(ITN)[3:7], asinh, transformationId="asinh") +dat <- trnasformList(ITN, tl) +bg <- backGating(dat, xy=c("FSC", "SSC"), channels="CD3") +} + +data(BackGating) +results <- flowStats:::idFeaturesByBackgating(bg=BackGating, nDim=2, + plot.workflow=TRUE, ask=TRUE) + +} + +\keyword{misc} diff --git a/man/landmarkMatrix.Rd b/man/landmarkMatrix.Rd new file mode 100644 index 0000000..1f95e0f --- /dev/null +++ b/man/landmarkMatrix.Rd @@ -0,0 +1,88 @@ +\name{landmarkMatrix} +\alias{landmarkMatrix} + + +\title{ Compute and cluster high density regions in 1D } + +\description{ + + This functions first identifies high-density regions for each + \code{\link[flowCore:flowFrame-class]{flowFrame}} in a + \code{\link[flowCore:flowSet-class]{flowSet}} and subsequently tries + to cluster these regions, yielding the landmarks matrix that needs to + be supplied to \code{\link[fda]{landmarkreg}}. The function is + considered to be internal. + +} + +\usage{ +landmarkMatrix(data, fres, parm, border=0.05, peakNr=NULL, densities = +NULL, n = 201, indices=FALSE) +} + + +\arguments{ + + \item{data}{ A \code{flowSet}. } + + \item{fres}{ A list of + \code{\link[flowCore:filterResultList-class]{filterResultList}} + objects generated by a filtering opration using a + \code{\link[flowCore:curv1Filter-class]{curv1Filter}}. Each list + item represents the results for one of the flow parameters in + \code{parm}.} + + \item{parm}{ Character scalar of flow paramater to compute landmarks + for. } + + \item{border}{ A numeric in \code{[0,1]}. Ignore all high-density + regions with mean values in the extreme percentiles of the data + range. } + + \item{peakNr}{ Force a fixed number of peaks. } + + \item{densities}{ An optional matrix of y values of the density + estimates for the \code{flowSet}. If this is not present, density + estimates will be calculated by the function. } + + \item{n}{ Number of bins used for the density estimation. } + + \item{indices}{ Return matrix of population indices instead of + landmark locations. These indices can be used to point into the + populations identified by the curv1Filter.} + +} + +\details{ + + In order to normalize the data using the \code{landmarkreg} function + in the \code{\link[fda:fda-package]{fda}}, a set of landmarks has to + be computed for each \code{flowFrame} in a \code{flowSet}. The number + of lansmarks has to be the same for each frame. This function + identifies high-density regions in each frame, computes a simple + clustering and returns a matrix of landmark locations. Missing + landmarks of individual frames are substituted by the mean landmark + location of the respective cluster. + +} + + +\value{ + + A matrix of landmark locations. Columns are landmarks and rows are + \code{flowFrames}. + +} + +\author{ Florian Hahne} + +\seealso{ \code{\link[fda]{landmarkreg}},\code{\link{warpSet}} } + + +\examples{ + +data(GvHD) +tmp <- list("FSC-H"=filter(GvHD[1:3], curv1Filter("FSC-H"))) +res <- flowStats:::landmarkMatrix(GvHD[1:3], tmp, "FSC-H") + +} \ No newline at end of file diff --git a/man/lymphGate.Rd b/man/lymphGate.Rd new file mode 100644 index 0000000..7ae317f --- /dev/null +++ b/man/lymphGate.Rd @@ -0,0 +1,199 @@ +\name{lymphFilter-class} +\docType{class} +\alias{lymphFilter-class} +\alias{\%in\%,flowFrame,lymphFilter-method} +\alias{lymphGate} +\alias{lymphFilter} + +\title{ Automated gating of elliptical cell populations in 2D. } + +\description{ + + Cell populations of roughly elliptical shape in two-dimensional projections + are of huge interest in many flow cytometry applications. This + function identifies a single such population, potentially from a mixture + of multiple populations. + +} + +\section{Extends}{ + + Class \code{\link[flowCore:parameterFilter-class]{parameterFilter}}, + directly. + + Class \code{\link[flowCore:concreteFilter-class]{concreteFilter}}, + by class "parameterFilter", distance 2. + + Class \code{\link[flowCore:filter-class]{filter}}, by class + "parameterFilter", distance 3. + +} + +\section{Slots}{ + + See \code{Arguments} section for details. + + \describe{ + + \item{\code{preselection}:}{Object of class \code{character}, the + name of the flow parameter used for preselection. } + + \item{\code{rectDef}:}{Object of class \code{list}, the initial + rectangular selection. } + + \item{\code{scale}:}{Object of class \code{numeric}.} + + \item{\code{bwFac}:}{Object of class \code{numeric}.} + + \item{\code{parameters}:}{Object of class \code{parameters}, the flow + parameters to operate on. } + + \item{\code{filterId}:}{Object of class \code{"character"}, the + filter identifier. } + + } +} + +\section{Objects from the Class}{ + + Objects can be created by calls of the form \code{new("lymphFilter", + parameters, ...)} or using the constructor \code{lymphFilter}. The + constructor is the recommended way of object instantiation. + +} + +\usage{ +lymphGate(x, channels, preselection=NULL, scale=2.5, bwFac=1.3, + filterId="defaultLymphGate", evaluate=TRUE, plot=FALSE, \dots) + +lymphFilter(channels, preselection=as.character(NULL), + scale=2.5, bwFac=1.3, filterId="defaultLymphFilter") + +} + +\arguments{ + + \item{x}{ An object of class \code{\link[flowCore:flowSet-class]{flowSet}}. } + + \item{channels}{ A character vector of length 2 of valid flow + parameters in \code{x}. } + + \item{preselection}{ Either \code{NULL}, in which case this boils down + to fitting a regular + \code{\link[flowCore:norm2Filter-class]{norm2Filter}}, a + character scalar giving one of the flow parameters in \code{x}, or a + named list of numerics specifying the initial rough + preselection. The latter gets passed on to + \code{\link[flowCore]{rectangleGate}}, see it's documentation for + details. } + + \item{scale}{ The \code{scaleFactor} parameter that gets passed on to + \code{norm2Filter}. } + + \item{bwFac}{ The bandwidth factor that gets passed on to + \code{\link[flowCore:curv1Filter-class]{curv1Filter}}. } + + \item{filterId}{ A character used as filterId. } + + \item{evaluate}{ A logical indicating wether the filter should be + resolved (computation of the + \code{\link[flowCore:filterResult-class]{filterResult}} + and the subset). } + + \item{plot}{Logical. Produce plots of filter results} + + \item{\dots}{ Additional arguments. } + +} + +\details{ + + This algorithm does not apply real mixture modelling, however it is + able to identify a single elliptical cell population from a mixture of + multiple such populations. The idea is to first define a rough + rectangular preselection and, in a second step, fit a bivariate normal + distribution to this subset only. + + Depending on the value of \code{preselection}, the initial rough + selection is either + + \describe{ + + \item{NULL:}{ No preselection at all} + + \item{character scalar}{ Preselection based on cells that are + positive for a single marker only. This allows for back-gating, + for instances by selecting \code{CD4+} T-cells and using this + information to back-gate lymphocytes in \code{FSC} and + \code{SSC}. Positive cells are identified using a + \code{curv1Filter}. } + + \item{a named list of numerics:}{ Preselection by a rectangular + gate. The items of the list have to be numerics of length one + giving the gate boundaries in the respective dimensions. } + + The \code{lymphFilter} class and constructor provide the means to + treat \code{lymphGates} as regular \code{flowCore} filters. + + } + +} + +\value{ + + A list with items + + \item{x }{The filtered \code{flowSet}. } + + \item{n2gate }{The \code{norm2Filter} object. } + + \item{n2gateResults }{The + \code{\link[flowCore:filterResult-class]{filterResult}} after + applying the \code{norm2Filter} on the \code{flowSet}. } + + for the \code{lymphGate} function. Note that \code{x} and + \code{n2gateResults} are \code{NULL} when \code{eval=FALSE}. + + The \code{lymphFilter} constructor returns and object of class + \code{lymphFilter}, which can be used as a regular \code{flowCore} + fitler. + +} + +\section{Methods}{ + + \describe{ + + \item{\%in\%}{\code{signature(x = + "flowFrame", table = "lymphFilter")}: the work horse for doing the + actual filtering. Internally, this simply calls the \code{lympghGate} + function. } + + } + +} + +\author{ Florian Hahne } + +\seealso{ \code{\link[flowCore:norm2Filter-class]{norm2Filter}}, + \code{\link[flowCore:curv1Filter-class]{curv1Filter}} } + +\examples{ + +data(GvHD) +dat <- GvHD[pData(GvHD)$Patient==10] +dat <- transform(dat, "FL4-H"=asinh(`FL4-H`)) +lg <- lymphGate(dat, channels=c("FSC-H", "SSC-H"), preselection="FL4-H",scale=1.5) + +if(require(flowViz)) +xyplot(`SSC-H`~`FSC-H`, dat, filter=lg$n2gate) + +## This is using the abstract lymphFilter class instead +lf <- lymphFilter(channels=c("FSC-H", "SSC-H"), preselection="FL4-H") +filter(dat, lf) + +} + +\keyword{misc} +\keyword{classes} + diff --git a/man/normQA.Rd b/man/normQA.Rd new file mode 100644 index 0000000..c298e35 --- /dev/null +++ b/man/normQA.Rd @@ -0,0 +1,60 @@ +\name{normQA} +\alias{normQA} + +\title{Normalization quality assessment} + +\description{Create QA plots for a flow cytometry normalization process.} + +\usage{ +normQA(data, morph = c("^fsc", "^ssc"), channels, odat = NULL, ask = names(dev.cur()) != "pdf", grouping = NULL, tag.outliers = FALSE, peaksOnly = TRUE) +} + +\arguments{ + + \item{data}{a normalized \code{\link[flowCore:flowSet-class]{flowSet}}.} + + \item{morph}{A character vector of channel names to use for the + backgating into the morphological channels.} + + \item{channels}{The channels for which to create plots. Defaults to + all normalized channels.} + + \item{odat}{The original data set, always needed if there are no + warping functions available.} + + \item{ask}{Ask before creating a new plot.} + + \item{grouping}{A grouping variable in \code{data}'s \code{phenoData} slot.} + + \item{tag.outliers}{Logical. Add sample name to outliers in the + plots.} + + \item{peaksOnly}{Logical. Only use data when a peak was detected in a + particular sample. If set to \code{FALSE}, a average peak location + is estimated.} + +} + +\details{ + + This function assumes that the necessary information has been added as + attributes to \code{data} during the normalization procedure. Depending + on the available information, a set of QA plots is generated. + Available plots are: + + Amount of peak adjustment + + Warping functions + + Landmark classification confidence + +Backgating of peak events in morphological channels + +} +\value{ +This function is called for its side effect of generating plots. +} + +\author{ +Florian Hahne +} diff --git a/man/normalize.Rd b/man/normalize.Rd new file mode 100644 index 0000000..08daca0 --- /dev/null +++ b/man/normalize.Rd @@ -0,0 +1,92 @@ +\name{normalize-methods} +\docType{methods} +\alias{normalize} +\alias{normalize-methods} +\alias{normalize,GatingSetInternal,missing-method} +\alias{normalize,GatingSet,missing-method} +\title{normalize a GatingSet imported with flowWorkspace, using sequential normalization on the manual gates in the GatingHierarchy.} +\description{ +The method will step through the gating hierarchy in a breadth first search manner and normalize each dimension and gate not explicitly excluded in skipdims,or skipgates. The normalization approach is based on warpSet, but uses sequential normalization to alternately normalize then perform gating of the cell populations. This often helps with feature registration of popualtions lower in the gating hierarchy. FSC and SSC, as well as time are generally excluded by default. The rule of thumb, is to only normalize a channel in a gate if it is absolutely warranted. +} +\usage{ +normalize(data,x,...) +} +\arguments{ + + \item{data}{ + The \code{GatingSet} to be normalized. +} +\item{x}{\code{missing}. Not used in here.} +\item{\dots}{ + Arguments passed to downstream functions. + + target: + The target sample to normalize the other samples in the gating set to. A \code{character} vector. Must match a sample name in \code{x}, otherwise \code{NULL} will use the mean (average) of the peaks identified in all samples + + skipgates: + A \code{numeric} vector of gate indices that are to be skipped (i.e. not normalized). + + + skipdims: + A \code{character} vector of parameter names to be omitted during sequential normalization. + + + chunksize: + For a memory-efficient implementation of normalization, set the \code{chunksize},(an \code{integer}), which will peform normalization on chunks of the data of size \code{chunksize}. + + nPeaks: + A \code{list} of \code{integer} or an \code{integer} vector that specifies the expected number of peaks for each sample. Can be omitted to keep all peaks. + + bwFac: + The bandwidth for density estimation, a \code{numeric}. Affects the sensitivity for smoothing and detecting distinct peaks. +} + + + +} +\details{ +This function implements sequential normalization using a GatingSet and a set of manual gates. For each gate in the gating hierarchy, the algorithm checks if the gate should be normalized, and which dimensions in the gate should be normalized. If normalization is warranted, this is performed prior to gating. After gating, the counts for the gate in the GatingSet are updated, and the next gate is processed. This is useful in the application of template gates to data that has staining variability in one or more channels. +} +\value{ +Returns a \code{GatingSet} of normalized data. +} +\author{ +Greg Finak +} + +\seealso{ +See also \code{\link[flowWorkspace]{GatingSet-class}}, \code{\link[flowWorkspace]{GatingHierarchy-class}}, \code{\link[ncdfFlow]{ncdfFlowSet}} +} +\examples{ +\dontrun{ +#open an xml flowJo workspace +ws<-openWorkspace("test.xml")) +#parse it into a GatingSet +G<-parseWorkspace(ws) + +#exclude some populations(gates) from normalization +skip.g<-(1:length(getNodes(G[[1]])))[-c(9,144)] +#exclude some channels(parameters) from normalization +skip.d<-colnames(getData(G[[1]]))[-c(9)] +#run the normalization method +G_norm<-normalize(data=G + ,target="517614.fcs" #reference sample + ,skipgates=skip.g + ,skipdims=skip.d +#show the population statistics for before and after normalization +getPopStats(G)) +getPopStats(G_norm) + +#plot the gate to see the effects of normalization +plotGate(G,9,xbin=64) +plotGate(G_norm,9,xbin=64) +} +} +\keyword{methods} + + + + + + + diff --git a/man/plotBins.Rd b/man/plotBins.Rd new file mode 100644 index 0000000..0540ed0 --- /dev/null +++ b/man/plotBins.Rd @@ -0,0 +1,66 @@ + +\name{plotBins} +\alias{plotBins} + +\title{ Plots the probability bins overlaid with flowFrame data} + +\description{ +This function is useful in visualizing the differences between the binned control +and sample datasets. The bins generated from the control dataset are +overlaid with the sample dataset. An optional argument residuals can be used to +shade each bin based on a calculated statistical measure of difference between +the number of events in each bin. +} + +\usage{ +plotBins(binRes,data,channels,title,residuals,shadeFactor) +} + +\arguments{ + + \item{binRes}{ The result generated by calling the \code{probBin} + function on a control dataset.} + + \item{data}{ An object of class + \code{\link[flowCore:flowFrame-class]{flowFrame}} + sample(dataset)} + + \item{channels}{ The flow parameters to be plotted.In cases where more + than two parameters are binned from the control set, the + \code{plotBins} function plots the projections of the hyperplanes in + 2 dimensions)} + + \item{title}{ Optional title for the plot generated} + + \item{residuals}{ A vector of length equal to the number of bins + generated that can be used to shade each bin. The residuals from the + \code{calcPearsonChi} function or the \code{calcPBChiSquare} + function can be used to highlight the bins that are different + between control and sample datasets} + + \item{shadeFactor}{ Optional argument between 0 and 1 that controls + the intensity of the shading of bins} + +} + +\author{Nishant Gopalakrishnan} + +\seealso{ \code{\link{proBin}}, \code{\link{calcPearsonChi}}, + \code{\link{calcPBChiSquare}}} + +\examples{ + +data(GvHD) +# flow frame 1 is treated as control dataset and used to generate bins +resCtrl<-proBin(GvHD[[1]],200,channels=c("FSC-H","SSC-H")) +plotBins(resCtrl,GvHD[[1]],channels=c("FSC-H","SSC-H"),title="Binned control data") +# Same bins are applied to flowFrame 16 +resSample<-binByRef(resCtrl,GvHD[[16]]) +stat<-calcPearsonChi(resCtrl,resSample) +dev.new() +plotBins(resCtrl,data=GvHD[[16]],channels=c("FSC-H","SSC-H","Time"),title="Comparision 1 & 16", +residuals=stat$residuals[2,],shadeFactor=0.7) + +} + +\keyword{misc} diff --git a/man/proBin.Rd b/man/proBin.Rd new file mode 100644 index 0000000..5934078 --- /dev/null +++ b/man/proBin.Rd @@ -0,0 +1,106 @@ +\name{proBin} +\alias{proBin} + +\title{ Probability binning - a metric for evaluating multivariate differences} + +\description{ + + This function divides the flowframe events into bins such that each + bin contains the same number of events. The number of events falling + into each bin can then be compared across the control and test samples + using statistical methods such as the Chi-squared test. + +} + +\usage{ +proBin(m, minEvents=500,channels=NULL) +} + +\arguments{ + + \item{m}{ An object of class + \code{\link[flowCore:flowFrame-class]{flowFrame}}} + + \item{minEvents}{ The \code{minEvents} The minimum number of events in + each bin. (i.e. the termination criterion for the probability + binning algorithm)} + + \item{channels}{ A character vector for the Flourescence channels on which + probability binning is to be performed. Defaults is NULL, in which case, + all flourescence channels are used for probability binning.( + Time information, if provided in the flowFrame is discarded) + } + +} + +\details{ + + The \code{flowSet} is first filtered using a \code{rectangleGate} and + the \code{norm2Filter} is subsequently fitted to the remaining subset. + +} + +\value{ + + A list with items: + + \item{table }{A \code{data.frame} that stores information regarding + each node of the tree generated during each stage of the probability + binning algorithm. Each row in the table represents a node, the + first row representing the original \code{flowFrame} matrix. + + The dataIndx column provides indexes for retrieving the matrices + during each stage of the binning process from the enviroment + \code{data} . + + The parent field indicates the row number in the table that holds + the parent information for the corresponding node. + + The left and right columns indicates the row numbers in the table + that stores information regarding the children of that particular + node. The leaf nodes that hold the binned data can be identified by + the nodes with the left of right values of zero( ie. no children + nodes) + + The visited column is used internally by the algorithm to check if a + particular node has been visited during the computation process. + + } + + \item{data }{An enviroment in which the matrices generated during each + stage of the probability binning process is stored. The matrices + stored at the leaf nodes represent the binned events obtained after + the stop criterion of minEvents has been achieved. These can be + identified by the corresponding dataIndx fields provided by the rows + in the table with the left or right column values of zero. } + + \item{limits }{A list containing the the boundaries of each hyperplane + generated during probability binning} + + \item{splitPars }{A \code{data.frame} containing two columns splitCol + - indicates the column number of the \code{flowFrame} , the split + was performed. + + splitMed - The median value which was used as the threshold for + splitting the \code{flowFrame} + + The splitCol and splitMed parameters are utilized by the plotBins + and shadeBins functions in visualizing the differences between + control and test sample cases. + + } + +} + + +\author{ Nishant Gopalakrishnan } + +\seealso{ \code{\link{plotBins}}, \code{\link{binByRef}}} + +\examples{ + +data(GvHD) +res<-proBin(GvHD[[1]],200,channels=c("FSC-H","SSC-H","FL1-H","FL4-H")) +} + +\keyword{misc} diff --git a/man/quadrantGate.Rd b/man/quadrantGate.Rd new file mode 100644 index 0000000..cdebc79 --- /dev/null +++ b/man/quadrantGate.Rd @@ -0,0 +1,99 @@ +\name{quadrantGate} +\alias{quadrantGate} + +\title{Automated quad gating} + +\description{ + This function tries to find the most likely separation of + two-dimensional flow cytometry in four quadrants. +} + +\usage{ +quadrantGate(x, stains, alpha=c("min", "min"), sd=c(2, 2), plot=FALSE, + filterId="defaultQuadGate", refLine.1=NULL, refLine.2=NULL + ,rare=c(FALSE,FALSE) + ,sig=c(NULL,NULL) + ,...) +} + +\arguments{ + + \item{x}{ A \code{\link[flowCore:flowSet-class]{flowSet}} or + \code{\link[flowCore:flowSet-class]{flowFrame}}. } + + \item{stains}{ A character vector of length two giving the two flow + parameters for which the quad gate is to be computed. } + + \item{alpha, sd}{ Tuning factors to control the computation of the + gate boundaries. See \code{\link{rangeGate}} for details. } + + \item{plot}{ Logical. Produce plots of intermediate results. } + + \item{filterId}{ Character, the name assigned to the resulting + filter. } + + \item{refLine.1}{Either \code{NULL} or a numeric of lenth 1. If + \code{NULL}, this parameter is ignored. When it is set to a numeric, the + minor sub-population (if any) below this reference line in the first + stain channel will be igored while determining the separator between + positive and negative.} + + \item{refLine.2}{Either \code{NULL} or a numeric of lenth 1. If + \code{NULL}, this parameter is ignored. When it is set to a numeric, the + minor sub-population (if any) below this reference line in the second + stain channel will be igored while determining the separator between + positive and negative.} + + \item{rare}{logical flags for two channels, Refer to \code{\link{density1d}} for more details. + } + + \item{sig}{parameters for two channels. Refer to \code{\link{density1d}} for more details. + } + \item{\dots}{ Additional arguments } +} + +\details{ + + The most likely separation between postitive and negative stains for + two-dimensional data is computed based on density + estimates. Essentially, the gate parameters are first fitted + separately for the two parameters and later combined. See the + documentation for \code{\link{rangeGate}} for details. There is a + certain amount of heuristics involved in this process. The algorithm + can be slightly tweaked using the \code{alpha} and \code{sd} + arguments. Their values will be recycled for the two dimensions unless + explicitely given as vectors of length 2. + +} + +\value{ + + An object of class \code{\link[flowCore]{quadGate}}. + +} + +\author{Florian Hahne } + +\seealso{ + + \code{\link[flowCore]{quadGate}}, + \code{\link{rangeGate}} + +} + +\examples{ +\dontrun{ +data(GvHD) +dat <- GvHD[pData(GvHD)$Patient==10] +dat <- transform(dat, "FL4-H"=asinh(`FL4-H`), "FL2-H"=asinh(`FL2-H`)) +qg <- quadrantGate(dat, c("FL2-H", "FL4-H")) +qg + +if(require(flowViz)) +xyplot(`FL2-H`~`FL4-H`, dat, filter=qg) + +qg <- quadrantGate(dat, c("FL2-H", "FL4-H"), alpha=c(0.1, 0.9), plot=TRUE) +qg +split(dat, qg) +} +} diff --git a/man/rangeGate.Rd b/man/rangeGate.Rd new file mode 100644 index 0000000..a4cf2bb --- /dev/null +++ b/man/rangeGate.Rd @@ -0,0 +1,164 @@ +\name{rangeGate} + +\alias{rangeGate} +\alias{rangeFilter} +\alias{rangeFilter-class} +\alias{\%in\%,flowFrame,rangeFilter-method} +\alias{oneDGate} + +\title{ Find most likely separation between positive and negative + populations in 1D } + + +\description{ + + The function tries to find a reasonable split point between the two + hypothetical cell populations "positive" and "negative". +} + +\usage{ +rangeGate(x, stain, alpha="min", sd=2, plot=FALSE, borderQuant=0.1, +absolute=TRUE, filterId="defaultRectangleGate", positive=TRUE, +refLine=NULL, simple = FALSE,...) + +rangeFilter(stain, alpha="min", sd=2, borderQuant=0.1, +filterId="defaultRangeFilter") +} + +\arguments{ + + \item{x}{ A \code{\link[flowCore:flowSet-class]{flowSet}} or + \code{\link[flowCore:flowFrame-class]{flowFrame}}. } + + \item{stain}{ A character scalar giving the flow parameter for which + to compute the separation. } + + \item{alpha}{ A tuning parameter that controls the location of the + split point between the two populations. This has to be a numeric in + the range \code{[0,1]}, where values closer to 0 will shift the + split point closer to the negative population and values closer to 1 + will shift towards the positive population. Additionally, the value + of \code{alpha} can be \code{"min"}, in which case the split point + will be selected as the area of lowest local density between the two + populations. } + + \item{sd}{For the case where there is only a single population, the + algorithm falls back to esitmating the mode of this population and a + robust measure of the variance of it distribution. The \code{sd} + tuning parameter controls how far away from the mode the split point + is set. } + + \item{plot}{ Create a plot of the results of the computation. } + + \item{borderQuant}{Usualy the instrument is set up in a way that the + positive population is somewhere on the high end of the measurement + range and the negative population is on the low end. This parameter + allows to disregard populations with mean values in the extreme + quantiles of the data range. It's value should be in the range + \code{[0,1]}. } + + \item{absolute}{Logical controling whether to classify a population + (positive or negative) relative to the theoretical measurment range + of the instrument or the actual range of the data. This can be set + to \code{TRUE} if the alignment of the measurment range is not + optimal and the bulk of the data is on one end of the theoretical + range.} + + \item{filterId}{ Character, the name assigned to the resulting + filter. } + + \item{positive}{ Create a range gate that includes the positive + (\code{TRUE}) or the negative (\code{FALSE}) population. } + + \item{refLine}{Either \code{NULL} or a numeric of lenth 1. If + \code{NULL}, this parameter is ignored. When it is set to a numeric, the + minor sub-population (if any) below this reference line + will be igored while determining the separator between positive and + negative. } + \item{simple}{ \code{logical} scalar indicating whether to use a simple peak finding version of density1d algorithm. + } + \item{\dots}{ Further arguments. } +} + +\details{ + + The algorithm first tries to identify high density regions in the + data. If the input is a \code{flowSet}, density regions will be + computed on the collapsed data, hence it should have been normalized + before (see \code{\link{warpSet}} for one possible normalization + technique). The high density regions are then clasified as positive + and negative populations, based on their mean value in the theoretical + (or absolute if argument \code{absolute=TRUE}) measurement range. In + case there are only two high-density regions the lower one is usually + clasified as the negative populations, however the heuristics in the + algorithm will force the classification towards a positive population + if the mean value is already very high. The \code{absolute} and + \code{borderQuant} arguments can be used to control this + behaviour. The split point between populations will be drawn at the + value of mimimum local density between the two populations, or, if the + \code{alpha} argument is used, somewhere between the two populations + where the value of alpha forces the point to be closer to the negative + (\code{0 - 0.5}) or closer to the positive population (\code{0.5 - + 1}). + + If there is only a single high-density region, the algorithm will fall + back to estimating the mode of the distribution + (\code{\link[MASS]{hubers}}) and a robust measure of it's variance + and, in combination with the \code{sd} argument, set the split point + somewhere in the right or left tail, depending on the classification + of the region. + + For more than two populations, the algorithm will still classify each + population into positive and negative and compute the split point + between those clusteres, similar to the two population case. + + + The \code{rangeFilter} class and constructor provide the means to + treat \code{rangeGate} as regular \code{flowCore} filters. + +} + +\section{Methods}{ + + \describe{ + + \item{\%in\%}{\code{signature(x = + "flowFrame", table = "rangeFilter")}: the work horse for doing the + actual filtering. Internally, this simply calls the \code{rangeGate} + function. } + } +} + +\value{ + + A range gate, more explicitely an object of class + \code{\link[flowCore]{rectangleGate}}. + +} + + +\author{ Florian Hahne, Kyongryun Lee } + +\seealso{ + + \code{\link{warpSet}}, \code{\link{rangeGate}}, + \code{\link[flowCore]{rectangleGate}} + +} + +\examples{ + +data(GvHD) +dat <- GvHD[pData(GvHD)$Patient==10] +dat <- transform(dat, "FL4-H"=asinh(`FL4-H`), "FL3-H"=asinh(`FL3-H`)) +rg <- rangeGate(dat, "FL4-H", plot=TRUE) +rg +split(dat, rg) + +## Test rangeGate when settting refLine=0; it does not do anything since +## there is no sub-population below zero. +rangeGate(dat, "FL4-H", plot=FALSE, refLine=0) + +rf <- rangeFilter("FL4-H") +filter(dat, rf) +} diff --git a/man/singletGate.Rd b/man/singletGate.Rd new file mode 100644 index 0000000..7bcc39b --- /dev/null +++ b/man/singletGate.Rd @@ -0,0 +1,85 @@ +\name{singletGate} +\alias{singletGate} + +\title{Creates a singlet polygon gate using the prediction bands from a robust linear model} + + +\description{ + + The function constructs a singlet \code{polygonGate} using the prediction bands + from a robust linear model trained on the area and height of one flow + parameter (usually forward scatter). + +} + +\usage{ +singletGate(x, area = "FSC-A", height = "FSC-H", sidescatter = NULL, + prediction_level = 0.99, subsample_pct = NULL, wider_gate = FALSE, + filterId = "singlet", ...) +} + +\arguments{ + + \item{x}{a \code{\link[flowCore:flowFrame-class]{flowFrame}} object} + + \item{area}{character giving the channel name that records the signal intensity as peak area} + + \item{height}{character giving the channel name that records the signal intensity as peak heightchannel name of height} + + \item{sidescatter}{character giving an optional channel name for the sidescatter signal. By default, ignored.} + + \item{prediction_level}{a numeric value between 0 and 1 specifying the level to use for the prediction bands} + + \item{subsample_pct}{a numeric value between 0 and 1 indicating the percentage of observations that should be randomly selected from \code{x} to construct the gate. By default, no subsampling is performed.} + + \item{wider_gate}{logical value. If \code{TRUE}, the prediction bands used to construct the singlet gate use the robust fitted weights, which increase prediction uncertainty, especially for large FSC-A. This leads to wider gates, which are sometimes desired.} + + \item{filterId}{the name for the filter that is returned} + + \item{...}{additional arguments passed to \code{\link[MASS]{rlm}}} +} + +\details{ + + We construct a singlet gate by applying a robust linear model with + \code{\link[MASS]{rlm}}. By default, we model the forward-scatter height + (FSC-H)as a function of forward-scatter area (FSC-A). If \code{sidescatter} + is given, forward-scatter height is as a function of \code{area} + + \code{sidescatter} + \code{sidescatter / area}. + + Because \code{\link[MASS]{rlm}} relies on iteratively reweighted least + squares (IRLS), the runtime to construct a singlet gate is dependent in part + on the number of observations in \code{x}. To improve the runtime, we provide + an option to subsample randomly a subset of \code{x}. A percentage of + observations to subsample can be given in \code{subsample_pct}. By default, no + subsampling is applied. + +} + + +\value{ + + \code{\link[flowCore]{polygonGate}}. + +} + + +\author{ Greg Finak, John Ramey } + +\seealso{ + + \code{\link{rangeGate}}, + \code{\link[flowCore]{polygonGate}} + +} + +\examples{ +\dontrun{ +# fr is a flowFrame +sg <- singletGate(fr, area = "FSC-A", height = "FSC-H") +sg +# plot the gate +xyplot(`FSC-H` ~ `FSC-A`, fr, filter = sg) +} + +} diff --git a/man/warpSet.Rd b/man/warpSet.Rd new file mode 100644 index 0000000..a16a3b5 --- /dev/null +++ b/man/warpSet.Rd @@ -0,0 +1,140 @@ +\name{warpSet} +\alias{warpSet} +\alias{warpSetNCDF} +\alias{warpSetGS} +\alias{warpSetNCDFLowMem} +\title{Normalization based on landmark registration} + +\description{ + + This function will perform a normalization of flow cytometry + data based on warping functions computed on high-density region + landmarks for individual flow channels. + +} + +\usage{ +warpSet(x, stains, grouping = NULL, monwrd = TRUE, subsample=NULL, + peakNr=NULL, clipRange=0.01, nbreaks=11, fres, bwFac=2, + warpFuns=FALSE,target=NULL, ...) +} + + +\arguments{ + + \item{x}{ A \code{\link[flowCore:flowSet-class]{flowSet}}. } + + \item{stains}{ A character vector of flow parameters in \code{x} to be + normalized. } + + \item{grouping}{ A character indicating one of the phenotypic + variables in the \code{phenoData} slot of \code{x} used as a grouping + factor. The within-group and between-group variance is computed and + a warning is issued in case the latter is bigger than the former, + indicating the likely removal of signal by the normalization + procedure. } + + \item{monwrd}{ Logical. Compute strictly monotone warping + functions. This gets directly passed on to + \code{\link[fda]{landmarkreg}}. } + + \item{subsample}{ Numeric. Reduce the number of events in each \code{flowSet} + by sub sampling for all density estimation steps and the calculation + of the warping functions. This can increase computation time for + large data sets, however it might reduce the accuracy of the density + estimates. To be used with care. } + + \item{peakNr}{ Numeric scalar. Force a fixed number of peaks to use + for the normalization.} + + \item{clipRange}{Only use peaks within a clipped data + range. Essentially, the number indicates the percent of clipping on + both sides of the data range, e.g. \code{min(x) - 0.01 * + diff(range(x))}.} + + \item{nbreaks}{The number of spline sections used to approximate the + data. Higher values produce more accurate results, however this + comes with the cost of increaseqd computing times. For most data, + the default setting is good enough.} + + \item{fres}{A named list of \code{filterResultList} objects. This can + be used to speed up the process since the \code{curv1Filter} step + can take quite some time.} + + \item{bwFac}{Numeric of lenght 1 used to set the bandwidth factor by + \code{\link{curv1Filter}} for smoothing of the density estimate. } + + \item{warpFuns}{Logical indcating whether to return the normalized + \code{flowSet} or a list of warping functions.} + \item{target}{Character vector specifying the target sample to which other samples in the \code{flowSet} should be normalized. If \code{NULL}, then the mean of the peaks is used.} + \item{\dots}{ Further arguments that are passed on to + \code{landmarkreg}. } + +} + +\details{ + + Normalization is achived by first identifying high-density regions + (landmarks) for each \code{\link[flowCore:flowFrame-class]{flowFrame}} + in the \code{flowSet} for a single channel and subsequently by + computing warping functions for each \code{flowFrame} that best align + these landmarks. This is based on the algorithm implemented in the + \code{landmarkreg} function in the \code{\link[fda:fda-package]{fda}} + package. An intermediate step classifies the high-density regions, see + \code{\link{landmarkMatrix}} for details. + + Please note that this normalization is on a channel-by-channel + basis. Multiple channels are normalized in a loop. + + +} + + +\value{ + + The normalized \code{flowSet} if \code{warpFuns} is \code{FALSE}, + otherwise a list of warping functions. Additional inforamtion is + attached as the \code{warping} attribute to the \code{flowSet} in form + of a list. + +} + +\references{ + + J.O. Ramsay and B.W. Silverman: Applied Functional Data Analysis, + Springer 2002 + +} + + +\author{ Florian Hahne } + +\note{ + + We currently use a patched fda version. + +} + +\seealso{ + + \code{\link[flowCore:curv1Filter-class]{curv1Filter}}, + \code{\link{landmarkMatrix}} + +} + +\examples{ + +data(ITN) +dat <- transform(ITN, "CD4"=asinh(CD4), "CD3"=asinh(CD3), "CD8"=asinh(CD8)) +lg <- lymphGate(dat, channels=c("CD3", "SSC"), +preselection="CD4",scale=1.5) +dat <- Subset(dat, lg$n2gate) +datr <- warpSet(dat, "CD8", grouping="GroupID", monwrd=TRUE) +if(require(flowViz)){ +d1 <- densityplot(~CD8, dat, main="original", filter=curv1Filter("CD8")) +d2 <- densityplot(~CD8, datr, main="normalized", filter=curv1Filter("CD8")) +plot(d1, split=c(1,1,2,1)) +plot(d2, split=c(2,1,2,1), newpage=FALSE) +} + +} diff --git a/vignettes/GettingStartedWithFlowStats.Rnw b/vignettes/GettingStartedWithFlowStats.Rnw new file mode 100644 index 0000000..8659b18 --- /dev/null +++ b/vignettes/GettingStartedWithFlowStats.Rnw @@ -0,0 +1,348 @@ +% NOTE -- ONLY EDIT THE .Rnw FILE!!! The .tex file is +% likely to be overwritten. +% +%\VignetteIndexEntry{flowStats Overview} +%\VignetteDepends{flowStats, flowViz} +%\VignetteKeywords{} +%\VignettePackage{flowStats} +\documentclass[11pt]{article} + +\usepackage{times} +\usepackage{hyperref} +\usepackage[authoryear,round]{natbib} +\usepackage{times} +\usepackage{comment} +\usepackage{graphicx} +\usepackage{subfigure} + +\textwidth=6.2in +\textheight=8.5in +\oddsidemargin=.1in +\evensidemargin=.1in +\headheight=-.3in + +\newcommand{\scscst}{\scriptscriptstyle} +\newcommand{\scst}{\scriptstyle} +\newcommand{\Rfunction}[1]{{\texttt{#1}}} +\newcommand{\Rcode}[1]{{\texttt{#1}}} +\newcommand{\Robject}[1]{{\texttt{#1}}} +\newcommand{\Rpackage}[1]{{\textsf{#1}}} +\newcommand{\Rclass}[1]{{\textit{#1}}} + + + +\title{Getting started with \Rpackage{flowStats}} +\author{F. Hahne, N. Gopalakrishnan} + +\begin{document} +\maketitle + +\begin{abstract} +\Rpackage{flowStats} is a collection of algorithms for the statistical +analysis of flow cytometry data. So far, the focus is on automated +gating and normalization. +\end{abstract} + + +\section{Introduction} +Since \Rpackage{flowStats} is more a collection of algorithms, writing +a coherent Vignette is somewhat difficult. Instead, we will present a +hypothetical data analysis process that also makes heavy use of the +functionality provided by \Rpackage{flowCore}, mainly the work flow +infrastructure. + +We start by loading the \Robject{GvHD} data set from the +\Rpackage{flowCore} package. +<>= +library(flowStats) +data(ITN) +@ +% +The data was acquired from blood samples by 3 groups of patients, each +group containing 5 samples. Each \Rclass{flowFrame} includes, in +addition to FSC and SSC, 5 fluoresence parameters: CD3, CD4, CD8, CD69 +and HLADR. + +First we need to tranform all the fluorescense channels. This is a +good point to start using a \Rclass{workFlow} object to keep track of +our progress. +<>= +wf <- workFlow(ITN) +tl <- transformList(colnames(ITN)[3:7], asinh, transformationId="asinh") +add(wf, tl) +@ +% +In a initial analysis step we first want to indentify and subset all +T-cells. This can be archived by gating in the CD3 and SSC dimensions, +however there are several other sub-populations, and we need to +either specify our selection further, or segment the individual +sub-populations. One solution for the latter aproach is to use the +mixture modelling infrastructure provided by the \Rclass{flowClust} +package. However, since we are only interested in one single +sub-population, the T-cell, it is much faster and easier to use the +\Rfunction{lymphGate} function in the \Rpackage{flowStats} +package. The idea here is to first do a rough preselection in the +two-dimensional projection of the data based on expert knowledge or +prior experience and subsequently to fit a \Rclass{norm2Filter} to +this subset. The function also allows to derive the pre-selection +through back-gating: we know that CD4 positive cells are a subset of +T-cells, so by estimating CD4 positive cells first we can get a rough +idea on where to find the T-cells in the CD3 SSC projection. + +<>= +lg <- lymphGate(Data(wf[["asinh"]]), channels=c("SSC", "CD3"), + preselection="CD4", filterId="TCells", eval=FALSE, + scale=2.5) +add(wf, lg$n2gate, parent="asinh") +<>= +library(flowViz) +print(xyplot(SSC ~ CD3| PatientID, wf[["TCells+"]], + par.settings=list(gate=list(col="red", + fill="red", alpha=0.3)))) +@ + +In the next step we want to separate T-helper and NK cells using the +CD4 and CD8 stains. A convenient way of doing this is to apply a +\Rclass{quadGate}, assuming that both CD4 and CD8 are binary markers +(cells are either positive or negative for CD4 and CD8). Often +investigators use negative samples to derive a split point between the +postive and negative populations, and apply this constant gate on all +their samples. This will only work if there are no unforseen shifts in +the fluorescence itensities between samples which are purely caused by +technical variation rather than biological phenotype. Let's take a +look at this variation for the T-cell subset and all 4 remaining +fluorescense channels: + +<>= +pars <- colnames(Data(wf[["base view"]]))[c(3,4,5,7)] +print(densityplot(PatientID~., Data(wf[["TCells+"]]), channels=pars, groups=GroupID, + scales=list(y=list(draw=F)), filter=lapply(pars, curv1Filter), + layout=c(4,1))) +@ + +Indeed the data, especially for CD4 and CD8, don't align well. At +this point we could decide to compute the \Rclass{quadGates} for each +sample separately. Alternatively, we can try to normalize the data and +then compute a common gate. The \Rfunction{warpSet} function can be +used to normalize data according to a set of landmarks, which +essentially are the peaks or high-density areas in the density +estimates shown before. The ideas here are simple: + +\begin{itemize} +\item High density areas represent particular sub-types of cells. +\item Markers are binary. Cells are either positive or negative for + a particular marker. +\item Peaks should aline if the above statements are true. +\end{itemize} + +The algorithm in \Rfunction{warpSet} performs the following steps: +\begin{enumerate} +\item Identify landmarks for each parameter using a \Rfunction{curv1Filter} +\item Estimate the most likely total number (k) of landmarks +\item Perform k-means clustering to classify landmarks +\item Estimate warping functions for each sample and parameter that + best align the landmarks, given the underlying data. This step uses + functionality from the \Rpackage{fda} package. +\item Transform the data using the warping functions. +\end{enumerate} + +The algorithm should be robust to missing peaks in some of the +samples, however the classification in step 3 becomes harder since it +is not clear which cell population it represents. + +<>= +norm <- normalization(normFun=function(x, parameters, ...) + warpSet(x, parameters, ...), + parameters=pars, + arguments=list(grouping="GroupID", monwrd=TRUE), + normalizationId="Warping") +add(wf, norm, parent="TCells+") +<>= +print(densityplot(PatientID~., Data(wf[["Warping"]]), channels=pars, groups=GroupID, + scales=list(y=list(draw=F)), filter=lapply(pars, curv1Filter), + layout=c(4,1))) +@ + +After normalization the data look much cleaner and we should be able +to use a single static gate for all \Rclass{flowFrames} in order to +separate CD4 and CD8 positive cells. Typically one would use a +\Rclass{quadGate}, and the \Rfunction{quadrantGate} function in +\Rpackage{flowStats} can be used to automatically estimate such a +gate. + +<>= +qgate <- quadrantGate(Data(wf[["Warping"]]), stains=c("CD4", "CD8"), + filterId="CD4CD8", sd=3) +add(wf, qgate, parent="Warping") +@ +<>= +print(xyplot(CD8 ~ CD4 | PatientID, wf[["CD4+CD8+"]], + par.settings=list(gate=list(fill="transparent", + col="red")))) +@ + +In a final step we might be interested in finding the proportion of +activated T-helper cells by means of the CD69 stain. The +\Rfunction{rangeGate} function is helpful in separating positive and +negative peaks in 1D. + +<>= +CD69rg <- rangeGate(Data(wf[["Warping"]]), stain="CD69", + alpha=0.75, filterId="CD4+CD8-CD69", sd=2.5) +add(wf, CD69rg, parent="CD4+CD8-") +@ +<>= +print(densityplot(PatientID ~ CD69, Data(wf[["CD4+CD8-"]]), main = "CD4+", + groups=GroupID, refline=CD69rg@min)) +@ + +\section{Probability Binning} +A probability binning algorithm for quantitating multivariate distribution +differences was described by Roederer et al. The algorithm identifies the flow +parameter in a flowFrame with the largest variance and divides the events in the +flowFrame into two subgroups based on the median of the parameter. This process +continues until the number of events in each subgroup is less than a user +specified threshold. + +For comparison across multiple samples, probability binning algorithm can be applied +to a control dataset to obtain the position of bins and the same bins can be +applied to the experimental dataset. The number of events in the control and sample +bins can then be compared using the Pearsons chi-square test or the probability +binning metric defined by Roederer et al. + +Although probability binning can be applied simultaneously to all parameters in +a flowFrame with bins in n dimensional hyperspace, we proceed with a two dimenstional +example from our previous discussion involving CD4 and CD8 populations. This helps +to simplify the demonstration of the method and interpretation of results. + +From the workflow object containing the warped data, we extract our data frame of +interest. We try to compare the panels using probability binning to identify patients +with CD4, CD8 populations different from a control flowFrame that we create using the +data from all the patients. + +<>= +dat <- Data(wf[["Warping"]]) + +@ + +The dat is visualized below +<>= +print( xyplot(CD8 ~ CD4 , dat, main= "Experimental data set")) +@ + +The control dataset is created by combining all the flowFrames in the flowSet. +The flowFrame is then subsetted after applying a sampleFilter so that the control +flowSet created has approximately the same number of events as the other flowSets +in our example. + +<>= +datComb <- as(dat,"flowFrame") +subCount <- nrow(exprs(datComb))/length(dat) + sf <- sampleFilter(filterId="mySampleFilter", size=subCount) + fres <- filter(datComb, sf) + ctrlData <- Subset(datComb, fres) + ctrlData <- ctrlData[,-ncol(ctrlData)] ##remove the column name "original" + +@ + +The probability binning algorithm can then applied to the control data. The +terminating condition for the algorithm is set so that the number of events +in each bin is approximately 5 percent of the total number of events in the +control data. +<>= +minRow=subCount*0.05 +refBins<-proBin(ctrlData,minRow,channels=c("CD4","CD8")) + +@ + +The binned control Data can be visualized using the \Rfunction{plotBins} function. +Areas in the scatter plot with a large number of data points have a higher +density of bins. Each bin also has approximately same number of events. + + +<>= +plotBins(refBins,ctrlData,channels=c("CD4","CD8"),title="Control Data") + +@ + +The same bin positions from the control data set are then applied to each flowFrame +in our sample Data set. + +<>= +sampBins <- fsApply(dat,function(x){ + binByRef(refBins,x) + }) +@ + +For each patient, the number events in the control and sample bins can be compared +using the \Rfunction{calcPearsonChi} or using Roederers probability binning +metric. + +<>= +pearsonStat <- lapply(sampBins,function(x){ + calcPearsonChi(refBins,x) + }) +@ + +<>= +sCount <- fsApply(dat,nrow) +pBStat <-lapply(seq_along(sampBins),function(x){ + calcPBChiSquare(refBins,sampBins[[x]],subCount,sCount[x]) + }) +@ + + +For each sample, the results can be visualized using the \Rfunction{plotBins} function. +The residuals from Roeders probability binning metric or the Pearsons chi square +test can be used to shade bins to highlight bins in each sample that differ +the most from the control sample. + +<>= +par(mfrow=c(4,4),mar=c(1.5,1.5,1.5,1.5)) + +plotBins(refBins,ctrlData,channels=c("CD4","CD8"),title="Control Data") +patNames <-sampleNames(dat) +tm<-lapply(seq_len(length(dat)),function(x){ + plotBins(refBins,dat[[x]],channels=c("CD4","CD8"), + title=patNames[x], + residuals=pearsonStat[[x]]$residuals[2,], + shadeFactor=0.7) + + } + ) +@ + +The patient with CD4/CD8 populations most different from that of the +control group can be identified from the magnitue of Pearson-chi square +statistic(or Probability binning statistic). + + +<>= +library(xtable) +chi_Square_Statistic <- unlist(lapply(pearsonStat,function(x){ + x$statistic + })) + +pBin_Statistic <-unlist(lapply(pBStat,function(x){ + x$pbStat + })) + +frame <- data.frame(chi_Square_Statistic, pBin_Statistic) +rownames(frame) <- patNames + +@ + +<>= +print(xtable(frame)) +@ + +\clearpage + + +<>= +toLatex(sessionInfo()) +@ + + +\end{document} +

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