R/testNhoods.R
In MikeDMorgan/miloR: Differential neighbourhood abundance testing on a graph
Defines functions
testNhoods
Documented in
testNhoods
#' Perform differential neighbourhood abundance testing
#'
#' This will perform differential neighbourhood abundance testing after cell
#' counting.
#' @param x A \code{\linkS4class{Milo}} object with a non-empty
#' \code{nhoodCounts} slot.
#' @param design A \code{formula} or \code{model.matrix} object describing the
#' experimental design for differential abundance testing. The last component
#' of the formula or last column of the model matrix are by default the test
#' variable. This behaviour can be overridden by setting the \code{model.contrasts}
#' argument
#' @param design.df A \code{data.frame} containing meta-data to which \code{design}
#' refers to
#' @param kinship (optional) An n X n \code{matrix} containing pair-wise relationships between
#' observations, such as expected relationships or computed from SNPs/SNVs/other genetic variants.
#' Row names and column names should correspond to the column names of \code{nhoods(x)} and rownames
#' of \code{design.df}.
#' @param min.mean A scalar used to threshold neighbourhoods on the minimum
#' average cell counts across samples.
#' @param model.contrasts A string vector that defines the contrasts used to perform
#' DA testing. For a specific comparison we recommend a single contrast be passed to
#' \code{testNhoods}. More details can be found in the vignette \code{milo_contrasts}.
#' @param fdr.weighting The spatial FDR weighting scheme to use. Choice from max,
#' neighbour-distance, graph-overlap or k-distance (default). If \code{none} is passed no
#' spatial FDR correction is performed and returns a vector of NAs.
#' @param robust If robust=TRUE then this is passed to edgeR and limma which use a robust
#' estimation for the global quasilikelihood dispersion distribution. See \code{edgeR} and
#' Phipson et al, 2013 for details.
#' @param norm.method A character scalar, either \code{"logMS"}, \code{"TMM"} or \code{"RLE"}.
#' The \code{"logMS"} method normalises the counts across samples using the log columns sums of
#' the count matrix as a model offset. \code{"TMM"} uses the trimmed mean of M-values normalisation
#' as described in Robinson & Oshlack, 2010, whilst \code{"RLE"} uses the relative log expression
#' method by Anders & Huber, 2010, to compute normalisation factors relative to a reference computed from
#' the geometric mean across samples. The latter methods provides a degree of robustness against false positives
#' when there are very large compositional differences between samples.
#' @param cell.sizes A named numeric vector of cell numbers per experimental samples. Names should correspond
#' to the columns of \code{nhoodCounts}. This can be used to define the model normalisation factors based on
#' a set of numbers instead of the \code{colSums(nhoodCounts(x))}. The example use-case is when performing an
#' analysis of a subset of nhoods while retaining the need to normalisation based on the numbers of cells
#' collected for each experimental sample to avoid compositional biases. Infinite or NA values will give an error.
#' @param reduced.dim A character scalar referring to the reduced dimensional slot used to compute distances for
#' the spatial FDR. This should be the same as used for graph building.
#' @param REML A logical scalar that controls the variance component behaviour to use either restricted maximum
#' likelihood (REML) or maximum likelihood (ML). The former is recommened to account for the bias in the ML
#' variance estimates.
#' @param glmm.solver A character scalar that determines which GLMM solver is applied. Must be one of: Fisher, HE
#' or HE-NNLS. HE or HE-NNLS are recommended when supplying a user-defined covariance matrix.
#' @param max.iters A scalar that determines the maximum number of iterations to run the GLMM solver if it does
#' not reach the convergence tolerance threshold.
#' @param max.tol A scalar that deterimines the GLMM solver convergence tolerance. It is recommended to keep
#' this number small to provide some confidence that the parameter estimates are at least in a feasible region
#' and close to a \emph{local} optimum
#' @param subset.nhoods A character, numeric or logical vector that will subset the analysis to the specific nhoods. If
#' a character vector these should correspond to row names of \code{nhoodCounts}. If a logical vector then
#' these should have the same \code{length} as \code{nrow} of \code{nhoodCounts}. If numeric, then these are assumed
#' to correspond to indices of \code{nhoodCounts} - if the maximal index is greater than \code{nrow(nhoodCounts(x))}
#' an error will be produced.
#' @param fail.on.error A logical scalar the determines the behaviour of the error reporting. Used for debugging only.
#' @param BPPARAM A \linkS4class{BiocParallelParam} object specifying the arguments for parallelisation. By default
#' this will evaluate using \code{SerialParam()}. See \code{details}on how to use parallelisation in \code{testNhoods}.
#'
#' @details
#' This function wraps up several steps of differential abundance testing using
#' the \code{edgeR} functions. These could be performed separately for users
#' who want to exercise more contol over their DA testing. By default this
#' function sets the \code{lib.sizes} to the colSums(x), and uses the
#' Quasi-Likelihood F-test in \code{glmQLFTest} for DA testing. FDR correction
#' is performed separately as the default multiple-testing correction is
#' inappropriate for neighbourhoods with overlapping cells.
#' The GLMM testing cannot be performed using \code{edgeR}, however, a separate
#' function \code{fitGLMM} can be used to fit a mixed effect model to each
#' nhood (see \code{fitGLMM} docs for details).
#'
#' Parallelisation is currently only enabled for the NB-LMM and uses the BiocParallel paradigm. In
#' general the GLM implementation in \code{glmQLFit} is sufficiently fast that it does not require
#' parallelisation. Parallelisation requires the user to pass a \linkS4class{BiocParallelParam} object
#' with the parallelisation arguments contained therein. This relies on the user to specify how
#' parallelisation - for details see the \code{BiocParallel} package.
#'
#' @return A \code{data.frame} of model results, which contain:
#' \describe{
#' \item{\code{logFC}:}{Numeric, the log fold change between conditions, or for
#' an ordered/continous variable the per-unit
#' change in (normalized) cell counts per unit-change in experimental variable.}
#' \item{\code{logCPM}:}{Numeric, the log counts per million (CPM), which equates
#' to the average log normalized cell counts
#' across all samples.}
#' \item{\code{F}:}{Numeric, the F-test statistic from the quali-likelihood F-test
#' implemented in \code{edgeR}.}
#' \item{\code{PValue}:}{Numeric, the unadjusted p-value from the quasi-likelihood F-test.}
#' \item{\code{FDR}:}{Numeric, the Benjamini & Hochberg false discovery weight
#' computed from \code{p.adjust}.}
#' \item{\code{Nhood}:}{Numeric, a unique identifier corresponding to the specific
#' graph neighbourhood.}
#' \item{\code{SpatialFDR}:}{Numeric, the weighted FDR, computed to adjust for spatial
#' graph overlaps between neighbourhoods. For details see \link{graphSpatialFDR}.}
#' }
#'
#' @author Mike Morgan
#'
#' @examples
#' library(SingleCellExperiment)
#' ux.1 <- matrix(rpois(12000, 5), ncol=400)
#' ux.2 <- matrix(rpois(12000, 4), ncol=400)
#' ux <- rbind(ux.1, ux.2)
#' vx <- log2(ux + 1)
#' pca <- prcomp(t(vx))
#'
#' sce <- SingleCellExperiment(assays=list(counts=ux, logcounts=vx),
#' reducedDims=SimpleList(PCA=pca$x))
#'
#' milo <- Milo(sce)
#' milo <- buildGraph(milo, k=20, d=10, transposed=TRUE)
#' milo <- makeNhoods(milo, k=20, d=10, prop=0.3)
#' milo <- calcNhoodDistance(milo, d=10)
#'
#' cond <- rep("A", ncol(milo))
#' cond.a <- sample(1:ncol(milo), size=floor(ncol(milo)*0.25))
#' cond.b <- setdiff(1:ncol(milo), cond.a)
#' cond[cond.b] <- "B"
#' meta.df <- data.frame(Condition=cond, Replicate=c(rep("R1", 132), rep("R2", 132), rep("R3", 136)))
#' meta.df$SampID <- paste(meta.df$Condition, meta.df$Replicate, sep="_")
#' milo <- countCells(milo, meta.data=meta.df, samples="SampID")
#'
#' test.meta <- data.frame("Condition"=c(rep("A", 3), rep("B", 3)), "Replicate"=rep(c("R1", "R2", "R3"), 2))
#' test.meta$Sample <- paste(test.meta$Condition, test.meta$Replicate, sep="_")
#' rownames(test.meta) <- test.meta$Sample
#' da.res <- testNhoods(milo, design=~Condition, design.df=test.meta[colnames(nhoodCounts(milo)), ], norm.method="TMM")
#' da.res
#'
#' @name testNhoods
NULL
#' @export
#' @importFrom Matrix colSums rowMeans
#' @importFrom MatrixGenerics colSums2
#' @importFrom utils tail
#' @importFrom stats dist median model.matrix
#' @importFrom limma makeContrasts
#' @importFrom BiocParallel bplapply SerialParam bptry bpok bpoptions
#' @importFrom edgeR DGEList estimateDisp glmQLFit glmQLFTest topTags calcNormFactors
testNhoods <- function(x, design, design.df, kinship=NULL,
fdr.weighting=c("k-distance", "neighbour-distance", "max", "graph-overlap", "none"),
min.mean=0, model.contrasts=NULL, robust=TRUE, reduced.dim="PCA", REML=TRUE,
norm.method=c("TMM", "RLE", "logMS"), cell.sizes=NULL,
max.iters = 50, max.tol = 1e-5, glmm.solver=NULL,
subset.nhoods=NULL, fail.on.error=FALSE, BPPARAM=SerialParam()){
is.lmm <- FALSE
geno.only <- FALSE
if(is(design, "formula")){
# parse to find random and fixed effects - need to double check the formula is valid
parse <- unlist(strsplit(gsub(design, pattern="~", replacement=""), split= "+", fixed=TRUE))
if(length(parse) < 1){
stop("Forumla ", design, " not a proper formula - variables should be separated by '+'")
}
find_re <- any(grepl(parse, pattern="1\\W?\\|"))
if(!find_re & any(grepl(parse, pattern="[0-9]?\\W?\\|\\W?"))){
stop(design, " is an invalid formula for random effects. Use (1 | VARIABLE) format.")
}
if(find_re | !is.null(kinship)){
message("Random effects found")
is.lmm <- TRUE
if(find_re | is.null(kinship)){
# make model matrices for fixed and random effects
z.model <- .parse_formula(design, design.df, vtype="re")
rownames(z.model) <- rownames(design.df)
} else if(find_re & !is.null(kinship)){
if(!all(rownames(kinship) == rownames(design.df))){
stop("Genotype rownames do not match design.df rownames")
}
z.model <- .parse_formula(design, design.df, vtype="re")
rownames(z.model) <- rownames(design.df)
} else if(!find_re & !is.null(kinship)){
z.model <- diag(nrow(kinship))
colnames(z.model) <- paste0("Genetic", seq_len(nrow(kinship)))
rownames(z.model) <- rownames(design.df)
geno.only <- TRUE
}
# this will always implicitly include an intercept term - perhaps
# this shouldn't be the case?
x.model <- .parse_formula(design, design.df, vtype="fe")
rownames(x.model) <- rownames(design.df)
max.iters <- max.iters
if(all(rownames(x.model) != rownames(z.model))){
stop("Discordant sample names for mixed model design matrices")
}
} else{
x.model <- model.matrix(design, data=design.df)
rownames(x.model) <- rownames(design.df)
}
} else if(is(design, "matrix")){
x.model <- design
if(nrow(x.model) != nrow(design.df)){
stop("Design matrix and model matrix are not the same dimensionality")
}
if(any(rownames(x.model) != rownames(design.df))){
warning("Design matrix and model matrix dimnames are not the same")
# check if rownames are a subset of the design.df
check.names <- any(rownames(x.model) %in% rownames(design.df))
if(isTRUE(check.names)){
rownames(x.model) <- rownames(design.df)
} else{
stop("Design matrix and model matrix rownames are not a subset")
}
}
} else{
stop("design must be either a formula or model matrix")
}
if(!is(x, "Milo")){
stop("Unrecognised input type - must be of class Milo")
} else if(.check_empty(x, "nhoodCounts")){
stop("Neighbourhood counts missing - please run countCells first")
}
if(!any(norm.method %in% c("TMM", "logMS", "RLE"))){
stop("Normalisation method ", norm.method, " not recognised. Must be either TMM, RLE or logMS")
}
if(!reduced.dim %in% reducedDimNames(x)){
stop(reduced.dim, " is not found in reducedDimNames. Avaiable options are ", paste(reducedDimNames(x), collapse=","))
}
subset.counts <- FALSE
if(ncol(nhoodCounts(x)) != nrow(x.model)){
# need to allow for design.df with a subset of samples only
if(all(rownames(x.model) %in% colnames(nhoodCounts(x)))){
message("Design matrix is a strict subset of the nhood counts")
subset.counts <- TRUE
} else{
stop("Design matrix (", nrow(x.model), ") and nhood counts (", ncol(nhoodCounts(x)), ") are not the same dimension")
}
}
if(is.lmm){
if(ncol(nhoodCounts(x)) != nrow(z.model)){
# need to allow for design.df with a subset of samples only
if(all(rownames(z.model) %in% colnames(nhoodCounts(x)))){
message("Random effects design matrix is a strict subset of the nhood counts")
subset.counts <- TRUE
} else{
stop("Random effects design matrix (", nrow(z.model), ") and nhood counts (",
ncol(nhoodCounts(x)), ") are not the same dimension")
}
}
}
# assume nhoodCounts and model are in the same order
# cast as DGEList doesn't accept sparse matrices
# what is the cost of cast a matrix that is already dense vs. testing it's class
if(min.mean > 0){
if(isTRUE(subset.counts)){
if(!is.null(subset.nhoods)){
mean.keep <- rowMeans(nhoodCounts(x)[, rownames(x.model)]) >= min.mean
if(is.character(subset.nhoods)){
if(all(subset.nhoods) %in% rownames(nhoodCounts(x))){
keep.nh <- rownames(nhoodCounts(x) %in% subset.nhoods)
} else{
stop("Nhood subsetting is illegal - use same names as in rownames of nhoodCounts")
}
} else if(is.logical(subset.nhoods)){
if(length(subset.nhoods) != nrow(nhoodCounts(x))){
stop("Logical subset vector must be same length as nrow nhoodCounts")
} else{
keep.nh <- subset.nhoods
}
} else if(is.numeric(subset.nhoods)){
if(max(subset.nhoods) > nrow(nhoodCounts(x))){
stop("Maximum index is out of bounds: ", max(subset.nhoods))
} else{
keep.nh <- seq_len(nrow(nhoodCounts(x))) %in% subset.nhoods
}
} else{
stop("Subsetting vector type not recognised: ", type(subset.nhoods))
}
keep.nh <- mean.keep & keep.nh
} else{
keep.nh <- rowMeans(nhoodCounts(x)[, rownames(x.model)]) >= min.mean
}
} else if(!is.null(subset.nhoods)){
mean.keep <- rowMeans(nhoodCounts(x)[, rownames(x.model)]) >= min.mean
if(is.character(subset.nhoods)){
if(all(subset.nhoods) %in% rownames(nhoodCounts(x))){
keep.nh <- rownames(nhoodCounts(x) %in% subset.nhoods)
} else{
stop("Nhood subsetting is illegal - use same names as in rownames of nhoodCounts")
}
} else if(is.logical(subset.nhoods)){
if(length(subset.nhoods) != nrow(nhoodCounts(x))){
stop("Logical subset vector must be same length as nrow nhoodCounts")
} else{
keep.nh <- subset.nhoods
}
} else if(is.numeric(subset.nhoods)){
if(max(subset.nhoods) > nrow(nhoodCounts(x))){
stop("Maximum index is out of bounds: ", max(subset.nhoods))
} else{
keep.nh <- seq_len(nrow(nhoodCounts(x))) %in% subset.nhoods
}
} else{
stop("Subsetting vector type not recognised: ", type(subset.nhoods))
}
keep.nh <- mean.keep & keep.nh
} else{
keep.nh <- rowMeans(nhoodCounts(x)) >= min.mean
}
} else if(!is.null(subset.nhoods)){
if(is.character(subset.nhoods)){
if(all(subset.nhoods) %in% rownames(nhoodCounts(x))){
keep.nh <- rownames(nhoodCounts(x) %in% subset.nhoods)
} else{
stop("Nhood subsetting is illegal - use same names as in rownames of nhoodCounts")
}
} else if(is.logical(subset.nhoods)){
if(length(subset.nhoods) != nrow(nhoodCounts(x))){
stop("Logical subset vector must be same length as nrow nhoodCounts")
} else{
keep.nh <- subset.nhoods
}
} else if(is.numeric(subset.nhoods)){
if(max(subset.nhoods) > nrow(nhoodCounts(x))){
stop("Maximum index is out of bounds: ", max(subset.nhoods))
} else{
keep.nh <- seq_len(nrow(nhoodCounts(x))) %in% subset.nhoods
}
} else{
stop("Subsetting vector type not recognised: ", type(subset.nhoods))
}
} else{
if(isTRUE(subset.counts)){
keep.nh <- rep(TRUE, nrow(nhoodCounts(x)[, rownames(x.model)]))
}else{
keep.nh <- rep(TRUE, nrow(nhoodCounts(x)))
}
}
if(isTRUE(subset.counts)){
keep.samps <- intersect(rownames(x.model), colnames(nhoodCounts(x)[keep.nh, ]))
} else{
keep.samps <- colnames(nhoodCounts(x)[keep.nh, ])
}
if(any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) != rownames(x.model)) & !any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) %in% rownames(x.model))){
stop("Sample names in design matrix and nhood counts are not matched.
Set appropriate rownames in design matrix.")
} else if(any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) != rownames(x.model)) & any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) %in% rownames(x.model))){
warning("Sample names in design matrix and nhood counts are not matched. Reordering")
x.model <- x.model[colnames(nhoodCounts(x)[keep.nh, keep.samps]), ]
if(is.lmm){
z.model <- z.model[colnames(nhoodCounts(x)[keep.nh, keep.samps]), , drop = FALSE]
}
}
if(isTRUE(is.null(cell.sizes))){
cell.sizes <- colSums(nhoodCounts(x)[keep.nh, keep.samps])
} else{
check_inf_na <- any(is.na(cell.sizes)) | any(is.infinite(cell.sizes))
if(isTRUE(check_inf_na)){
stop("NA or Infinite values found in cell.sizes - remove samples these and re-try")
}
}
if(length(norm.method) > 1){
message("Using TMM normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
dge <- calcNormFactors(dge, method="TMM")
} else if(norm.method %in% c("TMM")){
message("Using TMM normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
dge <- calcNormFactors(dge, method="TMM")
} else if(norm.method %in% c("RLE")){
message("Using RLE normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
dge <- calcNormFactors(dge, method="RLE")
}else if(norm.method %in% c("logMS")){
message("Using logMS normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
}
dge <- estimateDisp(dge, x.model)
if (is.lmm) {
message("Running GLMM model - this may take a few minutes")
if(isFALSE(geno.only)){
rand.levels <- lapply(seq_along(colnames(z.model)), FUN=function(X) {
zx <- unique(z.model[, X])
if(is.numeric(zx)){
paste0(colnames(z.model)[X], zx)
} else{
zx
}
})
names(rand.levels) <- colnames(z.model)
} else{
rand.levels <- list("Genetic"=colnames(z.model))
}
# extract tagwise dispersion for glmm
# re-scale these to allow for non-zero variances
dispersion <- dge$tagwise.dispersion
# I think these need to be logged
offsets <- log(dge$samples$norm.factors)
glmm.cont <- list(theta.tol=max.tol, max.iter=max.iters, solver=glmm.solver)
#wrapper function is the same for all analyses
glmmWrapper <- function(Y, disper, Xmodel, Zmodel, off.sets, randlevels,
reml, glmm.contr, genonly=FALSE, kin.ship=NULL,
BPPARAM=BPPARAM, error.fail=FALSE){
#bp.list <- NULL
# this needs to be able to run with BiocParallel
bp.list <- bptry({bplapply(seq_len(nrow(Y)), BPOPTIONS=bpoptions(stop.on.error = error.fail),
FUN=function(i, Xmodel, Zmodel, Y, off.sets,
randlevels, disper, genonly,
kin.ship, glmm.contr, reml){
fitGLMM(X=Xmodel, Z=Zmodel, y=Y[i, ], offsets=off.sets,
random.levels=randlevels, REML = reml,
dispersion=disper[i], geno.only=genonly,
Kin=kinship, glmm.control=glmm.contr)
}, BPPARAM=BPPARAM,
Xmodel=Xmodel, Zmodel=Zmodel, Y=Y, off.sets=off.sets,
randlevels=randlevels, disper=disper, genonly=genonly,
kin.ship=kin.ship, glmm.cont=glmm.cont, reml=reml)
}) # need to handle this output which is a bplist_error object
# parse the bplist_error object
if(all(bpok(bp.list))){
model.list <- as(bp.list, "list")
} else{
model.list <- list()
# set the failed results to all NA
for(x in seq_along(bp.list)){
if(!bpok(bp.list)[x]){
bperr <- attr(bp.list[[x]], "traceback")
if(isTRUE(error.fail)){
stop(bperr)
}
model.list[[x]] <- list("FE"=NA, "RE"=NA, "Sigma"=NA,
"converged"=FALSE, "Iters"=NA, "Dispersion"=NA,
"Hessian"=NA, "SE"=NA, "t"=NA, "PSVAR"=NA,
"COEFF"=NA, "P"=NA, "Vpartial"=NA, "Ginv"=NA,
"Vsinv"=NA, "Winv"=NA, "VCOV"=NA, "LOGLIHOOD"=NA,
"DF"=NA, "PVALS"=NA,
"ERROR"=bperr)
}else{
model.list[[x]] <- bp.list[[x]]
}
}
}
return(model.list)
}
if(!is.null(kinship)){
if(isTRUE(geno.only)){
message("Running genetic model with ", nrow(kinship), " individuals")
} else{
message("Running genetic model with ", nrow(z.model), " observations")
}
if(geno.only){
fit <- glmmWrapper(Y=dge$counts, disper = 1/dispersion, Xmodel=x.model, Zmodel=z.model,
off.sets=offsets, randlevels=rand.levels, reml=REML, glmm.contr = glmm.cont,
genonly = geno.only, kin.ship=kinship,
BPPARAM=BPPARAM, error.fail=fail.on.error)
} else{
fit <- glmmWrapper(Y=dge$counts, disper = 1/dispersion, Xmodel=x.model, Zmodel=z.model,
off.sets=offsets, randlevels=rand.levels, reml=REML, glmm.contr = glmm.cont,
genonly = geno.only, kin.ship=kinship,
BPPARAM=BPPARAM, error.fail=fail.on.error)
}
} else{
fit <- glmmWrapper(Y=dge$counts, disper = 1/dispersion, Xmodel=x.model, Zmodel=z.model,
off.sets=offsets, randlevels=rand.levels, reml=REML, glmm.contr = glmm.cont,
genonly = geno.only, kin.ship=kinship,
BPPARAM=BPPARAM, error.fail=fail.on.error)
}
# give warning about how many neighborhoods didn't converge and error if > 50% nhoods failed
n.nhoods <- length(fit)
half.n <- floor(n.nhoods * 0.5)
if (sum(!(unlist(lapply(fit, `[[`, "converged"))), na.rm = TRUE)/length(unlist(lapply(fit, `[[`, "converged"))) > 0){
if(sum(is.na(unlist(lapply(fit, `[[`, "FE")))) >= half.n){
err.list <- paste(unique(unlist(lapply(fit, `[[`, "ERROR"))), collapse="\n")
stop("Lowest traceback returned: ", err.list) # all unique error messages
} else{
warning(paste(sum(!unlist(lapply(fit, `[[`, "converged")), na.rm = TRUE), "out of", length(unlist(lapply(fit, `[[`, "converged"))),
"neighborhoods did not converge; increase number of iterations?"))
}
}
# res has to reflect output from glmQLFit - express variance as a proportion as well.
# this only reports the final fixed effect parameter
ret.beta <- ncol(x.model)
res <- cbind.data.frame("logFC" = unlist(lapply(lapply(fit, `[[`, "FE"), function(x) x[ret.beta])),
"logCPM"=log2((rowMeans(nhoodCounts(x)[keep.nh, ]/colSums2(nhoodCounts(x))))*1e6),
"SE"= unlist(lapply(lapply(fit, `[[`, "SE"), function(x) x[ret.beta])),
"tvalue" = unlist(lapply(lapply(fit, `[[`, "t"), function(x) x[ret.beta])),
"PValue" = unlist(lapply(lapply(fit, `[[`, "PVALS"), function(x) x[ret.beta])),
matrix(unlist(lapply(fit, `[[`, "Sigma")), ncol=length(rand.levels), byrow=TRUE),
"Converged"=unlist(lapply(fit, `[[`, "converged")), "Dispersion" = unlist(lapply(fit, `[[`, "Dispersion")),
"Logliklihood"=unlist(lapply(fit, `[[`, "LOGLIHOOD")))
rownames(res) <- 1:length(fit)
colnames(res)[6:(6+length(rand.levels)-1)] <- paste(names(rand.levels), "variance")
} else {
fit <- glmQLFit(dge, x.model, robust=robust)
if(!is.null(model.contrasts)){
mod.constrast <- makeContrasts(contrasts=model.contrasts, levels=x.model)
res <- as.data.frame(topTags(glmQLFTest(fit, contrast=mod.constrast),
sort.by='none', n=Inf))
} else{
n.coef <- ncol(x.model)
res <- as.data.frame(topTags(glmQLFTest(fit, coef=n.coef), sort.by='none', n=Inf))
}
}
res$Nhood <- as.numeric(rownames(res))
message("Performing spatial FDR correction with ", fdr.weighting[1], " weighting")
# res1 <- na.omit(res)
mod.spatialfdr <- graphSpatialFDR(x.nhoods=nhoods(x),
graph=graph(x),
weighting=fdr.weighting,
k=x@.k,
pvalues=res[order(res$Nhood), ]$PValue,
indices=nhoodIndex(x),
distances=nhoodDistances(x),
reduced.dimensions=reducedDim(x, reduced.dim))
res$SpatialFDR[order(res$Nhood)] <- mod.spatialfdr
res
}
MikeDMorgan/miloR documentation built on Feb. 29, 2024, 6:20 p.m.
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Defines functions testNhoods
Documented in testNhoods
#' Perform differential neighbourhood abundance testing
#'
#' This will perform differential neighbourhood abundance testing after cell
#' counting.
#' @param x A \code{\linkS4class{Milo}} object with a non-empty
#' \code{nhoodCounts} slot.
#' @param design A \code{formula} or \code{model.matrix} object describing the
#' experimental design for differential abundance testing. The last component
#' of the formula or last column of the model matrix are by default the test
#' variable. This behaviour can be overridden by setting the \code{model.contrasts}
#' argument
#' @param design.df A \code{data.frame} containing meta-data to which \code{design}
#' refers to
#' @param kinship (optional) An n X n \code{matrix} containing pair-wise relationships between
#' observations, such as expected relationships or computed from SNPs/SNVs/other genetic variants.
#' Row names and column names should correspond to the column names of \code{nhoods(x)} and rownames
#' of \code{design.df}.
#' @param min.mean A scalar used to threshold neighbourhoods on the minimum
#' average cell counts across samples.
#' @param model.contrasts A string vector that defines the contrasts used to perform
#' DA testing. For a specific comparison we recommend a single contrast be passed to
#' \code{testNhoods}. More details can be found in the vignette \code{milo_contrasts}.
#' @param fdr.weighting The spatial FDR weighting scheme to use. Choice from max,
#' neighbour-distance, graph-overlap or k-distance (default). If \code{none} is passed no
#' spatial FDR correction is performed and returns a vector of NAs.
#' @param robust If robust=TRUE then this is passed to edgeR and limma which use a robust
#' estimation for the global quasilikelihood dispersion distribution. See \code{edgeR} and
#' Phipson et al, 2013 for details.
#' @param norm.method A character scalar, either \code{"logMS"}, \code{"TMM"} or \code{"RLE"}.
#' The \code{"logMS"} method normalises the counts across samples using the log columns sums of
#' the count matrix as a model offset. \code{"TMM"} uses the trimmed mean of M-values normalisation
#' as described in Robinson & Oshlack, 2010, whilst \code{"RLE"} uses the relative log expression
#' method by Anders & Huber, 2010, to compute normalisation factors relative to a reference computed from
#' the geometric mean across samples. The latter methods provides a degree of robustness against false positives
#' when there are very large compositional differences between samples.
#' @param cell.sizes A named numeric vector of cell numbers per experimental samples. Names should correspond
#' to the columns of \code{nhoodCounts}. This can be used to define the model normalisation factors based on
#' a set of numbers instead of the \code{colSums(nhoodCounts(x))}. The example use-case is when performing an
#' analysis of a subset of nhoods while retaining the need to normalisation based on the numbers of cells
#' collected for each experimental sample to avoid compositional biases. Infinite or NA values will give an error.
#' @param reduced.dim A character scalar referring to the reduced dimensional slot used to compute distances for
#' the spatial FDR. This should be the same as used for graph building.
#' @param REML A logical scalar that controls the variance component behaviour to use either restricted maximum
#' likelihood (REML) or maximum likelihood (ML). The former is recommened to account for the bias in the ML
#' variance estimates.
#' @param glmm.solver A character scalar that determines which GLMM solver is applied. Must be one of: Fisher, HE
#' or HE-NNLS. HE or HE-NNLS are recommended when supplying a user-defined covariance matrix.
#' @param max.iters A scalar that determines the maximum number of iterations to run the GLMM solver if it does
#' not reach the convergence tolerance threshold.
#' @param max.tol A scalar that deterimines the GLMM solver convergence tolerance. It is recommended to keep
#' this number small to provide some confidence that the parameter estimates are at least in a feasible region
#' and close to a \emph{local} optimum
#' @param subset.nhoods A character, numeric or logical vector that will subset the analysis to the specific nhoods. If
#' a character vector these should correspond to row names of \code{nhoodCounts}. If a logical vector then
#' these should have the same \code{length} as \code{nrow} of \code{nhoodCounts}. If numeric, then these are assumed
#' to correspond to indices of \code{nhoodCounts} - if the maximal index is greater than \code{nrow(nhoodCounts(x))}
#' an error will be produced.
#' @param fail.on.error A logical scalar the determines the behaviour of the error reporting. Used for debugging only.
#' @param BPPARAM A \linkS4class{BiocParallelParam} object specifying the arguments for parallelisation. By default
#' this will evaluate using \code{SerialParam()}. See \code{details}on how to use parallelisation in \code{testNhoods}.
#'
#' @details
#' This function wraps up several steps of differential abundance testing using
#' the \code{edgeR} functions. These could be performed separately for users
#' who want to exercise more contol over their DA testing. By default this
#' function sets the \code{lib.sizes} to the colSums(x), and uses the
#' Quasi-Likelihood F-test in \code{glmQLFTest} for DA testing. FDR correction
#' is performed separately as the default multiple-testing correction is
#' inappropriate for neighbourhoods with overlapping cells.
#' The GLMM testing cannot be performed using \code{edgeR}, however, a separate
#' function \code{fitGLMM} can be used to fit a mixed effect model to each
#' nhood (see \code{fitGLMM} docs for details).
#'
#' Parallelisation is currently only enabled for the NB-LMM and uses the BiocParallel paradigm. In
#' general the GLM implementation in \code{glmQLFit} is sufficiently fast that it does not require
#' parallelisation. Parallelisation requires the user to pass a \linkS4class{BiocParallelParam} object
#' with the parallelisation arguments contained therein. This relies on the user to specify how
#' parallelisation - for details see the \code{BiocParallel} package.
#'
#' @return A \code{data.frame} of model results, which contain:
#' \describe{
#' \item{\code{logFC}:}{Numeric, the log fold change between conditions, or for
#' an ordered/continous variable the per-unit
#' change in (normalized) cell counts per unit-change in experimental variable.}
#' \item{\code{logCPM}:}{Numeric, the log counts per million (CPM), which equates
#' to the average log normalized cell counts
#' across all samples.}
#' \item{\code{F}:}{Numeric, the F-test statistic from the quali-likelihood F-test
#' implemented in \code{edgeR}.}
#' \item{\code{PValue}:}{Numeric, the unadjusted p-value from the quasi-likelihood F-test.}
#' \item{\code{FDR}:}{Numeric, the Benjamini & Hochberg false discovery weight
#' computed from \code{p.adjust}.}
#' \item{\code{Nhood}:}{Numeric, a unique identifier corresponding to the specific
#' graph neighbourhood.}
#' \item{\code{SpatialFDR}:}{Numeric, the weighted FDR, computed to adjust for spatial
#' graph overlaps between neighbourhoods. For details see \link{graphSpatialFDR}.}
#' }
#'
#' @author Mike Morgan
#'
#' @examples
#' library(SingleCellExperiment)
#' ux.1 <- matrix(rpois(12000, 5), ncol=400)
#' ux.2 <- matrix(rpois(12000, 4), ncol=400)
#' ux <- rbind(ux.1, ux.2)
#' vx <- log2(ux + 1)
#' pca <- prcomp(t(vx))
#'
#' sce <- SingleCellExperiment(assays=list(counts=ux, logcounts=vx),
#' reducedDims=SimpleList(PCA=pca$x))
#'
#' milo <- Milo(sce)
#' milo <- buildGraph(milo, k=20, d=10, transposed=TRUE)
#' milo <- makeNhoods(milo, k=20, d=10, prop=0.3)
#' milo <- calcNhoodDistance(milo, d=10)
#'
#' cond <- rep("A", ncol(milo))
#' cond.a <- sample(1:ncol(milo), size=floor(ncol(milo)*0.25))
#' cond.b <- setdiff(1:ncol(milo), cond.a)
#' cond[cond.b] <- "B"
#' meta.df <- data.frame(Condition=cond, Replicate=c(rep("R1", 132), rep("R2", 132), rep("R3", 136)))
#' meta.df$SampID <- paste(meta.df$Condition, meta.df$Replicate, sep="_")
#' milo <- countCells(milo, meta.data=meta.df, samples="SampID")
#'
#' test.meta <- data.frame("Condition"=c(rep("A", 3), rep("B", 3)), "Replicate"=rep(c("R1", "R2", "R3"), 2))
#' test.meta$Sample <- paste(test.meta$Condition, test.meta$Replicate, sep="_")
#' rownames(test.meta) <- test.meta$Sample
#' da.res <- testNhoods(milo, design=~Condition, design.df=test.meta[colnames(nhoodCounts(milo)), ], norm.method="TMM")
#' da.res
#'
#' @name testNhoods
NULL
#' @export
#' @importFrom Matrix colSums rowMeans
#' @importFrom MatrixGenerics colSums2
#' @importFrom utils tail
#' @importFrom stats dist median model.matrix
#' @importFrom limma makeContrasts
#' @importFrom BiocParallel bplapply SerialParam bptry bpok bpoptions
#' @importFrom edgeR DGEList estimateDisp glmQLFit glmQLFTest topTags calcNormFactors
testNhoods <- function(x, design, design.df, kinship=NULL,
fdr.weighting=c("k-distance", "neighbour-distance", "max", "graph-overlap", "none"),
min.mean=0, model.contrasts=NULL, robust=TRUE, reduced.dim="PCA", REML=TRUE,
norm.method=c("TMM", "RLE", "logMS"), cell.sizes=NULL,
max.iters = 50, max.tol = 1e-5, glmm.solver=NULL,
subset.nhoods=NULL, fail.on.error=FALSE, BPPARAM=SerialParam()){
is.lmm <- FALSE
geno.only <- FALSE
if(is(design, "formula")){
# parse to find random and fixed effects - need to double check the formula is valid
parse <- unlist(strsplit(gsub(design, pattern="~", replacement=""), split= "+", fixed=TRUE))
if(length(parse) < 1){
stop("Forumla ", design, " not a proper formula - variables should be separated by '+'")
}
find_re <- any(grepl(parse, pattern="1\\W?\\|"))
if(!find_re & any(grepl(parse, pattern="[0-9]?\\W?\\|\\W?"))){
stop(design, " is an invalid formula for random effects. Use (1 | VARIABLE) format.")
}
if(find_re | !is.null(kinship)){
message("Random effects found")
is.lmm <- TRUE
if(find_re | is.null(kinship)){
# make model matrices for fixed and random effects
z.model <- .parse_formula(design, design.df, vtype="re")
rownames(z.model) <- rownames(design.df)
} else if(find_re & !is.null(kinship)){
if(!all(rownames(kinship) == rownames(design.df))){
stop("Genotype rownames do not match design.df rownames")
}
z.model <- .parse_formula(design, design.df, vtype="re")
rownames(z.model) <- rownames(design.df)
} else if(!find_re & !is.null(kinship)){
z.model <- diag(nrow(kinship))
colnames(z.model) <- paste0("Genetic", seq_len(nrow(kinship)))
rownames(z.model) <- rownames(design.df)
geno.only <- TRUE
}
# this will always implicitly include an intercept term - perhaps
# this shouldn't be the case?
x.model <- .parse_formula(design, design.df, vtype="fe")
rownames(x.model) <- rownames(design.df)
max.iters <- max.iters
if(all(rownames(x.model) != rownames(z.model))){
stop("Discordant sample names for mixed model design matrices")
}
} else{
x.model <- model.matrix(design, data=design.df)
rownames(x.model) <- rownames(design.df)
}
} else if(is(design, "matrix")){
x.model <- design
if(nrow(x.model) != nrow(design.df)){
stop("Design matrix and model matrix are not the same dimensionality")
}
if(any(rownames(x.model) != rownames(design.df))){
warning("Design matrix and model matrix dimnames are not the same")
# check if rownames are a subset of the design.df
check.names <- any(rownames(x.model) %in% rownames(design.df))
if(isTRUE(check.names)){
rownames(x.model) <- rownames(design.df)
} else{
stop("Design matrix and model matrix rownames are not a subset")
}
}
} else{
stop("design must be either a formula or model matrix")
}
if(!is(x, "Milo")){
stop("Unrecognised input type - must be of class Milo")
} else if(.check_empty(x, "nhoodCounts")){
stop("Neighbourhood counts missing - please run countCells first")
}
if(!any(norm.method %in% c("TMM", "logMS", "RLE"))){
stop("Normalisation method ", norm.method, " not recognised. Must be either TMM, RLE or logMS")
}
if(!reduced.dim %in% reducedDimNames(x)){
stop(reduced.dim, " is not found in reducedDimNames. Avaiable options are ", paste(reducedDimNames(x), collapse=","))
}
subset.counts <- FALSE
if(ncol(nhoodCounts(x)) != nrow(x.model)){
# need to allow for design.df with a subset of samples only
if(all(rownames(x.model) %in% colnames(nhoodCounts(x)))){
message("Design matrix is a strict subset of the nhood counts")
subset.counts <- TRUE
} else{
stop("Design matrix (", nrow(x.model), ") and nhood counts (", ncol(nhoodCounts(x)), ") are not the same dimension")
}
}
if(is.lmm){
if(ncol(nhoodCounts(x)) != nrow(z.model)){
# need to allow for design.df with a subset of samples only
if(all(rownames(z.model) %in% colnames(nhoodCounts(x)))){
message("Random effects design matrix is a strict subset of the nhood counts")
subset.counts <- TRUE
} else{
stop("Random effects design matrix (", nrow(z.model), ") and nhood counts (",
ncol(nhoodCounts(x)), ") are not the same dimension")
}
}
}
# assume nhoodCounts and model are in the same order
# cast as DGEList doesn't accept sparse matrices
# what is the cost of cast a matrix that is already dense vs. testing it's class
if(min.mean > 0){
if(isTRUE(subset.counts)){
if(!is.null(subset.nhoods)){
mean.keep <- rowMeans(nhoodCounts(x)[, rownames(x.model)]) >= min.mean
if(is.character(subset.nhoods)){
if(all(subset.nhoods) %in% rownames(nhoodCounts(x))){
keep.nh <- rownames(nhoodCounts(x) %in% subset.nhoods)
} else{
stop("Nhood subsetting is illegal - use same names as in rownames of nhoodCounts")
}
} else if(is.logical(subset.nhoods)){
if(length(subset.nhoods) != nrow(nhoodCounts(x))){
stop("Logical subset vector must be same length as nrow nhoodCounts")
} else{
keep.nh <- subset.nhoods
}
} else if(is.numeric(subset.nhoods)){
if(max(subset.nhoods) > nrow(nhoodCounts(x))){
stop("Maximum index is out of bounds: ", max(subset.nhoods))
} else{
keep.nh <- seq_len(nrow(nhoodCounts(x))) %in% subset.nhoods
}
} else{
stop("Subsetting vector type not recognised: ", type(subset.nhoods))
}
keep.nh <- mean.keep & keep.nh
} else{
keep.nh <- rowMeans(nhoodCounts(x)[, rownames(x.model)]) >= min.mean
}
} else if(!is.null(subset.nhoods)){
mean.keep <- rowMeans(nhoodCounts(x)[, rownames(x.model)]) >= min.mean
if(is.character(subset.nhoods)){
if(all(subset.nhoods) %in% rownames(nhoodCounts(x))){
keep.nh <- rownames(nhoodCounts(x) %in% subset.nhoods)
} else{
stop("Nhood subsetting is illegal - use same names as in rownames of nhoodCounts")
}
} else if(is.logical(subset.nhoods)){
if(length(subset.nhoods) != nrow(nhoodCounts(x))){
stop("Logical subset vector must be same length as nrow nhoodCounts")
} else{
keep.nh <- subset.nhoods
}
} else if(is.numeric(subset.nhoods)){
if(max(subset.nhoods) > nrow(nhoodCounts(x))){
stop("Maximum index is out of bounds: ", max(subset.nhoods))
} else{
keep.nh <- seq_len(nrow(nhoodCounts(x))) %in% subset.nhoods
}
} else{
stop("Subsetting vector type not recognised: ", type(subset.nhoods))
}
keep.nh <- mean.keep & keep.nh
} else{
keep.nh <- rowMeans(nhoodCounts(x)) >= min.mean
}
} else if(!is.null(subset.nhoods)){
if(is.character(subset.nhoods)){
if(all(subset.nhoods) %in% rownames(nhoodCounts(x))){
keep.nh <- rownames(nhoodCounts(x) %in% subset.nhoods)
} else{
stop("Nhood subsetting is illegal - use same names as in rownames of nhoodCounts")
}
} else if(is.logical(subset.nhoods)){
if(length(subset.nhoods) != nrow(nhoodCounts(x))){
stop("Logical subset vector must be same length as nrow nhoodCounts")
} else{
keep.nh <- subset.nhoods
}
} else if(is.numeric(subset.nhoods)){
if(max(subset.nhoods) > nrow(nhoodCounts(x))){
stop("Maximum index is out of bounds: ", max(subset.nhoods))
} else{
keep.nh <- seq_len(nrow(nhoodCounts(x))) %in% subset.nhoods
}
} else{
stop("Subsetting vector type not recognised: ", type(subset.nhoods))
}
} else{
if(isTRUE(subset.counts)){
keep.nh <- rep(TRUE, nrow(nhoodCounts(x)[, rownames(x.model)]))
}else{
keep.nh <- rep(TRUE, nrow(nhoodCounts(x)))
}
}
if(isTRUE(subset.counts)){
keep.samps <- intersect(rownames(x.model), colnames(nhoodCounts(x)[keep.nh, ]))
} else{
keep.samps <- colnames(nhoodCounts(x)[keep.nh, ])
}
if(any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) != rownames(x.model)) & !any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) %in% rownames(x.model))){
stop("Sample names in design matrix and nhood counts are not matched.
Set appropriate rownames in design matrix.")
} else if(any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) != rownames(x.model)) & any(colnames(nhoodCounts(x)[keep.nh, keep.samps]) %in% rownames(x.model))){
warning("Sample names in design matrix and nhood counts are not matched. Reordering")
x.model <- x.model[colnames(nhoodCounts(x)[keep.nh, keep.samps]), ]
if(is.lmm){
z.model <- z.model[colnames(nhoodCounts(x)[keep.nh, keep.samps]), , drop = FALSE]
}
}
if(isTRUE(is.null(cell.sizes))){
cell.sizes <- colSums(nhoodCounts(x)[keep.nh, keep.samps])
} else{
check_inf_na <- any(is.na(cell.sizes)) | any(is.infinite(cell.sizes))
if(isTRUE(check_inf_na)){
stop("NA or Infinite values found in cell.sizes - remove samples these and re-try")
}
}
if(length(norm.method) > 1){
message("Using TMM normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
dge <- calcNormFactors(dge, method="TMM")
} else if(norm.method %in% c("TMM")){
message("Using TMM normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
dge <- calcNormFactors(dge, method="TMM")
} else if(norm.method %in% c("RLE")){
message("Using RLE normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
dge <- calcNormFactors(dge, method="RLE")
}else if(norm.method %in% c("logMS")){
message("Using logMS normalisation")
dge <- DGEList(counts=nhoodCounts(x)[keep.nh, keep.samps],
lib.size=cell.sizes)
}
dge <- estimateDisp(dge, x.model)
if (is.lmm) {
message("Running GLMM model - this may take a few minutes")
if(isFALSE(geno.only)){
rand.levels <- lapply(seq_along(colnames(z.model)), FUN=function(X) {
zx <- unique(z.model[, X])
if(is.numeric(zx)){
paste0(colnames(z.model)[X], zx)
} else{
zx
}
})
names(rand.levels) <- colnames(z.model)
} else{
rand.levels <- list("Genetic"=colnames(z.model))
}
# extract tagwise dispersion for glmm
# re-scale these to allow for non-zero variances
dispersion <- dge$tagwise.dispersion
# I think these need to be logged
offsets <- log(dge$samples$norm.factors)
glmm.cont <- list(theta.tol=max.tol, max.iter=max.iters, solver=glmm.solver)
#wrapper function is the same for all analyses
glmmWrapper <- function(Y, disper, Xmodel, Zmodel, off.sets, randlevels,
reml, glmm.contr, genonly=FALSE, kin.ship=NULL,
BPPARAM=BPPARAM, error.fail=FALSE){
#bp.list <- NULL
# this needs to be able to run with BiocParallel
bp.list <- bptry({bplapply(seq_len(nrow(Y)), BPOPTIONS=bpoptions(stop.on.error = error.fail),
FUN=function(i, Xmodel, Zmodel, Y, off.sets,
randlevels, disper, genonly,
kin.ship, glmm.contr, reml){
fitGLMM(X=Xmodel, Z=Zmodel, y=Y[i, ], offsets=off.sets,
random.levels=randlevels, REML = reml,
dispersion=disper[i], geno.only=genonly,
Kin=kinship, glmm.control=glmm.contr)
}, BPPARAM=BPPARAM,
Xmodel=Xmodel, Zmodel=Zmodel, Y=Y, off.sets=off.sets,
randlevels=randlevels, disper=disper, genonly=genonly,
kin.ship=kin.ship, glmm.cont=glmm.cont, reml=reml)
}) # need to handle this output which is a bplist_error object
# parse the bplist_error object
if(all(bpok(bp.list))){
model.list <- as(bp.list, "list")
} else{
model.list <- list()
# set the failed results to all NA
for(x in seq_along(bp.list)){
if(!bpok(bp.list)[x]){
bperr <- attr(bp.list[[x]], "traceback")
if(isTRUE(error.fail)){
stop(bperr)
}
model.list[[x]] <- list("FE"=NA, "RE"=NA, "Sigma"=NA,
"converged"=FALSE, "Iters"=NA, "Dispersion"=NA,
"Hessian"=NA, "SE"=NA, "t"=NA, "PSVAR"=NA,
"COEFF"=NA, "P"=NA, "Vpartial"=NA, "Ginv"=NA,
"Vsinv"=NA, "Winv"=NA, "VCOV"=NA, "LOGLIHOOD"=NA,
"DF"=NA, "PVALS"=NA,
"ERROR"=bperr)
}else{
model.list[[x]] <- bp.list[[x]]
}
}
}
return(model.list)
}
if(!is.null(kinship)){
if(isTRUE(geno.only)){
message("Running genetic model with ", nrow(kinship), " individuals")
} else{
message("Running genetic model with ", nrow(z.model), " observations")
}
if(geno.only){
fit <- glmmWrapper(Y=dge$counts, disper = 1/dispersion, Xmodel=x.model, Zmodel=z.model,
off.sets=offsets, randlevels=rand.levels, reml=REML, glmm.contr = glmm.cont,
genonly = geno.only, kin.ship=kinship,
BPPARAM=BPPARAM, error.fail=fail.on.error)
} else{
fit <- glmmWrapper(Y=dge$counts, disper = 1/dispersion, Xmodel=x.model, Zmodel=z.model,
off.sets=offsets, randlevels=rand.levels, reml=REML, glmm.contr = glmm.cont,
genonly = geno.only, kin.ship=kinship,
BPPARAM=BPPARAM, error.fail=fail.on.error)
}
} else{
fit <- glmmWrapper(Y=dge$counts, disper = 1/dispersion, Xmodel=x.model, Zmodel=z.model,
off.sets=offsets, randlevels=rand.levels, reml=REML, glmm.contr = glmm.cont,
genonly = geno.only, kin.ship=kinship,
BPPARAM=BPPARAM, error.fail=fail.on.error)
}
# give warning about how many neighborhoods didn't converge and error if > 50% nhoods failed
n.nhoods <- length(fit)
half.n <- floor(n.nhoods * 0.5)
if (sum(!(unlist(lapply(fit, `[[`, "converged"))), na.rm = TRUE)/length(unlist(lapply(fit, `[[`, "converged"))) > 0){
if(sum(is.na(unlist(lapply(fit, `[[`, "FE")))) >= half.n){
err.list <- paste(unique(unlist(lapply(fit, `[[`, "ERROR"))), collapse="\n")
stop("Lowest traceback returned: ", err.list) # all unique error messages
} else{
warning(paste(sum(!unlist(lapply(fit, `[[`, "converged")), na.rm = TRUE), "out of", length(unlist(lapply(fit, `[[`, "converged"))),
"neighborhoods did not converge; increase number of iterations?"))
}
}
# res has to reflect output from glmQLFit - express variance as a proportion as well.
# this only reports the final fixed effect parameter
ret.beta <- ncol(x.model)
res <- cbind.data.frame("logFC" = unlist(lapply(lapply(fit, `[[`, "FE"), function(x) x[ret.beta])),
"logCPM"=log2((rowMeans(nhoodCounts(x)[keep.nh, ]/colSums2(nhoodCounts(x))))*1e6),
"SE"= unlist(lapply(lapply(fit, `[[`, "SE"), function(x) x[ret.beta])),
"tvalue" = unlist(lapply(lapply(fit, `[[`, "t"), function(x) x[ret.beta])),
"PValue" = unlist(lapply(lapply(fit, `[[`, "PVALS"), function(x) x[ret.beta])),
matrix(unlist(lapply(fit, `[[`, "Sigma")), ncol=length(rand.levels), byrow=TRUE),
"Converged"=unlist(lapply(fit, `[[`, "converged")), "Dispersion" = unlist(lapply(fit, `[[`, "Dispersion")),
"Logliklihood"=unlist(lapply(fit, `[[`, "LOGLIHOOD")))
rownames(res) <- 1:length(fit)
colnames(res)[6:(6+length(rand.levels)-1)] <- paste(names(rand.levels), "variance")
} else {
fit <- glmQLFit(dge, x.model, robust=robust)
if(!is.null(model.contrasts)){
mod.constrast <- makeContrasts(contrasts=model.contrasts, levels=x.model)
res <- as.data.frame(topTags(glmQLFTest(fit, contrast=mod.constrast),
sort.by='none', n=Inf))
} else{
n.coef <- ncol(x.model)
res <- as.data.frame(topTags(glmQLFTest(fit, coef=n.coef), sort.by='none', n=Inf))
}
}
res$Nhood <- as.numeric(rownames(res))
message("Performing spatial FDR correction with ", fdr.weighting[1], " weighting")
# res1 <- na.omit(res)
mod.spatialfdr <- graphSpatialFDR(x.nhoods=nhoods(x),
graph=graph(x),
weighting=fdr.weighting,
k=x@.k,
pvalues=res[order(res$Nhood), ]$PValue,
indices=nhoodIndex(x),
distances=nhoodDistances(x),
reduced.dimensions=reducedDim(x, reduced.dim))
res$SpatialFDR[order(res$Nhood)] <- mod.spatialfdr
res
}
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