# Tests for functions dependent on a seurat object set.seed(42) pbmc.file <- system.file('extdata', 'pbmc_raw.txt', package = 'Seurat') pbmc.test <- as.sparse(x = as.matrix(read.table(pbmc.file, sep = "\t", row.names = 1))) # Tests for object creation (via CreateSeuratObject) # -------------------------------------------------------------------------------- context("Object creation") fake.meta.data <- data.frame(rep(1, ncol(pbmc.test))) rownames(fake.meta.data) <- colnames(pbmc.test) colnames(fake.meta.data) <- "FMD" object <- CreateSeuratObject(counts = pbmc.test, meta.data = fake.meta.data) test_that("object initialization actually creates seurat object", { expect_is(object, "Seurat") }) #this should be moved to seurat object # test_that("meta.data slot generated correctly", { # expect_equal(dim(object[[]]), c(80, 4)) # expect_equal(colnames(object[[]]), c("orig.ident", "nCount_RNA", "nFeature_RNA", "FMD")) # expect_equal(rownames(object[[]]), colnames(object)) # expect_equal(object[["nFeature_RNA"]][1:5, ], c(47, 52, 50, 56, 53)) # expect_equal(object[["nCount_RNA"]][75:80, ], c(228, 527, 202, 157, 150, 233)) # }) object.filtered <- CreateSeuratObject( counts = pbmc.test, min.cells = 10, min.features = 30 ) test_that("Filtering handled properly", { expect_equal(nrow(x = LayerData(object = object.filtered, layer = "counts")), 163) expect_equal(ncol(x = LayerData(object = object.filtered, layer = "counts")), 77) }) #this should be moved to seurat object # test_that("Metadata check errors correctly", { # pbmc.md <- pbmc_small[[]] # pbmc.md.norownames <- as.matrix(pbmc.md) # rownames(pbmc.md.norownames) <- NULL # expect_error(CreateSeuratObject(counts = pbmc.test, meta.data = pbmc.md.norownames), # "Row names not set in metadata. Please ensure that rownames of metadata match column names of data matrix") # }) # Tests for NormalizeData # -------------------------------------------------------------------------------- context("NormalizeData") test_that("NormalizeData error handling", { expect_error(NormalizeData(object = object, assay = "FAKE")) expect_equal( object = LayerData( object = NormalizeData( object = object, normalization.method = NULL, verbose = FALSE ), layer = "data" ), expected = LayerData(object = object, layer = "counts") ) }) object <- NormalizeData(object = object, verbose = FALSE, scale.factor = 1e6) test_that("NormalizeData scales properly", { expect_equal(LayerData(object = object, layer = "data")[2, 1], 9.567085, tolerance = 1e-6) expect_equal(LayerData(object = object, layer = "data")[161, 55], 8.415309, tolerance = 1e-6) expect_equal(Command(object = object, command = "NormalizeData.RNA", value = "scale.factor"), 1e6) expect_equal(Command(object = object, command = "NormalizeData.RNA", value = "normalization.method"), "LogNormalize") }) normalized.data <- LogNormalize(data = GetAssayData(object = object[["RNA"]], layer = "counts"), verbose = FALSE) test_that("LogNormalize normalizes properly", { expect_equal( as.matrix(LogNormalize(data = GetAssayData(object = object[["RNA"]], layer = "counts"), verbose = FALSE)), as.matrix(LogNormalize(data = as.data.frame(as.matrix(GetAssayData(object = object[["RNA"]], layer = "counts"))), verbose = FALSE)) ) }) clr.counts <- NormalizeData(object = pbmc.test, normalization.method = "CLR", verbose = FALSE) test_that("CLR normalization returns expected values", { expect_equal(dim(clr.counts), c(dim(pbmc.test))) expect_equal(clr.counts[2, 1], 0.5517828, tolerance = 1e-6) expect_equal(clr.counts[228, 76], 0.5971381, tolerance = 1e-6) expect_equal(clr.counts[230, 80], 0) }) rc.counts <- NormalizeData(object = pbmc.test, normalization.method = "RC", verbose = FALSE) test_that("Relative count normalization returns expected values", { expect_equal(rc.counts[2, 1], 142.8571, tolerance = 1e-6) expect_equal(rc.counts[228, 76], 18.97533, tolerance = 1e-6) expect_equal(rc.counts[230, 80], 0) rc.counts <- NormalizeData(object = pbmc.test, normalization.method = "RC", verbose = FALSE, scale.factor = 1e6) expect_equal(rc.counts[2, 1], 14285.71, tolerance = 1e-6) }) # Tests for v5 NormalizeData # -------------------------------------------------------------------------------- context("v5 NormalizeData") if(class(object[['RNA']]) == "Assay5") { fake.groups <- c(rep(1, floor(ncol(pbmc.test)/2)), rep(2, ncol(pbmc.test) - (floor(ncol(pbmc.test)/2))) ) object$groups <- fake.groups object.split <- CreateSeuratObject(split(object[["RNA"]], f = object$groups)) object.split <- NormalizeData(object = object.split) group1 <- subset(object, groups==1) group1 <- NormalizeData(group1) test_that("Normalization is performed for each layer", { expect_equal(Layers(object.split),c("counts.1", "counts.2", "data.1", "data.2")) expect_equal(group1[['RNA']]$data, LayerData(object.split, layer="data.1")) }) object.split <- NormalizeData(object = object.split, normalization.method = "CLR", verbose = FALSE) group1 <- NormalizeData(object = group1, normalization.method = "CLR", verbose = FALSE) test_that("CLR normalization works with multiple layers", { expect_equal(Layers(object.split),c("counts.1", "counts.2", "data.1", "data.2")) expect_equal(group1[['RNA']]$data, LayerData(object.split, layer="data.1")) }) object.split <- NormalizeData(object = object.split, normalization.method = "RC", verbose = FALSE) group1 <- NormalizeData(object = group1, normalization.method = "RC", verbose = FALSE) test_that("RC normalization works with multiple layers", { expect_equal(Layers(object.split),c("counts.1", "counts.2", "data.1", "data.2")) expect_equal(group1[['RNA']]$data, LayerData(object.split, layer="data.1")) }) } test_that("NormalizeData scales properly for BPcells", { # Tests for BPCells NormalizeData # -------------------------------------------------------------------------------- skip_on_cran() library(Matrix) skip_if_not_installed("BPCells") library(BPCells) mat_bpcells <- t(as(t(object[['RNA']]$counts ), "IterableMatrix")) object[['RNAbp']] <- CreateAssay5Object(counts = mat_bpcells) object <- NormalizeData(object = object, verbose = FALSE, scale.factor = 1e6, assay = "RNAbp") object <- NormalizeData(object = object, verbose = FALSE, scale.factor = 1e6, assay = "RNA") expect_equal(as.matrix(object[['RNAbp']]$data), as.matrix(object[['RNA']]$data), tolerance = 1e-6) expect_equal(Command(object = object, command = "NormalizeData.RNAbp", value = "scale.factor"), 1e6) expect_equal(Command(object = object, command = "NormalizeData.RNAbp", value = "normalization.method"), "LogNormalize") }) test_that("LogNormalize normalizes properly for BPCells", { skip_on_cran() library(Matrix) skip_if_not_installed("BPCells") library(BPCells) mat_bpcells <- t(as(t(object[['RNA']]$counts ), "IterableMatrix")) object[['RNAbp']] <- CreateAssay5Object(counts = mat_bpcells) object <- NormalizeData(object = object, verbose = FALSE, scale.factor = 1e6, assay = "RNAbp") object <- NormalizeData(object = object, verbose = FALSE, scale.factor = 1e6, assay = "RNA") normalized.data.bp <- LogNormalize(data = GetAssayData(object = object[["RNAbp"]], layer = "counts"), verbose = FALSE) normalized.data <- LogNormalize(data = GetAssayData(object = object[["RNA"]], layer = "counts"), verbose = FALSE) expect_equal( as.matrix(normalized.data.bp), as.matrix(normalized.data), tolerance = 1e-6 ) }) # Tests for ScaleData # -------------------------------------------------------------------------------- context("ScaleData") object <- ScaleData(object, verbose = FALSE) test_that("ScaleData returns expected values when input is a sparse matrix", { expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[1, 1], -0.4148587, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[75, 25], -0.2562305, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[162, 59], -0.4363939, tolerance = 1e-6) }) new.data <- as.matrix(GetAssayData(object = object[["RNA"]], layer = "data")) new.data[1, ] <- rep(x = 0, times = ncol(x = new.data)) object2 <- object object2 <- SetAssayData( object = object, assay = "RNA", slot = "data", new.data = new.data ) object2 <- ScaleData(object = object2, verbose = FALSE) object <- ScaleData(object = object, verbose = FALSE) test_that("ScaleData returns expected values when input is not sparse", { expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[75, 25], -0.2562305, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[162, 59], -0.4363939, tolerance = 1e-6) }) test_that("ScaleData handles zero variance features properly", { expect_equal(GetAssayData(object = object2[["RNA"]], layer = "scale.data")[1, 1], 0) expect_equal(GetAssayData(object = object2[["RNA"]], layer = "scale.data")[1, 80], 0) }) ng1 <- rep(x = "g1", times = round(x = ncol(x = object) / 2)) object$group <- c(ng1, rep(x = "g2", times = ncol(x = object) - length(x = ng1))) g1 <- subset(x = object, group == "g1") g1 <- ScaleData(object = g1, features = rownames(x = g1), verbose = FALSE) g2 <- subset(x = object, group == "g2") g2 <- ScaleData(object = g2, features = rownames(x = g2), verbose = FALSE) object <- ScaleData(object = object, features = rownames(x = object), verbose = FALSE, split.by = "group") #move to SeuratObject # test_that("split.by option works", { # expect_equal(GetAssayData(object = object, layer = "scale.data")[, Cells(x = g1)], # GetAssayData(object = g1, layer = "scale.data")) # expect_equal(GetAssayData(object = object, layer = "scale.data")[, Cells(x = g2)], # GetAssayData(object = g2, layer = "scale.data")) # }) g1 <- ScaleData(object = g1, features = rownames(x = g1), vars.to.regress = "nCount_RNA", verbose = FALSE) g2 <- ScaleData(object = g2, features = rownames(x = g2), vars.to.regress = "nCount_RNA", verbose = FALSE) object <- ScaleData(object = object, features = rownames(x = object), verbose = FALSE, split.by = "group", vars.to.regress = "nCount_RNA") test_that("split.by option works with regression", { expect_equal(LayerData(object = object, layer = "scale.data")[, Cells(x = g1)], LayerData(object = g1, layer = "scale.data")) expect_equal(LayerData(object = object, layer = "scale.data")[, Cells(x = g2)], LayerData(object = g2, layer = "scale.data")) }) # Tests for various regression techniques context("Regression") suppressWarnings({ object <- ScaleData( object = object, vars.to.regress = "nCount_RNA", features = rownames(x = object)[1:10], verbose = FALSE, model.use = "linear") }) test_that("Linear regression works as expected", { expect_equal(dim(x = GetAssayData(object = object[["RNA"]], layer = "scale.data")), c(10, 80)) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[1, 1], -0.6436435, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[5, 25], -0.09035383, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[10, 80], -0.2723782, tolerance = 1e-6) }) object <- ScaleData( object, vars.to.regress = "nCount_RNA", features = rownames(x = object)[1:10], verbose = FALSE, model.use = "negbinom") test_that("Negative binomial regression works as expected", { expect_equal(dim(x = GetAssayData(object = object[["RNA"]], layer = "scale.data")), c(10, 80)) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[1, 1], -0.5888811, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[5, 25], -0.2553394, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[10, 80], -0.1921429, tolerance = 1e-6) }) test_that("Regression error handling checks out", { expect_error(ScaleData(object, vars.to.regress = "nCount_RNA", model.use = "not.a.model", verbose = FALSE)) }) object <- ScaleData( object, vars.to.regress = "nCount_RNA", features = rownames(x = object)[1:10], verbose = FALSE, model.use = "poisson") test_that("Poisson regression works as expected", { expect_equal(dim(x = GetAssayData(object = object[["RNA"]], layer = "scale.data")), c(10, 80)) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[1, 1], -1.011717, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[5, 25], 0.05575307, tolerance = 1e-6) expect_equal(GetAssayData(object = object[["RNA"]], layer = "scale.data")[10, 80], -0.1662119, tolerance = 1e-6) }) #Tests for SampleUMI #-------------------------------------------------------------------------------- context("SampleUMI") downsampled.umis <- SampleUMI( data = LayerData(object = object, layer = "counts"), max.umi = 100, verbose = FALSE ) downsampled.umis.p.cell <- SampleUMI( data = LayerData(object = object, layer = "counts"), max.umi = seq(50, 1640, 20), verbose = FALSE, upsample = TRUE ) test_that("SampleUMI gives reasonable downsampled/upsampled UMI counts", { expect_true(!any(colSums(x = downsampled.umis) < 30, colSums(x = downsampled.umis) > 120)) expect_error(SampleUMI(data = LayerData(object = object, layer = "counts"), max.umi = rep(1, 5))) expect_true(!is.unsorted(x = colSums(x = downsampled.umis.p.cell))) expect_error(SampleUMI( data = LayerData(object = object, layer = "counts"), max.umi = seq(50, 900, 10), verbose = FALSE, upsample = TRUE )) }) # Tests for FindVariableFeatures # -------------------------------------------------------------------------------- context("FindVariableFeatures") object <- FindVariableFeatures(object = object, selection.method = "mean.var.plot", verbose = FALSE) test_that("mean.var.plot selection option returns expected values", { expect_equal(VariableFeatures(object = object)[1:4], c("PTGDR", "SATB1", "ZNF330", "S100B")) expect_equal(length(x = VariableFeatures(object = object)), 20) hvf_info <- HVFInfo(object = object[["RNA"]], method = 'mvp') expect_equal(hvf_info[[grep("mean$", colnames(hvf_info), value = TRUE)]][1:2], c(8.328927, 8.444462), tolerance = 1e-6) expect_equal(hvf_info[[grep("dispersion$", colnames(hvf_info), value = TRUE)]][1:2], c(10.552507, 10.088223), tolerance = 1e-6) expect_equal(as.numeric(hvf_info[[grep("dispersion.scaled$", colnames(hvf_info), value = TRUE)]][1:2]), c(0.1113214, -0.1332181523), tolerance = 1e-6) }) object <- FindVariableFeatures(object, selection.method = "dispersion", verbose = FALSE) test_that("dispersion selection option returns expected values", { expect_equal(VariableFeatures(object = object)[1:4], c("PCMT1", "PPBP", "LYAR", "VDAC3")) expect_equal(length(x = VariableFeatures(object = object)), 230) hvf_info <- HVFInfo(object = object[["RNA"]], method = 'mvp') expect_equal(hvf_info[[grep("mean$", colnames(hvf_info), value = TRUE)]][1:2], c(8.328927, 8.444462), tolerance = 1e-6) expect_equal(hvf_info[[grep("dispersion$", colnames(hvf_info), value = TRUE)]][1:2], c(10.552507, 10.088223), tolerance = 1e-6) expect_equal(as.numeric(hvf_info[[grep("dispersion.scaled$", colnames(hvf_info), value = TRUE)]][1:2]), c(0.1113214, -0.1332181523), tolerance = 1e-6) expect_true(!is.unsorted(rev(hvf_info[VariableFeatures(object = object), "dispersion"]))) }) object <- FindVariableFeatures(object, selection.method = "vst", verbose = FALSE) test_that("vst selection option returns expected values", { expect_equal(VariableFeatures(object = object)[1:4], c("PPBP", "IGLL5", "VDAC3", "CD1C")) expect_equal(length(x = VariableFeatures(object = object)), 230) hvf_info <- HVFInfo(object = object[["RNA"]], method = 'vst') expect_equal(hvf_info[[grep("variance$", colnames(hvf_info), value = TRUE)]][1:2], c(1.0251582, 1.2810127), tolerance = 1e-6) expect_equal(hvf_info[[grep("variance.standardized$", colnames(hvf_info), value = TRUE)]][1:2], c(0.8983463, 0.4731134), tolerance = 1e-6) expect_true(!is.unsorted(rev(hvf_info[VariableFeatures(object = object), grep("variance.standardized$", colnames(hvf_info))]))) }) #object <- FindVariableFeatures(object, assay = "RNAbp") #this breaks currently # Tests for internal functions # ------------------------------------------------------------------------------ norm.fxn <- function(x) {x / mean(x)} test_that("CustomNormalize works as expected", { expect_equal( CustomNormalize(data = pbmc.test, custom_function = norm.fxn, margin = 2), apply(X = pbmc.test, MARGIN = 2, FUN = norm.fxn) ) expect_equal( CustomNormalize(data = as.matrix(pbmc.test), custom_function = norm.fxn, margin = 2), apply(X = pbmc.test, MARGIN = 2, FUN = norm.fxn) ) expect_equal( CustomNormalize(data = as.data.frame(as.matrix(pbmc.test)), custom_function = norm.fxn, margin = 2), apply(X = pbmc.test, MARGIN = 2, FUN = norm.fxn) ) expect_equal( CustomNormalize(data = pbmc.test, custom_function = norm.fxn, margin = 1), t(apply(X = pbmc.test, MARGIN = 1, FUN = norm.fxn)) ) expect_error(CustomNormalize(data = pbmc.test, custom_function = norm.fxn, margin = 10)) }) # Tests for SCTransform # -------------------------------------------------------------------------------- context("SCTransform") object <- suppressWarnings(SCTransform(object = object, verbose = FALSE, vst.flavor = "v1", seed.use = 1448145)) test_that("SCTransform v1 works as expected", { expect_true("SCT" %in% names(object)) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[1]), 11.40288448) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[5]), 0) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "data"))[1]), 57.7295742, tolerance = 1e-6) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "data"))[5]), 11.74403719, tolerance = 1e-6) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[1]), 129) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[5]), 28) expect_equal(length(VariableFeatures(object[["SCT"]])), 220) fa <- SCTResults(object = object, assay = "SCT", slot = "feature.attributes") expect_equal(fa["MS4A1", "detection_rate"], 0.15) expect_equal(fa["MS4A1", "gmean"], 0.2027364, tolerance = 1e-6) expect_equal(fa["MS4A1", "variance"], 1.025158, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_mean"], 0.2362887, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_variance"], 2.875761, tolerance = 1e-6) }) test_that("SCTransform v2 works as expected", { skip_on_cran() skip_if_not_installed("glmGamPoi") object <- suppressWarnings(SCTransform(object = object, verbose = FALSE, vst.flavor = "v2", seed.use = 1448145)) expect_true("SCT" %in% names(object)) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[1]), 24.5183, tolerance = 1e-2) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[5]), 0) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "data"))[1]), 58.65829, tolerance = 1e-6) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "data"))[5]), 13.75449, tolerance = 1e-6) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[1]), 141) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[5]), 40) expect_equal(length(VariableFeatures(object[["SCT"]])), 220) fa <- SCTResults(object = object, assay = "SCT", slot = "feature.attributes") expect_equal(fa["MS4A1", "detection_rate"], 0.15) expect_equal(fa["MS4A1", "gmean"], 0.2027364, tolerance = 1e-6) expect_equal(fa["MS4A1", "variance"], 1.025158, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_mean"], 0.2763993, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_variance"], 3.023062, tolerance = 1e-6) }) suppressWarnings(RNGversion(vstr = "3.5.0")) object <- suppressWarnings(SCTransform(object = object, vst.flavor = "v1", ncells = 80, verbose = FALSE, seed.use = 42)) test_that("SCTransform ncells param works", { expect_true("SCT" %in% names(object)) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[1]), 11.40288, tolerance = 1e-6) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[5]), 0) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "data"))[1]), 57.72957, tolerance = 1e-6) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "data"))[5]), 11.74404, tolerance = 1e-6) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[1]), 129) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[5]), 28) expect_equal(length(VariableFeatures(object[["SCT"]])), 220) fa <- SCTResults(object = object, assay = "SCT", slot = "feature.attributes") expect_equal(fa["MS4A1", "detection_rate"], 0.15) expect_equal(fa["MS4A1", "gmean"], 0.2027364, tolerance = 1e-6) expect_equal(fa["MS4A1", "variance"], 1.025158, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_mean"], 0.2362887, tolerance = 1e-3) expect_equal(fa["MS4A1", "residual_variance"], 2.875761, tolerance = 1e-3) }) suppressWarnings(object[["SCT_SAVE"]] <- object[["SCT"]]) object[["SCT"]] <- suppressWarnings({SetAssayData(object = object[["SCT"]], slot = "scale.data", new.data = GetAssayData(object = object[["SCT"]], layer = "scale.data")[1:100, ])}) object <- GetResidual(object = object, features = rownames(x = object), verbose = FALSE) test_that("GetResidual works", { expect_equal(dim(GetAssayData(object = object[["SCT"]], layer = "scale.data")), c(220, 80)) expect_equal( GetAssayData(object = object[["SCT"]], layer = "scale.data"), GetAssayData(object = object[["SCT_SAVE"]], layer = "scale.data") ) expect_warning(GetResidual(object, features = "asd")) }) test_that("SCTransform v2 works as expected", { skip_on_cran() skip_if_not_installed("glmGamPoi") object <- suppressWarnings(SCTransform(object = object, verbose = FALSE, vst.flavor = "v2", seed.use = 1448145)) expect_true("SCT" %in% names(object)) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[1]), 24.5813, tolerance = 1e-4) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "scale.data"))[5]), 0) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "data"))[1]), 58.65829, tolerance = 1e-6) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "data"))[5]), 13.75449, tolerance = 1e-6) expect_equal(as.numeric(colSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[1]), 141) expect_equal(as.numeric(rowSums(GetAssayData(object = object[["SCT"]], layer = "counts"))[5]), 40) expect_equal(length(VariableFeatures(object[["SCT"]])), 220) fa <- SCTResults(object = object, assay = "SCT", slot = "feature.attributes") expect_equal(fa["MS4A1", "detection_rate"], 0.15) expect_equal(fa["MS4A1", "gmean"], 0.2027364, tolerance = 1e-6) expect_equal(fa["MS4A1", "variance"], 1.025158, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_mean"], 0.2763993, tolerance = 1e-6) expect_equal(fa["MS4A1", "residual_variance"], 3.023062, tolerance = 1e-6) expect_equal(fa["FCER2", "theta"], Inf) }) test_that("SCTransform `vars.to.regress` param works as expected", { # make a copy of the testing data test.data <- object # add a fake mitochondrial gene to the counts matrix counts <- LayerData(test.data, assay = "RNA", layer = "counts") counts <- rbind(counts, 5) rownames(counts)[nrow(counts)] <- "MT-TEST" # use the fake feature to populate a new meta.data column test.data[[ "percent.mt" ]] <- PercentageFeatureSet( test.data, pattern="^MT-" ) # make sure that `ncells` is smaller than the datset being transformed # so tha the regression model is trained on a subset of the data - make sure # the regression is applied to the entire dataset left <- suppressWarnings( SCTransform( test.data, vars.to.regress = NULL, ncells = ncol(test.data) / 2, verbose = FALSE ) ) right <- suppressWarnings( SCTransform( test.data, vars.to.regress = "percent.mt", ncells = ncol(test.data) / 2, verbose = FALSE ) ) expect_false(identical(left[["SCT"]]$scale.data, right[["SCT"]]$scale.data)) # if the `assay` points to an `Assay5` instance the regression is handled # using separate logic test.data[["RNAv5"]] <- CreateAssay5Object( counts = LayerData( test.data, assay = "RNA", layer = "counts" ) ) left <- suppressWarnings( SCTransform( test.data, assay = "RNAv5", vars.to.regress = NULL, ncells = ncol(test.data) / 2, verbose = FALSE ) ) right <- suppressWarnings( SCTransform( test.data, assay = "RNAv5", vars.to.regress = "percent.mt", ncells = ncol(test.data) / 2, verbose = FALSE ) ) expect_false(identical(left[["SCT"]]$scale.data, right[["SCT"]]$scale.data)) }) test_that("SCTransform is equivalent for BPcells ", { skip_on_cran() skip_on_cran() skip_if_not_installed("glmGamPoi") library(Matrix) skip_if_not_installed("BPCells") library(BPCells) mat_bpcells <- t(as(t(object[['RNA']]$counts ), "IterableMatrix")) object[['RNAbp']] <- CreateAssay5Object(counts = mat_bpcells) object <- suppressWarnings(SCTransform(object = object, assay = "RNA", new.assay.name = "SCT", verbose = FALSE, vst.flavor = "v2", seed.use = 1448145)) object <- suppressWarnings(SCTransform(object = object, assay = "RNAbp", new.assay.name = "SCTbp", verbose = FALSE, vst.flavor = "v2", seed.use = 1448145)) expect_equal(as.matrix(LayerData(object = object[["SCT"]], layer = "data")), as.matrix(LayerData(object = object[["SCTbp"]], layer = "data")), tolerance = 1e-6) })