## ----echo=FALSE, results="hide", message=FALSE-------------------------------- knitr::opts_chunk$set(error=FALSE, message=FALSE, warning=FALSE) library(BiocStyle) ## ----------------------------------------------------------------------------- suppressPackageStartupMessages(library(escape)) suppressPackageStartupMessages(library(SingleCellExperiment)) suppressPackageStartupMessages(library(scran)) suppressPackageStartupMessages(library(Seurat)) suppressPackageStartupMessages(library(SeuratObject)) suppressPackageStartupMessages(library(RColorBrewer)) suppressPackageStartupMessages(library(ggplot2)) pbmc_small <- get("pbmc_small") sce.pbmc <- as.SingleCellExperiment(pbmc_small, assay = "RNA") ## ----------------------------------------------------------------------------- GS.hallmark <- getGeneSets(library = "H") ## ----eval = FALSE------------------------------------------------------------- # data("escape.gene.sets", package="escape") # gene.sets <- escape.gene.sets ## ----eval=FALSE, tidy=FALSE--------------------------------------------------- # gene.sets <- list(Bcells = c("MS4A1","CD79B","CD79A","IGH1","IGH2") # Myeloid = c("SPI1","FCER1G","CSF1R"), # Tcells = c("CD3E", "CD3D", "CD3G", "CD7","CD8A")) ## ----tidy = FALSE------------------------------------------------------------- enrichment.scores <- escape.matrix(pbmc_small, gene.sets = GS.hallmark, groups = 1000, min.size = 5) ggplot(data = as.data.frame(enrichment.scores), mapping = aes(enrichment.scores[,1], enrichment.scores[,2])) + geom_point() + theme_classic() + theme(axis.title = element_blank()) ## ----tidy=FALSE, eval=FALSE--------------------------------------------------- # enrichment.scores <- escape.matrix(pbmc_small, # gene.sets = GS.hallmark, # groups = 1000, # min.size = 5, # BPPARAM = SnowParam(workers = 2)) ## ----tidy = FALSE------------------------------------------------------------- pbmc_small <- runEscape(pbmc_small, method = "ssGSEA", gene.sets = GS.hallmark, groups = 1000, min.size = 5, new.assay.name = "escape.ssGSEA") sce.pbmc <- runEscape(sce.pbmc, method = "UCell", gene.sets = GS.hallmark, groups = 1000, min.size = 5, new.assay.name = "escape.UCell") ## ----tidy = FALSE------------------------------------------------------------- #Define color palette colorblind_vector <- hcl.colors(n=7, palette = "inferno", fixup = TRUE) FeaturePlot(pbmc_small, "HALLMARK-APOPTOSIS") + scale_color_gradientn(colors = colorblind_vector) + theme(plot.title = element_blank()) ## ----------------------------------------------------------------------------- pbmc_small <- performNormalization(pbmc_small, assay = "escape.ssGSEA", gene.sets = GS.hallmark) ## ----------------------------------------------------------------------------- pbmc_small <- performNormalization(pbmc_small, assay = "escape.ssGSEA", gene.sets = GS.hallmark, scale.factor = pbmc_small$nFeature_RNA) ## ----------------------------------------------------------------------------- heatmapEnrichment(pbmc_small, group.by = "ident", gene.set.use = "all", assay = "escape.ssGSEA") ## ----------------------------------------------------------------------------- heatmapEnrichment(sce.pbmc, group.by = "ident", assay = "escape.UCell", scale = TRUE, cluster.rows = TRUE, cluster.columns = TRUE) ## ----------------------------------------------------------------------------- hcl.pals() ## ----------------------------------------------------------------------------- heatmapEnrichment(pbmc_small, assay = "escape.ssGSEA", palette = "Spectral") ## ----------------------------------------------------------------------------- heatmapEnrichment(sce.pbmc, group.by = "ident", assay = "escape.UCell") + scale_fill_gradientn(colors = rev(brewer.pal(11, "RdYlBu"))) ## ----------------------------------------------------------------------------- geyserEnrichment(pbmc_small, assay = "escape.ssGSEA", gene.set = "HALLMARK-INTERFERON-GAMMA-RESPONSE") ## ----------------------------------------------------------------------------- geyserEnrichment(pbmc_small, assay = "escape.ssGSEA", gene.set = "HALLMARK-INTERFERON-GAMMA-RESPONSE", order.by = "mean") ## ----------------------------------------------------------------------------- geyserEnrichment(pbmc_small, assay = "escape.ssGSEA", gene.set = "HALLMARK-INTERFERON-GAMMA-RESPONSE", facet.by = "groups") ## ----------------------------------------------------------------------------- geyserEnrichment(pbmc_small, assay = "escape.ssGSEA", gene.set = "HALLMARK-INTERFERON-GAMMA-RESPONSE", color.by = "HALLMARK-INTERFERON-GAMMA-RESPONSE") ## ----------------------------------------------------------------------------- ridgeEnrichment(sce.pbmc, assay = "escape.UCell", gene.set = "HALLMARK-IL2-STAT5-SIGNALING") ## ----------------------------------------------------------------------------- ridgeEnrichment(sce.pbmc, assay = "escape.UCell", gene.set = "HALLMARK-IL2-STAT5-SIGNALING", add.rug = TRUE, scale = TRUE) ## ----------------------------------------------------------------------------- splitEnrichment(pbmc_small, assay = "escape.ssGSEA", gene.set = "HALLMARK-IL2-STAT5-SIGNALING", split.by = "groups") ## ----------------------------------------------------------------------------- densityEnrichment(pbmc_small, gene.set.use = "HALLMARK-IL6-JAK-STAT3-SIGNALING", gene.sets = GS.hallmark) ## ----------------------------------------------------------------------------- scatterEnrichment(pbmc_small, assay = "escape.ssGSEA", x.axis = "HALLMARK-INTERFERON-GAMMA-RESPONSE", y.axis = "HALLMARK-IL6-JAK-STAT3-SIGNALING") ## ----------------------------------------------------------------------------- scatterEnrichment(sce.pbmc, assay = "escape.UCell", x.axis = "HALLMARK-INTERFERON-GAMMA-RESPONSE", y.axis = "HALLMARK-IL6-JAK-STAT3-SIGNALING", style = "hex") ## ----------------------------------------------------------------------------- pbmc_small <- performPCA(pbmc_small, assay = "escape.ssGSEA", n.dim = 1:10) ## ----------------------------------------------------------------------------- pcaEnrichment(pbmc_small, dimRed = "escape.PCA", x.axis = "PC1", y.axis = "PC2") ## ----------------------------------------------------------------------------- pcaEnrichment(pbmc_small, dimRed = "escape.PCA", x.axis = "PC1", y.axis = "PC2", add.percent.contribution = TRUE, display.factors = TRUE, number.of.factors = 10) ## ----------------------------------------------------------------------------- pbmc_small <- performNormalization(pbmc_small, assay = "escape.ssGSEA", gene.sets = GS.hallmark, make.positive = TRUE) all.markers <- FindAllMarkers(pbmc_small, assay = "escape.ssGSEA_normalized", min.pct = 0, logfc.threshold = 0) head(all.markers) ## ----------------------------------------------------------------------------- sessionInfo()