From a5ea14c6b08ab73ee3d3f44cf9ac84403c5dafb4 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 11:34:24 +0100 Subject: [PATCH 01/17] Added first draft of scGSEA wrapper --- R/scGSEA.R | 81 ++++++++++++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 81 insertions(+) create mode 100644 R/scGSEA.R diff --git a/R/scGSEA.R b/R/scGSEA.R new file mode 100644 index 0000000..d12497b --- /dev/null +++ b/R/scGSEA.R @@ -0,0 +1,81 @@ +#' @include internal.R +#' +NULL + +#' Run Single cell Gene Set Enrichement Analysis on GF-ICF on a Seurat object +#' +#' This function run runScGSEA function from GF-ICF package on Seurat object. +#' It computes GSEA for each cells across a set of input pathways by using NMF. +#' +#' @param object Seurat object +#' @param assay Assay to use, defaults to the default assay +#' @param filterGenes Rank of NMF to use for the enrichment +#' @param nmf.dim Rank of NMF to use for the enrichment +#' @param geneID The type of gene names in rownames of \code{object}. It can be either 'ensamble' or 'symbol'. Default: 'ensamble' +#' @param species The type of species in \code{object}. It can be either 'mouse' or 'human'. Default: 'human' +#' @param category MSigDB collection abbreviation, such as H or C1 +#' @param subcategory MSigDB sub-collection abbreviation, such as CGP or BP +#' @param rescale If different by none, pathway's activity scores are resealed as Z-score. Possible values are none, byGS or byCell. Default is none +#' +#' @export + +gficfScGSEA <- function( + object, + assay = NULL, + filterGenes = TRUE, + nmf.dim = 100, + geneID = c("ensamble","symbol"), + species = c("mouse", "human"), + category = "H", + subcategory = NULL, + verbose = F) { + + SeuratWrappers:::CheckPackage(package = 'gficf', repository = 'CRAN') + assay <- assay %||% DefaultAssay(object = object) + + # Get raw count matrix from Seurat object + M <- GetAssayData(object = object, slot = "counts") + # Data normalization and gene filtering + data <- gficf::gficf(M = M, filterGenes = filterGenes) + # Create NMF-subspace + data <- gficf::runNMF(data = data, dim = nmf.dim) + # Create t-UMAP space + data <- gficf::runReduction(data = data, reduction = "umap", verbose = verbose) + # Compute GSEA for each cells across a set of input pathways by using NMF + data <- gficf::runScGSEA(data = data, + geneID = geneID, + species = species, + category = category, + subcategory = subcategory, + nmf.k = nmf.dim, + fdr.th = .1, + rescale = 'none', + verbose = verbose) + + # Add cell names to enrichment results + raw_enrich <- t(data$scgsea$x) + colnames(raw_enrich) <- colnames(object) + # Normalize enrichment results by computing pathway's activity z-scores + norm_enrich <- Matrix::Matrix((raw_enrich - rowMeans(raw_enrich)) / apply(raw_enrich, 1, sd), sparse=T) + # Create a new Seurat object containing the raw pathway x cell matrix in counts slot + path_obj <- CreateSeuratObject(counts = raw_enrich, meta.data = object@meta.data) + # And the z-score-normalized one in data and scale.data slots + path_obj <- SetAssayData(object = path_obj, slot = 'data', new.data = norm_enrich) + path_obj <- SetAssayData(object = path_obj, slot = 'scale.data', new.data = as.matrix(norm_enrich)) + # Store pathways metadata + feat.meta <- data.frame(data$scgsea$stat, row.names = 1) + feat.meta$genes <- do.call(c, lapply(data$scgsea$pathways[data$scgsea$stat$pathway], function(x) paste(x, collapse = ','))) + path_obj[[assay]]@meta.features <- feat.meta + + # Store dimensionality reduction results computed on genes x cells matrix + path_obj@reductions <- object@reductions + names(path_obj@reductions) <- paste0(names(path_obj@reductions), '_byGenes') + + return(path_obj) + +} + +#'umap'%in%names(object@reductions) + + + From d3a63e92cf574bf4bcb3bdeb0e913958b46b64f6 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 13:59:48 +0100 Subject: [PATCH 02/17] Added scGSEA wrapper code to DESCRIPTION file --- DESCRIPTION | 1 + 1 file changed, 1 insertion(+) diff --git a/DESCRIPTION b/DESCRIPTION index d779fcb..4aec4f7 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -55,6 +55,7 @@ Collate: 'miqc.R' 'monocle3.R' 'presto.R' + 'scGSEA.R' 'tricycle.R' 'velocity.R' Encoding: UTF-8 From 30bb35b3dce34bcb3c63f97b31d889011b03f8a0 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 14:06:32 +0100 Subject: [PATCH 03/17] Resolved bug scGSEA wrapper --- R/scGSEA.R | 30 ++++++++++++++---------------- 1 file changed, 14 insertions(+), 16 deletions(-) diff --git a/R/scGSEA.R b/R/scGSEA.R index d12497b..6b024cf 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -18,6 +18,7 @@ NULL #' @param rescale If different by none, pathway's activity scores are resealed as Z-score. Possible values are none, byGS or byCell. Default is none #' #' @export +#' gficfScGSEA <- function( object, @@ -30,27 +31,27 @@ gficfScGSEA <- function( subcategory = NULL, verbose = F) { - SeuratWrappers:::CheckPackage(package = 'gficf', repository = 'CRAN') + SeuratWrappers:::CheckPackage(package = 'gambalab/gficf', repository = 'github') assay <- assay %||% DefaultAssay(object = object) # Get raw count matrix from Seurat object M <- GetAssayData(object = object, slot = "counts") # Data normalization and gene filtering - data <- gficf::gficf(M = M, filterGenes = filterGenes) + M <- gficf::gficf(M = M, filterGenes = filterGenes) # Create NMF-subspace - data <- gficf::runNMF(data = data, dim = nmf.dim) + M <- gficf::runNMF(data = M, dim = nmf.dim) # Create t-UMAP space - data <- gficf::runReduction(data = data, reduction = "umap", verbose = verbose) + M <- gficf::runReduction(data = M, reduction = "umap", verbose = verbose) # Compute GSEA for each cells across a set of input pathways by using NMF - data <- gficf::runScGSEA(data = data, - geneID = geneID, - species = species, - category = category, - subcategory = subcategory, - nmf.k = nmf.dim, - fdr.th = .1, - rescale = 'none', - verbose = verbose) + M <- gficf::runScGSEA(data = M, + geneID = geneID, + species = species, + category = category, + subcategory = subcategory, + nmf.k = nmf.dim, + fdr.th = .1, + rescale = 'none', + verbose = verbose) # Add cell names to enrichment results raw_enrich <- t(data$scgsea$x) @@ -75,7 +76,4 @@ gficfScGSEA <- function( } -#'umap'%in%names(object@reductions) - - From bdf1846e345b189a7e6f9fa18b28b72d6a55dbff Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 17:48:37 +0100 Subject: [PATCH 04/17] Changed function name --- R/scGSEA.R | 8 ++++---- 1 file changed, 4 insertions(+), 4 deletions(-) diff --git a/R/scGSEA.R b/R/scGSEA.R index 6b024cf..88896d9 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -20,7 +20,7 @@ NULL #' @export #' -gficfScGSEA <- function( +RunScGSEA <- function( object, assay = NULL, filterGenes = TRUE, @@ -54,7 +54,7 @@ gficfScGSEA <- function( verbose = verbose) # Add cell names to enrichment results - raw_enrich <- t(data$scgsea$x) + raw_enrich <- t(M$scgsea$x) colnames(raw_enrich) <- colnames(object) # Normalize enrichment results by computing pathway's activity z-scores norm_enrich <- Matrix::Matrix((raw_enrich - rowMeans(raw_enrich)) / apply(raw_enrich, 1, sd), sparse=T) @@ -64,8 +64,8 @@ gficfScGSEA <- function( path_obj <- SetAssayData(object = path_obj, slot = 'data', new.data = norm_enrich) path_obj <- SetAssayData(object = path_obj, slot = 'scale.data', new.data = as.matrix(norm_enrich)) # Store pathways metadata - feat.meta <- data.frame(data$scgsea$stat, row.names = 1) - feat.meta$genes <- do.call(c, lapply(data$scgsea$pathways[data$scgsea$stat$pathway], function(x) paste(x, collapse = ','))) + feat.meta <- data.frame(M$scgsea$stat, row.names = 1) + feat.meta$genes <- do.call(c, lapply(M$scgsea$pathways[M$scgsea$stat$pathway], function(x) paste(x, collapse = ','))) path_obj[[assay]]@meta.features <- feat.meta # Store dimensionality reduction results computed on genes x cells matrix From 5cd7503c395ceef9c35c90c9e2999a64f27617e3 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 18:32:58 +0100 Subject: [PATCH 05/17] Fixed installation problems --- DESCRIPTION | 4 ++-- NAMESPACE | 1 + R/scGSEA.R | 2 +- man/PlotMiQC.Rd | 3 --- man/RunPresto.Rd | 1 - man/RunPrestoAll.Rd | 1 - 6 files changed, 4 insertions(+), 8 deletions(-) diff --git a/DESCRIPTION b/DESCRIPTION index 4aec4f7..e43dd26 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -45,7 +45,7 @@ Collate: 'internal.R' 'alevin.R' 'alra.R' - 'banksy.R' + 'banksy.R' 'cellbrowser.R' 'cogaps.R' 'conos.R' @@ -60,7 +60,7 @@ Collate: 'velocity.R' Encoding: UTF-8 LazyData: true -RoxygenNote: 7.1.1 +RoxygenNote: 7.2.3 Suggests: cipr, conos, diff --git a/NAMESPACE b/NAMESPACE index f593c28..61059a4 100644 --- a/NAMESPACE +++ b/NAMESPACE @@ -23,6 +23,7 @@ export(RunPrestoAll) export(RunQuantileAlignSNF) export(RunQuantileNorm) export(RunSNF) +export(RunScGSEA) export(RunVelocity) export(Runtricycle) export(StopCellbrowser) diff --git a/R/scGSEA.R b/R/scGSEA.R index 88896d9..6d175f8 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -17,8 +17,8 @@ NULL #' @param subcategory MSigDB sub-collection abbreviation, such as CGP or BP #' @param rescale If different by none, pathway's activity scores are resealed as Z-score. Possible values are none, byGS or byCell. Default is none #' +#' @return returns a Seurat object with pathways x cell matrix #' @export -#' RunScGSEA <- function( object, diff --git a/man/PlotMiQC.Rd b/man/PlotMiQC.Rd index 07e9d46..64d43a3 100644 --- a/man/PlotMiQC.Rd +++ b/man/PlotMiQC.Rd @@ -14,9 +14,6 @@ PlotMiQC( } \arguments{ \item{object}{Seurat object} -} -\value{ - } \description{ Run miQC on a Seurat object diff --git a/man/RunPresto.Rd b/man/RunPresto.Rd index 18972d9..e32299f 100644 --- a/man/RunPresto.Rd +++ b/man/RunPresto.Rd @@ -25,7 +25,6 @@ RunPresto( latent.vars = NULL, min.cells.feature = 3, min.cells.group = 3, - pseudocount.use = 1, mean.fxn = NULL, fc.name = NULL, base = 2, diff --git a/man/RunPrestoAll.Rd b/man/RunPrestoAll.Rd index 5cf5c91..9c64c4f 100644 --- a/man/RunPrestoAll.Rd +++ b/man/RunPrestoAll.Rd @@ -22,7 +22,6 @@ RunPrestoAll( latent.vars = NULL, min.cells.feature = 3, min.cells.group = 3, - pseudocount.use = 1, mean.fxn = NULL, fc.name = NULL, base = 2, From 6c013aad300ad6923316107c79ecebe63a6d60c4 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 18:45:32 +0100 Subject: [PATCH 06/17] Fixed installation problems attempt2 --- DESCRIPTION | 3 ++- NAMESPACE | 1 + R/scGSEA.R | 1 + 3 files changed, 4 insertions(+), 1 deletion(-) diff --git a/DESCRIPTION b/DESCRIPTION index e43dd26..2e5d8b0 100644 --- a/DESCRIPTION +++ b/DESCRIPTION @@ -24,7 +24,8 @@ Remotes: welch-lab/liger, mojaveazure/seurat-disk, powellgenomicslab/Nebulosa, atakanekiz/CIPR-Package, - prabhakarlab/Banksy + prabhakarlab/Banksy, + gambalab/gficf Depends: R (>= 3.5.0) biocViews: diff --git a/NAMESPACE b/NAMESPACE index 61059a4..b390a47 100644 --- a/NAMESPACE +++ b/NAMESPACE @@ -28,6 +28,7 @@ export(RunVelocity) export(Runtricycle) export(StopCellbrowser) export(as.cell_data_set) +import(RcppML) importFrom(BiocManager,install) importFrom(Matrix,Matrix) importFrom(Matrix,writeMM) diff --git a/R/scGSEA.R b/R/scGSEA.R index 6d175f8..ae69936 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -18,6 +18,7 @@ NULL #' @param rescale If different by none, pathway's activity scores are resealed as Z-score. Possible values are none, byGS or byCell. Default is none #' #' @return returns a Seurat object with pathways x cell matrix +#' @import RcppML #' @export RunScGSEA <- function( From 865a03a13da4435475c457fc98208f686b9280a7 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 19:19:45 +0100 Subject: [PATCH 07/17] fix --- R/scGSEA.R | 3 ++- 1 file changed, 2 insertions(+), 1 deletion(-) diff --git a/R/scGSEA.R b/R/scGSEA.R index ae69936..258bad8 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -57,8 +57,9 @@ RunScGSEA <- function( # Add cell names to enrichment results raw_enrich <- t(M$scgsea$x) colnames(raw_enrich) <- colnames(object) + print(dim(raw_enrich)) # Normalize enrichment results by computing pathway's activity z-scores - norm_enrich <- Matrix::Matrix((raw_enrich - rowMeans(raw_enrich)) / apply(raw_enrich, 1, sd), sparse=T) + norm_enrich <- Matrix::Matrix((raw_enrich - Matrix::rowMeans(raw_enrich)) / apply(raw_enrich, 1, sd), sparse=T) # Create a new Seurat object containing the raw pathway x cell matrix in counts slot path_obj <- CreateSeuratObject(counts = raw_enrich, meta.data = object@meta.data) # And the z-score-normalized one in data and scale.data slots From ca0c256793f4a67299780f9272f90d6d93dbf5e3 Mon Sep 17 00:00:00 2001 From: melania15 Date: Wed, 11 Jan 2023 20:04:35 +0100 Subject: [PATCH 08/17] Added preservation genes-based clustering --- R/scGSEA.R | 6 ++++-- 1 file changed, 4 insertions(+), 2 deletions(-) diff --git a/R/scGSEA.R b/R/scGSEA.R index 258bad8..a5ee9b2 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -57,10 +57,12 @@ RunScGSEA <- function( # Add cell names to enrichment results raw_enrich <- t(M$scgsea$x) colnames(raw_enrich) <- colnames(object) - print(dim(raw_enrich)) # Normalize enrichment results by computing pathway's activity z-scores norm_enrich <- Matrix::Matrix((raw_enrich - Matrix::rowMeans(raw_enrich)) / apply(raw_enrich, 1, sd), sparse=T) - # Create a new Seurat object containing the raw pathway x cell matrix in counts slot + # Create a new Seurat object containing the raw pathway x cell matrix in counts slot and preserving meta data (adding _byGenes tag only if clusters were already computed) + if('seurat_clusters' %in% colnames(object@meta.data)) { + colnames(object@meta.data) <- gsub('seurat_clusters', 'seurat_clusters_byGenes', colnames(object@meta.data)) + } path_obj <- CreateSeuratObject(counts = raw_enrich, meta.data = object@meta.data) # And the z-score-normalized one in data and scale.data slots path_obj <- SetAssayData(object = path_obj, slot = 'data', new.data = norm_enrich) From 4ef563cf13ad5a0a83a39fd9ed1e8f0ae1286d2a Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 12:25:09 +0100 Subject: [PATCH 09/17] Feature metadata bug fix --- R/scGSEA.R | 7 +- docs/scGSEA.Rmd | 168 ++++++++++++++++++++++++++++++++++++++++++++++++ 2 files changed, 172 insertions(+), 3 deletions(-) create mode 100644 docs/scGSEA.Rmd diff --git a/R/scGSEA.R b/R/scGSEA.R index a5ee9b2..c057622 100644 --- a/R/scGSEA.R +++ b/R/scGSEA.R @@ -67,9 +67,10 @@ RunScGSEA <- function( # And the z-score-normalized one in data and scale.data slots path_obj <- SetAssayData(object = path_obj, slot = 'data', new.data = norm_enrich) path_obj <- SetAssayData(object = path_obj, slot = 'scale.data', new.data = as.matrix(norm_enrich)) - # Store pathways metadata - feat.meta <- data.frame(M$scgsea$stat, row.names = 1) - feat.meta$genes <- do.call(c, lapply(M$scgsea$pathways[M$scgsea$stat$pathway], function(x) paste(x, collapse = ','))) + # Store metadata of pathways retained after significance filtering + feat.meta <- M$scgsea$stat[M$scgsea$stat$pathway%in%colnames(M$scgsea$x), ] + feat.meta <- data.frame(feat.meta, row.names = 1) + feat.meta$genes <- do.call(c, lapply(M$scgsea$pathways[rownames(feat.meta)], function(x) paste(x, collapse = ','))) path_obj[[assay]]@meta.features <- feat.meta # Store dimensionality reduction results computed on genes x cells matrix diff --git a/docs/scGSEA.Rmd b/docs/scGSEA.Rmd new file mode 100644 index 0000000..abe588b --- /dev/null +++ b/docs/scGSEA.Rmd @@ -0,0 +1,168 @@ +--- +title: "Running ScGSEA from gficf on Seurat Objects" +date: 'Compiled: `r format(Sys.Date(), "%B %d, %Y")`' +output: + github_document: + html_preview: true + toc: true + html_document: + df_print: kable + theme: united +--- + +This vigettte demonstrates how to run single-cell gene set enrichment analysis with gficf on Seurat objects. + +> *Single-cell gene set enrichment analysis and transfer learning for functional annotation of scRNA-seq data* +> +> Pellecchia Simona , Viscido Gaetano, Franchini Melania, Gambardella Gennaro +> +> bioRxiv, 2022 +> +> doi: +> +> +> GitHub: +> + +```{r setup, include=FALSE} +knitr::opts_chunk$set( + tidy = TRUE, + tidy.opts = list(width.cutoff = 95), + message = FALSE, + warning = FALSE, + fig.height = 20, + fig.width = 16 +) +``` + + +Prerequisites to install: + +* [Seurat](https://satijalab.org/seurat/install) +* [SeuratData](https://github.com/satijalab/seurat-data) +* [SeuratWrappers](https://github.com/satijalab/seurat-wrappers) +* [gficf](https://github.com/gambalab/gficf) + +```{r looad libraries} +library(Seurat) +library(SeuratData) +library(SeuratWrappers) +library(gficf) +``` + + + +# Introduction +GF-ICF is an R package for normalization, visualization and analysis of of single-cell RNA sequencing data, based on a data transformation model called term frequency–inverse document frequency (TF-IDF), which has been extensively used in the field of text mining. Here, we show one of the latest implementation which enables the pathway activity computation at single-cell level. + +## Load examle data from SeuratData +To demonstrate how to run runScGSEA() function on a single-cell RNA-seq dataset. +We will use the pbmc3k dataset from the SeuratData package which contains 2,700 Peripheral Blood Mononuclear Cells (PBMC) which were sequenced on the Illumina NextSeq 500. + +```{r load example data} + +InstallData("pbmc3k") +data("pbmc3k.final") +pbmc3k.final + +``` + +## Compute pathways activity at single-cell resolution +Single cell gene set enrichment analysis is performed by the function runScGSEA() of gficf package which computes GSEA scores for each cell across a set of input pathways by using NMF. +The list of gene sets to use can can be specified trough the category and subcategory parameters. + +```{r runScGsea, results='hide'} + +pbmc.ptw <- RunScGSEA(object = pbmc3k.final, geneID = "symbol", species = "human", category = "H") + +``` + + + +# PART 1 : Evaluate pathways activity in single-cell data +## Visualize UMAP plot computed on genes expression +As dimensionality reduction results computed on genes expression are preserved, let's graph the UMAP computed on genes x cells matrix. In this case cells are colored by their cell type. + +```{r DimPlotGenes} + +DimPlot(pbmc.ptw, reduction = "umap_byGenes", group.by = "seurat_annotations") + +``` + +## Identify how pathways activity change across cell type +To evaluate how pathways activity change across cell type, we utilize DotPlot() function where dot size encodes for the percentage of cells of each cell type in which the given pathway is active. Finally, cell types are ordered (by hierarchical clustering) based on given features by setting cluster.idents = TRUE. + +```{r DotPlotPathways} + +library(ggplot2) +library(RColorBrewer) + +all.pathways = rownames(pbmc.ptw) + +DotPlot(object = pbmc.ptw, + features = all.pathways, + split.by = 'seurat_annotations', + cols = RColorBrewer::brewer.pal(n = length(unique(pbmc.ptw$seurat_annotations)), name = 'Set1'), + cluster.idents = TRUE) + + theme(axis.text.x = element_text(angle = 60, vjust = 1, hjust = 1, size = 10)) + +``` + +## Inspect pathways activation in each cell of a given cell type +To inspect pathways activation in each cell of a given cell type, we use DoHeatmap() function. In this case, we are plotting all given pathways for each cell type. + +```{r DoHeatmapPathways} + +DoHeatmap(pbmc.ptw, features = all.pathways, group.by = "seurat_annotations", angle = 90) + NoLegend() + +``` + + + +# PART 2 : Analyze data based on pathways activity scores +## Visualize UMAP plot computed on pathways activity scores +Now, let's see the UMAP computed on pathways activity scores. To this end, we follow the standard Seurat workflow which requires the computation of PCA on scaled data (i.e. pathway scores normalized by gene-set) before run the UMAP reduction. Results will be stored in separate reduction slot of the Seurat object. +Finally, we display the UMAP plot where cells are colored by their cell type. + +```{r UMAPcellTypePathways, results='hide'} + +pbmc.ptw <- RunPCA(pbmc.ptw, features = all.pathways) +pbmc.ptw <- RunUMAP(pbmc.ptw, dims = 1:5) +DimPlot(pbmc.ptw, reduction = "umap", group.by = 'seurat_annotations') + +``` + +## Cluster cells based on pathways activity scores +Now that we reconstructed pathway’s activity at single cell level we can try to cluster cell according to these values using Seurat functions FindNeighbors() and FindClusters() and, finally, we graph results ad UMAP plot form. + +```{r UMAPclustersPathways} + +DimPlot(pbmc.ptw, reduction = "umap", group.by = 'seurat_clusters_byGenes') +pbmc.ptw <- FindNeighbors(pbmc.ptw, dims = 1:10) +pbmc.ptw <- FindClusters(pbmc.ptw, resolution = 0.5) +DimPlot(pbmc.ptw, reduction = "umap", group.by = 'seurat_clusters') + +``` + + +# TIPS +## Perform linear dimensional reduction (PCA) +Next we perform PCA on the scaled data. By default, only the previously determined variable features are used as input, but can be defined using features argument if you wish to choose a different subset. + +```{r runFunction2, results='hide'} + +# pbmc.ptw.kegg <- gficfScGSEA(object = pbmc3k.final, +# geneID = "symbol", +# species = "human", +# category = "C2", +# subcategory = 'CP:KEGG') +# +# feat.meta <- pbmc.ptw.kegg[['RNA']]@meta.features +# pbmc.ptw.kegg[['RNA']]@meta.features <- NULL +# pbmc.ptw.kegg <- FindVariableFeatures(pbmc.ptw.kegg, selection.method = "vst", nfeatures = ..) +# plot1 <- VariableFeaturePlot(pbmc.ptw.kegg) +# +# pbmc.ptw.kegg <- RunPCA(pbmc.ptw.kegg, features = VariableFeatures(object = pbmc.ptw.kegg)) + +``` + From 9b3d596f9dcdf4da1ce88835c912f978f40f1513 Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 14:56:32 +0100 Subject: [PATCH 10/17] Added scGSEA vignette --- docs/scGSEA.Rmd | 57 +++++++++++++++++++++++++++---------------------- 1 file changed, 31 insertions(+), 26 deletions(-) diff --git a/docs/scGSEA.Rmd b/docs/scGSEA.Rmd index abe588b..e735c88 100644 --- a/docs/scGSEA.Rmd +++ b/docs/scGSEA.Rmd @@ -5,12 +5,12 @@ output: github_document: html_preview: true toc: true + toc_depth: 3 html_document: df_print: kable - theme: united --- -This vigettte demonstrates how to run single-cell gene set enrichment analysis with gficf on Seurat objects. +This vignette demonstrates how to run single-cell gene set enrichment analysis with gficf on Seurat objects. > *Single-cell gene set enrichment analysis and transfer learning for functional annotation of scRNA-seq data* > @@ -25,14 +25,16 @@ This vigettte demonstrates how to run single-cell gene set enrichment analysis w > ```{r setup, include=FALSE} + knitr::opts_chunk$set( tidy = TRUE, tidy.opts = list(width.cutoff = 95), message = FALSE, warning = FALSE, - fig.height = 20, - fig.width = 16 + fig.height = 10, + fig.width = 20 ) + ``` @@ -44,10 +46,12 @@ Prerequisites to install: * [gficf](https://github.com/gambalab/gficf) ```{r looad libraries} + library(Seurat) library(SeuratData) library(SeuratWrappers) library(gficf) + ``` @@ -57,7 +61,7 @@ GF-ICF is an R package for normalization, visualization and analysis of of singl ## Load examle data from SeuratData To demonstrate how to run runScGSEA() function on a single-cell RNA-seq dataset. -We will use the pbmc3k dataset from the SeuratData package which contains 2,700 Peripheral Blood Mononuclear Cells (PBMC) which were sequenced on the Illumina NextSeq 500. +We will use the pbmc3k.final dataset from the SeuratData package which contains 2,638 PBMCs processed using the standard Seurat workflow as demonstrated in Seurat's guided clustering tutorial. ```{r load example data} @@ -133,36 +137,37 @@ DimPlot(pbmc.ptw, reduction = "umap", group.by = 'seurat_annotations') ``` ## Cluster cells based on pathways activity scores -Now that we reconstructed pathway’s activity at single cell level we can try to cluster cell according to these values using Seurat functions FindNeighbors() and FindClusters() and, finally, we graph results ad UMAP plot form. - +Now that we reconstructed pathway’s activity at single cell level we can try to cluster cell according to these values using Seurat functions FindNeighbors() and FindClusters(). Finally, we graph clustering results computed both on gene expression and pathway activity scores as UMAP plot form. ```{r UMAPclustersPathways} -DimPlot(pbmc.ptw, reduction = "umap", group.by = 'seurat_clusters_byGenes') pbmc.ptw <- FindNeighbors(pbmc.ptw, dims = 1:10) pbmc.ptw <- FindClusters(pbmc.ptw, resolution = 0.5) -DimPlot(pbmc.ptw, reduction = "umap", group.by = 'seurat_clusters') +DimPlot(pbmc.ptw, reduction = "umap", group.by = c('seurat_clusters_byGenes', 'seurat_clusters')) ``` # TIPS -## Perform linear dimensional reduction (PCA) -Next we perform PCA on the scaled data. By default, only the previously determined variable features are used as input, but can be defined using features argument if you wish to choose a different subset. - -```{r runFunction2, results='hide'} - -# pbmc.ptw.kegg <- gficfScGSEA(object = pbmc3k.final, -# geneID = "symbol", -# species = "human", -# category = "C2", -# subcategory = 'CP:KEGG') -# -# feat.meta <- pbmc.ptw.kegg[['RNA']]@meta.features -# pbmc.ptw.kegg[['RNA']]@meta.features <- NULL -# pbmc.ptw.kegg <- FindVariableFeatures(pbmc.ptw.kegg, selection.method = "vst", nfeatures = ..) -# plot1 <- VariableFeaturePlot(pbmc.ptw.kegg) -# -# pbmc.ptw.kegg <- RunPCA(pbmc.ptw.kegg, features = VariableFeatures(object = pbmc.ptw.kegg)) +## How can I handle collection with an high number of genesets? +In the case you are handling collections containing a lot of gene-sets, it could be useful to perform a feature selection step. To this end, we first store pathways metadata in a separate variable (i.e. feat.meta), then we create an empty dataframe in the meta.features slot to enable the storing of the output of the Seurat function FindVariableFeatures() setting nfeatures equal to 10% of the number of genesets used. + +```{r PCAselection, results='hide'} + +pbmc.ptw.kegg <- RunScGSEA(object = pbmc3k.final, + geneID = "symbol", + species = "human", + category = "C2", + subcategory = 'CP:KEGG') + +feat.meta <- pbmc.ptw.kegg[['RNA']]@meta.features +pbmc.ptw.kegg[['RNA']]@meta.features <- data.frame(row.names = rownames(pbmc.ptw.kegg)) +nfeatures <- round(nrow(pbmc.ptw.kegg)*0.10) +pbmc.ptw.kegg <- FindVariableFeatures(pbmc.ptw.kegg, selection.method = "vst", nfeatures = nfeatures) +top <- head(VariableFeatures(pbmc.ptw.kegg), nfeatures) +LabelPoints(plot = VariableFeaturePlot(pbmc.ptw.kegg), points = top, repel = TRUE) + +pbmc.ptw.kegg <- RunPCA(pbmc.ptw.kegg, features = VariableFeatures(object = pbmc.ptw.kegg)) +VizDimLoadings(pbmc.ptw.kegg, dims = 1:2, reduction = "pca") ``` From ab915f430be053a09dcb9156cd162e28143a78ef Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 15:46:34 +0100 Subject: [PATCH 11/17] Added scGSEA vignette all outputs --- docs/scGSEA.Rmd | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/docs/scGSEA.Rmd b/docs/scGSEA.Rmd index e735c88..647f015 100644 --- a/docs/scGSEA.Rmd +++ b/docs/scGSEA.Rmd @@ -2,12 +2,12 @@ title: "Running ScGSEA from gficf on Seurat Objects" date: 'Compiled: `r format(Sys.Date(), "%B %d, %Y")`' output: + html_document: + df_print: kable github_document: html_preview: true toc: true toc_depth: 3 - html_document: - df_print: kable --- This vignette demonstrates how to run single-cell gene set enrichment analysis with gficf on Seurat objects. From 3326eb345b3b51f0c1f5bce2ad66b2f5355883c3 Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 15:53:30 +0100 Subject: [PATCH 12/17] Added html vignette --- docs/scGSEA.html | 565 +++++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 565 insertions(+) create mode 100644 docs/scGSEA.html diff --git a/docs/scGSEA.html b/docs/scGSEA.html new file mode 100644 index 0000000..1514417 --- /dev/null +++ b/docs/scGSEA.html @@ -0,0 +1,565 @@ + + + + + + + + + + + + + +Running ScGSEA from gficf on Seurat Objects + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
+ + + + + + + +

This vignette demonstrates how to run single-cell gene set enrichment +analysis with gficf on Seurat objects.

+
+

Single-cell gene set enrichment analysis and transfer learning +for functional annotation of scRNA-seq data

+

Pellecchia Simona , Viscido Gaetano, Franchini Melania, Gambardella +Gennaro

+

bioRxiv, 2022

+

doi: https://doi.org/10.1101/2022.10.24.513476

+

GitHub: https://github.com/gambalab/gficf

+
+

Prerequisites to install:

+ +
library(Seurat)
+library(SeuratData)
+library(SeuratWrappers)
+library(gficf)
+
+

Introduction

+

GF-ICF is an R package for normalization, visualization and analysis +of of single-cell RNA sequencing data, based on a data transformation +model called term frequency–inverse document frequency (TF-IDF), which +has been extensively used in the field of text mining. Here, we show one +of the latest implementation which enables the pathway activity +computation at single-cell level.

+
+

Load examle data from SeuratData

+

To demonstrate how to run runScGSEA() function on a single-cell +RNA-seq dataset. We will use the pbmc3k.final dataset from the +SeuratData package which contains 2,638 PBMCs processed using the +standard Seurat workflow as demonstrated in Seurat’s guided clustering +tutorial.

+
InstallData("pbmc3k")
+data("pbmc3k.final")
+pbmc3k.final
+
## An object of class Seurat 
+## 13714 features across 2638 samples within 1 assay 
+## Active assay: RNA (13714 features, 2000 variable features)
+##  2 dimensional reductions calculated: pca, umap
+
+
+

Compute pathways activity at single-cell resolution

+

Single cell gene set enrichment analysis is performed by the function +runScGSEA() of gficf package which computes GSEA scores for each cell +across a set of input pathways by using NMF. The list of gene sets to +use can can be specified trough the category and subcategory +parameters.

+
pbmc.ptw <- RunScGSEA(object = pbmc3k.final, geneID = "symbol", species = "human", category = "H")
+
+
+
+

PART 1 : Evaluate pathways activity in single-cell data

+
+

Visualize UMAP plot computed on genes expression

+

As dimensionality reduction results computed on genes expression are +preserved, let’s graph the UMAP computed on genes x cells matrix. In +this case cells are colored by their cell type.

+
DimPlot(pbmc.ptw, reduction = "umap_byGenes", group.by = "seurat_annotations")
+

+
+
+

Identify how pathways activity change across cell type

+

To evaluate how pathways activity change across cell type, we utilize +DotPlot() function where dot size encodes for the percentage of cells of +each cell type in which the given pathway is active. Finally, cell types +are ordered (by hierarchical clustering) based on given features by +setting cluster.idents = TRUE.

+
library(ggplot2)
+library(RColorBrewer)
+
+all.pathways = rownames(pbmc.ptw)
+
+DotPlot(object = pbmc.ptw, features = all.pathways, split.by = "seurat_annotations", cols = RColorBrewer::brewer.pal(n = length(unique(pbmc.ptw$seurat_annotations)),
+    name = "Set1"), cluster.idents = TRUE) + theme(axis.text.x = element_text(angle = 60, vjust = 1,
+    hjust = 1, size = 10))
+

+
+
+

Inspect pathways activation in each cell of a given cell type

+

To inspect pathways activation in each cell of a given cell type, we +use DoHeatmap() function. In this case, we are plotting all given +pathways for each cell type.

+
DoHeatmap(pbmc.ptw, features = all.pathways, group.by = "seurat_annotations", angle = 90) + NoLegend()
+

+
+
+
+

PART 2 : Analyze data based on pathways activity scores

+
+

Visualize UMAP plot computed on pathways activity scores

+

Now, let’s see the UMAP computed on pathways activity scores. To this +end, we follow the standard Seurat workflow which requires the +computation of PCA on scaled data (i.e. pathway scores normalized by +gene-set) before run the UMAP reduction. Results will be stored in +separate reduction slot of the Seurat object. Finally, we display the +UMAP plot where cells are colored by their cell type.

+
pbmc.ptw <- RunPCA(pbmc.ptw, features = all.pathways)
+pbmc.ptw <- RunUMAP(pbmc.ptw, dims = 1:5)
+DimPlot(pbmc.ptw, reduction = "umap", group.by = "seurat_annotations")
+

+
+
+

Cluster cells based on pathways activity scores

+

Now that we reconstructed pathway’s activity at single cell level we +can try to cluster cell according to these values using Seurat functions +FindNeighbors() and FindClusters(). Finally, we graph clustering results +computed both on gene expression and pathway activity scores as UMAP +plot form.

+
pbmc.ptw <- FindNeighbors(pbmc.ptw, dims = 1:10)
+pbmc.ptw <- FindClusters(pbmc.ptw, resolution = 0.5)
+
## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
+## 
+## Number of nodes: 2638
+## Number of edges: 85312
+## 
+## Running Louvain algorithm...
+## Maximum modularity in 10 random starts: 0.8276
+## Number of communities: 9
+## Elapsed time: 0 seconds
+
DimPlot(pbmc.ptw, reduction = "umap", group.by = c("seurat_clusters_byGenes", "seurat_clusters"))
+

+
+
+
+

TIPS

+
+

How can I handle collection with an high number of genesets?

+

In the case you are handling collections containing a lot of +gene-sets, it could be useful to perform a feature selection step. To +this end, we first store pathways metadata in a separate variable +(i.e. feat.meta), then we create an empty dataframe in the meta.features +slot to enable the storing of the output of the Seurat function +FindVariableFeatures() setting nfeatures equal to 10% of the number of +genesets used.

+
pbmc.ptw.kegg <- RunScGSEA(object = pbmc3k.final, geneID = "symbol", species = "human", category = "C2",
+    subcategory = "CP:KEGG")
+
+feat.meta <- pbmc.ptw.kegg[["RNA"]]@meta.features
+pbmc.ptw.kegg[["RNA"]]@meta.features <- data.frame(row.names = rownames(pbmc.ptw.kegg))
+nfeatures <- round(nrow(pbmc.ptw.kegg) * 0.1)
+pbmc.ptw.kegg <- FindVariableFeatures(pbmc.ptw.kegg, selection.method = "vst", nfeatures = nfeatures)
+top <- head(VariableFeatures(pbmc.ptw.kegg), nfeatures)
+LabelPoints(plot = VariableFeaturePlot(pbmc.ptw.kegg), points = top, repel = TRUE)
+

+
pbmc.ptw.kegg <- RunPCA(pbmc.ptw.kegg, features = VariableFeatures(object = pbmc.ptw.kegg))
+VizDimLoadings(pbmc.ptw.kegg, dims = 1:2, reduction = "pca")
+

+
+
+ + + + +
+ + + + + + + + + + + + + + + From 504e108cc6aa3152ad3aea9877706fbdc64a661f Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 15:55:39 +0100 Subject: [PATCH 13/17] Added html vignette --- docs/scGSEA.md | 220 +++++++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 220 insertions(+) create mode 100644 docs/scGSEA.md diff --git a/docs/scGSEA.md b/docs/scGSEA.md new file mode 100644 index 0000000..5d71040 --- /dev/null +++ b/docs/scGSEA.md @@ -0,0 +1,220 @@ +Running ScGSEA from gficf on Seurat Objects +================ +Compiled: gennaio 12, 2023 + +- [Introduction](#introduction) + - [Load examle data from + SeuratData](#load-examle-data-from-seuratdata) + - [Compute pathways activity at single-cell + resolution](#compute-pathways-activity-at-single-cell-resolution) +- [PART 1 : Evaluate pathways activity in single-cell + data](#part-1--evaluate-pathways-activity-in-single-cell-data) + - [Visualize UMAP plot computed on genes + expression](#visualize-umap-plot-computed-on-genes-expression) + - [Identify how pathways activity change across cell + type](#identify-how-pathways-activity-change-across-cell-type) + - [Inspect pathways activation in each cell of a given cell + type](#inspect-pathways-activation-in-each-cell-of-a-given-cell-type) +- [PART 2 : Analyze data based on pathways activity + scores](#part-2--analyze-data-based-on-pathways-activity-scores) + - [Visualize UMAP plot computed on pathways activity + scores](#visualize-umap-plot-computed-on-pathways-activity-scores) + - [Cluster cells based on pathways activity + scores](#cluster-cells-based-on-pathways-activity-scores) +- [TIPS](#tips) + - [How can I handle collection with an high number of + genesets?](#how-can-i-handle-collection-with-an-high-number-of-genesets) + +This vignette demonstrates how to run single-cell gene set enrichment +analysis with gficf on Seurat objects. + +> *Single-cell gene set enrichment analysis and transfer learning for +> functional annotation of scRNA-seq data* +> +> Pellecchia Simona , Viscido Gaetano, Franchini Melania, Gambardella +> Gennaro +> +> bioRxiv, 2022 +> +> doi: +> +> GitHub: + +Prerequisites to install: + +- [Seurat](https://satijalab.org/seurat/install) +- [SeuratData](https://github.com/satijalab/seurat-data) +- [SeuratWrappers](https://github.com/satijalab/seurat-wrappers) +- [gficf](https://github.com/gambalab/gficf) + +``` r +library(Seurat) +library(SeuratData) +library(SeuratWrappers) +library(gficf) +``` + +# Introduction + +GF-ICF is an R package for normalization, visualization and analysis of +of single-cell RNA sequencing data, based on a data transformation model +called term frequency–inverse document frequency (TF-IDF), which has +been extensively used in the field of text mining. Here, we show one of +the latest implementation which enables the pathway activity computation +at single-cell level. + +## Load examle data from SeuratData + +To demonstrate how to run runScGSEA() function on a single-cell RNA-seq +dataset. We will use the pbmc3k.final dataset from the SeuratData +package which contains 2,638 PBMCs processed using the standard Seurat +workflow as demonstrated in Seurat’s guided clustering tutorial. + +``` r +InstallData("pbmc3k") +data("pbmc3k.final") +pbmc3k.final +``` + + ## An object of class Seurat + ## 13714 features across 2638 samples within 1 assay + ## Active assay: RNA (13714 features, 2000 variable features) + ## 2 dimensional reductions calculated: pca, umap + +## Compute pathways activity at single-cell resolution + +Single cell gene set enrichment analysis is performed by the function +runScGSEA() of gficf package which computes GSEA scores for each cell +across a set of input pathways by using NMF. The list of gene sets to +use can can be specified trough the category and subcategory parameters. + +``` r +pbmc.ptw <- RunScGSEA(object = pbmc3k.final, geneID = "symbol", species = "human", category = "H") +``` + +# PART 1 : Evaluate pathways activity in single-cell data + +## Visualize UMAP plot computed on genes expression + +As dimensionality reduction results computed on genes expression are +preserved, let’s graph the UMAP computed on genes x cells matrix. In +this case cells are colored by their cell type. + +``` r +DimPlot(pbmc.ptw, reduction = "umap_byGenes", group.by = "seurat_annotations") +``` + +![](scGSEA_files/figure-gfm/DimPlotGenes-1.png) + +## Identify how pathways activity change across cell type + +To evaluate how pathways activity change across cell type, we utilize +DotPlot() function where dot size encodes for the percentage of cells of +each cell type in which the given pathway is active. Finally, cell types +are ordered (by hierarchical clustering) based on given features by +setting cluster.idents = TRUE. + +``` r +library(ggplot2) +library(RColorBrewer) + +all.pathways = rownames(pbmc.ptw) + +DotPlot(object = pbmc.ptw, features = all.pathways, split.by = "seurat_annotations", cols = RColorBrewer::brewer.pal(n = length(unique(pbmc.ptw$seurat_annotations)), + name = "Set1"), cluster.idents = TRUE) + theme(axis.text.x = element_text(angle = 60, vjust = 1, + hjust = 1, size = 10)) +``` + +![](scGSEA_files/figure-gfm/DotPlotPathways-1.png) + +## Inspect pathways activation in each cell of a given cell type + +To inspect pathways activation in each cell of a given cell type, we use +DoHeatmap() function. In this case, we are plotting all given pathways +for each cell type. + +``` r +DoHeatmap(pbmc.ptw, features = all.pathways, group.by = "seurat_annotations", angle = 90) + NoLegend() +``` + +![](scGSEA_files/figure-gfm/DoHeatmapPathways-1.png) + +# PART 2 : Analyze data based on pathways activity scores + +## Visualize UMAP plot computed on pathways activity scores + +Now, let’s see the UMAP computed on pathways activity scores. To this +end, we follow the standard Seurat workflow which requires the +computation of PCA on scaled data (i.e. pathway scores normalized by +gene-set) before run the UMAP reduction. Results will be stored in +separate reduction slot of the Seurat object. Finally, we display the +UMAP plot where cells are colored by their cell type. + +``` r +pbmc.ptw <- RunPCA(pbmc.ptw, features = all.pathways) +pbmc.ptw <- RunUMAP(pbmc.ptw, dims = 1:5) +DimPlot(pbmc.ptw, reduction = "umap", group.by = "seurat_annotations") +``` + +![](scGSEA_files/figure-gfm/UMAPcellTypePathways-1.png) + +## Cluster cells based on pathways activity scores + +Now that we reconstructed pathway’s activity at single cell level we can +try to cluster cell according to these values using Seurat functions +FindNeighbors() and FindClusters(). Finally, we graph clustering results +computed both on gene expression and pathway activity scores as UMAP +plot form. + +``` r +pbmc.ptw <- FindNeighbors(pbmc.ptw, dims = 1:10) +pbmc.ptw <- FindClusters(pbmc.ptw, resolution = 0.5) +``` + + ## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck + ## + ## Number of nodes: 2638 + ## Number of edges: 85312 + ## + ## Running Louvain algorithm... + ## Maximum modularity in 10 random starts: 0.8276 + ## Number of communities: 9 + ## Elapsed time: 0 seconds + +``` r +DimPlot(pbmc.ptw, reduction = "umap", group.by = c("seurat_clusters_byGenes", "seurat_clusters")) +``` + +![](scGSEA_files/figure-gfm/UMAPclustersPathways-1.png) + +# TIPS + +## How can I handle collection with an high number of genesets? + +In the case you are handling collections containing a lot of gene-sets, +it could be useful to perform a feature selection step. To this end, we +first store pathways metadata in a separate variable (i.e. feat.meta), +then we create an empty dataframe in the meta.features slot to enable +the storing of the output of the Seurat function FindVariableFeatures() +setting nfeatures equal to 10% of the number of genesets used. + +``` r +pbmc.ptw.kegg <- RunScGSEA(object = pbmc3k.final, geneID = "symbol", species = "human", category = "C2", + subcategory = "CP:KEGG") + +feat.meta <- pbmc.ptw.kegg[["RNA"]]@meta.features +pbmc.ptw.kegg[["RNA"]]@meta.features <- data.frame(row.names = rownames(pbmc.ptw.kegg)) +nfeatures <- round(nrow(pbmc.ptw.kegg) * 0.1) +pbmc.ptw.kegg <- FindVariableFeatures(pbmc.ptw.kegg, selection.method = "vst", nfeatures = nfeatures) +top <- head(VariableFeatures(pbmc.ptw.kegg), nfeatures) +LabelPoints(plot = VariableFeaturePlot(pbmc.ptw.kegg), points = top, repel = TRUE) +``` + +![](scGSEA_files/figure-gfm/PCAselection-1.png) + +``` r +pbmc.ptw.kegg <- RunPCA(pbmc.ptw.kegg, features = VariableFeatures(object = pbmc.ptw.kegg)) +VizDimLoadings(pbmc.ptw.kegg, dims = 1:2, reduction = "pca") +``` + +![](scGSEA_files/figure-gfm/PCAselection-2.png) From fd5fd4eb7d9148415ce724bb445e61d623efb36f Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 15:58:03 +0100 Subject: [PATCH 14/17] Added man RunScGsea --- man/RunScGSEA.Rd | 44 ++++++++++++++++++++++++++++++++++++++++++++ 1 file changed, 44 insertions(+) create mode 100644 man/RunScGSEA.Rd diff --git a/man/RunScGSEA.Rd b/man/RunScGSEA.Rd new file mode 100644 index 0000000..6f03452 --- /dev/null +++ b/man/RunScGSEA.Rd @@ -0,0 +1,44 @@ +% Generated by roxygen2: do not edit by hand +% Please edit documentation in R/scGSEA.R +\name{RunScGSEA} +\alias{RunScGSEA} +\title{Run Single cell Gene Set Enrichement Analysis on GF-ICF on a Seurat object} +\usage{ +RunScGSEA( + object, + assay = NULL, + filterGenes = TRUE, + nmf.dim = 100, + geneID = c("ensamble", "symbol"), + species = c("mouse", "human"), + category = "H", + subcategory = NULL, + verbose = F +) +} +\arguments{ +\item{object}{Seurat object} + +\item{assay}{Assay to use, defaults to the default assay} + +\item{filterGenes}{Rank of NMF to use for the enrichment} + +\item{nmf.dim}{Rank of NMF to use for the enrichment} + +\item{geneID}{The type of gene names in rownames of \code{object}. It can be either 'ensamble' or 'symbol'. Default: 'ensamble'} + +\item{species}{The type of species in \code{object}. It can be either 'mouse' or 'human'. Default: 'human'} + +\item{category}{MSigDB collection abbreviation, such as H or C1} + +\item{subcategory}{MSigDB sub-collection abbreviation, such as CGP or BP} + +\item{rescale}{If different by none, pathway's activity scores are resealed as Z-score. Possible values are none, byGS or byCell. Default is none} +} +\value{ +returns a Seurat object with pathways x cell matrix +} +\description{ +This function run runScGSEA function from GF-ICF package on Seurat object. +It computes GSEA for each cells across a set of input pathways by using NMF. +} From 6ca71feb5e068bdeb0d7b7ac1876c3b5c62230b4 Mon Sep 17 00:00:00 2001 From: melania15 Date: Thu, 12 Jan 2023 16:01:22 +0100 Subject: [PATCH 15/17] fix --- .../scGSEA_files/figure-gfm/DimPlotGenes-1.png | Bin 0 -> 170175 bytes .../figure-gfm/DoHeatmapPathways-1.png | Bin 0 -> 206835 bytes .../figure-gfm/DotPlotPathways-1.png | Bin 0 -> 157764 bytes .../scGSEA_files/figure-gfm/PCAselection-1.png | Bin 0 -> 55424 bytes .../scGSEA_files/figure-gfm/PCAselection-2.png | Bin 0 -> 49549 bytes .../figure-gfm/UMAPcellTypePathways-1.png | Bin 0 -> 193876 bytes .../figure-gfm/UMAPclustersPathways-1.png | Bin 0 -> 301954 bytes 7 files changed, 0 insertions(+), 0 deletions(-) create mode 100644 docs/scGSEA_files/figure-gfm/DimPlotGenes-1.png 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Objects" -date: 'Compiled: `r format(Sys.Date(), "%B %d, %Y")`' +title: "Running scGSEA from gficf on Seurat Objects" +date: 'Compiled: `r format(Sys.Date(), "%m %d %y")`' output: html_document: df_print: kable @@ -14,12 +14,12 @@ This vignette demonstrates how to run single-cell gene set enrichment analysis w > *Single-cell gene set enrichment analysis and transfer learning for functional annotation of scRNA-seq data* > -> Pellecchia Simona , Viscido Gaetano, Franchini Melania, Gambardella Gennaro +> Franchini Melania, Pellecchia Simona , Viscido Gaetano, Gambardella Gennaro > -> bioRxiv, 2022 +> NAR Genomics and Bioinformatics, Volume 5, Issue 1, March 2023, lqad024 > > doi: -> +> > > GitHub: > @@ -47,13 +47,22 @@ Prerequisites to install: ```{r looad libraries} -library(Seurat) -library(SeuratData) -library(SeuratWrappers) -library(gficf) +require(Seurat) +require(SeuratData) +require(SeuratWrappers) +require(gficf) ``` +# Installation +Please install the Seurat wrapper which enables to run single-cell gene set enrichment analysis with gficf on Seurat objects as reported here: + +```{r install wrapper from gficf} + +if(!require(devtools)){install.packages("devtools")} +devtools::install_github("gambalab/seurat-wrappers") + +``` # Introduction diff --git a/docs/scGSEA.html b/docs/scGSEA.html index 1514417..71f9756 100644 --- a/docs/scGSEA.html +++ b/docs/scGSEA.html @@ -11,7 +11,7 @@ -Running ScGSEA from gficf on Seurat Objects +Running scGSEA from gficf on Seurat Objects

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