--- title: 【迁移】使用metacell进行分群聚类 urlname: shi-yong-metacell-jin-hang-fen-qun-ju-lei date: 2022-10-01 17:42:22 tags: metacell --- ## 预处理 ```R f_QC_plot <- function(sce){ options(repr.plot.width = 12, repr.plot.height = 6) print(Seurat::VlnPlot(sce, features = c("nFeature_RNA", "nCount_RNA", "percent.mt"), ncol = 3)) plot1 <- Seurat::FeatureScatter(sce, feature1 = "nCount_RNA", feature2 = "percent.mt") plot2 <- Seurat::FeatureScatter(sce, feature1 = "nCount_RNA", feature2 = "nFeature_RNA") plot1 + plot2 } f_read10x <- function(sce, project='sce'){ sce <- Seurat::CreateSeuratObject(sce, project = project, min.cells = 3, min.features = 200) sce[["percent.mt"]] <- Seurat::PercentageFeatureSet(sce, pattern = "^MT-") sce[["percent.rp"]] <- Seurat::PercentageFeatureSet(sce, pattern = "^RP[SL]") sce <- subset(sce, nFeature_RNA >= quantile(nFeature_RNA, 0.025) & nFeature_RNA <= quantile(nFeature_RNA, 0.975) & nCount_RNA >= quantile(nCount_RNA, 0.025) & nCount_RNA <= quantile(nCount_RNA, 0.975) & percent.mt <= quantile(percent.mt, 0.975)) sce <- Seurat::NormalizeData(sce) g2m_genes <- Seurat::CaseMatch(search=Seurat::cc.genes$g2m.genes, match=rownames(sce)) s_genes <- Seurat::CaseMatch(search=Seurat::cc.genes$s.genes, match=rownames(sce)) sce <- Seurat::CellCycleScoring(sce, g2m.features=g2m_genes, s.features=s_genes) sce$CC.Difference <- sce$S.Score - sce$G2M.Score sce <- sce[!grepl(pattern = "(^MT-^RP[SL])",x = rownames(sce)),] sce <- Seurat::SCTransform(sce, vst.flavor = "v2", vars.to.regress = c("CC.Difference", "percent.mt", "percent.rp"), verbose = F) sce } ``` ```R sce <- Seurat::Read10X('filtered_feature_bc_matrix') sce <- f_read10x(sce, project = 'SRX8890106') ``` ## 安装补充包 * [conda activate seurat](/shi-yong-SCTransform-biao-zhun-hua) * ~/dev/xray/xray -c ~/etc/xui2.json & * wget -e "https\_proxy=http://127.0.0.1:20809" https://github.com/tanaylab/metacell/archive/refs/heads/master.zip -O metacell-master.zip * devtools::install\_local('metacell-master.zip') * conda install -c bioconda bioconductor-singlecellexperiment -y ## seurat转metacell ```R ###### 构建metacell对象 ## 初始化 # 设置存放数据的目录 if(!dir.exists("scdb")){dir.create("scdb")} metacell::scdb_init("scdb", force_reinit=T) # 设置存放图形的目录 if(!dir.exists("figs")){dir.create("figs")} metacell::scfigs_init("figs") ## 提取高变基因 var.genes <- Seurat::VariableFeatures(sce) var.genes <- structure(rep(1:length(var.genes)), names=var.genes) var.genes <- metacell::gset_new_gset(sets = var.genes, desc = "seurat variable genes") metacell::scdb_add_gset("SRX8890106", var.genes) ## 提取counts矩阵 mat <- Seurat::as.SingleCellExperiment(sce) mat <- metacell::scm_import_sce_to_mat(mat) metacell::scdb_add_mat("SRX8890106", mat) ``` ## 聚类MetaCell ```R ## 构建平衡KNN图 metacell::mcell_add_cgraph_from_mat_bknn(mat_id = "SRX8890106", gset_id = "SRX8890106", graph_id = "SRX8890106_k100", K = 100, dsamp = F) # 20,000 cells之内不必抽样 ## 共聚类 metacell::mcell_coclust_from_graph_resamp(coc_id = "SRX8890106_n1000", graph_id = "SRX8890106_k100", min_mc_size = 20, p_resamp = 0.75, n_resamp=1000) ## 生成初级metacell metacell::mcell_mc_from_coclust_balanced(coc_id = "SRX8890106_n1000", mat_id = "SRX8890106", mc_id = "SRX8890106", K = 20, min_mc_size = 20, alpha = 2) ## 修剪metacell metacell::mcell_plot_outlier_heatmap(mc_id = "SRX8890106", mat_id = "SRX8890106", T_lfc = 3) metacell::mcell_mc_split_filt(new_mc_id = "SRX8890106", mc_id = "SRX8890106", mat_id = "SRX8890106", T_lfc = 3, plot_mats = T) ## 2D图展示Cells与MCs metacell::mc_colorize_default('SRX8890106') metacell::mcell_mc2d_force_knn(mc2d_id="SRX8890106", mc_id="SRX8890106", graph_id="SRX8890106_k100") tgconfig::set_param("mcell_mc2d_height", 1000, "metacell") tgconfig::set_param("mcell_mc2d_width", 1000, "metacell") metacell::mcell_mc2d_plot(mc2d_id = "SRX8890106") ``` ## 导出MetaCell到seurat ```R mc <- metacell::scdb_mc('SRX8890106') sce$metacell <- 0 sce$metacell[names(mc@mc)] <- mc@mc saveRDS(sce@meta.data, 'SRX8890106_meta.rds') ``` ## 确定最佳分群 读入之前使用metacell进行分群聚类中的数据 ```R f_getBestPcs <- function(stdev){ # Determine percent of variation associated with each PC pct <- stdev / sum(stdev) * 100 # Calculate cumulative percents for each PC cumu <- cumsum(pct) # Determine which PC exhibits cumulative percent greater than 90% and % variation associated with the PC as less than 5 co1 <- which(cumu > 90 & pct < 5)[1] co1 # Determine the difference between variation of PC and subsequent PC co2 <- sort(which((pct[1:length(pct) - 1] - pct[2:length(pct)]) > 0.1), decreasing = T)[1] + 1 # Minimum of the two calculation pcs <- min(co1, co2) pcs } f_plotBestClusters <- function(sce){ sce <- Seurat::FindClusters( object = sce, resolution = c(seq(.1,1.6,.1)) #起始粒度，结束粒度，间隔 ) options(repr.plot.width = 12, repr.plot.height = 16) require(clustree) clustree::clustree(sce@meta.data, prefix = "SCT_snn_res.") } ``` ```R sce <- readRDS('SRX8890106.rds') sce@meta.data <- readRDS('SRX8890106_meta.rds') sce <- Seurat::RunPCA(sce, assay="SCT", verbose = FALSE) pcs <- f_getBestPcs(sce [["pca"]]@stdev) sce <- Seurat::FindNeighbors(sce, reduction = "pca", dims = 1:pcs, verbose = FALSE) f_plotBestClusters(sce) ``` ## 进行分群 ```R sce <- Seurat::FindClusters( object = sce, resolution = 1.3 #读图得到最佳分辨率 ) sce <- Seurat::RenameIdents(sce, '6'='6', '12'='6', '16'='6', '2'='6', '5'='6' ) sce <- Seurat::RenameIdents(sce, '0'='0', '1'='0', '9'='0' ) table(Seurat::Idents(sce)) ``` ## 标注Doublet 读表获取先验的Doublet占比 ```R f_Doublet_get_pK <- function(sce, pcs){ sweep.res <- DoubletFinder::paramSweep_v3(sce, PCs = 1:pcs, sct = T, num.cores=4) sweep.stats <- DoubletFinder::summarizeSweep(sweep.res, GT = FALSE) bcmvn <- DoubletFinder::find.pK(sweep.stats) pK_bcmvn <- as.numeric(as.character(bcmvn$pK[which.max(bcmvn$BCmetric)])) pK_bcmvn } f_DoubletFinder <- function(sce, pcs, pK_bcmvn, DoubletRate, seurat_clusters){ homotypic.prop <- DoubletFinder::modelHomotypic(seurat_clusters) # 最好提供celltype nExp_poi <- round(DoubletRate*length(seurat_clusters)) nExp_poi.adj <- round(nExp_poi*(1-homotypic.prop)) sce <- DoubletFinder::doubletFinder_v3(sce, PCs = 1:pcs, pN = 0.25, pK = pK_bcmvn, nExp = nExp_poi.adj, reuse.pANN = FALSE, sct = T) sce } ``` ```R pK_bcmvn <- f_Doublet_get_pK(sce, pcs) sce$seurat_clusters <- Idents(sce) # ~8000 cells ~6.1% DoubletRate sce <- f_DoubletFinder(sce, pcs, pK_bcmvn, 0.061, sce$seurat_clusters) saveRDS(sce@meta.data, 'SRX8890106_meta.rds') ``` ## 可视化 ```R sce <- Seurat::RunUMAP(sce, reduction = "pca", dims = 1:30, verbose = FALSE) options(repr.plot.width = 12, repr.plot.height = 6) DimPlot(sce, reduction = "umap", label = T, repel = T, group.by = c("DF.classifications_0.25_0.04_416", 'seurat_clusters')) ``` ![](https://img.limour.top/2023/09/01/64f1b3487b4a7.webp)