scRNA-seq_analysis

pipelines/05_subset_seurat/subset_seurat.R

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+# import libraries
+library(Seurat)
+
+seurat.obj.addr = "../../data/test_yolk_sac_subset.RDS"
+save.at = "../../data/test_yolk_sac_subset_cluster_1.RDS"
+process = T
+add.dr  = T
+filter.args = list("cell.labels" = c("HSC/MPP" , "Neutrophil-myeloid progenitor", "Monocyte-DC precursor", "DC2"), "gender" = c("male"))
+
+
+#######################################################################################################
+#######################################################################################################
+#######################################################################################################
+
+# load the seurat object
+print("Loading the data ... ")
+seurat.obj = readRDS(seurat.obj.addr)
+print("Data loaded")
+
+source("../../tools/bunddle_utils.R")
+
+cells.to.keep = rep(T, length(seurat.obj@ident))
+for(k in 1:length(filter.args)){
+  cat = filter.args[k]
+  conditions = as.vector(unlist(cat))
+  cat = as.vector(names(cat))
+  satisfy = seurat.obj@meta.data[, cat] %in% conditions
+  cells.to.keep = cells.to.keep & satisfy
+}
+
+if(!any(cells.to.keep)){
+  print("No cells have been selected. Relax the conditions.")
+}else{
+  seurat.obj = SubsetData(object=seurat.obj, cells.use=names(seurat.obj@ident)[cells.to.keep], subset.raw=T, do.clean=T)
+  # add processing
+  
+  if (process){
+    # normaliza data
+    print("Normalizing data ...")
+    seurat.obj = NormalizeData(object = seurat.obj, normalization.method = "LogNormalize", scale.factor = 10000)
+    
+    print("Computing variable genes ...")
+    # find variable genes
+    seurat.obj = FindVariableGenes(object = seurat.obj, mean.function = ExpMean, 
+                                   dispersion.function = LogVMR, x.low.cutoff = .0125, 
+                                   x.high.cutoff = 3, y.cutoff = .625)
+    # calculate percentage of variable genes
+    print(paste("Percentage of variable genes:", round(100 * length(seurat.obj@var.genes) / dim(seurat.obj@data)[1], digits = 2), sep = " "))
+    
+    # scale data in variable genes, otherwise pca is not possible
+    print("Scaling data ...")
+    seurat.obj = ScaleData(object=seurat.obj)
+    
+    # run PCA
+    print("Performing PCA ...")
+    seurat.obj = RunPCA(object = seurat.obj, pc.genes = seurat.obj@var.genes, do.print = TRUE, pcs.print = 1:20, genes.print = 10)
+  }
+  
+  if(add.dr){
+    # run TSNE
+    print("Performing TSNE")
+    seurat.obj = RunTSNE(object=seurat.obj, dims.use=1:20, seed.use=42, do.fast=TRUE)
+    
+    # run umap
+    print("running UMAP")
+    umap.coordinates = RunUMAP(pca.df=seurat.obj@dr$pca@cell.embeddings, tool_addr=tool_addr, python.addr=python.addr)
+    rownames(umap.coordinates) = names(seurat.obj@ident)
+    seurat.obj = SetDimReduction(object=seurat.obj, reduction.type="umap", slot="cell.embeddings", new.data=as.matrix(umap.coordinates))
+    seurat.obj = SetDimReduction(object=seurat.obj, reduction.type="umap", slot="key", new.data="umap")
+    
+    # run force-directed graph
+    print("Running force directed graph")
+    seurat.obj = BuildSNN(object=seurat.obj, reduction.type="pca", dims.use=1:20, plot.SNN=F, force.recalc=TRUE, prune.SNN=.1)
+    fdg_coordinates = runFDG(pca.df=seurat.obj@dr$pca@cell.embeddings, snn=seurat.obj@snn, iterations=2000, tool_addr=tool_addr, python.addr=python.addr)
+    seurat.obj = SetDimReduction(object=seurat.obj, reduction.type="fdg", slot="cell.embeddings", new.data=as.matrix(fdg_coordinates))
+    seurat.obj = SetDimReduction(object=seurat.obj, reduction.type="fdg", slot = "key", new.data = "fdg")
+  }
+  
+  # save seurat object
+  print(sprintf("saving data at: %s", save.at))
+  saveRDS(seurat.obj, save.at)
+}
+
+file.remove("Rplots.pdf")
+
+print("Ended beautifully ... ")

pipelines/05_subset_seurat/subset_seurat.sh

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+#!/bin/bash
+
+#$ -cwd
+#$ -N subset_seurat
+#$ -V
+#$ -l h_rt=47:59:59
+#$ -l h_vmem=300G
+
+Rscript subset_seurat.R
+
+echo "End on `date`"