class: center, middle, inverse, title-slide # Single-cell RNA sequencing ~ Session 2
### Rockefeller University, Bioinformatics Resource Centre ###
https://rockefelleruniversity.github.io/scRNA-seq/
--- ## Data analysis with [Seurat](https://satijalab.org/seurat/) [Seurat](https://satijalab.org/seurat/) is a powerful package for single-cell data analysis. It supports many common analysis of single-cell RNAseq and cross connection with many useful packages through [SeuratWrapper](https://github.com/satijalab/seurat-wrappers). In this session, we will demonstrate our regular workflow for single-cell RNAseq data analysis with Seurat. --- ## Outline - Read Cell Ranger results into Seurat object - Generate QC - Evaluate cell cycle - Normalize data and do clustering - Marker genes for each cluster - Advanced visualization --- class: inverse, center, middle # Create Seurat object and Generare QC plots <html><div style='float:left'></div><hr color='#EB811B' size=1px width=720px></html> --- ## Cell Ranger counting matrix - The data is available on [DropBox](https://www.dropbox.com/sh/ztu0pucvu21szxm/AABGUhTryKp1T1CsoEUFRPcwa?dl=0) ```r tar_dir <- "~/path/to/raw/data" ``` --- ## Load Cell Ranger results into Seurat - Loading data - Estimate mitochondrial contents ```r library(Seurat) samID <- "CTRL" X10_file <- Seurat::Read10X(tar_dir) obj <- CreateSeuratObject(counts = X10_file, project = samID, min.cells = 5, min.features = 200) obj$dset <- samID obj <- Seurat::RenameCells(obj, add.cell.id = samID) # obj[["percent.mt"]] <- PercentageFeatureSet(obj, pattern = "^mt-") ``` --- ## Subset dataset We have a small subset version of this dataset that you can load in from the *data/* directory to try this out. --- ## Cell information The object metadata contains cell barcode, mitochondrial contents, datasets, and clustering etc ```r head(obj@meta.data) ``` ``` ## orig.ident nCount_RNA nFeature_RNA dset percent.mt ## CTRL_CACTCCATCTTATCTG-1 CTRL 7748 2372 CTRL 5.201342 ## CTRL_CCTCTGACAGTAGAGC-1 CTRL 9301 2715 CTRL 5.053220 ## CTRL_GACGTTAGTAGAAGGA-1 CTRL 8213 2412 CTRL 4.699866 ## CTRL_TGATTTCCAGCCTGTG-1 CTRL 8363 2308 CTRL 5.177568 ## CTRL_ATCGAGTCAATCTGCA-1 CTRL 3392 1303 CTRL 20.725236 ## CTRL_TGACTTTTCACCGGGT-1 CTRL 7034 2166 CTRL 7.094114 ``` --- ## Raw count matrix ```r head(obj@assays$RNA@counts) ``` ``` ## 6 x 1000 sparse Matrix of class "dgCMatrix" ``` ``` ## [[ suppressing 1000 column names 'CTRL_CACTCCATCTTATCTG-1', 'CTRL_CCTCTGACAGTAGAGC-1', 'CTRL_GACGTTAGTAGAAGGA-1' ... ]] ``` ``` ## ## Mrpl15 . 1 3 . 1 1 1 . . . 5 . . 5 1 1 2 . . . 1 1 . 1 2 1 2 . 2 . 2 1 1 . . 1 ## Lypla1 . 1 . . . . 2 . 1 1 . . . 2 . . 1 . . . . . 1 1 . 1 . 2 1 1 1 . . . . 1 ## Tcea1 . 1 . . . 1 . . . . 1 . 1 1 . . . . . . . 3 . 1 . . . 1 . 1 . . 1 . . 2 ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . 3 . . . . . 1 . . . 1 3 . . 1 . . . 1 . . . . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . . 1 1 1 1 1 1 2 . . 1 1 4 . . . . 1 . . . 2 2 . . . 2 3 . 1 . 1 1 . . ## Lypla1 . . 1 . . 1 . . 1 . . . . 1 . . 1 . 1 . . 1 . 1 . . . 2 . . . . . 2 . . ## Tcea1 . . 1 . . 1 . 2 . . 1 . . 2 . . . . . . 1 . 2 2 . . . . . . 1 . 2 . . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . 1 1 . . . . . . . . . . 1 . . 1 . 1 . . . . 1 . . . . . . . 1 1 . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 2 3 1 1 4 1 4 1 . . . 1 3 . . . 1 . 1 . 1 . 2 3 . 1 . . . 1 . 2 . . 3 . ## Lypla1 . . 1 . . . 1 . . . 1 . 1 . 1 . 1 1 . 1 3 . 1 1 2 . . . . . 1 . . 1 . 1 ## Tcea1 2 2 . . 1 1 3 . . . 1 . . . . . 1 . . 1 2 . . . . . 1 . . . . . . 1 2 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 1 2 . . . 2 . 1 . . . . . . . . . . . . . . . 1 . . . 1 . . . 1 . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 2 2 . . . . 2 . . 1 . . 3 . . . . 1 3 . . 1 . 1 . 2 2 1 . 2 1 . 1 . 2 . ## Lypla1 . . 1 1 . . 2 . . 1 . . . . . . . 1 . . . 1 . 4 . . 1 2 . 1 . . . . 1 . ## Tcea1 4 1 1 . . . . 1 1 . . . . . . . . . . . . . 1 5 . 1 1 . . 1 . . 1 . . 1 ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . 1 1 . . . . . 1 . . . 1 . . . . . . . . . . . . 1 . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . 1 . . . 1 . . 2 . 2 . . 1 . . . . . 2 1 . 2 . . 1 1 . . 2 . . . . 1 2 ## Lypla1 . . . . 2 . . . . . . . . . . . . . 2 . . . 1 . 1 1 . . 1 . 1 . 1 . . 1 ## Tcea1 1 . . . . . . . . . 1 1 . . . 1 . . 1 . . 1 . . . . . . . 5 . . . 1 . 2 ## Rgs20 . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . 1 . . . . . . . . 1 1 1 . . . . . . . . . . . . 1 1 . . 1 . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . . . . . . 1 1 . . 2 . . . 1 3 1 . . 1 1 . 3 1 . 2 1 1 . 2 . . . 2 1 4 ## Lypla1 . . . 1 . 1 . . 1 . . 1 . . 1 2 1 . . . . . . . . 1 1 1 . 1 . 1 . . 1 3 ## Tcea1 . . . 1 . . . . 1 . 1 . . . . . . . . 1 1 1 . . . . . . 1 . . . . 2 . 3 ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . 1 . . 1 . . . . . . . . . . . . . 2 . . . 1 . 1 . . 1 . . 1 . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . 3 1 2 2 . . . 3 1 6 . 3 . . 2 . 2 . . 4 . 1 . 1 . . . . 1 . 2 2 1 . . ## Lypla1 . . 1 . . . . . 1 . 1 . . . . 1 . 1 1 . 1 . . . 1 . . . 2 . 1 . . . 1 . ## Tcea1 . . . . . . . . 2 . 1 . . . . . . . . . . . 2 . 1 . 1 . . . 1 . . . . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 1 . 1 . 1 . . . . . . . . . . . . . . 1 . . . . . 1 . 1 . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . 1 2 . . 1 1 . . . 3 . . . . . . . 1 . . . 1 1 . 3 . 1 . . . 5 . . . 1 ## Lypla1 . . . . . . . . . . 1 . . 1 1 . . . . 1 . . 1 1 . 1 . . 1 . . . . 1 1 . ## Tcea1 1 . . 1 . . . . . . . 1 . . . 1 . . 1 . 1 . . 2 . 1 . . . . 1 1 . . . . ## Rgs20 . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . . 1 2 . . . . . . . . . . . . . . . . . 1 . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 2 2 1 1 2 . 4 2 2 . 2 . . 2 4 . . 1 . 2 2 . . 1 2 1 1 1 . 3 1 4 1 . 2 2 ## Lypla1 . . . . 2 . 1 . . . . . . . 1 . 2 . . . 1 . . 1 1 . . . 2 . 1 2 . . 1 1 ## Tcea1 . . 1 . . . 1 . . . . . . . 1 1 . 1 1 1 2 . . 1 . 1 . . 1 1 1 1 . . 2 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . 1 . 1 1 . . . . . . . . . . . . . 1 1 1 . . 3 . . 1 . . . 1 . . . . . ## Npbwr1 . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 3 . . . . . 2 . . . 2 1 . . . 2 . . . . 2 . . 4 1 . . 1 . 1 6 . . . 3 1 ## Lypla1 1 . . . . . . . . . . . 2 . . . . . . . 1 . 1 1 . . . . . . . . 2 . . 1 ## Tcea1 1 1 1 . 1 . 1 . . . 1 . 1 . . 3 . . . . 1 . . 1 1 . . . . 2 . 1 1 2 1 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 2 . . 1 . . 1 . . . . . . . . . . 1 1 . . . 1 . . . . . . . 1 . 1 . 2 1 ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 . 1 1 1 . . . . . . 1 . . . 1 . . . 1 . 3 1 1 3 . 1 . 1 3 1 1 1 . . 1 ## Lypla1 . . . 3 1 . . . . . . . . . . . . . 1 1 . . . 1 . . . . . . 1 . . . . . ## Tcea1 . . . 1 . . . . . 1 . . . . 1 . 1 . 2 1 . 2 . 1 1 . 1 . . 1 . . 1 . . 1 ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 1 . . . . . . . . . . . . . . . . . . 1 . 2 1 . 1 . . . . 1 . . . . . 1 ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 3 1 1 1 . . . 2 . . . . 1 . . 4 3 . 4 . 3 . . 2 1 1 . 1 . 2 . 1 . . . 1 ## Lypla1 . . 1 . . 1 . 1 . . 1 1 . . . 1 2 . 1 . . 1 . 1 . . . 1 1 . 2 . 1 . 1 . ## Tcea1 4 . 2 . 2 2 1 1 . . . . . . . . . . . . . 1 . 1 . 1 1 . . 1 . 1 . . . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . 1 . 1 . 1 . . . . . . . 1 . . . 1 1 . . . . 1 1 . . . . 1 . . . 1 ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 . . . . 1 . . 1 . . 2 . . 1 . 1 1 . 1 . . 1 1 2 . 1 3 1 . 1 . 2 . 1 . ## Lypla1 1 . . . 1 . 1 . . 1 . . . 1 1 1 . 1 . . . . . 1 . . 1 2 1 . . . . 1 . . ## Tcea1 . 1 . . . 1 . . . 2 . . . 1 . . . . 1 . . . 1 1 1 . . 1 4 . . . . . 2 1 ## Rgs20 . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . 1 . . . . 1 . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . ## ## Mrpl15 . . . 1 1 2 . . 1 1 1 1 1 1 . . 1 2 3 1 . . . . 2 . 5 1 . 1 . 1 1 2 2 1 ## Lypla1 1 . 1 1 . 1 . 2 1 . . . 1 2 . . . 2 . . . 1 . 2 1 . 2 . 1 1 . . 1 2 1 . ## Tcea1 . 1 . . . . 2 2 . . . . 1 . . . 2 4 2 1 . . . . . . 3 2 1 . . . . 1 . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . 2 . 1 . 1 . . . . . . . . 1 . 1 . . 1 . 1 1 . 1 . . . . . 1 . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . 3 . . . 1 . 4 6 . . . . . . . 1 1 2 . 1 1 . 1 3 . 2 3 . . 6 1 . 1 2 4 ## Lypla1 . . . . 1 1 . 1 1 1 . . 2 . . . 1 . 1 . . 1 . 1 . . 2 2 . . . 1 . . . . ## Tcea1 . 1 . . 2 . . . 4 . 1 . . 1 . 1 2 . 1 . . . . . 2 1 . . . . 3 2 . . . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 1 1 . 1 . 1 1 . 1 . . . . . 1 . 1 1 . . . . . . 1 . . . . 1 . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 2 . . . 5 . 3 2 . 1 1 . 2 1 . . 1 . . 1 . . . . 1 . 1 . 5 1 1 . . 7 . 1 ## Lypla1 1 1 . 1 . 2 . . . . . . 4 . 1 . 1 . . . . . . . . . . . 3 . 1 . . 1 . . ## Tcea1 2 2 1 . . . . 1 1 . . 1 3 1 . . 1 . . . . . . . 1 1 . . 1 1 1 . . . 1 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . 1 . . . . . . . . . . . . . . . . . . . . . 1 . . 2 . 1 . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 . . 1 . . . . . 2 . . 3 . 1 3 4 . . 1 . . . . . 2 1 1 2 . 4 . 1 . . . ## Lypla1 . . . . . 2 1 2 1 1 . . . 3 . 1 1 1 1 . . 2 . . . 1 . . 1 . 1 . . . 2 . ## Tcea1 2 . . 1 1 . . 1 . 1 . 2 1 2 . 4 2 1 . . . . 1 1 . 1 . . 1 . . . 2 . . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . . . 1 1 . . . 1 1 . . . . . . . . . . 1 . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . . 1 . 3 2 . . . 1 1 1 . . 1 . . . 4 . 3 . 3 3 . . 1 . 2 . . 2 1 . . . ## Lypla1 . 1 1 . 2 1 . . . . . . 1 1 1 . . . 1 . . . 2 1 . 1 . . . . 1 . 1 1 . . ## Tcea1 1 . . . 2 3 . . . . . 1 . . . . 1 . . . 2 1 1 . 2 . . . . . . 1 . 1 . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . . 2 1 1 . . . 1 . . 1 . . . . . . . . 1 1 . 2 . 1 . . . . . . 1 ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 . . . 1 . 1 4 2 . 3 2 . . . 1 . . . . 2 . . 1 3 . . . 2 . 1 . . . 1 2 ## Lypla1 . . . 1 . 1 . 1 . . 1 . . . . 1 . . 1 . . . . . . 1 . 1 2 . . 1 . . . . ## Tcea1 . . 1 . 1 1 . . 1 . 2 3 . . . . 1 . 1 2 . 1 . . 3 . . 1 . 1 . . . 2 . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . 1 . . . 1 . . . 2 1 . . 1 . . . . 1 . . . . . . . . 1 . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . 1 2 . . 1 1 . 3 2 . . . . 3 1 1 2 6 . 1 1 . 1 4 2 . 2 . 1 2 . . 3 1 . ## Lypla1 . . . . . . 1 . . . . 1 . . 1 . . . . 1 . . . 1 2 2 1 . . 1 . 2 . . . . ## Tcea1 . 1 . . . . 1 1 . . . 1 . . . . . . 1 1 . 1 . . 2 3 . . . 2 1 . . . 1 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 1 1 . . . . 1 . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 3 1 . . 2 . 1 . 1 . 1 1 . 1 . . 1 1 1 3 . 3 . . . 1 . . . . 1 1 . . . . ## Lypla1 2 1 . . 1 . . . . . . . . . . . 2 . . 1 . . 1 . . . 1 . . . . 1 . . . . ## Tcea1 1 . 2 . 2 . 1 . . . . 1 . 1 . . 1 . . . . . . . . . . . . . . . . 1 . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . 1 . . . . . . . . . 1 . 1 . . . . . . . . . . . . . . ## Npbwr1 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 . 1 . 1 . 4 . . . . . 3 . . 2 2 1 . 1 1 . . . . . . . . 1 2 1 . . 1 5 ## Lypla1 1 . 2 . . 1 1 . 1 . . 1 1 . 2 . 1 1 . . . . . 1 1 . 1 . 2 . 1 . 1 . 2 . ## Tcea1 1 . 2 . . . 1 . . . . 2 1 1 . 1 . . . . . . . . 1 2 1 1 . 1 . 1 1 . . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . . 2 . . . 2 1 . . . 3 1 1 . . . . . 1 2 . . 1 1 . . 2 . . 1 . . . 2 2 ## Lypla1 . . 1 . . . 1 1 . . . . . . . 1 2 . . 1 . . . 1 . . . 1 . . . . . . 1 . ## Tcea1 . . 1 . 2 . . . . 1 . . . 1 2 2 . . . 2 . . . 1 . . . . . . . . . 1 1 . ## Rgs20 . . . . . . 1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . 2 . . . . . . . . . . . . 1 . . . . . . . 1 . . . . . 1 . . . . . 1 ## Npbwr1 . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 . 4 . . 2 1 . 3 1 . . 7 . 2 1 . . . . 3 . . . 1 . . . 2 . . 1 . 3 . 1 ## Lypla1 . . . . . . 2 . . 2 . . 1 . 3 . . 1 . . . . . . . . 1 . 2 . 1 1 . 2 2 1 ## Tcea1 . . 1 2 . . . . . . . . 1 . 1 . . . . . . . . . . . 2 . . . . . 1 1 . 2 ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h 1 . . . . . . . 1 2 . 1 1 . . . . . . . . . 1 . . . . . . . 1 . 1 . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 1 1 3 . . . . 1 . . 1 . . 4 1 . 1 2 . . 4 2 2 3 . . 1 . . . . . . . 2 . ## Lypla1 1 2 . . 1 . . 3 . 1 1 . . . . . . . . . . 1 1 1 . . . . . . . . . . . . ## Tcea1 . 3 1 . . 1 . . 1 . . 1 . . 1 1 1 . . 3 1 1 2 4 . . . . . . . . . 1 . . ## Rgs20 . . . . . . . . . . . . . . . . 1 . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . 3 . . . . . . . . . . . . . . . . . 1 . . . . . 1 . . . 1 . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . . 1 . 2 3 2 . 1 . . 1 1 . . 1 . . 1 1 2 . . 1 1 . 3 . . . . . . 1 1 . ## Lypla1 . . . . 1 . 1 . . . . 2 2 . . . . . 3 . 1 . . . 1 . 1 1 . . . . . . . 1 ## Tcea1 . . . . . . . . 1 2 . . 2 2 . . . . 1 . 1 . . 1 . 1 1 . . . . . . . 1 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . 1 . 2 . . . . . . 1 . 2 . . . . 1 . . . . 1 . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . 2 . 2 . . . . 1 3 . . . . . . . . . 7 . . . 1 1 1 . 3 2 . . . . 4 . . ## Lypla1 1 . . 2 1 . . . . 1 1 . . 1 1 1 . . . 1 . . . . 1 1 . . . . . . 1 1 . . ## Tcea1 . . . 1 . . . . . 1 . . . . . 1 . . . 1 . . . 3 2 1 . 1 . 1 1 . 1 2 . . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . . . 1 . . . . . . . . . . . . . . . 1 . . 1 . . . . 1 . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## ## Mrpl15 . . . 2 . . . . . . 1 2 1 . . . . . . 3 2 . 1 2 2 1 5 2 ## Lypla1 . 1 . 1 1 . . 2 . 2 2 1 . . . . . . . . . . 1 1 1 1 3 1 ## Tcea1 . . 1 1 . . 2 . . . 1 1 1 . 2 . . . . . . . 1 . 1 1 1 . ## Rgs20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . ## Atp6v1h . . 1 . 1 . . 1 . . 1 . . . . 1 . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . . . . . 1 . . . . . . 1 . . . ``` --- ## Scaled or normalized data ```r head(obj@assays$RNA@data) ``` ``` ## 6 x 1000 sparse Matrix of class "dgCMatrix" ``` ``` ## [[ suppressing 1000 column names 'CTRL_CACTCCATCTTATCTG-1', 'CTRL_CCTCTGACAGTAGAGC-1', 'CTRL_GACGTTAGTAGAAGGA-1' ... ]] ``` ``` ## ## Mrpl15 . 0.730035 1.537458 . 1.373238 0.8844558 0.6195407 . . . ## Lypla1 . 0.730035 . . . . 0.9992151 . 1.104379 3.559047 ## Tcea1 . 0.730035 . . . 0.8844558 . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . 1.537458 . . . . . 1.104379 . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.2145144 . . 1.1217778 0.6051149 2.285045 0.8713536 . . . ## Lypla1 . . . 0.6032898 . . 0.5277252 . . . ## Tcea1 0.3877977 . 0.6290406 0.3464661 . . . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . 0.6290406 0.8074507 . . 0.5277252 . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 0.6674739 0.7474484 . 0.5769045 1.323142 1.00434 1.082541 ## Lypla1 . . 1.010965 0.5769045 . 1.00434 . ## Tcea1 . 1.4666790 . 0.5769045 . . . ## Rgs20 . . . . . . . ## Atp6v1h 0.6674739 . . . . . . ## Npbwr1 . . . . . . . ## ## Mrpl15 . 1.4637782 . 1.0300889 0.8535947 0.7490319 . . ## Lypla1 0.9045438 0.9787507 1.127986 0.6421999 . . . . ## Tcea1 0.5512392 . 1.127986 . . 0.7490319 . . ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . . . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.6376245 . . 0.7682868 0.7993703 1.112263 0.7582599 1.0414 ## Lypla1 0.6376245 . . 0.7682868 . . 0.7582599 . ## Tcea1 1.0238752 . . 0.7682868 . . 0.7582599 . ## Rgs20 . . . . . . . . ## Atp6v1h . . 2.569763 0.7682868 . . . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 1.027490 0.9686507 . . 1.269255 0.8926312 1.1808316 . . ## Lypla1 . 0.5972934 . . . . 0.4474195 . . ## Tcea1 1.523463 . . 1.603068 . . 0.7554367 . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . 0.4474195 . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . . 1.007861 . . . 1.162086 1.0003006 . . . ## Lypla1 0.7882174 . 1.007861 . . 0.9601634 . 0.6203343 . . . ## Tcea1 . . . . 0.8686539 . 1.162086 1.0003006 . . . ## Rgs20 . . . . . . . . . . . ## Atp6v1h 0.7882174 . 1.007861 . . . . 0.6203343 . . . ## Npbwr1 . . . . . . . . . . . ## ## Mrpl15 0.8231349 1.55706 . 0.8450944 . 0.6176447 1.024390 . . 0.8342935 ## Lypla1 0.8231349 . . . . . 1.519686 . . . ## Tcea1 . . . 0.8450944 . 0.9966200 . . . 0.8342935 ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . 1.034569 0.6176447 . . . 0.5017404 ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.406582 0.5226733 0.8209195 1.6592407 0.5452607 1.1100416 0.6174872 . ## Lypla1 . 0.5226733 . . . 0.4111961 . . ## Tcea1 1.116666 . . 0.7245634 0.5452607 0.9265829 . . ## Rgs20 . . . . . . . . ## Atp6v1h 1.116666 . . . 0.8961354 . 0.6174872 . ## Npbwr1 . . . . . . . . ## ## Mrpl15 . . 1.105054 1.1222247 . . . 0.5832715 . ## Lypla1 . 0.5196728 . 0.5250599 . 0.8779965 . 0.5832715 0.9746219 ## Tcea1 . 0.5196728 . . . . . 0.5832715 . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.6622919 . 1.234842 . 1.0357402 1.3665974 . 2.974884 ## Lypla1 . 0.5272411 2.117881 . 0.6463675 0.6800591 1.512336 . ## Tcea1 . 0.5272411 1.770820 . . . . . ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . 0.6800591 . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 . . . 1.271097 . 1.4760271 . . 1.389981 ## Lypla1 . . . . 2.084093 . . 0.6916505 . ## Tcea1 1.08093 . . . . . . 0.6916505 1.101890 ## Rgs20 . . . . . . . . . ## Atp6v1h . 0.8642599 . . . 0.9887095 . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.7676675 1.1516997 . . . . 1.387834 . ## Lypla1 0.86978 . . 0.8047306 0.9070905 . . 1.387834 . ## Tcea1 . 1.1967877 0.7332246 0.8047306 . . . . 0.6537198 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.7414139 . . 1.390132 . . . . 0.6963626 ## Lypla1 . 0.7414139 . . . . . . . 0.6963626 ## Tcea1 2.362304 . . . . . . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . 2.365796 1.424929 . . . . . 0.6963626 ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.402177 . . 0.8095438 . 0.6176447 . 1.4533954 1.145157 1.443925 ## Lypla1 . . . 0.8095438 . 1.4857367 . . 0.728257 2.011509 ## Tcea1 . . . . 2.35623 1.6625566 . 0.9703269 0.728257 . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . 0.8095438 . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . 1.255538 1.510959 . 0.8306603 . 0.6484289 . . 1.49627 ## Lypla1 . 0.813089 . . . . 0.3758762 . . . ## Tcea1 . 0.813089 . . 0.8306603 . . 2.133039 0.8344041 . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . . . 0.3758762 . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . . 1.18452 . . 1.125097 . 0.9141959 . . 0.5906094 ## Lypla1 . . 1.4613570 . . . . . . . . . ## Tcea1 . . . . . . . . 0.5581199 1.305131 . . ## Rgs20 . . . . . . . . 0.5581199 . . . ## Atp6v1h . . 0.9767848 . . . . . . . . 0.5906094 ## Npbwr1 . . . . . . . . . . . . ## ## Mrpl15 . . . . . 1.574381 1.316809 . 1.0575019 . ## Lypla1 . . . . 1.663388 . . . 0.6624739 . ## Tcea1 . 1.176703 . . 1.143770 . . 1.067727 . . ## Rgs20 . . . . . . . . . . ## Atp6v1h 0.7269846 1.176703 . . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . 1.512061 1.203926 . . 0.6915759 . . . ## Lypla1 2.286632 1.512061 . . 0.7010908 . 1.557454 . 0.7959497 ## Tcea1 . . . . . 1.2505175 . . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . 0.7010908 0.4044179 . . 0.7959497 ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 2.290613 1.206217 . . . . . . 0.4287466 ## Lypla1 . . 0.774917 . . . 1.270728 . 1.344869 . ## Tcea1 2.639336 . 1.206217 . . . 1.270728 . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . . . 1.270728 . . 0.4287466 ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 0.8448672 . . 0.7544508 . . . 1.174226 1.1169427 ## Lypla1 . 0.5444391 . . 1.089104 . . 1.174226 0.8630302 ## Tcea1 . 0.5444391 . 0.4467488 . . . . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.7307041 . . 1.622036 0.6812627 . 1.33564 1.297734 . ## Lypla1 0.7307041 . . . . . . . . ## Tcea1 . . . 1.622036 0.6812627 1.136392 . . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . 1.0827348 . . . 1.563098 ## Npbwr1 . . . . . . . . . ## ## Mrpl15 1.1872043 0.6716618 0.8364748 . 1.1742255 . . . ## Lypla1 0.7603161 0.6716618 0.8364748 . 0.7503863 . 2.107231 . ## Tcea1 . . . 0.6679366 . . . . ## Rgs20 . . . . . . . . ## Atp6v1h . 0.6716618 . . 0.7503863 . . 0.9095639 ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.9406024 0.7531106 1.327861 . 1.212431 1.227117 1.434354 ## Lypla1 . 0.7531106 1.124840 . . 1.227117 . ## Tcea1 0.9406024 . 1.124840 . . . . ## Rgs20 . . . . . . . ## Atp6v1h . . . 1.089889 . 1.227117 . ## Npbwr1 . . . . . . . ## ## Mrpl15 1.740916 . . . 1.0027344 1.563396 1.5276391 . 1.131673 . . 0.8406863 . ## Lypla1 . . . . 0.4544087 . 0.4707624 . . . . 0.5062021 . ## Tcea1 . . . . 0.7656926 . 0.4707624 . . . . . . ## Rgs20 . . . . . . . . . . . . . ## Atp6v1h 1.209343 . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . ## ## Mrpl15 1.2861506 . . 1.4705822 . 0.899669 . 0.8018036 . ## Lypla1 0.8369941 2.278727 . 0.6086465 . . . 0.8018036 . ## Tcea1 . . . . . 1.365474 . 0.8018036 . ## Rgs20 . . . . . . . . . ## Atp6v1h . . 1.051105 . . . . . 1.60402 ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . . . 1.126857 . 1.103166 0.9301154 ## Lypla1 . . 1.239529 . 0.653764 . . ## Tcea1 0.8177259 . . . 0.653764 . . ## Rgs20 . . . . . . . ## Atp6v1h . 0.8576395 . . . . . ## Npbwr1 . . . . . . . ## ## Mrpl15 0.7003747 . . . 0.980621 2.144269 . . 1.476418 ## Lypla1 . 1.574836 . . . . . . . ## Tcea1 . . . 1.752056 . . 1.26122 . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 1.044972 . . . 1.0229805 . . . . . ## Lypla1 . . . . 0.4661363 . . 0.3874174 1.301226 . ## Tcea1 . . . . . 1.597702 . . . 1.616192 ## Rgs20 . . . . 0.4661363 . . . . . ## Atp6v1h . . . . . . 2.048564 0.6659519 . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . 0.5027192 . . . 0.9210571 0.5418993 . 1.4373643 . ## Lypla1 . . . 1.075047 . . 0.9210571 0.5418993 . 0.7274819 . ## Tcea1 . . 0.5027192 . 1.119855 . . 0.8913989 . 0.7274819 . ## Rgs20 . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . ## ## Mrpl15 0.6449434 . . . 1.1860974 . . . 0.9890276 ## Lypla1 . 0.8227767 . . . . 1.21315 1.549518 . ## Tcea1 . . . 0.8500502 0.3749066 . . . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . 0.3749066 . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 1.475767 0.8991068 1.089365 0.6784292 1.2035180 . 1.3058662 0.8183979 ## Lypla1 . . . . 1.2035180 . 0.5144517 . ## Tcea1 . . 1.089365 . . . 0.5144517 . ## Rgs20 . . . . . . . . ## Atp6v1h . 0.5473718 . 0.6784292 0.7728401 . . . ## Npbwr1 . . . . . . 0.5144517 . ## ## Mrpl15 1.292344 . 0.995543 . . 1.413408 1.813439 . . 0.9427973 ## Lypla1 . . . . . . 0.825435 . 1.707412 . ## Tcea1 . . . . . . 0.825435 1.490915 . 0.9427973 ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . 1.1059998 1.3755538 . . 1.141880 1.526557 0.697982 0.621808 ## Lypla1 . . 0.9077075 . . 1.141880 1.030031 . . ## Tcea1 1.339023 0.6986929 1.3755538 . . 1.141880 . 0.697982 . ## Rgs20 . . . . . . . . . ## Atp6v1h 1.339023 0.6986929 0.9077075 . . 2.001203 . . 0.621808 ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.8329317 . 1.6645446 0.4498345 1.1939347 1.780187 . 1.1227839 ## Lypla1 . 1.919221 . 0.4498345 0.7654772 . . 0.7113304 ## Tcea1 . 1.362980 0.8869672 0.4498345 0.4542617 . . 1.1227839 ## Rgs20 . . . . . . . . ## Atp6v1h . . . 0.4498345 . . . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 1.0259948 1.3455690 . . . . . 1.4015230 . ## Lypla1 0.6391844 0.6661821 . . . . . . . ## Tcea1 . 0.6661821 0.8317578 3.341021 . 0.8146986 . 0.9284921 . ## Rgs20 . . . . . . . . . ## Atp6v1h . 1.0624948 . . 1.780187 . . 0.9284921 . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . . 0.8620016 0.8653739 . . . 1.035623 . . . . ## Lypla1 . . . . 1.3233438 . . . . . . . ## Tcea1 . . 0.5211409 . 0.8662513 . . 1.315145 . . . . ## Rgs20 . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . 0.8089907 1.003026 . ## Npbwr1 . . . . . . . . . . . . ## ## Mrpl15 1.2193324 . . 1.4889182 1.144646 . . 1.540812 . 1.048811 ## Lypla1 0.7850259 . 0.8876395 0.6195407 . . . . . . ## Tcea1 0.7850259 . . 0.6195407 1.144646 . . . . 1.549372 ## Rgs20 . . . . . . . . . . ## Atp6v1h . . 0.8876395 . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.5050698 . . . 1.5704975 0.8493603 0.4597013 . ## Lypla1 . . 1.613611 . . 0.8493603 . . ## Tcea1 . 1.126575 1.102091 1.419795 0.8196389 . . . ## Rgs20 . . . . . . . . ## Atp6v1h 0.4600026 . 1.102091 . 1.2639459 0.8493603 0.4597013 . ## Npbwr1 . . . . . . . . ## ## Mrpl15 1.591132 0.5056008 0.7390057 . . . . . . 2.962621 . . . ## Lypla1 . 1.0898890 0.7390057 . . . . . . . . . . ## Tcea1 . 0.5056008 . . . . . 0.8693774 . . . . 1.32466 ## Rgs20 . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . ## ## Mrpl15 0.7980252 . . . 0.7655695 . 1.0297609 0.6526147 ## Lypla1 . . . 0.7178295 0.7655695 . . . ## Tcea1 . 1.277593 . 1.1313886 0.7655695 . 0.7885774 . ## Rgs20 . . . . . . . . ## Atp6v1h . . . . 0.7655695 . 0.7885774 0.6526147 ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.6341122 1.2945191 . 0.5968895 . 1.167777 1.310380 0.7798091 0.7843131 ## Lypla1 0.6341122 . . . . . . 0.7798091 . ## Tcea1 0.6341122 0.6329088 . 0.5968895 . . 0.643183 . . ## Rgs20 . . . . . . . . . ## Atp6v1h . 0.6329088 . . . . 0.643183 . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.6184727 . . 0.9159908 1.180024 0.9639603 0.6772359 1.443183 . ## Lypla1 . . . . . . 0.6772359 . . ## Tcea1 0.6184727 . . 0.9159908 1.387777 . 1.0773404 . 0.8724465 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . 0.9159908 . . . 1.443183 . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . . 0.9371888 . . . . 0.9702251 . . ## Lypla1 0.8002472 . 0.5745874 . . 2.07126 1.119994 . . . ## Tcea1 1.2390066 0.785675 0.5745874 . . . . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h 0.8002472 . 0.5745874 . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.5079391 1.816775 . 1.5691664 . 1.2132612 . . 1.1421700 ## Lypla1 0.6309275 1.489450 . 0.6681681 . . 1.438507 . 0.7259921 ## Tcea1 . . . . . . 1.438507 . 0.7259921 ## Rgs20 . . . . . . . . . ## Atp6v1h 0.6309275 . . . 1.219837 0.5811795 . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.7942841 0.6916505 . 0.8319043 . 1.979118 . ## Lypla1 . . . 0.8319043 1.03986 . 1.499775 ## Tcea1 . 0.6916505 1.780187 . . 1.415411 . ## Rgs20 . . . . . . . ## Atp6v1h . 0.6916505 1.780187 . . . . ## Npbwr1 . . . . . . . ## ## Mrpl15 1.133956 . . . 0.6158779 1.230194 . . . ## Lypla1 . 0.8889873 . 1.022676 . 1.230194 . . . ## Tcea1 1.133956 . . . . . 0.7677294 . . ## Rgs20 . . . . . . . . . ## Atp6v1h 1.133956 . . . 0.6158779 . 0.7677294 . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.7077087 . . 1.41329 . . 1.243993 . . ## Lypla1 0.8345517 . 1.017573 . . 0.666044 . . . 0.5380458 ## Tcea1 . 0.7077087 . . . 1.062309 . . . 0.5380458 ## Rgs20 . 0.7077087 . . . . . . . . ## Atp6v1h . . 1.017573 . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.30337 . 0.8540593 0.8716366 . 1.021993 . . 0.6383824 ## Lypla1 1.30337 0.7703381 . 0.8716366 . . . . . ## Tcea1 . . . . 0.9058592 . . . 0.6383824 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . 0.6383824 ## ## Mrpl15 0.588784 1.0634871 . 1.414232 1.0723838 0.5829567 . 1.257425 . 1.046108 ## Lypla1 0.588784 . . 1.414232 0.8248587 0.5829567 . . . . ## Tcea1 0.588784 0.6669196 . . 0.4951712 1.4267903 . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . 0.4951712 . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . 1.055360 . . . . 0.762628 ## Lypla1 0.9461585 . . 1.17176 . 1.061504 0.762628 ## Tcea1 . 1.557306 1.085714 . 0.8460807 . . ## Rgs20 . . . . . . . ## Atp6v1h . . . . . . 1.190220 ## Npbwr1 . . . . . . . ## ## Mrpl15 2.952241 0.4667123 . . 0.8554565 0.6893193 1.338816 ## Lypla1 . 0.2603401 . 2.559796 0.8554565 . . ## Tcea1 . . 1.273964 2.559796 . . . ## Rgs20 . . . . . . . ## Atp6v1h . 0.2603401 . 1.941125 . . . ## Npbwr1 . . . . . . . ## ## Mrpl15 0.6808289 0.8044573 0.7517459 . . 0.8301493 0.9494484 1.3478844 ## Lypla1 . 0.8044573 1.1760049 . . . 0.9494484 . ## Tcea1 . 0.8044573 . . . 1.2774060 1.4275725 1.0645437 ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . 0.8301493 . 0.6677052 ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.6901606 . . . . 0.9860149 . 1.1914317 0.8235651 ## Lypla1 . . 0.8008555 . 2.513194 0.6099099 . 0.6506135 . ## Tcea1 0.6901606 . . . . . . 0.8650288 1.2689787 ## Rgs20 . . . . . . . . . ## Atp6v1h . . 0.8008555 . 1.897942 0.6099099 . 0.3773113 . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.4589069 . 2.824224 1.093175 1.0991459 1.1091898 1.087015 ## Lypla1 0.7676675 0.4589069 . . 1.093175 1.0991459 0.7010908 . ## Tcea1 0.7676675 . . . . 0.6935474 . . ## Rgs20 . . . . . . . . ## Atp6v1h . . . . 1.093175 . . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 . 1.0412023 . . . 0.61141 . 1.7303318 ## Lypla1 . . . . 0.8730144 0.61141 . 0.7704005 ## Tcea1 . 0.4767766 . . 1.3318999 . . . ## Rgs20 . . . . . . . . ## Atp6v1h 0.9538407 0.4767766 . 1.108034 . 0.61141 1.371918 . ## Npbwr1 . . . . . . . . ## ## Mrpl15 1.5884398 . . . . . . . ## Lypla1 0.5003817 2.1456 . . 0.9936644 . . . ## Tcea1 1.2802121 . 0.960462 . . 1.170685 . 0.9951129 ## Rgs20 . . . . . . . . ## Atp6v1h 0.5003817 . . . . . 2.229881 . ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.5124308 0.6550954 0.9629580 . 2.184526 1.017799 . 0.7723383 1.3575955 ## Lypla1 0.5124308 . 0.5931703 . . 1.017799 . 0.7723383 . ## Tcea1 0.8495901 . 0.5931703 . . . . . 1.0731431 ## Rgs20 . . . . . . . . . ## Atp6v1h 0.5124308 0.6550954 . . . . . . 0.6741064 ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.9971055 1.390509 . . 1.3190793 0.9926477 . 0.904719 ## Lypla1 . 0.9971055 1.102360 . . . 0.9926477 . . ## Tcea1 1.025421 . . . . 0.8628849 1.4808632 . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . 2.076734 . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.9564972 1.415175 0.9748275 . . . 1.610142 ## Lypla1 . . 0.6017744 1.474337 . 1.689895 . ## Tcea1 . . 0.9748275 2.045920 1.473299 . . ## Rgs20 . . . . . . . ## Atp6v1h . . . . . 1.689895 . ## Npbwr1 . . . . . . . ## ## Mrpl15 . 1.274923 1.196063 . 0.8751305 0.9895582 . ## Lypla1 1.301615 . . . . . . ## Tcea1 . . 0.767111 1.124746 . . 0.9848049 ## Rgs20 . . . . . . . ## Atp6v1h . . . . . . . ## Npbwr1 . . . . . . . ## ## Mrpl15 1.354388 0.807749 . . 0.8739084 . . 0.740552 . . . . 0.5821887 ## Lypla1 1.909362 . 1.014201 . 0.8739084 . . . . . . . . ## Tcea1 1.669890 0.807749 . . 0.8739084 . . . . . . . 0.5821887 ## Rgs20 . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . ## ## Mrpl15 . 1.340688 . 1.1075448 0.6021852 0.7602554 . . 1.5857730 ## Lypla1 . . . 0.7957717 . 0.7602554 . . 0.4412977 ## Tcea1 0.9123192 . . 0.3403102 0.6021852 0.7602554 . . . ## Rgs20 . . . . . . . . . ## Atp6v1h 0.9123192 . . 0.5937505 . 0.7602554 . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.8648166 0.630393 . . 0.9400316 . . . ## Lypla1 . . . . . . . 1.379211 0.6306396 ## Tcea1 0.7772597 . 1.014033 . . 0.9400316 0.8303682 . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . 0.6306396 ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . . 1.0953563 . . 1.0753009 . 1.119577 ## Lypla1 1.4320444 0.8910172 0.6907062 . . . 1.443595 . ## Tcea1 0.9530567 . 0.6907062 . 1.785537 0.4969041 1.149706 . ## Rgs20 . . . . . . . . ## Atp6v1h 0.9530567 . . . 1.247432 0.4969041 . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.7084055 1.5598986 . . 1.54515 . . . ## Lypla1 0.2953404 0.6624739 0.9482845 1.229167 . . 1.779368 . ## Tcea1 0.8647171 1.0575019 0.9482845 . . . . 0.8576395 ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . . . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 . . 0.8076801 0.9721626 1.93047 1.2438177 . 1.4712279 . ## Lypla1 . . 0.4832589 . . 0.8039797 . 0.6090288 . ## Tcea1 0.7132885 . 0.4832589 . . 0.8039797 . . . ## Rgs20 . . . . . . . . . ## Atp6v1h 0.7132885 . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.675952 . . . . . 0.5674849 . 0.7564953 ## Lypla1 . . 2.168246 . . 0.7317661 0.5674849 . 0.5618211 ## Tcea1 1.075619 . . . 0.5774525 . . . 0.5618211 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.7896921 . . . 1.4192 0.969615 0.6999662 . ## Lypla1 0.4708527 . . . . . . 1.586788 ## Tcea1 1.0310745 . . . . . 0.6999662 . ## Rgs20 . . . . . . . . ## Atp6v1h 0.7896921 0.9563985 1.510409 . . . 0.6999662 . ## Npbwr1 . . . . . . . . ## ## Mrpl15 . 0.6190656 . . . 1.571126 . 1.333853 . 1.2107744 ## Lypla1 0.6480155 0.6190656 . . . 0.669375 . . . 0.9441867 ## Tcea1 . . . 1.597073 . . . 1.052135 1.965536 0.5796206 ## Rgs20 . . . . . . . . . . ## Atp6v1h . 0.6190656 . . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 1.7748776 . . 0.6652625 . 1.209755 . . 0.7162467 ## Lypla1 0.9681947 . 0.8092672 . . . . 1.751268 . ## Tcea1 . 1.4676204 . . . . . . 0.4209239 ## Rgs20 . . . . . . . . . ## Atp6v1h 0.9681947 0.9818721 . 1.0612572 . 0.777641 . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.6517775 . . . 0.9025349 . . . ## Lypla1 0.6517775 1.171606 . . . . . 1.576052 ## Tcea1 . 1.171606 . . . . 0.6779513 . ## Rgs20 . . . . . . . . ## Atp6v1h . . . 0.982081 . 0.7350281 . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.9952204 . 0.6619736 1.6588963 1.1033008 . 1.0728899 ## Lypla1 . 1.759183 . 0.7243441 . . 0.4954717 ## Tcea1 0.9952204 1.759183 . . 0.6966657 . 0.8252909 ## Rgs20 . . . . . . . ## Atp6v1h . 1.759183 . . . 2.382582 0.4954717 ## Npbwr1 . . . . . . . ## ## Mrpl15 1.067665 . . . 1.164128 . . . . 1.084222 ## Lypla1 . . . . 1.164128 . . 1.290622 . . ## Tcea1 1.351410 . . . . 1.839566 . 1.290622 2.086253 . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . 3.690441 . . . . 1.290622 . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . 1.025421 1.353394 . . . 1.008027 . ## Lypla1 . . . . 3.01824 . 0.7293116 1.008027 . ## Tcea1 1.085974 . . 1.353394 . . 0.7293116 . 0.8589706 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . 0.7293116 . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 1.856686 . . . 0.8316845 0.7443023 . 0.9445227 ## Lypla1 . 2.783436 . . . . . . ## Tcea1 . . . 1.405343 . . . 0.9445227 ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . . 0.7901192 0.9445227 ## Npbwr1 . . . . . . . . ## ## Mrpl15 2.301057 . . 0.9240708 0.7821186 . 1.880784 1.587717 . . ## Lypla1 . . . . 0.7821186 . . . . 0.6254633 ## Tcea1 . . . . 0.7821186 1.886934 . . . 0.6254633 ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . 0.7821186 . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . 1.6072017 1.327912 0.7794895 1.1693820 1.9655356 . ## Lypla1 . . 0.8457011 . . . . 0.9741085 ## Tcea1 . . . . . . 0.7046421 0.9741085 ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . 0.7466885 . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 0.9605615 0.8616461 . 0.5691437 1.4345971 1.166710 . 1.676337 . ## Lypla1 . . . 0.5691437 0.9551178 1.166710 2.208684 . . ## Tcea1 . 0.8616461 . . 0.9551178 1.462588 . . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.9267366 1.640550 . . 1.916002 1.384796 . 1.1633709 0.6605679 ## Lypla1 0.9267366 . 1.715405 . . . . 0.9030580 0.6605679 ## Tcea1 1.3993347 1.124606 . . . 1.384796 . 0.5501818 . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . 0.5501818 . ## ## Mrpl15 . . 1.0824120 . 1.94883 . 1.330158 . 0.6534984 0.9248045 ## Lypla1 . . 0.6810217 . . . . . . . ## Tcea1 1.062123 . 1.0824120 . 1.94883 . . . . 0.9248045 ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . . . . 1.191336 . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . 1.001498 . . 0.7127052 0.8619621 0.7042802 1.4108635 . 1.3210901 ## Lypla1 . . . . 1.1246055 . . 0.7095935 . . ## Tcea1 . 1.001498 . . 0.7127052 . . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . . . . 0.7095935 . 0.6501534 ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . . 0.8798907 . . . . 1.233288 0.8737457 . . . ## Lypla1 0.924621 . . . 1.040452 . . . . 0.8737457 . . . ## Tcea1 . . . . . . . . . . . 0.9940395 . ## Rgs20 . . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . . ## ## Mrpl15 . 0.702939 . 0.4265739 . 1.164734 . 1.5025528 . . . . ## Lypla1 . 0.702939 . 0.7246456 . . 1.127845 0.6276942 . 0.9874395 . . ## Tcea1 . 0.702939 . 0.7246456 . . . 0.6276942 . . . . ## Rgs20 . . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . . ## ## Mrpl15 . 1.1756176 . . 1.0487508 0.9860676 0.785935 . ## Lypla1 0.9809334 0.5577366 . 2.689671 . 0.6099482 0.785935 . ## Tcea1 1.4664653 0.5577366 0.6498471 . 0.6559861 . . . ## Rgs20 . . . . . . . . ## Atp6v1h . . . . . . . . ## Npbwr1 . . . . . . . . ## ## Mrpl15 1.744999 1.298314 . . . . . . . ## Lypla1 . . . . 1.557454 0.7182062 . 2.125857 . ## Tcea1 . . . . . 0.7182062 1.3511979 2.125857 1.981001 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . 0.8883128 2.125857 . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 1.096749 1.0145374 0.9835457 . . 0.8159629 1.393341 . ## Lypla1 1.497212 . 0.6307629 . 0.8226335 . 1.2592290 . . ## Tcea1 . 1.096749 . 0.9835457 0.8226335 . . . . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 0.7874659 . . . 0.6715213 1.124325 . . . 1.579509 ## Lypla1 . 0.4693221 . . . 0.3911000 1.124325 . . . . ## Tcea1 . 0.4693221 . 1.263491 . . . . 0.729534 . . ## Rgs20 . . . . . 0.3911000 . . . . . ## Atp6v1h . 0.7874659 . . . . . . . . . ## Npbwr1 . . . . . . . . . . . ## ## Mrpl15 1.171452 0.6917003 . . . . . 0.835660 1.219753 ## Lypla1 . . . 0.8964788 1.0692327 . . 0.835660 . ## Tcea1 . 0.6917003 0.6959338 1.3614675 . . . 1.284448 . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . 0.8964788 . . . . . ## Npbwr1 . . . . 0.6711938 . . . . ## ## Mrpl15 . . 0.430565 0.7663086 . . 1.2056437 . . 1.071121 . . . ## Lypla1 . . 0.430565 . . . 0.7744755 . . . . . . ## Tcea1 . . 0.430565 . . . . . . . . . 0.7840543 ## Rgs20 . . . . . . . . . . . . . ## Atp6v1h . . 0.430565 . . . . . 1.20589 . . . . ## Npbwr1 . . . . . . . . . . . . . ## ## Mrpl15 1.494256 0.7145380 0.5948052 . 1.515308 . . 1.237963 0.6319578 . ## Lypla1 1.003573 . . . . . . . 1.0161652 . ## Tcea1 1.003573 . . . 0.635362 1.267327 . . . . ## Rgs20 . . . . . . . . . . ## Atp6v1h . 0.4197763 0.5948052 . . . . . . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 0.9463514 0.5882849 . . 1.4021992 . 0.8777911 1.018137 . ## Lypla1 . 0.9562012 . . 0.3630673 . 1.1340985 . . ## Tcea1 . . . . 0.3630673 . 0.5322681 . . ## Rgs20 . . . . . . . . . ## Atp6v1h 0.4222769 0.9562012 . 2.384369 0.3630673 . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . . . 1.676572 . . . 0.6372462 . . . 0.9507616 . ## Lypla1 0.6903093 . . . . . . . . 0.5852028 . 0.9507616 . ## Tcea1 . . . . . . . . . 0.9519321 . . . ## Rgs20 . . . . . . . . . . . . . ## Atp6v1h . . . . . 1.895055 . . . . . . . ## Npbwr1 . . . . . . . . . . . . . ## ## Mrpl15 . 0.6133831 . 1.0199477 . 0.7277032 0.8558454 ## Lypla1 1.074919 0.6133831 . 0.7802571 1.571957 0.7277032 0.8558454 ## Tcea1 . . 0.8923759 0.4643732 . 1.1444266 . ## Rgs20 . . . . . . . ## Atp6v1h 1.074919 . 0.8923759 . . . . ## Npbwr1 . . . . . . . ## ## Mrpl15 0.6664124 0.8019167 . . . . 0.8498201 . . ## Lypla1 1.0628046 . . 0.857874 . . 1.6129672 . 0.9796849 ## Tcea1 1.3459192 0.3435449 . . 0.9657701 . . 1.203763 . ## Rgs20 . . . . . . . . . ## Atp6v1h . 0.8019167 . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 0.7943506 . . 1.121387 0.6386354 . 0.494297 1.682904 . ## Lypla1 0.7943506 . . . . . . . . ## Tcea1 . 0.4995395 . . 0.6386354 1.754424 0.494297 . . ## Rgs20 . . . . . . 0.494297 . . ## Atp6v1h . . . . . . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 . 1.6051319 1.0674143 0.9241624 1.4528933 . . 0.5986563 . . . ## Lypla1 . . 0.6698404 0.5652447 0.7380356 . . . . . . ## Tcea1 1.421978 0.6904581 0.6698404 0.9241624 1.6803229 . . . . . . ## Rgs20 . . . . . . . . . . . ## Atp6v1h . 0.6904581 . . . . . 0.5986563 . . . ## Npbwr1 . . . . . . . . . . . ## ## Mrpl15 . . . . 1.522202 . . . 2.226923 . 0.9408881 1.856079 ## Lypla1 . . . . . . . . . . 0.5772468 . ## Tcea1 . . . 0.9968357 . . . . . . . . ## Rgs20 . . . . . . . . . . . . ## Atp6v1h 0.5550367 . . . . . . . 2.226923 . 0.9408881 . ## Npbwr1 . . . . . . . . . . . . ## ## Mrpl15 1.1650380 . 1.515377 . . 0.8330052 0.4773100 . . ## Lypla1 0.7433755 . . . . 1.2810566 0.7990673 . . ## Tcea1 . . 1.515377 1.366017 . . 0.7990673 0.8794369 . ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . 0.8330052 . 0.8794369 . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 1.861259 . . 0.5515511 0.8717176 1.3316947 . . 0.5271558 0.7582599 ## Lypla1 . . . 1.1655948 . 0.8728521 . . . 0.7582599 ## Tcea1 . . . 0.5515511 . 0.8728521 . . 0.5271558 . ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . 0.5515511 . . . . 0.5271558 . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . 1.0179115 . . . . . . 0.9610597 0.6371203 . ## Lypla1 . 0.4631907 0.694751 . . . . . . . 1.593003 ## Tcea1 1.615223 0.4631907 . . . . . . . 0.6371203 . ## Rgs20 . . . . . . . . . . . ## Atp6v1h . . . . . . . . . . . ## Npbwr1 . . . . . . . . . . . ## ## Mrpl15 . 1.005822 . 0.9038440 . . . . 0.755736 1.3220982 . . ## Lypla1 0.937 . . 0.9038440 0.7441284 . . . . 0.6508108 1.44121 . ## Tcea1 . . . 0.5507411 . . . . . 0.6508108 . . ## Rgs20 . . . . . . . . . . . . ## Atp6v1h . . . . 0.7441284 . . . . . . . ## Npbwr1 . . . . . . . . . . . . ## ## Mrpl15 . . . . . . . 1.6909256 . . . 0.4317165 ## Lypla1 . 2.395172 1.318812 0.8031622 . . . 0.4898499 . . . . ## Tcea1 . . . 0.8031622 . . . 0.4898499 . . . 0.9630581 ## Rgs20 . . . . . . . . . . . . ## Atp6v1h . . . . . . . . 0.8590491 . . 0.4317165 ## Npbwr1 . . . . . . . . . . . . ## ## Mrpl15 0.8579523 1.066726 . 1.5641923 1.201076 . . . . ## Lypla1 0.8579523 1.066726 . . . . . . 1.520384 ## Tcea1 1.3128236 1.066726 . 0.8151898 . 0.5284101 1.110349 . 1.520384 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . 0.770962 . . . . ## Npbwr1 . . . . . . . . . ## ## Mrpl15 1.7012490 . . . . . 1.1584761 . . . ## Lypla1 0.7515091 . . . 0.5679815 . 0.7383777 1.459199 . . ## Tcea1 1.1756950 . . . . 0.8894938 0.7383777 . . 1.396353 ## Rgs20 . . . . . . . . . . ## Atp6v1h . . . . . 0.8894938 . 1.459199 . . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . . 0.8811307 0.8905088 0.5679815 . . . . ## Lypla1 1.2165669 . 1.56864 1.3421478 0.5412682 . . . . . ## Tcea1 . . . 0.8811307 0.5412682 0.5679815 . 0.873827 . . ## Rgs20 . . . . . . . . . . ## Atp6v1h 0.7828917 . . 0.8811307 . . . . 2.146934 . ## Npbwr1 . . . . . . . . . . ## ## Mrpl15 . . 1.366743 1.272667 . 1.291004 1.352259 1.0938352 0.7656926 ## Lypla1 . . . . . 1.291004 0.889156 0.6895665 0.7656926 ## Tcea1 . . . . . 1.291004 . 0.6895665 0.7656926 ## Rgs20 . . . . . . . . . ## Atp6v1h . . . . . . . . . ## Npbwr1 1.476418 . . . . . . 0.6895665 . ## ## Mrpl15 1.1473383 1.0423498 ## Lypla1 0.8284989 0.6512499 ## Tcea1 0.3576462 . ## Rgs20 . . ## Atp6v1h . . ## Npbwr1 . . ``` --- ## Evaluate data Seurat has in-built functions to easily compare features. - read counts: nCount_RNA - gene counts: nFeature_RNA - mitochondrial content: percent.mt ```r VlnPlot(obj, features = c("nCount_RNA", "nFeature_RNA", "percent.mt"), pt.size = 0.2) ``` ![](Session2_files/figure-html/eval_readCount_geneCount_mitoCont-1.png)<!-- --> --- ## Read counts vs genes counts *FeatureScatter* allows pairwise comparisons of features, like counts... ```r FeatureScatter(obj, feature1 = "nCount_RNA", feature2 = "nFeature_RNA") ``` <img src="Session2_files/figure-html/eval_readCount_geneCount-1.png" style="display: block; margin: auto;" /> --- ## Read counts vs mitochondrial content ...or mitochondiral content. ```r FeatureScatter(obj, feature1 = "nCount_RNA", feature2 = "percent.mt") ``` <img src="Session2_files/figure-html/eval_readCount_mitoCont-1.png" style="display: block; margin: auto;" /> --- ## Remove potential cell debris - High mitochondrial content and low read counts can be filtered out. ```r summary(obj@meta.data$percent.mt) ``` ``` ## Min. 1st Qu. Median Mean 3rd Qu. Max. ## 0.000 4.249 5.986 10.804 12.428 59.680 ``` ```r mt_cutH <- 10 obj_unfiltered <- obj obj <- subset(obj, subset = percent.mt < mt_cutH) ``` --- ## Remove potential cell debris .pull-left[ Unfiltered ```r VlnPlot(obj_unfiltered, features = c("nCount_RNA", "nFeature_RNA", "percent.mt"), pt.size = 0.2) ``` ![](Session2_files/figure-html/rmMito_vln1-1.png)<!-- --> ] .pull-right[ Filtered ```r VlnPlot(obj, features = c("nCount_RNA", "nFeature_RNA", "percent.mt"), pt.size = 0.2) ``` ![](Session2_files/figure-html/rmMito_vln2-1.png)<!-- --> ] --- ## Remove potential cell debris .pull-left[ ```r FeatureScatter(obj_unfiltered, feature1 = "nCount_RNA", feature2 = "percent.mt") ``` ![](Session2_files/figure-html/rmMito_scatter1-1.png)<!-- --> ] .pull-right[ ```r FeatureScatter(obj, feature1 = "nCount_RNA", feature2 = "percent.mt") ``` ![](Session2_files/figure-html/rmMito_scatter2-1.png)<!-- --> ] --- class: inverse, center, middle # Cell cycle phase determination <html><div style='float:left'></div><hr color='#EB811B' size=1px width=720px></html> --- ## Cell cycle ~ covert gene list - In Seurat, the default marker genes for S phase and G2/M phase are stored in *cc.gene* list - This list is based on human data. - We can convert it into mouse gene symbol by using *bioMart* ```r convertHumanGeneList <- function(x) { require("biomaRt") human = useMart("ensembl", dataset = "hsapiens_gene_ensembl", host = "http://useast.ensembl.org") mouse = useMart("ensembl", dataset = "mmusculus_gene_ensembl", host = "http://useast.ensembl.org") genesV2 = getLDS(attributes = c("hgnc_symbol"), filters = "hgnc_symbol", values = x, mart = human, attributesL = c("mgi_symbol"), martL = mouse, uniqueRows = T) humanx <- unique(genesV2[, 2]) return(humanx) } # s_gene <- convertHumanGeneList(cc.genes$s.genes) ``` ``` ## Loading required package: biomaRt ``` ```r g2m_gene <- convertHumanGeneList(cc.genes$g2m.genes) ``` --- ## Cell cycle ~ estimation ```r obj <- CellCycleScoring(obj, s.features = s_gene, g2m.features = g2m_gene, set.ident = TRUE) obj@meta.data[1:2, ] ``` ``` ## orig.ident nCount_RNA nFeature_RNA dset percent.mt ## CTRL_CACTCCATCTTATCTG-1 CTRL 7748 2372 CTRL 5.201342 ## CTRL_CCTCTGACAGTAGAGC-1 CTRL 9301 2715 CTRL 5.053220 ## S.Score G2M.Score Phase old.ident ## CTRL_CACTCCATCTTATCTG-1 -0.2222360 0.2924512 G2M CTRL ## CTRL_CCTCTGACAGTAGAGC-1 -0.1435008 0.4806422 G2M CTRL ``` --- ## Cell cycle ~ phase ```r yd_dat <- as.data.frame(table(obj@meta.data$dset, obj@meta.data$Phase)) head(yd_dat) ``` ``` ## Var1 Var2 Freq ## 1 CTRL G1 246 ## 2 CTRL G2M 225 ## 3 CTRL S 221 ``` --- ## Cell cycle ~ phase ```r library(ggplot2) ggplot(yd_dat, aes(x = Var1, y = Freq, fill = Var2)) + geom_bar(stat = "identity", position = "stack") + labs(x = "", y = "Counts", fill = "Phase") + theme_classic() ``` <img src="Session2_files/figure-html/cellCycle_plot2-1.png" style="display: block; margin: auto;" /> --- class: inverse, center, middle # Regression and clustering <html><div style='float:left'></div><hr color='#EB811B' size=1px width=720px></html> --- ## Data normalization Re-scale and regress data to **percent.mt**, **S.score**, and **G2M.score** ```r obj <- ScaleData(obj, vars.to.regress = c("percent.mt", "S.score", "G2M.score", "Phase")) ``` ``` ## Warning: Requested variables to regress not in object: S.score, G2M.score ``` ``` ## Regressing out percent.mt, Phase ``` ``` ## Centering and scaling data matrix ``` --- ## Principle Component Analysis ```r set.seed(1000) obj <- RunPCA(obj, npcs = 30, verbose = FALSE) ``` --- ## How many PCs? ~ Elbow Plot - Elbow plots are useful to estimate number of PCs to take. You need to look for the *elbow* i.e. the point start were the curve turns flat. This approximation minimizes deviation and PC number. ```r ElbowPlot(obj, ndims = 30) ``` <img src="Session2_files/figure-html/pcSel-1.png" style="display: block; margin: auto;" /> ```r # numPC <- 15 ``` --- ## Clustering ```r set.seed(1000) maxPC <- numPC obj <- FindNeighbors(obj, reduction = "pca", dims = 1:maxPC) ``` ``` ## Computing nearest neighbor graph ``` ``` ## Computing SNN ``` ```r obj <- FindClusters(obj, resolution = 0.5) ``` ``` ## Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck ## ## Number of nodes: 692 ## Number of edges: 21274 ## ## Running Louvain algorithm... ## Maximum modularity in 10 random starts: 0.7747 ## Number of communities: 7 ## Elapsed time: 0 seconds ``` --- ## Clustering ```r obj <- RunUMAP(obj, reduction = "pca", dims = 1:maxPC) ``` ``` ## Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric ## To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation' ## This message will be shown once per session ``` ``` ## 16:21:49 UMAP embedding parameters a = 0.9922 b = 1.112 ``` ``` ## 16:21:49 Read 692 rows and found 15 numeric columns ``` ``` ## 16:21:49 Using Annoy for neighbor search, n_neighbors = 30 ``` ``` ## 16:21:49 Building Annoy index with metric = cosine, n_trees = 50 ``` ``` ## 0% 10 20 30 40 50 60 70 80 90 100% ``` ``` ## [----|----|----|----|----|----|----|----|----|----| ``` ``` ## **************************************************| ## 16:21:49 Writing NN index file to temp file /tmp/RtmpMsibtn/file176945121b8d ## 16:21:49 Searching Annoy index using 1 thread, search_k = 3000 ## 16:21:49 Annoy recall = 100% ## 16:21:50 Commencing smooth kNN distance calibration using 1 thread ## 16:21:51 Initializing from normalized Laplacian + noise ## 16:21:51 Commencing optimization for 500 epochs, with 25698 positive edges ## 16:21:52 Optimization finished ``` ```r obj <- RunTSNE(obj, reduction = "pca", dims = 1:maxPC) ``` --- ## UMAP plot ```r DimPlot(obj, reduction = "umap") # default it 'umap' ``` ![](Session2_files/figure-html/seurat_UMAP-1.png)<!-- --> --- ## tSNE plot ```r DimPlot(obj, reduction = "tsne") ``` ![](Session2_files/figure-html/seurat_tSNE-1.png)<!-- --> --- class: inverse, center, middle # Identify marker genes for each cluster <html><div style='float:left'></div><hr color='#EB811B' size=1px width=720px></html> --- ## Marker genes ```r obj <- SetIdent(obj, value = "seurat_clusters") clust.markers <- FindAllMarkers(obj, only.pos = TRUE, min.pct = 0.25, logfc.threshold = 0.25) ``` ``` ## Calculating cluster 0 ``` ``` ## Calculating cluster 1 ``` ``` ## Calculating cluster 2 ``` ``` ## Calculating cluster 3 ``` ``` ## Calculating cluster 4 ``` ``` ## Calculating cluster 5 ``` ``` ## Calculating cluster 6 ``` ```r head(clust.markers) ``` ``` ## p_val avg_log2FC pct.1 pct.2 p_val_adj cluster gene ## Krt14 3.374000e-28 0.7427281 1.000 0.980 4.894999e-24 0 Krt14 ## Igfbp2 4.871443e-28 0.8042739 0.810 0.404 7.067489e-24 0 Igfbp2 ## Serpinb10 8.757815e-26 0.6780455 0.754 0.350 1.270584e-21 0 Serpinb10 ## H19 1.392904e-24 0.7646044 0.969 0.784 2.020825e-20 0 H19 ## Fth1 1.126329e-22 0.4766948 1.000 0.935 1.634079e-18 0 Fth1 ## Col17a1 8.362472e-22 0.6835676 0.844 0.506 1.213227e-17 0 Col17a1 ``` --- ## Select marker genes for each cluster Here we will select the top 5 marker genes for each cluster. ```r topG <- clust.markers %>% group_by(cluster) %>% top_n(n = 5, wt = avg_log2FC) head(topG) ``` ``` ## # A tibble: 6 × 7 ## # Groups: cluster [2] ## p_val avg_log2FC pct.1 pct.2 p_val_adj cluster gene ## <dbl> <dbl> <dbl> <dbl> <dbl> <fct> <chr> ## 1 3.37e-28 0.743 1 0.98 4.89e-24 0 Krt14 ## 2 4.87e-28 0.804 0.81 0.404 7.07e-24 0 Igfbp2 ## 3 8.76e-26 0.678 0.754 0.35 1.27e-21 0 Serpinb10 ## 4 1.39e-24 0.765 0.969 0.784 2.02e-20 0 H19 ## 5 8.36e-22 0.684 0.844 0.506 1.21e-17 0 Col17a1 ## 6 2.10e-81 1.77 0.841 0.076 3.04e-77 1 Sox9 ``` --- class: inverse, center, middle # Advanced plots <html><div style='float:left'></div><hr color='#EB811B' size=1px width=720px></html> --- ## Dimplots ~ adjust point size ```r DimPlot(obj, pt.size = 0.2) ``` ![](Session2_files/figure-html/dimPlot_ptSize-1.png)<!-- --> --- ## Dimplots with labeling clusters ```r DimPlot(obj, pt.size = 0.2, label = TRUE) + NoLegend() ``` ![](Session2_files/figure-html/dimPlot_label-1.png)<!-- --> --- ## Marker Gene expression ~ heatmap ```r DoHeatmap(obj, features = topG$gene) + NoLegend() ``` ![](Session2_files/figure-html/markGene_heatmap2-1.png)<!-- --> --- ## Marker Gene Expression ~ FeaturePlot ```r gene_marker <- c("Krt1", "Pthlh", "Krt14", "Cenpa", "Shh") FeaturePlot(obj, features = gene_marker, pt.size = 0.2) ``` ![](Session2_files/figure-html/makGene_featurePlot-1.png)<!-- --> --- ## Marker Gene Expression ~ RidgePlot ```r RidgePlot(obj, features = gene_marker) ``` ![](Session2_files/figure-html/makGene_RidgePlott2-1.png)<!-- --> --- ## Cell cycle phases by cluster ```r tbl <- table(obj@meta.data$seurat_clusters, obj@meta.data$Phase) tbl_dat <- as.data.frame(tbl) to <- rowSums(tbl) names(to) <- rownames(tbl) tbl_dat$to <- to[match(names(to), tbl_dat$Var1)] tbl_dat$prop <- tbl_dat$Freq/tbl_dat$to tbl_dat[1:2, ] ``` ``` ## Var1 Var2 Freq to prop ## 1 0 G1 136 289 0.4705882 ## 2 1 G1 38 113 0.3362832 ``` --- ## Cell cycle phases by cluster ~ plot - The second cluster may need to be removed, as there seems to be cell cycle bias. ![](Session2_files/figure-html/cellcycle_cluster_plot2-1.png)<!-- --> --- ## Save file First we filter out the second cluster, and then save the data object. ```r cellID <- rownames(obj@meta.data)[!obj@meta.data$seurat_clusters == 1] obj_sub <- obj[, cellID] saveRDS(obj_sub, "scSeq_Seurat_clean.rds") ``` --- ## SeuratWrappers - Seurat objects can be converted into different objects for further analysis with other tools i.e. Monocle or Velocity. - For detailed information please refer to the Github repository for [SeuratWrappers](https://github.com/satijalab/seurat-wrappers). --- ## Time for an exercise! Exercise on Seurat can be found [here](../../exercises/exercises/exercise1_exercise.html). Answers can be found [here](../../exercises/answers/exercise1_answers.html).