Single-cell RNA sequencing ~ Session 2
Rockefeller University, Bioinformatics Resource Centre
https://rockefelleruniversity.github.io/scRNA-seq/
Data analysis with Seurat
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.
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
Create Seurat object and Generare QC plots
Load Cell Ranger results into Seurat
- Loading data
- Estimate mitochondrial contents
library(Seurat)
samID <- "CTRL"
X10_file <- Seurat::Read10X(tar_dir)
obj <- CreateSeuratObject(counts = X10_file, project = samID, min.cells = 5, min.features = 200)
obj$dset <- samID
obj <- Seurat::RenameCells(obj, add.cell.id = samID)
#
obj[["percent.mt"]] <- PercentageFeatureSet(obj, pattern = "^mt-")Subset dataset
We have a small subset version of this dataset that you can load in from the data/ directory to try this out.
Cell information
The object metadata contains cell barcode, mitochondrial contents, datasets, and clustering etc
head(obj@meta.data)## orig.ident nCount_RNA nFeature_RNA dset percent.mt
## CTRL_CACTCCATCTTATCTG-1 CTRL 7748 2372 CTRL 5.201342
## CTRL_CCTCTGACAGTAGAGC-1 CTRL 9301 2715 CTRL 5.053220
## CTRL_GACGTTAGTAGAAGGA-1 CTRL 8213 2412 CTRL 4.699866
## CTRL_TGATTTCCAGCCTGTG-1 CTRL 8363 2308 CTRL 5.177568
## CTRL_ATCGAGTCAATCTGCA-1 CTRL 3392 1303 CTRL 20.725236
## CTRL_TGACTTTTCACCGGGT-1 CTRL 7034 2166 CTRL 7.094114Raw count matrix
head(obj@assays$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
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
VlnPlot(obj, features = c("nCount_RNA", "nFeature_RNA", "percent.mt"), pt.size = 0.2)
Read counts vs genes counts
FeatureScatter allows pairwise comparisons of features, like counts…
FeatureScatter(obj, feature1 = "nCount_RNA", feature2 = "nFeature_RNA")
Read counts vs mitochondrial content
…or mitochondiral content.
FeatureScatter(obj, feature1 = "nCount_RNA", feature2 = "percent.mt")
Remove potential cell debris
- High mitochondrial content and low read counts can be filtered out.
summary(obj@meta.data$percent.mt)## Min. 1st Qu. Median Mean 3rd Qu. Max.
## 0.000 4.249 5.986 10.804 12.428 59.680mt_cutH <- 10
obj_unfiltered <- obj
obj <- subset(obj, subset = percent.mt < mt_cutH)Remove potential cell debris
Unfiltered
VlnPlot(obj_unfiltered, features = c("nCount_RNA", "nFeature_RNA", "percent.mt"),
pt.size = 0.2)
Filtered
VlnPlot(obj, features = c("nCount_RNA", "nFeature_RNA", "percent.mt"), pt.size = 0.2)
Remove potential cell debris
FeatureScatter(obj_unfiltered, feature1 = "nCount_RNA", feature2 = "percent.mt")
FeatureScatter(obj, feature1 = "nCount_RNA", feature2 = "percent.mt")
Cell cycle phase determination
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
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$s.genes)
g2m_gene <- convertHumanGeneList(cc.genes$g2m.genes)Cell cycle ~ estimation
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 -0.2260109 0.2533774 G2M CTRL
## CTRL_CCTCTGACAGTAGAGC-1 -0.1392145 0.4500886 G2M CTRLCell cycle ~ phase
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 214
## 3 CTRL S 232Cell cycle ~ phase
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()
Regression and clustering
Data normalization
Re-scale and regress data to percent.mt, S.score, and G2M.score
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 matrixPrinciple Component Analysis
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.
ElbowPlot(obj, ndims = 30)
#
numPC <- 15Clustering
set.seed(1000)
maxPC <- numPC
obj <- FindNeighbors(obj, reduction = "pca", dims = 1:maxPC)## Computing nearest neighbor graph## Computing SNNobj <- 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: 21643
##
## Running Louvain algorithm...
## Maximum modularity in 10 random starts: 0.7679
## Number of communities: 7
## Elapsed time: 0 secondsClustering
obj <- RunUMAP(obj, reduction = "pca", dims = 1:maxPC)## 16:22:41 UMAP embedding parameters a = 0.9922 b = 1.112## 16:22:41 Read 692 rows and found 15 numeric columns## 16:22:41 Using Annoy for neighbor search, n_neighbors = 30## 16:22:41 Building Annoy index with metric = cosine, n_trees = 50## 0% 10 20 30 40 50 60 70 80 90 100%## [----|----|----|----|----|----|----|----|----|----|## **************************************************|
## 16:22:41 Writing NN index file to temp file /tmp/RtmpMsibtn/file17694e1f1b09
## 16:22:41 Searching Annoy index using 1 thread, search_k = 3000
## 16:22:41 Annoy recall = 100%
## 16:22:41 Commencing smooth kNN distance calibration using 1 thread
## 16:22:42 Initializing from normalized Laplacian + noise
## 16:22:42 Commencing optimization for 500 epochs, with 25644 positive edges
## 16:22:44 Optimization finishedobj <- RunTSNE(obj, reduction = "pca", dims = 1:maxPC)UMAP plot
DimPlot(obj, reduction = "umap") # default it 'umap'
tSNE plot
DimPlot(obj, reduction = "tsne")
Identify marker genes for each cluster
Marker genes
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 6head(clust.markers)## p_val avg_log2FC pct.1 pct.2 p_val_adj cluster gene
## Igfbp2 5.690084e-29 0.8136702 0.820 0.403 8.255174e-25 0 Igfbp2
## Krt14 8.448994e-28 0.7369650 1.000 0.980 1.225780e-23 0 Krt14
## H19 2.916693e-26 0.7943296 0.972 0.785 4.231539e-22 0 H19
## Serpinb10 3.994589e-25 0.6534384 0.756 0.355 5.795350e-21 0 Serpinb10
## Fth1 4.408103e-22 0.4731999 1.000 0.936 6.395275e-18 0 Fth1
## Col17a1 1.542743e-21 0.6859089 0.845 0.511 2.238212e-17 0 Col17a1Select marker genes for each cluster
Here we will select the top 5 marker genes for each cluster.
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 5.69e-29 0.814 0.82 0.403 8.26e-25 0 Igfbp2
## 2 8.45e-28 0.737 1 0.98 1.23e-23 0 Krt14
## 3 2.92e-26 0.794 0.972 0.785 4.23e-22 0 H19
## 4 1.54e-21 0.686 0.845 0.511 2.24e-17 0 Col17a1
## 5 2.72e-17 0.655 0.728 0.452 3.94e-13 0 Cdkn1c
## 6 2.02e-80 1.76 0.833 0.076 2.93e-76 1 Sox9Advanced plots
Dimplots ~ adjust point size
DimPlot(obj, pt.size = 0.2)
Dimplots with labeling clusters
DimPlot(obj, pt.size = 0.2, label = TRUE) + NoLegend()
Marker Gene expression ~ heatmap
DoHeatmap(obj, features = topG$gene) + NoLegend()
Marker Gene Expression ~ FeaturePlot
gene_marker <- c("Krt1", "Pthlh", "Krt14", "Cenpa", "Shh")
FeaturePlot(obj, features = gene_marker, pt.size = 0.2)
Marker Gene Expression ~ RidgePlot
RidgePlot(obj, features = gene_marker)
Cell cycle phases by cluster
tbl <- table(obj@meta.data$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 134 283 0.4734982
## 2 1 G1 39 114 0.3421053Cell cycle phases by cluster ~ plot
- The second cluster may need to be removed, as there seems to be cell cycle bias.
Save file
First we filter out the second cluster, and then save the data object.
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.