Cell quality control

Load libraries

library(Seurat)
library(dplyr)
library(RColorBrewer)
library(ggplot2)
library(ggExtra)
library(cowplot)
library(reticulate)
library(wesanderson)
use_python("/usr/bin/python3")

#Set ggplot theme as classic
theme_set(theme_classic())

This dataset was generated from the sequencing of two 10X V3 libraries run in parallel from the same tissue dissociation prep

Process the first library

Load the raw filtered matrix output from Cellranger

Countdata <- Read10X(data.dir = "../../RawData/Hem_1_filtered_feature_bc_matrix/")

Raw.data <- CreateSeuratObject(raw.data = Countdata,
                              min.cells = 3,
                              min.genes = 800,
                              project = "Hem1") ; rm(Countdata)

Raw.data@meta.data$Barcodes <- rownames(Raw.data@meta.data)

dim(Raw.data@data)

## [1] 18098 10180

Compute mito and ribo gene content per cell

mito.genes <- grep(pattern = "^mt-", x = rownames(x = Raw.data@data), value = TRUE)
percent.mito <- Matrix::colSums(Raw.data@raw.data[mito.genes, ])/Matrix::colSums(Raw.data@raw.data)
Raw.data <- AddMetaData(object = Raw.data, metadata = percent.mito, col.name = "percent.mito")

ribo.genes <- grep(pattern = "(^Rpl|^Rps|^Mrp)", x = rownames(x = Raw.data@data), value = TRUE)
percent.ribo <- Matrix::colSums(Raw.data@raw.data[ribo.genes, ])/Matrix::colSums(Raw.data@raw.data)
Raw.data <- AddMetaData(object = Raw.data, metadata = percent.ribo, col.name = "percent.ribo")

rm(mito.genes, percent.mito,ribo.genes,percent.ribo)

VlnPlot(object = Raw.data, features.plot = c("nGene","nUMI", "percent.mito", "percent.ribo"), nCol = 2 )

Inspect cell based on relation between nUMI and nGene detected

# Relation between nUMI and nGene detected
Cell.QC.Stat <- Raw.data@meta.data
Cell.QC.Stat$Barcodes <- rownames(Cell.QC.Stat)

p1 <- ggplot(Cell.QC.Stat, aes(x=nUMI, y=nGene)) + geom_point() + geom_smooth(method="lm")
p1 <- ggMarginal(p1, type = "histogram", fill="lightgrey")

p2 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) + geom_point() + geom_smooth(method="lm")
p2 <- ggMarginal(p2, type = "histogram", fill="lightgrey")

plot_grid(plotlist = list(p1,p2), ncol=2, align='h', rel_widths = c(1, 1)) ; rm(p1,p2)

Cells with deviating nGene/nUMI ratio display an Erythrocyte signature

genes.list <- list(c("Hbb-bt", "Hbq1a", "Isg20", "Fech", "Snca", "Rec114"))
enrich.name <- "Erythrocyte.signature"
Raw.data <- AddModuleScore(Raw.data,
                           genes.list = genes.list,
                           genes.pool = NULL,
                           n.bin = 5,
                           seed.use = 1,
                           ctrl.size = length(genes.list),
                           use.k = FALSE,
                           enrich.name = enrich.name,
                           random.seed = 1)

Cell.QC.Stat$Erythrocyte.signature <- Raw.data@meta.data$Erythrocyte.signature1

gradient <- colorRampPalette(brewer.pal(n =11, name = "Spectral"))(100)

p1 <- ggplot(Cell.QC.Stat, aes(x= log10(nUMI), y= log10(nGene))) +
      geom_point(aes(color= Erythrocyte.signature))  + 
      scale_color_gradientn(colours=rev(gradient), name='Erythrocyte score') +
      geom_smooth(method="lm")


p1 <- ggMarginal(p1, type = "histogram", fill="lightgrey")
p1

Low quality cell filtering

Filtering cells based on percentage of mitochondrial transcripts

We applied a high and low median absolute deviation (mad) thresholds to exclude outlier cells

max.mito.thr <- median(Cell.QC.Stat$percent.mito) + 3*mad(Cell.QC.Stat$percent.mito)
min.mito.thr <- median(Cell.QC.Stat$percent.mito) - 3*mad(Cell.QC.Stat$percent.mito)

p1 <- ggplot(Cell.QC.Stat, aes(x=nGene, y=percent.mito)) +
  geom_point() +
  geom_hline(aes(yintercept = max.mito.thr), colour = "red", linetype = 2) +
  geom_hline(aes(yintercept = min.mito.thr), colour = "red", linetype = 2) +
  annotate(geom = "text", label = paste0(as.numeric(table(Cell.QC.Stat$percent.mito > max.mito.thr | Cell.QC.Stat$percent.mito < min.mito.thr)[2])," cells removed\n",
                                         as.numeric(table(Cell.QC.Stat$percent.mito > max.mito.thr | Cell.QC.Stat$percent.mito < min.mito.thr)[1])," cells remain"),
           x = 6000, y = 0.4)

ggMarginal(p1, type = "histogram", fill="lightgrey", bins=100) 

# Filter cells based on these thresholds
Cell.QC.Stat <- Cell.QC.Stat %>% filter(percent.mito < max.mito.thr) %>% filter(percent.mito > min.mito.thr)

Filtering cells based on number of genes and transcripts detected

Remove cells with to few gene detected or with to many UMI counts

We filter cells which are likely to be doublet based on their higher content of transcript detected as well as cell with to few genes/UMI sequenced

# Set low and hight thresholds on the number of detected genes
min.Genes.thr <- median(log10(Cell.QC.Stat$nGene)) - 3*mad(log10(Cell.QC.Stat$nGene))
max.Genes.thr <- median(log10(Cell.QC.Stat$nGene)) + 3*mad(log10(Cell.QC.Stat$nGene))

# Set hight threshold on the number of transcripts
max.nUMI.thr <- median(log10(Cell.QC.Stat$nUMI)) + 3*mad(log10(Cell.QC.Stat$nUMI))

# Gene/UMI scatter plot before filtering
p1 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) +
  geom_point() +
  geom_smooth(method="lm") +
  geom_hline(aes(yintercept = min.Genes.thr), colour = "green", linetype = 2) +
  geom_hline(aes(yintercept = max.Genes.thr), colour = "green", linetype = 2) +
  geom_vline(aes(xintercept = max.nUMI.thr), colour = "red", linetype = 2)

ggMarginal(p1, type = "histogram", fill="lightgrey")

# Filter cells base on both metrics
Cell.QC.Stat <- Cell.QC.Stat %>% filter(log10(nGene) > min.Genes.thr) %>% filter(log10(nUMI) < max.nUMI.thr)

Filter cells below the main population nUMI/nGene relationship

lm.model <- lm(data = Cell.QC.Stat, formula = log10(nGene) ~ log10(nUMI))

p2 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) +
  geom_point() +
  geom_smooth(method="lm") +
  geom_hline(aes(yintercept = min.Genes.thr), colour = "green", linetype = 2) +
  geom_hline(aes(yintercept = max.Genes.thr), colour = "green", linetype = 2) +
  geom_vline(aes(xintercept = max.nUMI.thr), colour = "red", linetype = 2) +
  geom_abline(intercept = lm.model$coefficients[1] - 0.09 , slope = lm.model$coefficients[2], color="orange") +
  annotate(geom = "text", label = paste0(dim(Cell.QC.Stat)[1], " QC passed cells"), x = 4, y = 3.8)

ggMarginal(p2, type = "histogram", fill="lightgrey")

# Cells to exclude lie below an intercept offset of -0.09
Cell.QC.Stat$valideCells <- log10(Cell.QC.Stat$nGene) > (log10(Cell.QC.Stat$nUMI) * lm.model$coefficients[2] + (lm.model$coefficients[1] - 0.09))

p3 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) +
  geom_point(aes(colour = valideCells)) +
  geom_smooth(method="lm") +
  geom_abline(intercept = lm.model$coefficients[1] - 0.09 , slope = lm.model$coefficients[2], color="orange") + 
  theme(legend.position="none") +
  annotate(geom = "text", label = paste0(as.numeric(table(Cell.QC.Stat$valideCells)[2]), " QC passed cells\n",
                                         as.numeric(table(Cell.QC.Stat$valideCells)[1]), " QC filtered"), x = 4, y = 3.8)

ggMarginal(p3, type = "histogram", fill="lightgrey")

# Remove invalid cells
Cell.QC.Stat <- Cell.QC.Stat %>% filter(valideCells)

Keep only the valid cells in the Seurat object

Raw.data <- SubsetData(Raw.data, cells.use = Cell.QC.Stat$Barcodes , subset.raw = T,  do.clean = F)

# Plot final QC metrics
VlnPlot(object = Raw.data, features.plot = c("nGene","nUMI", "percent.mito", "percent.ribo"), nCol = 2 )

p1 <- ggplot(Raw.data@meta.data, aes(x=log10(nUMI), y=log10(nGene))) + geom_point() + geom_smooth(method="lm")
ggMarginal(p1, type = "histogram", fill="lightgrey")

rm(list = ls()[!ls() %in% "Raw.data"])

Use Scrublet to detect obvious doublets

Run Scrublet with default parameter

Export raw count matrix as input to Scrublet

#Export filtered matrix
dir.create("../../Scrublet_inputs")

exprData <- Matrix(as.matrix(Raw.data@raw.data), sparse = TRUE)
writeMM(exprData, "../../Scrublet_inputs/matrix1.mtx")

## NULL

import scrublet as scr
import scipy.io
import numpy as np
import os

#Load raw counts matrix and gene list
input_dir = '../../Scrublet_inputs'
counts_matrix = scipy.io.mmread(input_dir + '/matrix1.mtx').T.tocsc()

#Initialize Scrublet
scrub = scr.Scrublet(counts_matrix,
                     expected_doublet_rate=0.1,
                     sim_doublet_ratio=2,
                     n_neighbors = 8)

#Run the default pipeline
doublet_scores, predicted_doublets = scrub.scrub_doublets(min_counts=1, 
                                                          min_cells=3, 
                                                          min_gene_variability_pctl=85, 
                                                          n_prin_comps=25)

## Preprocessing...
## Simulating doublets...
## Embedding transcriptomes using PCA...
## Calculating doublet scores...
## Automatically set threshold at doublet score = 0.20
## Detected doublet rate = 7.4%
## Estimated detectable doublet fraction = 68.6%
## Overall doublet rate:
##  Expected   = 10.0%
##  Estimated  = 10.8%
## Elapsed time: 28.5 seconds

# Import scrublet's doublet score
Raw.data@meta.data$Doubletscore <- py$doublet_scores

# Plot doublet score
ggplot(Raw.data@meta.data, aes(x = Doubletscore, stat(ndensity))) +
  geom_histogram(bins = 200, colour ="lightgrey")+
  geom_vline(xintercept = 0.20, colour = "red", linetype = 2)

# Manually set threshold at doublet score to 0.2
Raw.data@meta.data$Predicted_doublets <- ifelse(py$doublet_scores > 0.2, "Doublet","Singlet" )
table(Raw.data@meta.data$Predicted_doublets)

## 
## Doublet Singlet 
##     658    8184

Filter doublets

#Remove Scrublet inferred doublets
Valid.Cells <- rownames(Raw.data@meta.data[Raw.data@meta.data$Predicted_doublets == "Singlet",])

QC.data.1 <- SubsetData(Raw.data,  cells.use = Valid.Cells, subset.raw = T, do.clean = F)

rm(list = ls()[!ls() %in% "QC.data.1"])

Process the second library

Load the raw filtered matrix output from Cellranger

Countdata <- Read10X(data.dir = "../../RawData/Hem_2_filtered_feature_bc_matrix/")

Raw.data <- CreateSeuratObject(raw.data = Countdata,
                              min.cells = 3,
                              min.genes = 800,
                              project = "Hem2") ; rm(Countdata)

Raw.data@meta.data$Barcodes <- rownames(Raw.data@meta.data)

dim(Raw.data@data)

## [1] 18048  8817

Compute mito and ribo gene content per cell

mito.genes <- grep(pattern = "^mt-", x = rownames(x = Raw.data@data), value = TRUE)
percent.mito <- Matrix::colSums(Raw.data@raw.data[mito.genes, ])/Matrix::colSums(Raw.data@raw.data)
Raw.data <- AddMetaData(object = Raw.data, metadata = percent.mito, col.name = "percent.mito")

ribo.genes <- grep(pattern = "(^Rpl|^Rps|^Mrp)", x = rownames(x = Raw.data@data), value = TRUE)
percent.ribo <- Matrix::colSums(Raw.data@raw.data[ribo.genes, ])/Matrix::colSums(Raw.data@raw.data)
Raw.data <- AddMetaData(object = Raw.data, metadata = percent.ribo, col.name = "percent.ribo")

rm(mito.genes, percent.mito,ribo.genes,percent.ribo)

VlnPlot(object = Raw.data, features.plot = c("nGene","nUMI", "percent.mito", "percent.ribo"), nCol = 2 )

Inspect cell based on relation between nUMI and nGene detected

# Relation between nUMI and nGene detected
Cell.QC.Stat <- Raw.data@meta.data
Cell.QC.Stat$Barcodes <- rownames(Cell.QC.Stat)

p1 <- ggplot(Cell.QC.Stat, aes(x=nUMI, y=nGene)) + geom_point() + geom_smooth(method="lm")
p1 <- ggMarginal(p1, type = "histogram", fill="lightgrey")

p2 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) + geom_point() + geom_smooth(method="lm")
p2 <- ggMarginal(p2, type = "histogram", fill="lightgrey")

plot_grid(plotlist = list(p1,p2), ncol=2, align='h', rel_widths = c(1, 1)) ; rm(p1,p2)

Cells with deviating nGene/nUMI ratio display an Erythrocyte signature

genes.list <- list(c("Hbb-bt", "Hbq1a", "Isg20", "Fech", "Snca", "Rec114"))
enrich.name <- "Erythrocyte.signature"
Raw.data <- AddModuleScore(Raw.data,
                           genes.list = genes.list,
                           genes.pool = NULL,
                           n.bin = 5,
                           seed.use = 1,
                           ctrl.size = length(genes.list),
                           use.k = FALSE,
                           enrich.name = enrich.name,
                           random.seed = 1)

Cell.QC.Stat$Erythrocyte.signature <- Raw.data@meta.data$Erythrocyte.signature1

gradient <- colorRampPalette(brewer.pal(n =11, name = "Spectral"))(100)

p1 <- ggplot(Cell.QC.Stat, aes(x= log10(nUMI), y= log10(nGene))) +
      geom_point(aes(color= Erythrocyte.signature))  + 
      scale_color_gradientn(colours=rev(gradient), name='Erythrocyte score') +
      geom_smooth(method="lm")


p1 <- ggMarginal(p1, type = "histogram", fill="lightgrey")
p1

Low quality cell filtering

Filtering cells based on percentage of mitochondrial transcripts

We applied a high and low median absolute deviation (mad) thresholds to exclude outlier cells

max.mito.thr <- median(Cell.QC.Stat$percent.mito) + 3*mad(Cell.QC.Stat$percent.mito)
min.mito.thr <- median(Cell.QC.Stat$percent.mito) - 3*mad(Cell.QC.Stat$percent.mito)

p1 <- ggplot(Cell.QC.Stat, aes(x=nGene, y=percent.mito)) +
  geom_point() +
  geom_hline(aes(yintercept = max.mito.thr), colour = "red", linetype = 2) +
  geom_hline(aes(yintercept = min.mito.thr), colour = "red", linetype = 2) +
  annotate(geom = "text", label = paste0(as.numeric(table(Cell.QC.Stat$percent.mito > max.mito.thr | Cell.QC.Stat$percent.mito < min.mito.thr)[2])," cells removed\n",
                                         as.numeric(table(Cell.QC.Stat$percent.mito > max.mito.thr | Cell.QC.Stat$percent.mito < min.mito.thr)[1])," cells remain"),
           x = 6000, y = 0.4)

ggMarginal(p1, type = "histogram", fill="lightgrey", bins=100) 

# Filter cells based on these thresholds
Cell.QC.Stat <- Cell.QC.Stat %>% filter(percent.mito < max.mito.thr) %>% filter(percent.mito > min.mito.thr)

Filtering cells based on number of genes and transcripts detected

Remove cells with to few gene detected or with to many UMI counts

We filter cells which are likely to be doublet based on their higher content of transcript detected as well as cell with to few genes/UMI sequenced

# Set low and hight thresholds on the number of detected genes
min.Genes.thr <- median(log10(Cell.QC.Stat$nGene)) - 3*mad(log10(Cell.QC.Stat$nGene))
max.Genes.thr <- median(log10(Cell.QC.Stat$nGene)) + 3*mad(log10(Cell.QC.Stat$nGene))

# Set hight threshold on the number of transcripts
max.nUMI.thr <- median(log10(Cell.QC.Stat$nUMI)) + 3*mad(log10(Cell.QC.Stat$nUMI))

# Gene/UMI scatter plot before filtering
p1 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) +
  geom_point() +
  geom_smooth(method="lm") +
  geom_hline(aes(yintercept = min.Genes.thr), colour = "green", linetype = 2) +
  geom_hline(aes(yintercept = max.Genes.thr), colour = "green", linetype = 2) +
  geom_vline(aes(xintercept = max.nUMI.thr), colour = "red", linetype = 2)

ggMarginal(p1, type = "histogram", fill="lightgrey")

# Filter cells base on both metrics
Cell.QC.Stat <- Cell.QC.Stat %>% filter(log10(nGene) > min.Genes.thr) %>% filter(log10(nUMI) < max.nUMI.thr)

Filter cells below the main population nUMI/nGene relationship

lm.model <- lm(data = Cell.QC.Stat, formula = log10(nGene) ~ log10(nUMI))

p2 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) +
  geom_point() +
  geom_smooth(method="lm") +
  geom_hline(aes(yintercept = min.Genes.thr), colour = "green", linetype = 2) +
  geom_hline(aes(yintercept = max.Genes.thr), colour = "green", linetype = 2) +
  geom_vline(aes(xintercept = max.nUMI.thr), colour = "red", linetype = 2) +
  geom_abline(intercept = lm.model$coefficients[1] - 0.09 , slope = lm.model$coefficients[2], color="orange") +
  annotate(geom = "text", label = paste0(dim(Cell.QC.Stat)[1], " QC passed cells"), x = 4, y = 3.8)

ggMarginal(p2, type = "histogram", fill="lightgrey")

# Cells to exclude lie below an intercept offset of -0.09
Cell.QC.Stat$valideCells <- log10(Cell.QC.Stat$nGene) > (log10(Cell.QC.Stat$nUMI) * lm.model$coefficients[2] + (lm.model$coefficients[1] - 0.09))

p3 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) +
  geom_point(aes(colour = valideCells)) +
  geom_smooth(method="lm") +
  geom_abline(intercept = lm.model$coefficients[1] - 0.09 , slope = lm.model$coefficients[2], color="orange") + 
  theme(legend.position="none") +
  annotate(geom = "text", label = paste0(as.numeric(table(Cell.QC.Stat$valideCells)[2]), " QC passed cells\n",
                                         as.numeric(table(Cell.QC.Stat$valideCells)[1]), " QC filtered"), x = 4, y = 3.8)

ggMarginal(p3, type = "histogram", fill="lightgrey")

# Remove invalid cells
Cell.QC.Stat <- Cell.QC.Stat %>% filter(valideCells)

Keep only the valid cells in the Seurat object

Raw.data <- SubsetData(Raw.data, cells.use = Cell.QC.Stat$Barcodes , subset.raw = T,  do.clean = F)

# Plot final QC metrics
VlnPlot(object = Raw.data, features.plot = c("nGene","nUMI", "percent.mito", "percent.ribo"), nCol = 2 )

p1 <- ggplot(Raw.data@meta.data, aes(x=log10(nUMI), y=log10(nGene))) + geom_point() + geom_smooth(method="lm")
ggMarginal(p1, type = "histogram", fill="lightgrey")

rm(list = ls()[!ls() %in% c("Raw.data", "QC.data.1")])

Use Scrublet to detect obvious doublets

Run Scrublet with default parameter

Export raw count matrix as input to Scrublet

#Export filtered matrix
exprData <- Matrix(as.matrix(Raw.data@raw.data), sparse = TRUE)
writeMM(exprData, "../../Scrublet_inputs/matrix2.mtx")

## NULL

import scrublet as scr
import scipy.io
import numpy as np
import os

#Load raw counts matrix and gene list
input_dir = '../../Scrublet_inputs'
counts_matrix = scipy.io.mmread(input_dir + '/matrix2.mtx').T.tocsc()

#Initialize Scrublet
scrub = scr.Scrublet(counts_matrix,
                     expected_doublet_rate=0.1,
                     sim_doublet_ratio=2,
                     n_neighbors = 8)

#Run the default pipeline
doublet_scores, predicted_doublets = scrub.scrub_doublets(min_counts=1, 
                                                          min_cells=3, 
                                                          min_gene_variability_pctl=85, 
                                                          n_prin_comps=25)

## Preprocessing...
## Simulating doublets...
## Embedding transcriptomes using PCA...
## Calculating doublet scores...
## Automatically set threshold at doublet score = 0.24
## Detected doublet rate = 5.1%
## Estimated detectable doublet fraction = 59.0%
## Overall doublet rate:
##  Expected   = 10.0%
##  Estimated  = 8.7%
## Elapsed time: 9.8 seconds
## 
## /home/matthieu/.local/lib/python3.6/site-packages/scrublet/helper_functions.py:239: RuntimeWarning: invalid value encountered in log
##   gLog = lambda input: np.log(input[1] * np.exp(-input[0]) + input[2])

# Import scrublet's doublet score
Raw.data@meta.data$Doubletscore <- py$doublet_scores

# Plot doublet score
ggplot(Raw.data@meta.data, aes(x = Doubletscore, stat(ndensity))) +
  geom_histogram(bins = 200, colour ="lightgrey")+
  geom_vline(xintercept = 0.24, colour = "red", linetype = 2)

# Manually set threshold at doublet score to 0.2
Raw.data@meta.data$Predicted_doublets <- ifelse(py$doublet_scores > 0.24, "Doublet","Singlet" )
table(Raw.data@meta.data$Predicted_doublets)

## 
## Doublet Singlet 
##     385    7149

Filter doublets

#Remove Scrublet inferred doublets
Valid.Cells <- rownames(Raw.data@meta.data[Raw.data@meta.data$Predicted_doublets == "Singlet",])

QC.data.2 <- SubsetData(Raw.data,  cells.use = Valid.Cells, subset.raw = T, do.clean = F)

rm(list = ls()[!ls() %in% c("QC.data.1", "QC.data.2")])

Merge the two libraries

Hem.data <- MergeSeurat(QC.data.1, QC.data.2,
                        do.normalize = F,
                        add.cell.id1 = "Hem1",
                        add.cell.id2 = "Hem2")

Hem.data@meta.data$Barcodes <- rownames(Hem.data@meta.data)

Cell.QC.Stat <- Hem.data@meta.data
Cell.QC.Stat$Barcodes <- rownames(Cell.QC.Stat)

p1 <- ggplot(Cell.QC.Stat, aes(x=nUMI, y=nGene)) + geom_point() + geom_smooth(method="lm")
p1 <- ggMarginal(p1, type = "histogram", fill="lightgrey")

p2 <- ggplot(Cell.QC.Stat, aes(x=log10(nUMI), y=log10(nGene))) + geom_point() + geom_smooth(method="lm")
p2 <- ggMarginal(p2, type = "histogram", fill="lightgrey")

plot_grid(plotlist = list(p1,p2), ncol=2, align='h', rel_widths = c(1, 1)) ; rm(p1,p2)

rm(list = ls()[!ls() %in% "Hem.data"])

Filter gene expression matrix

# Filter genes expressed by less than 3 cells
num.cells <- Matrix::rowSums(Hem.data@data > 0)
genes.use <- names(x = num.cells[which(x = num.cells >= 3)])
Hem.data@raw.data <- Hem.data@raw.data[genes.use, ]
Hem.data@data <- Hem.data@data[genes.use, ]

# log-normalize the gene expression matrix
Hem.data<- NormalizeData(object = Hem.data,
                          normalization.method = "LogNormalize", 
                          scale.factor = round(median(Hem.data@meta.data$nUMI)),
                          display.progress = F)

Generate SRING dimentionality reduction

dir.create("../../SpringCoordinates")

# Export raw expression matrix and gene list to regenerate a spring plot
exprData <- Matrix(as.matrix(Hem.data@raw.data), sparse = TRUE)
writeMM(exprData, "../../SpringCoordinates/ExprData.mtx")

## NULL

Genelist <- row.names(Hem.data@raw.data)
write.table(Genelist, "../../SpringCoordinates/Genelist.csv", sep="\t", col.names = F, row.names = F)

Spring coordinates were generated using the online version of SPRING with the following parameters :

Number of cells: 15333
Number of genes that passed filter: 874
Min expressing cells (gene filtering): 3
Min number of UMIs (gene filtering): 3
Gene variability %ile (gene filtering): 95
Number of principal components: 20
Number of nearest neighbors: 8
Number of force layout iterations: 500

Import the new coordinates

# Import Spring coordinates
Coordinates <-read.table("../SpringCoordinates/hem_spring.csv", sep=",", header = T)
rownames(Coordinates) <- colnames(Hem.data@data)

Hem.data <- SetDimReduction(Hem.data,
                            reduction.type = "spring",
                            slot = "cell.embeddings",
                            new.data = as.matrix(Coordinates))

Hem.data@dr$spring@key <- "spring"
colnames(Hem.data@dr$spring@cell.embeddings) <- paste0(GetDimReduction(object= Hem.data, reduction.type = "spring",slot = "key"), c(1,2))

Assign cell state scores

Cell-Cycle Scores

s.genes <- c("Mcm5", "Pcna", "Tym5", "Fen1", "Mcm2", "Mcm4", "Rrm1", "Ung", "Gins2", "Mcm6", "Cdca7", "Dtl", "Prim1", "Uhrf1", "Mlf1ip", "Hells", "Rfc2", "Rap2", "Nasp", "Rad51ap1", "Gmnn", "Wdr76", "Slbp", "Ccne2", "Ubr7", "Pold3", "Msh2", "Atad2", "Rad51", "Rrm2", "Cdc45", "Cdc6", "Exo1", "Tipin", "Dscc1", "Blm", " Casp8ap2", "Usp1", "Clspn", "Pola1", "Chaf1b", "Brip1", "E2f8")
g2m.genes <- c("Hmgb2", "Ddk1","Nusap1", "Ube2c", "Birc5", "Tpx2", "Top2a", "Ndc80", "Cks2", "Nuf2", "Cks1b", "Mki67", "Tmpo", " Cenpk", "Tacc3", "Fam64a", "Smc4", "Ccnb2", "Ckap2l", "Ckap2", "Aurkb", "Bub1", "Kif11", "Anp32e", "Tubb4b", "Gtse1", "kif20b", "Hjurp", "Cdca3", "Hn1", "Cdc20", "Ttk", "Cdc25c", "kif2c", "Rangap1", "Ncapd2", "Dlgap5", "Cdca2", "Cdca8", "Ect2", "Kif23", "Hmmr", "Aurka", "Psrc1", "Anln", "Lbr", "Ckap5", "Cenpe", "Ctcf", "Nek2", "G2e3", "Gas2l3", "Cbx5", "Cenpa")

Hem.data <- CellCycleScoring(object = Hem.data,
                             s.genes = s.genes,
                             g2m.genes = g2m.genes,
                             set.ident = TRUE)

Hem.data@meta.data$CC.Difference <- Hem.data@meta.data$S.Score - Hem.data@meta.data$G2M.Score

DimPlot(Hem.data,
        reduction.use = "spring",
        group.by = "Phase",
        cols.use = wes_palette("GrandBudapest1", 3, type = "discrete")[3:1],
        dim.1 = 1, 
        dim.2 = 2,
        do.label=T,
        label.size = 4,
        no.legend = F )

We assigned broad transcriptional cell state score based on known and manually curated marker genes

Apical progenitors

APgenes <- c("Rgcc", "Sparc", "Hes5","Hes1", "Slc1a3",
             "Ddah1", "Ldha", "Hmga2","Sfrp1", "Id4",
             "Creb5", "Ptn", "Lpar1", "Rcn1","Zfp36l1",
             "Sox9", "Sox2", "Nr2e1", "Ttyh1", "Trip6")
genes.list <- list(APgenes)
enrich.name <- "AP_signature"
Hem.data <- AddModuleScore(Hem.data,
                                  genes.list = genes.list,
                                  genes.pool = NULL,
                                  n.bin = 5,
                                  seed.use = 1,
                                  ctrl.size = length(genes.list),
                                  use.k = FALSE,
                                  enrich.name = enrich.name,
                                  random.seed = 1)

plot <- FeaturePlot(object = Hem.data,
                    features.plot = APgenes,
                    cols.use = c("grey90", brewer.pal(9,"YlGnBu")),
                    reduction.use = "spring",
                    no.legend = T,
                    overlay = F,
                    dark.theme = F,
                    do.return =T,
                    no.axes = T)

for (i in 1:length(plot)){
  plot[[i]]$data <- plot[[i]]$data[order(plot[[i]]$data$gene),]
}

cowplot::plot_grid(plotlist = plot[1:20], ncol = 5)

Apical progenitors gene expression

Basal progenitors

BPgenes <- c("Eomes", "Igsf8", "Insm1", "Elavl2", "Elavl4",
             "Hes6","Gadd45g", "Neurog2", "Btg2", "Neurog1")
genes.list <- list(BPgenes)
enrich.name <- "BP_signature"
Hem.data <- AddModuleScore(Hem.data,
                                  genes.list = genes.list,
                                  genes.pool = NULL,
                                  n.bin = 5,
                                  seed.use = 1,
                                  ctrl.size = length(genes.list),
                                  use.k = FALSE,
                                  enrich.name = enrich.name,
                                  random.seed = 1)

plot <- FeaturePlot(object = Hem.data,
                    features.plot = BPgenes,
                    cols.use = c("grey90", brewer.pal(9,"YlGnBu")),
                    reduction.use = "spring",
                    no.legend = T,
                    overlay = F,
                    dark.theme = F,
                    do.return =T,
                    no.axes = T)

for (i in 1:length(plot)){
  plot[[i]]$data <- plot[[i]]$data[order(plot[[i]]$data$gene),]
}

cowplot::plot_grid(plotlist = plot[1:10], ncol = 5)

Basal progenitors gene expression

Early pallial neurons

ENgenes <- c("Mfap4", "Nhlh2", "Nhlh1", "Ppp1r14a", "Nav1",
             "Neurod1", "Sorl1", "Svip", "Cxcl12", "Tenm4",
             "Dll3", "Rgmb", "Cntn2", "Vat1")
genes.list <- list(ENgenes)
enrich.name <- "EN_signature"
Hem.data <- AddModuleScore(Hem.data,
                                  genes.list = genes.list,
                                  genes.pool = NULL,
                                  n.bin = 5,
                                  seed.use = 1,
                                  ctrl.size = length(genes.list),
                                  use.k = FALSE,
                                  enrich.name = enrich.name,
                                  random.seed = 1)

plot <- FeaturePlot(object = Hem.data,
                    features.plot = ENgenes,
                    cols.use = c("grey90", brewer.pal(9,"YlGnBu")),
                    reduction.use = "spring",
                    no.legend = T,
                    overlay = F,
                    dark.theme = F,
                    do.return =T,
                    no.axes = T)

for (i in 1:length(plot)){
  plot[[i]]$data <- plot[[i]]$data[order(plot[[i]]$data$gene),]
}

cowplot::plot_grid(plotlist = plot[1:14], ncol = 5)

Early pallial neurons gene expression

Late pallial neurons

LNgenes <- c("Snhg11", "Pcsk1n", "Mapt", "Ina", "Stmn4",
             "Gap43", "Tubb2a", "Ly6h","Ptprd", "Mef2c")
genes.list <- list(LNgenes)
enrich.name <- "LN_signature"
Hem.data <- AddModuleScore(Hem.data,
                                  genes.list = genes.list,
                                  genes.pool = NULL,
                                  n.bin = 5,
                                  seed.use = 1,
                                  ctrl.size = length(genes.list),
                                  use.k = FALSE,
                                  enrich.name = enrich.name,
                                  random.seed = 1)

plot <- FeaturePlot(object = Hem.data,
                    features.plot = LNgenes,
                    cols.use = c("grey90", brewer.pal(9,"YlGnBu")),
                    reduction.use = "spring",
                    no.legend = T,
                    overlay = F,
                    dark.theme = F,
                    do.return =T,
                    no.axes = T)

for (i in 1:length(plot)){
  plot[[i]]$data <- plot[[i]]$data[order(plot[[i]]$data$gene),]
}

cowplot::plot_grid(plotlist = plot[1:10], ncol = 5)

Late pallial neurons gene expression

Meninges cells

Mgenes <- c("Lum", "Lgals1", "Foxc1")
genes.list <- list(Mgenes)
enrich.name <- "Meninges_signature"
Hem.data <- AddModuleScore(Hem.data,
                                  genes.list = genes.list,
                                  genes.pool = NULL,
                                  n.bin = 5,
                                  seed.use = 1,
                                  ctrl.size = length(genes.list),
                                  use.k = FALSE,
                                  enrich.name = enrich.name,
                                  random.seed = 1)

plot <- FeaturePlot(object = Hem.data,
                    features.plot = Mgenes,
                    cols.use = c("grey90", brewer.pal(9,"YlGnBu")),
                    reduction.use = "spring",
                    no.legend = T,
                    overlay = F,
                    dark.theme = F,
                    do.return =T,
                    no.axes = T)

for (i in 1:length(plot)){
  plot[[i]]$data <- plot[[i]]$data[order(plot[[i]]$data$gene),]
}

cowplot::plot_grid(plotlist = plot[1:3], ncol = 3)

Meninges gene expression

Immune cells

Immunegenes <- c("Fcer1g", "C1qb", "Tyrobp")
genes.list <- list(Immunegenes)
enrich.name <- "Immune_signature"
Hem.data <- AddModuleScore(Hem.data,
                           genes.list = genes.list,
                           genes.pool = NULL,
                           n.bin = 5,
                           seed.use = 1,
                           ctrl.size = length(genes.list),
                           use.k = FALSE,
                           enrich.name = enrich.name,
                           random.seed = 1)

plot <- FeaturePlot(object = Hem.data,
                    features.plot = Immunegenes,
                    cols.use = c("grey90", brewer.pal(9,"YlGnBu")),
                    reduction.use = "spring",
                    no.legend = T,
                    overlay = F,
                    dark.theme = F,
                    do.return =T,
                    no.axes = T)

for (i in 1:length(plot)){
  plot[[i]]$data <- plot[[i]]$data[order(plot[[i]]$data$gene),]
}

cowplot::plot_grid(plotlist = plot[1:3], ncol = 3)

Immune gene expression

FeaturePlot(object = Hem.data,
            features.plot = c("AP_signature1", "BP_signature1",
                              "EN_signature1", "LN_signature1",
                              "Meninges_signature1","Immune_signature1"),
            cols.use = rev(brewer.pal(10,"Spectral")),
            reduction.use = "spring",
            no.legend = T,
            overlay = F,
            dark.theme = F,
            no.axes = T)

Save Seurat object

saveRDS(Hem.data, "../QC.filtered.cells.RDS")

Session Info

#date
format(Sys.time(), "%d %B, %Y, %H,%M")

## [1] "15 septembre, 2021, 09,47"

#Packages used
sessionInfo()

## R version 3.6.3 (2020-02-29)
## Platform: x86_64-pc-linux-gnu (64-bit)
## Running under: Ubuntu 18.04.5 LTS
## 
## Matrix products: default
## BLAS:   /usr/lib/x86_64-linux-gnu/atlas/libblas.so.3.10.3
## LAPACK: /usr/lib/x86_64-linux-gnu/atlas/liblapack.so.3.10.3
## 
## locale:
##  [1] LC_CTYPE=fr_FR.UTF-8       LC_NUMERIC=C              
##  [3] LC_TIME=fr_FR.UTF-8        LC_COLLATE=fr_FR.UTF-8    
##  [5] LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=fr_FR.UTF-8   
##  [7] LC_PAPER=fr_FR.UTF-8       LC_NAME=C                 
##  [9] LC_ADDRESS=C               LC_TELEPHONE=C            
## [11] LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C       
## 
## attached base packages:
## [1] stats     graphics  grDevices utils     datasets  methods   base     
## 
## other attached packages:
## [1] wesanderson_0.3.6  reticulate_1.18    ggExtra_0.9        RColorBrewer_1.1-2
## [5] dplyr_1.0.6        Seurat_2.3.4       Matrix_1.2-17      cowplot_1.1.1     
## [9] ggplot2_3.3.3     
## 
## loaded via a namespace (and not attached):
##   [1] Rtsne_0.15          colorspace_2.0-1    ellipsis_0.3.2     
##   [4] class_7.3-17        modeltools_0.2-22   ggridges_0.5.1     
##   [7] mclust_5.4.5        htmlTable_1.13.2    base64enc_0.1-3    
##  [10] rstudioapi_0.11     proxy_0.4-23        farver_2.1.0       
##  [13] npsurv_0.4-0        flexmix_2.3-15      bit64_4.0.2        
##  [16] fansi_0.5.0         codetools_0.2-16    splines_3.6.3      
##  [19] R.methodsS3_1.7.1   lsei_1.2-0          robustbase_0.93-5  
##  [22] knitr_1.26          jsonlite_1.7.2      Formula_1.2-3      
##  [25] ica_1.0-2           cluster_2.1.0       kernlab_0.9-29     
##  [28] png_0.1-7           R.oo_1.23.0         shiny_1.4.0        
##  [31] compiler_3.6.3      httr_1.4.2          backports_1.1.5    
##  [34] fastmap_1.0.1       later_1.2.0         lars_1.2           
##  [37] acepack_1.4.1       htmltools_0.5.1.1   tools_3.6.3        
##  [40] igraph_1.2.5        gtable_0.3.0        glue_1.4.2         
##  [43] reshape2_1.4.3      RANN_2.6.1          rappdirs_0.3.1     
##  [46] Rcpp_1.0.6          vctrs_0.3.8         gdata_2.18.0       
##  [49] ape_5.3             nlme_3.1-141        iterators_1.0.12   
##  [52] fpc_2.2-3           lmtest_0.9-37       gbRd_0.4-11        
##  [55] xfun_0.18           stringr_1.4.0       mime_0.10          
##  [58] miniUI_0.1.1.1      lifecycle_1.0.0     irlba_2.3.3        
##  [61] gtools_3.8.1        DEoptimR_1.0-8      zoo_1.8-6          
##  [64] MASS_7.3-53         scales_1.1.1        promises_1.2.0.1   
##  [67] doSNOW_1.0.18       parallel_3.6.3      yaml_2.2.1         
##  [70] pbapply_1.4-2       gridExtra_2.3       segmented_1.0-0    
##  [73] rpart_4.1-15        latticeExtra_0.6-28 stringi_1.4.6      
##  [76] highr_0.8           foreach_1.4.7       checkmate_1.9.4    
##  [79] caTools_1.17.1.2    bibtex_0.4.2        Rdpack_0.11-0      
##  [82] SDMTools_1.1-221.1  rlang_0.4.11        pkgconfig_2.0.3    
##  [85] dtw_1.21-3          prabclus_2.3-1      bitops_1.0-6       
##  [88] evaluate_0.14       lattice_0.20-41     ROCR_1.0-7         
##  [91] purrr_0.3.4         labeling_0.4.2      htmlwidgets_1.5.3  
##  [94] bit_4.0.4           tidyselect_1.1.1    plyr_1.8.4         
##  [97] magrittr_2.0.1      R6_2.5.0            snow_0.4-3         
## [100] gplots_3.0.1.1      generics_0.1.0      Hmisc_4.3-0        
## [103] DBI_1.0.0           mgcv_1.8-33         pillar_1.6.1       
## [106] foreign_0.8-72      withr_2.4.2         mixtools_1.1.0     
## [109] fitdistrplus_1.0-14 survival_2.44-1.1   nnet_7.3-14        
## [112] tsne_0.1-3          tibble_3.1.2        crayon_1.4.1       
## [115] hdf5r_1.3.2.9000    KernSmooth_2.23-15  utf8_1.2.1         
## [118] rmarkdown_2.5       grid_3.6.3          data.table_1.14.0  
## [121] metap_1.1           digest_0.6.27       diptest_0.75-7     
## [124] xtable_1.8-4        httpuv_1.5.2        tidyr_1.1.3        
## [127] R.utils_2.9.0       stats4_3.6.3        munsell_0.5.0

---

1. Institute of Psychiatry and Neuroscience of Paris, INSERM U1266, 75014, Paris, France, matthieu.moreau@inserm.fr↩︎