In waldronlab/SingleCellMultiModal: Integrating Multi-modal Single Cell Experiment datasets

Installation

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("SingleCellMultiModal")

Load

library(SingleCellMultiModal)
library(MultiAssayExperiment)
library(scran)
library(scater)

Description

This data set consists of about 10K Peripheral Blood Mononuclear Cells (PBMCs) derived from a single healthy donor. It is available from the 10x Genomics website.

Provided are the RNA expression counts quantified at the gene level and the chromatin accessibility levels quantified at the peak level. Here we provide the default peaks called by the CellRanger software. If you want to explore other peak definitions or chromatin accessibility quantifications (at the promoter level, etc.), you have download the fragments.tsv.gz file from the 10x Genomics website.

Downloading datasets

The user can see the available dataset by using the default options

mae <- scMultiome("pbmc_10x", mode = "*", dry.run = FALSE, format = "MTX")

gg_color_hue <- function(n) {
  hues = seq(15, 375, length = n + 1)
  hcl(h = hues, l = 65, c = 100)[1:n]
}
colors <- gg_color_hue(length(unique(mae$celltype)))
names(colors) <- unique(mae$celltype)

Exploring the data structure

There are two assays: rna and atac, stored as SingleCellExperiment objects

mae

where the cells are the same in both assays:

upsetSamples(mae)

Cell metadata

Columns:

nCount_RNA: number of read counts
nFeature_RNA: number of genes with at least one read count
nCount_ATAC: number of ATAC read counts
nFeature_ATAC: number of ATAC peaks with at least one read count
celltype: The cell types have been annotated by the 10x Genomics R&D team using gene markers. They provide a rough characterisation of the cell type diversity, but keep in mind that they are not ground truth labels.
broad_celltype: Lymphoid or Myeloid origin

The cells have not been QC-ed, choosing a minimum number of genes/peaks per cell depends is left to you! In addition, there are further quality control criteria that you may want to apply, including mitochondrial coverage, fraction of reads overlapping ENCODE Blacklisted regions, Transcription start site enrichment, etc. See suggestions below for software that can perform a semi-automated quality control pipeline

head(colData(mae))

RNA expression

The RNA expression consists of 36,549 genes and 10,032 cells, stored using the dgCMatrix sparse matrix format

dim(experiments(mae)[["rna"]])

names(experiments(mae))

Let's do some standard dimensionality reduction plot:

sce.rna <- experiments(mae)[["rna"]]

# Normalisation
sce.rna <- logNormCounts(sce.rna)

# Feature selection
decomp <- modelGeneVar(sce.rna)
hvgs <- rownames(decomp)[decomp$mean>0.01 & decomp$p.value <= 0.05]
sce.rna <- sce.rna[hvgs,]

# PCA
sce.rna <- runPCA(sce.rna, ncomponents = 25)

# UMAP
set.seed(42)
sce.rna <- runUMAP(sce.rna, dimred="PCA", n_neighbors = 25, min_dist = 0.3)
plotUMAP(sce.rna, colour_by="celltype", point_size=0.5, point_alpha=1)

Chromatin Accessibility

The ATAC expression consists of 108,344 peaks and 10,032 cells:

dim(experiments(mae)[["atac"]])

Let's do some standard dimensionality reduction plot. Note that scATAC-seq data is sparser than scRNA-seq, almost binary. The log normalisation + PCA approach that scater implements for scRNA-seq is not a good strategy for scATAC-seq data. Topic modelling or TFIDF+SVD are a better strategy. Please see the package recommendations below.

sce.atac <- experiments(mae)[["atac"]]

# Normalisation
sce.atac <- logNormCounts(sce.atac)

# Feature selection
decomp <- modelGeneVar(sce.atac)
hvgs <- rownames(decomp)[decomp$mean>0.25]
sce.atac <- sce.atac[hvgs,]

# PCA
sce.atac <- runPCA(sce.atac, ncomponents = 25)

# UMAP
set.seed(42)
sce.atac <- runUMAP(sce.atac, dimred="PCA", n_neighbors = 25, min_dist = 0.3)
plotUMAP(sce.atac, colour_by="celltype", point_size=0.5, point_alpha=1)

Suggested software for the downstream analysis

These are my personal recommendations of R-based analysis software:

RNA expression: scater, scran
ATAC accessibility: archR, snapATAC, cisTopic, Signac, chromVar, Cicero
Integrative analysis: MOFA+, Seurat. Note that both methods have released vignettes in their website where they analysed this same data set.