LRcell tutorial

Package version: 0.99.5

Introduction

The goal of LRcell is to identify specific sub-cell types that drives the changes observed in a bulk RNA-seq differential gene expression experiment. To achieve this, LRcell utilizes sets of cell marker genes acquired from single-cell RNA-sequencing (scRNA-seq) as indicators for various cell types in the tissue of interest. Next, for each cell type, using its marker genes as indicators, we apply Logistic Regression on the complete set of genes with differential expression p-values to calculate a cell-type significance p-value. Finally, these p-values are compared to predict which one(s) are likely to be responsible for the differential gene expression pattern observed in the bulk RNA-seq experiments. LRcell is inspired by the LRpath algorithm developed by Sartor et al., originally designed for pathway/gene set enrichment analysis. LRcell contains three major components: LRcell analysis, plot generation and marker gene selection. All modules in this package are written in R.

Pre-loaded marker genes

LRcell provides marker genes in the Prefrontal Cortex (pFC) human brain region, human PBMC and nine mouse brain regions (Frontal Cortex, Cerebellum, Globus Pallidus, Hippocampus, Entopeduncular, Posterior Cortex, Striatum, Substantia Nigra and Thalamus). The human brain dataset describes single-cell gene expression profiles of control samples in Major Depressive Disorder (MDD) disease studies. The mouse brain dataset comes from a whole mouse brain analysis and the data is also available at DropViz.

Workflow

Installation

This is a R Bioconductor package and it can be installed by using BiocManager.

if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager") ## this will install the BiocManager package
BiocManager::install("LRcell")

To check whether LRcell package is successfully installed:

library(LRcell)

LRcell usage

Once we have LRcell package loaded, we can start using it to analyze the transcriptional engagement of cell types or clusters. LRcell takes both single-cell marker genes list and p-values of bulk DE genes as input to calculate the enrichment of cell-type specific marker genes in the ranked DE genes.

As mentioned above, LRcell provides single-cell marker genes list in 1 human brain region (Prefrontal Cortex), 1 human PBMC and 9 mouse brain regions (Frontal Cortex, Cerebellum, Globus Pallidus, Hippocampus, Entopeduncular, Posterior Cortex, Striatum, Substantia Nigra and Thalamus).

• The human brain data comes from control samples in Major Depressive Disorder studies.
• The human PBMC data comes from volunteers in HIV vaccine trial at time point of immediately before.
• The mouse data comes from the whole brain single-cell RNA-seq experiments. Another resource for this dataset is DropViz.

The data is stored in another Bioconductor ExperimentHub package named LRcellTypeMarkers. Users can access the data through ExperimentHub by:

## for installing ExperimentHub
BiocManager::install("ExperimentHub")

## query data
library(ExperimentHub)
eh <- ExperimentHub::ExperimentHub()
eh <- AnnotationHub::query(eh, "LRcellTypeMarkers")
eh  ## this will list out EH number to access the calculated gene enrichment scores

## get mouse brain Frontal Cortex enriched genes
enriched.g <- eh[["EH4548"]]
marker.g <- get_markergenes(enriched_genes, method="LR", topn=100)

Users are also encouraged to process a read count matrix with cell annotation information into a gene enrichment scores matrix.

enriched.g <- LRcell_gene_enriched_scores(expr, annot, power=1, parallel=TRUE, n.cores=4)

Here, expr is a read count matrix with rows as genes and columns as cells. annot is a named-vector with names as cell names (which is in accordance with the column names of expr) and values as annotated cell types. power is a hyper-parameter controlling how much penalty for the proportion of cells expressing certain gene. parallel and n.cores are two parameters for executing function in parallel to accelerate the calculation.

Directly indicate species and brain region in LRcell

Compared to processing data yourself, a much easier way is to indicate species and brain region or tissue. In this way, marker genes are extracted from ExperimentHub accordingly. For example, we can use mouse Frontal Cortex marker genes to do LRcell analysis on the example bulk experiment. (The example contains 23, 420 genes along with p-values calculated from DESeq2. Data is processed from a mouse Alzheimer's disease model (GEO: GSE90693), which is 6 months after treatment in Frontal Cortex brain region.)

Here, we take method as Linear Regression as example:

data("example_gene_pvals")
res <- LRcell(gene.p = example_gene_pvals,
              marker.g = NULL,
              species = "mouse",
              region = "FC",
              method = "LiR")
FC_res <- res$FC
# exclude leading genes for a better view
sub_FC_res <- subset(FC_res, select=-lead_genes)
head(sub_FC_res[order(sub_FC_res$p.value), ])

The top 6 significant lines of the result:

ID	genes_num	coef	p.value	FDR	cell_type
FC_8-2.Astrocytes	100	0.019701817	4.761701e-121	3.856978e-119	Astrocytes
FC_11-4.unknown	90	0.089774550	1.327954e-93	5.378213e-92	unknown
FC_11-1.Microglia	98	0.056507809	2.718086e-91	7.338832e-90	Microglia
FC_9-1.Oligodendrocytes	100	0.008969389	6.032076e-40	1.221495e-38	Oligodendrocytes
FC_9-3.Oligodendrocytes	98	0.006969152	1.660017e-35	2.689227e-34	Oligodendrocytes
FC_9-4.Oligodendrocytes	97	0.006447281	6.246377e-31	8.432608e-30	Oligodendrocytes

You can also try plotting out the result:

g <- plot_manhattan_enrich(FC_res, sig.cutoff = .05, label.topn = 5) ## a ggplot2 object is returned
g

Calculate gene enrichment scores from expression dataframe

The gene enrichment scores calculation is based on algorithm in this science paper which takes both enrichment of genes in certain cell types and fraction of cells expressing the gene into consideration.

LRcell_gene_enriched_scores() function takes the gene-by-cell expression matrix and a cell-type annotation as input, which means a clustering algorithm and/or cell-type annotations should be done first. The columns of the expression matrix should match with cell names in the annotation vector.

Below is a naive example on how to utilize this function to select marker genes.

Example gene-by-cell expression matrix:

	cell1	cell2	cell3	cell4	cell5	cell6	cell7	cell8	cell9	cell10
gene1	3	0	2	8	10	6	1	0	0	2
gene2	7	5	8	1	0	5	2	3	2	1
gene3	8	10	6	7	8	9	5	8	6	8

Example cell annotation

cell ID	cell1	cell2	cell3	cell4	cell5	cell6	cell7	cell8	cell9	cell10
Annotation	celltype1	celltype1	celltype1	celltype2	celltype2	celltype2	celltype3	celltype3	celltype3	celltype3

This toy example contains 3 genes and 10 cells. As you can tell from the matrix, gene1 is a marker gene of celltype2; gene2 is a marker gene of celltype1; gene3 is a house keeping gene.

# generate a simulated gene*cell read counts matrix
n.row <- 3; n.col <- 10
sim.expr <- matrix(0, nrow=n.row, ncol=n.col)
rownames(sim.expr) <- paste0("gene", 1:n.row)
colnames(sim.expr) <- paste0("cell", 1:n.col)

# generate a simulated annotation for cells
sim.annot <- c(rep("celltype1", 3), rep("celltype2", 3), rep("celltype3", 4))
names(sim.annot) <- colnames(sim.expr)

sim.expr['gene1', ] <- c(3, 0, 2, 8, 10, 6, 1, 0, 0, 2) # marker gene for celltype2
sim.expr['gene2', ] <- c(7, 5, 8, 1, 0, 5, 2, 3, 2, 1) # marker gene for celltype1
sim.expr['gene3', ] <- c(8, 10, 6, 7, 8, 9, 5, 8, 6, 8) # house keeping

# generating the enrichment score 
enriched_res <- LRcell_gene_enriched_scores(expr = sim.expr,
                            annot = sim.annot, parallel = FALSE)
enriched_res

After using LRcell_gene_enriched_scores() function, we get the enriched scores for each gene in each cell type, which is:

	celltype1	celltype2	celltype3
gene1	0.3472222	2.5	0.1171875
gene2	1.960784	0.3921569	0.5882353
gene3	1.066667	1.066667	0.9

For example, gene2 is a marker gene of celltype1, after calculation, it has the highest enrichment score in celltype1 among all three cell types.

Then we can choose marker genes using top 1 as threshold:

marker_res <- get_markergenes(enriched.g = enriched_res,
                method = "LR", topn=1)

The result becomes:

celltype	marker genes
celltype1	gene2
celltype2	gene1
celltype3	gene3