Multi-subject scRNA-seq Analysis

In aggregateBioVar: Differential Gene Expression Analysis for Multi-subject scRNA-seq

knitr::opts_chunk$set(
    collapse = TRUE,
    comment = "#>"
)
knitr::opts_knit$set(
    eval.after = "fig.cap"
)

# For analysis of scRNAseq data
library(aggregateBioVar)
library(SummarizedExperiment, quietly = TRUE)
library(SingleCellExperiment, quietly = TRUE)
library(DESeq2, quietly = TRUE)

# For data transformation and visualization
library(magrittr, quietly = TRUE)
library(dplyr, quietly = TRUE)
library(ggplot2, quietly = TRUE)
library(cowplot, quietly = TRUE)
library(ggtext, quietly = TRUE)

link_sce <-
    paste0(
        "https://bioconductor.org/packages/release/bioc/",
        "vignettes/SingleCellExperiment/inst/doc/intro.html"
    )
link_se <-
    paste0(
        "https://bioconductor.org/packages/release/bioc/",
        "vignettes/SummarizedExperiment/inst/doc/SummarizedExperiment.html"
    )
link_ncbi <-
    paste0(
        "https://www.ncbi.nlm.nih.gov/Taxonomy/Browser/",
        "wwwtax.cgi?mode=Info&id=9823"
    )
link_DESeq2 <-
    paste0(
        "https://bioconductor.org/packages/devel/bioc/",
        "vignettes/DESeq2/inst/doc/DESeq2.html"
    )

cap_metadata <-
    SummarizedExperiment::colData(x = small_airway) %>% tibble::as_tibble() %>%
    dplyr::select("orig.ident", "Genotype") %>%
    dplyr::distinct() %>% dplyr::arrange(.data$orig.ident)

# Figure 1
cap_pheatmanp_cell <-
    paste0(
        "Gene-by-cell Count Matrix. ",
        "Heatmap of *secretory cell* gene expression with log~2~ counts ",
        "per million cells. Includes ",
        length(grep("Secretory cell", small_airway$celltype)),
        " cells from ", length(unique(small_airway$orig.ident)), " subjects",
        " with genotypes `WT` (n=", length(grep("WT", cap_metadata$Genotype)),
        ", cells=",
        length(
            intersect(
                grep("WT", small_airway$Genotype),
                grep("Secretory cell", small_airway$celltype)
            )
        ),
        ") and `CFTRKO` (n=", length(grep("CFTRKO", cap_metadata$Genotype)),
        ", cells=",
        length(
            intersect(
                grep("CFTRKO", small_airway$Genotype),
                grep("Secretory cell", small_airway$celltype)
            )
        ),
        ")."
    )

# Figure 2
cap_pheatmanp_subj <-
    paste0(
        "Gene-by-subject Count Matrix. ",
        "Heatmap of gene counts aggregated by ",
        "subject with `aggregateBioVar()`.",
        "The `SummarizedExperiment` object with the *Secretory cells* subset ",
        "contains gene counts summed by subject. Aggregate gene-by-subject ",
        "counts are used as input for bulk RNA-seq tools."
    )

# Figure 3
cap_volcano <-
    paste0(
        "Differential expression analysis of scRNA-seq data. ",
        "Comparison of differential expression in ",
        "secretory cells from small airway epithelium ",
        "without aggregation (A) and after aggregation of gene counts ",
        "by subject (biological replicates; B). ",
        "Genes with an absolute log~2~ fold change greater than 1 and an ",
        "adjusted P value less than 0.05 are highlighted in red. Aggregation ",
        "of counts by subject reduced the number of differentially expressed ",
        "genes to CD36 and CFTR for the secretory cell subset."
    )

# Figure 4
cap_counts <-
    paste0(
        "Normalized within-subject gene counts. ",
        "Gene counts aggregated by subject for significantly differentially ",
        "expressed genes from the secretory cell subset."
    )

Introduction

Single cell RNA sequencing (scRNA-seq) studies allow gene expression quantification at the level of individual cells, and these studies introduce multiple layers of biological complexity. These include variations in gene expression between cell states within a sample (e.g., T cells versus macrophages), between samples within a population (e.g., biological or technical replicates), and between populations (e.g., healthy versus diseased individuals). Because many early scRNA-seq studies involved analysis of only a single sample, many bioinformatics tools operate on the first layer, comparing gene expression between cells within a sample. This software is aimed at organizing scRNA-seq data to permit analysis in the latter two layers, comparing gene expression between samples and between populations. An example is given with an implementation of differential gene expression analysis between populations. From scRNA-seq data stored as a SingleCellExperiment [@R-SingleCellExperiment] object with pre-defined cell states, aggregateBioVar() stratifies data as a list of SummarizedExperiment [@R-SummarizedExperiment] objects, a standard Bioconductor data structure for downstream analysis of RNA-seq data.

Case Study: Small Airway Epithelium in Cystic Fibrosis

To illustrate the utility of biological replication for scRNA-seq sequencing experiments, consider a set of single cell data from porcine small airway epithelium. In this study, small airway (< 2 mm) tissue samples were collected from newborn pigs (Sus scrofa) to investigate gene expression patterns and cellular composition in a cystic fibrosis phenotype. Single cell sequencing samples were prepared using a 10X Genomics Chromium controller and sequenced on an Illumina HiSeq4000. Data obtained from seven individuals include both non-CF (CFTR+/+; genotype WT; n=4) and CFTR-knockout subjects expressing a cystic fibrosis phenotype (CFTR-/-; genotype CFTRKO; n=3). Cell types were determined following a standard scRNA-seq pipeline using Seurat [@Seurat2019], including cell count normalization, scaling, determination of highly variable genes, dimension reduction via principal components analysis, and shared nearest neighbor clustering. Both unsupervised marker detection (via Seurat::FindMarkers()) and a list of known marker genes were used to annotate cell types. The full data set has been uploaded to the Gene Expression Omnibus as accession number GSE150211.

The small_airway Dataset

A subset of r nrow(small_airway) genes from r ncol(small_airway) cells assigned as secretory, endothelial, and immune cell types are available in the small_airway data set. The data are formatted as a SingleCellExperiment class S4 object [@SingleCellExperiment2020], an extension of the RangedSummarizedExperiment class from the SummarizedExperiment package.

small_airway

The primary data in SingleCellExperiment objects are stored in the assays slot. Here, a single assay counts contains gene counts from the single cell sequencing data. Each column of the assay count matrix represents a cell and each row a feature (e.g., gene). Assay slot data can be obtained by SummarizedExperiment::assay(), indicating theSingleCellExperiment object and name of the assay slot (here, "counts"). In the special case of the assay being named "counts", the data can be accessed with SingleCellExperiment::counts().

assays(small_airway)

# Dimensions of gene-by-cell count matrix
dim(counts(small_airway))

# Access dgCMatrix with gene counts
counts(small_airway)[1:5, 1:30]

sbj_cf <-
    grep(pattern = "CF", x = unique(small_airway$orig.ident), value = TRUE)
sbj_wt <-
    grep(pattern = "WT", x = unique(small_airway$orig.ident), value = TRUE)

Cell Metadata

SingleCellExperiment objects may also include column metadata with additional information annotating individual cells. Here, the metadata variable orig.ident identifies the biological sample of the cell while celltype indicates the assigned cellular identity. Of the r length(unique(small_airway$orig.ident)) individual subjects, 3 are CF (r sbj_cf) and 4 are non-CF (r sbj_wt). Genotype indicates the sample genotype, one of WT or CFTRKO for non-CF and CF subjects, respectively. Column metadata from SingleCellExperiment objects can be accessed with the $ operator, where the length of a metadata column variable is equal to the number of columns (i.e., cells) in the feature count matrix from the assays slot.

# Subject values
table(small_airway$orig.ident)

# Cell type values
table(small_airway$celltype)

# Subject genotype
table(small_airway$Genotype)

The experiment metadata are included as an S4 DataFrame object. To access the full column metadata from SingleCellExperiment objects, use colData() from the SummarizedExperiment package. Here, metadata include r ncol(colData(small_airway)) variables for each of r nrow(colData(small_airway)) cells. In addition to the biological sample identifier, cell type, and genotype, metadata include total unique molecular identifiers (UMIs) and number of detected features.

colData(small_airway)

Aggregating Gene Counts

The main functionality of this package involves two generalizable operations:

1. Aggregate within-subject gene counts
2. Summarize metadata to retain inter-subject variation

A wrapper function aggregateBioVar() abstracts away these operations and applies them on a by-cell type basis. For input, a SingleCellExperiment object containing gene counts should contain metadata variables for the subject by which to aggregate cells (e.g., biological sample) and the assigned cell types.

Gene-by-subject Count Matrix

The first operation involves summing all gene counts by subject. For each gene, counts from all cells within each subject are combined. A gene-by-cell count matrix is converted into a gene-by-subject count matrix.

countsBySubject(scExp = small_airway, subjectVar = "orig.ident")

Subject Metadata

The second operation removes metadata variables with intrasubject variation. This effectively retains inter-subject metadata and eliminates variables with intrasubject (i.e., intercellular) variation (e.g., feature or gene counts by cell). This summarized metadata is used for modeling a differential expression design matrix.

subjectMetaData(scExp = small_airway, subjectVar = "orig.ident")

Return SummarizedExperiment

Both the gene count aggregation and metadata collation steps are combined in summarizedCounts(). This function returns a SummarizedExperiment object with the gene-by-subject count matrix in the assays slot, and the summarized inter-subject metadata as colData. Notice the column names now correspond to the subject level, replacing the cellular barcodes in the SingleCellExperiment following aggregation of gene counts from within-subject cells.

summarizedCounts(scExp = small_airway, subjectVar = "orig.ident")

aggregateBioVar()

These operations are applied to each cell type subset with aggregateBioVar(). The full SingleCellExperiment object is subset by cell type (e.g., secretory, endothelial, and immune cell), the gene-by-subject aggregate count matrix and collated metadata are tabulated, and a SummarizedExperiment object for that cell type is constructed. A list of SummarizedExperiment objects output by summarizedCounts() is the returned to the user. The first element contains the aggregate SummarizedExperiment across all cells, and subsequent list elements correspond to the cell type indicated by the metadata variable cellVar:

aggregateBioVar(scExp = small_airway,
                subjectVar = "orig.ident", cellVar = "celltype")

Application to Differential Gene Expression (DGE)

In this case, we want to test for differential expression between non-CF and CF pigs in the Secretory cell subset. To do so, aggregateBioVar() is run on the SingleCellExperiment object by indicated the metadata variables representing the subject-level (subjectVar) and assigned cell type (cellVar). If multiple assays are included in the input scExp object, the first assay slot is used.

# Perform aggregation of counts and metadata by subject and cell type.
aggregate_counts <-
    aggregateBioVar(
        scExp = small_airway,
        subjectVar = "orig.ident", cellVar = "celltype"
    )

Exploratory Data Analysis

To visualize the gene-by-subject count aggregation, consider a function to calculate log₂ counts per million cells and display a heatmap of normalized expression using pheatmap [@R-pheatmap]. RColorBrewer [@R-RColorBrewer] and viridis [@R-viridis] are used to generate discrete and continuous color scales, respectively.

#' Single-cell Counts `pheatmap`
#'
#' @param sumExp `SummarizedExperiment` or `SingleCellExperiment` object
#'   with individual cell or aggregate counts by-subject.
#' @param logSample Subset of log2 values to include for clustering.
#' @param ... Forwarding arguments to pheatmap
#' @inheritParams aggregateBioVar
#'
scPHeatmap <- function(sumExp, subjectVar, gtVar, logSample = 1:100, ...) {
    orderSumExp <- sumExp[, order(sumExp[[subjectVar]])]
    sumExpCounts <- as.matrix(
        SummarizedExperiment::assay(orderSumExp, "counts")
    )
    logcpm <- log2(
        1e6*t(t(sumExpCounts) / colSums(sumExpCounts)) + 1
    )
    annotations <- data.frame(
        Genotype = orderSumExp[[gtVar]],
        Subject = orderSumExp[[subjectVar]]
    )
    rownames(annotations) <- colnames(orderSumExp)

    singleCellpHeatmap <- pheatmap::pheatmap(
        mat = logcpm[logSample, ], annotation_col = annotations,
        cluster_cols = FALSE, show_rownames = FALSE, show_colnames = FALSE,
        scale = "none", ...
    )
    return(singleCellpHeatmap)
}

Without aggregation, bulk RNA-seq methods for differential expression analysis would be applied at the cell level (here, secretory cells; Figure \@ref(fig:pheatmapCells)).

# Subset `SingleCellExperiment` secretory cells.
sumExp <- small_airway[, small_airway$celltype == "Secretory cell"]

# List of annotation color specifications for pheatmap.
ann_colors <- list(
    Genotype = c(CFTRKO = "red", WT = "black"),
    Subject = c(RColorBrewer::brewer.pal(7, "Accent"))
)
ann_names <- unique(sumExp[["orig.ident"]])
names(ann_colors$Subject) <- ann_names[order(ann_names)]

# Heatmap of log2 expression across all cells.
scPHeatmap(
    sumExp = sumExp, logSample = 1:100,
    subjectVar = "orig.ident", gtVar = "Genotype",
    color = viridis::viridis(75), annotation_colors = ann_colors,
    treeheight_row = 0, treeheight_col = 0
)

---

Summation of gene counts across all cells creates a "pseudo-bulk" data set on which a subject-level test of differential expression is applied (Figure \@ref(fig:pheatmapSubject)).

# List of `SummarizedExperiment` objects with aggregate subject counts.
scExp <-
    aggregateBioVar(
        scExp = small_airway,
        subjectVar = "orig.ident", cellVar = "celltype"
    )

# Heatmap of log2 expression from aggregate gene-by-subject count matrix.
scPHeatmap(
    sumExp = aggregate_counts$`Secretory cell`, logSample = 1:100,
    subjectVar = "orig.ident", gtVar = "Genotype",
    color = viridis::viridis(75), annotation_colors = ann_colors,
    treeheight_row = 0, treeheight_col = 0
)

DGE with DESeq2

To run DESeq2 [@DESeq2], a DESeqDataSet object can be constructed using DESeqDataSetFromMatrix(). Here, the aggregate counts and subject metadata from the secretory cell subset are modeled by the variable Genotype. Differential expression analysis is performed with DESeq and a results table is extracted by results() to obtain log~2~ fold changes with p-values and adjusted p-values.

subj_dds_dataset <-
    DESeqDataSetFromMatrix(
        countData = assay(aggregate_counts$`Secretory cell`, "counts"),
        colData = colData(aggregate_counts$`Secretory cell`),
        design = ~ Genotype
    )

subj_dds <- DESeq(subj_dds_dataset)

subj_dds_results <-
    results(subj_dds, contrast = c("Genotype", "WT", "CFTRKO"))

For comparison of differential expression with and without aggregation of gene-by-subject counts, a subset of all secretory cells is used to construct a DESeqDataSet and analysis of differential expression is repeated.

cells_secretory <-
    small_airway[, which(
        as.character(small_airway$celltype) == "Secretory cell")]
cells_secretory$Genotype <- as.factor(cells_secretory$Genotype)

cell_dds_dataset <-
    DESeqDataSetFromMatrix(
        countData = assay(cells_secretory, "counts"),
        colData = colData(cells_secretory),
        design = ~ Genotype
    )

cell_dds <- DESeq(cell_dds_dataset)

cell_dds_results <-
    results(cell_dds, contrast = c("Genotype", "WT", "CFTRKO"))

Add a new variable with log~10~-transformed adjusted P-values.

subj_dds_transf <- as.data.frame(subj_dds_results) %>%
    bind_cols(feature = rownames(subj_dds_results)) %>%
    mutate(log_padj = - log(.data$padj, base = 10))

cell_dds_transf <- as.data.frame(cell_dds_results) %>%
    bind_cols(feature = rownames(cell_dds_results)) %>%
    mutate(log_padj = - log(.data$padj, base = 10))

Results

dge_cells <-
    filter(
        .data = cell_dds_transf,
        abs(.data$log2FoldChange) > 1, .data$padj < 0.05
    )
dge_subj <-
    filter(
        .data = subj_dds_transf,
        abs(.data$log2FoldChange) > 1, .data$padj < 0.05
    )

DGE is summarized by volcano plot ggplot [@R-ggplot2] to show cell-level (Figure \@ref(fig:plotVolcano)A) and subject-level tests (Figure \@ref(fig:plotVolcano)B). Aggregation of gene counts by subject reduced the number of genes with both an adjusted p-value < 0.05 and an absolute log~2~ fold change > 1 from r nrow(dge_cells) genes to r nrow(dge_subj) (Figure \@ref(fig:plotVolcano)).

# Function to add theme for ggplots of DESeq2 results.
deseq_themes <- function() {
    list(
        theme_classic(),
        lims(x = c(-4, 5), y = c(0, 80)),
        labs(
            x = "log<sub>2</sub> (fold change)",
            y = "-log<sub>10</sub> (p<sub>adj</sub>)"
        ),
        ggplot2::theme(
            axis.title.x = ggtext::element_markdown(),
            axis.title.y = ggtext::element_markdown())
    )
}

# Build ggplots to visualize subject-level differential expression in scRNA-seq
ggplot_full <- ggplot(data = cell_dds_transf) +
    geom_point(aes(x = log2FoldChange, y = log_padj), na.rm = TRUE) +
    geom_point(
        data = filter(
            .data = cell_dds_transf,
            abs(.data$log2FoldChange) > 1, .data$padj < 0.05
        ),
        aes(x = log2FoldChange, y = log_padj), color = "red"
    ) +
    deseq_themes()

ggplot_subj <- ggplot(data = subj_dds_transf) +
    geom_point(aes(x = log2FoldChange, y = log_padj), na.rm = TRUE) +
    geom_point(
        data = filter(
            .data = subj_dds_transf,
            abs(.data$log2FoldChange) > 1, .data$padj < 0.05
        ),
        aes(x = log2FoldChange, y = log_padj), color = "red"
    ) +
    geom_label(
        data = filter(
            .data = subj_dds_transf,
            abs(.data$log2FoldChange) > 1, .data$padj < 0.05
        ),
        aes(x = log2FoldChange + 0.5, y = log_padj + 5, label = feature)
    ) +
    deseq_themes()

cowplot::plot_grid(ggplot_full, ggplot_subj, ncol = 2, labels = c("A", "B"))

---

From the significantly differentially expressed genes CFTR and CD36, the aggregate counts by subject are plotted in Figure \@ref(fig:plotGeneCounts).

# Extract counts subset by gene to plot normalized counts.
ggplot_counts <- function(dds_obj, gene) {
    norm_counts <-
        counts(dds_obj, normalized = TRUE)[grepl(gene, rownames(dds_obj)), ]
    sc_counts <-
        data.frame(
            norm_count = norm_counts,
            subject = colData(dds_obj)[["orig.ident"]],
            genotype = factor(
                colData(dds_obj)[["Genotype"]],
                levels = c("WT", "CFTRKO")
            )
        )

    count_ggplot <- ggplot(data = sc_counts) +
        geom_jitter(
            aes(x = genotype, y = norm_count, color = genotype),
            height = 0, width = 0.05
        ) +
        scale_color_manual(
            "Genotype", values = c("WT" = "blue", "CFTRKO" = "red")
        ) +
        lims(x = c("WT", "CFTRKO"), y = c(0, 350)) +
        labs(x = "Genotype", y = "Normalized Counts") +
        ggtitle(label = gene) +
        theme_classic()
    return(count_ggplot)
}

cowplot::plot_grid(
    ggplot_counts(dds_obj = subj_dds, gene = "CFTR") +
        theme(legend.position = "FALSE"),
    ggplot_counts(dds_obj = subj_dds, gene = "CD36") +
        theme(legend.position = "FALSE"),
    cowplot::get_legend(
        plot = ggplot_counts(dds_obj = subj_dds, gene = "CD36")
    ),
    ncol = 3, rel_widths = c(4, 4, 1)
)

References

Session Info

sessionInfo()

aggregateBioVar documentation built on Nov. 8, 2020, 7:46 p.m.