This article demonstrates the data visualization tools in Chevreul. We’ll introduce included functions, their usage, and resulting plots

First step is to load Chevreul package and all other packages required

library(chevreul)
library(scater)
library(scran)
library(tidyverse)
library(ggraph)
library(patchwork)

The different plotting functions within Chevreul allows for visualization of data, these plots can be customized for interactive or non-interactive display.

1 Plot expression

Expression of a feature (genes or transcripts) can be plotted on a given embedding resulting in an interactive feature plot.

When plotting only one feature, output is identical to SingleCellExperiment::FeaturePlot

plot_feature(chevreul_sce,
    embedding = "UMAP",
    features = "NRL", return_plotly = FALSE
)

An interactive output plot can be generated by specifying return_plotly = TRUE which uses ggplotly allowing identification of individual cells for further investigation.

1.1 Plot read count or other QC measurements

The plot_readcount function displays a histogram of cell read counts colored according to a categorical variable using the argument color.by. Here we can see that read counts for this dataset are not distinctly different depending on the method of organoid preparation used (Kuwahara or Zhong)

plot_readcount(chevreul_sce,
    group_by = "nCount_RNA",
    color.by = "Prep.Method"
)

1.2 Plot metadata variable

Make an interactive scatter plot of a metadata variable, where each point in the plot represents a cell whose position on the plot is given by the cell embedding determined by the dimensional reduction technique by default, “UMAP”. The group argument specifies the colData variable by which to group the cells by, by default, “batch”.

plot_var(chevreul_sce,
    group = "gene_snn_res.0.2",
    embedding = "UMAP"
)

This function utilizes a SingleCellExperiment function, DimPlot(), as sub function which produces the dimensional reduction plot. The interactive parameter, return_plotly, in plot_var when set to TRUE will convert the plot into an interactive plot using ggplotly function from R’s plotly package

1.3 Plot cluster marker genes

Marker genes of louvain clusters or additional experimental metadata can be plotted using plot_markers. This allows visualization of n marker features grouped by the metadata of interest. Marker genes are identified using wilcoxon rank-sum test as implemented in presto. In the resulting dot plot the size of the dot corresponds to the percentage of cells expressing the feature in each cluster and the color represents the average expression level of the feature.

plot_markers(chevreul_sce,
    group_by = "gene_snn_res.0.2",
    marker_method = "wilcox"
)

1.4 Violin plot

To visualize the distribution of expression level of a feature in different groups of cells Chevreul draws a violin plot. This function uses SingleCellExperiment’s VInPlot() function as a sub-function to create the violin plot, where the metadata variable, provided to the function through the variable plot_var, is used to group the cells and based on the level of feature expression a violin plot is produced.

plot_violin(chevreul_sce, plot_var = "Kit_ID", features = "NRL")

1.5 Plotting all transcripts

Expression level of all the transcripts present in a given SingleCellExperiment object for a gene of interest. specified by the argument ‘features’. By default the data is superimposed on UMAP embedding.

plot_all_transcripts(chevreul_sce, features = "NRL")

1.6 Plotting transcript composition

plot_transcript_composition() plots the proportion of reads of a given gene map to each transcript. The gene of interest is specified by the argument ‘gene_symbol’.

1.7 Plot Complex heatmap from SingleCellExperiment object

We can plot an annotated complex heat map from the SingleCellExperiment object with a dendogram added to the left side and the top.

top_10_features <- getTopHVGs(chevreul_sce)[1:10]

make_complex_heatmap(chevreul_sce,
    features = top_10_features,
    group.by = "gene_snn_res.0.2"
)


make_complex_heatmap(chevreul_sce,
    features = top_10_features,
    group.by = "gene_snn_res.0.2", col_arrangement = "average"
)


make_complex_heatmap(chevreul_sce,
    features = top_10_features,
    group.by = c("Prep.Method", "gene_snn_res.0.2"),
    col_arrangement = "gene_snn_res.0.2"
)

1.8 Session information

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#>  date     2024-08-11
#>  pandoc   2.7.3 @ /usr/bin/ (via rmarkdown)
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