knitr::opts_chunk$set( collapse = TRUE, comment = "#>" ) library(ggplot2) theme_set(theme_classic())
Reduced dimension plotting is one of the essential tools for the analysis of
single cell data. However, as the number of cells/nuclei in these plots
increases, the usefulness of these plots decreases. Many cells are plotted
on top of each other obscuring information, even when taking advantage of
transparency settings. This package provides binning strategies of cells/nuclei
into hexagon cells. Plotting summarized information of all cells/nuclei in their
respective hexagon cells presents information without obstructions. The
package seemlessly works with the two most common object classes for the storage
of single cell data;
SingleCellExperiment from the
Seurat from the Seurat package. In
this vignette I will be presenting the use of
library(schex) library(dplyr) library(scater) library(Seurat) library(TENxPBMCData)
In order to demonstrate the capabilities of the schex package, I will use the
a dataset of Peripheral Blood Mononuclear Cells (PBMC) freely available from
10x Genomics. There are 2,700 single cells that were sequenced on the
Illumina NextSeq 500. This data is handly available in the
Note that we will then have to convert the
SingleCellExperiment object to a
Seurat object first.
tenx_pbmc3k <- TENxPBMCData(dataset = "pbmc3k") rownames(tenx_pbmc3k) <- uniquifyFeatureNames(rowData(tenx_pbmc3k)$ENSEMBL_ID, rowData(tenx_pbmc3k)$Symbol_TENx) pbmc <- as.Seurat(tenx_pbmc3k, data = NULL)
In the next few sections, I will perform some simple quality control steps outlined in the Seurat vignette. I will then calculate various dimension reductions and cluster the data also outlined in the vignette.
Next a global-scaling normalization method is employed to normalizes the feature expression measurements for each cell.
pbmc <- NormalizeData(pbmc, normalization.method = "LogNormalize", scale.factor = 10000, verbose=FALSE)
Many of the downstream methods are based on only the highly variable genes, hence we require their identification.
pbmc <- FindVariableFeatures(pbmc, selection.method = "vst", nfeatures = 2000, verbose = FALSE)
Prior to dimension reduction the data is scaled.
all.genes <- rownames(pbmc) pbmc <- ScaleData(pbmc, features = all.genes, verbose = FALSE)
First a PCA is applied to the data. Using the PCA you will have to decide on the dimensionality of the data. Here the dimensionality was decided to be 10. Please refer to the original Seurat vignette for methods on how this is assessed.
pbmc <- RunPCA(pbmc, features = VariableFeatures(object = pbmc), verbose = FALSE)
Next a UMAP dimensionality reduction is also run. Since there is a random component in the UMAP, we will set a seed.
set.seed(10) pbmc <- RunUMAP(pbmc, dims = 1:10, verbose=FALSE)
At this stage in the workflow we usually would like to plot aspects of our data in one of the reduced dimension representations. Instead of plotting this in an ordinary fashion, I will demonstrate how schex can provide a better way of plotting this.
First, I will calculate the hexagon cell representation for each cell for
a specified dimension reduction representation. I decide to use
specifies that I divide my x range into 40 bins. Note that this might be a
parameter that you want to play around with depending on the number of cells/
nuclei in your dataset. Generally, for more cells/nuclei,
nbins should be
pbmc <- make_hexbin(pbmc, nbins = 40, dimension_reduction = "UMAP")
First I plot how many cells are in each hexagon cell. This should be
relatively even, otherwise change the
nbins parameter in the previous
Next I colour the hexagon cells by some meta information, such as the median total count in each hexagon cell.
pbmc$nCount_RNA <- colSums(GetAssayData(pbmc, assay="RNA", "data")) plot_hexbin_meta(pbmc, col="nCount_RNA", action="median")
Finally, I will visualize the gene expression of the CD19 gene in the hexagon cell representation.
gene_id <-"CD19" schex::plot_hexbin_feature(pbmc, type="scale.data", feature=gene_id, action="mean", xlab="UMAP1", ylab="UMAP2", title=paste0("Mean of ", gene_id))
schex packages renders ordinary
ggplot objects and thus these can be
treated and manipulated using the
ggplot grammar. For example the non-data
components of the plots can be changed using the function
gene_id <-"CD19" gg <- schex::plot_hexbin_feature(pbmc, type="scale.data", feature=gene_id, action="mean", xlab="UMAP1", ylab="UMAP2", title=paste0("Mean of ", gene_id)) gg + theme_void()
The fact that
ggplot objects can also be used to save these
plots. Simply use
ggsave in order to save any created plot.
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