---
title: "Overview of the tidySingleCellExperiment package"
author: "Stefano Mangiola"
date: "`r Sys.Date()`"
package: tidySingleCellExperiment
output:
BiocStyle::html_document:
toc_float: true
bibliography: tidySingleCellExperiment.bib
vignette: >
%\VignetteEngine{knitr::knitr}
%\VignetteIndexEntry{Overview of the tidySingleCellExperiment package}
%\usepackage[UTF-8]{inputenc}
---
```{r, echo=FALSE, include=FALSE}
library(knitr)
knitr::opts_chunk$set(
cache=TRUE, warning=FALSE,
message=FALSE, cache.lazy=FALSE
)
```
# Introduction
tidySingleCellExperiment provides a bridge between Bioconductor single-cell packages [@amezquita2019orchestrating] and the tidyverse [@wickham2019welcome]. It creates an invisible layer that enables viewing the
Bioconductor *SingleCellExperiment* object as a tidyverse tibble, and provides SingleCellExperiment-compatible *dplyr*, *tidyr*, *ggplot* and *plotly* functions. This allows users to get the best of both Bioconductor and tidyverse worlds.
## Functions/utilities available
SingleCellExperiment-compatible Functions | Description
------------ | -------------
`all` | After all `tidySingleCellExperiment` is a SingleCellExperiment object, just better
tidyverse Packages | Description
------------ | -------------
`dplyr` | All `dplyr` tibble functions (e.g. `tidySingleCellExperiment::select`)
`tidyr` | All `tidyr` tibble functions (e.g. `tidySingleCellExperiment::pivot_longer`)
`ggplot2` | `ggplot` (`tidySingleCellExperiment::ggplot`)
`plotly` | `plot_ly` (`tidySingleCellExperiment::plot_ly`)
Utilities | Description
------------ | -------------
`tidy` | Add `tidySingleCellExperiment` invisible layer over a SingleCellExperiment object
`as_tibble` | Convert cell-wise information to a `tbl_df`
`join_features` | Add feature-wise information, returns a `tbl_df`
## Installation
```{r, eval=FALSE}
if (!requireNamespace("BiocManager", quietly=TRUE))
install.packages("BiocManager")
BiocManager::install("tidySingleCellExperiment")
```
Load libraries used in this vignette.
```{r message=FALSE}
# Bioconductor single-cell packages
library(scater)
library(scran)
library(SingleR)
library(SingleCellSignalR)
# Tidyverse-compatible packages
library(ggplot2)
library(purrr)
library(tidyHeatmap)
# Both
library(tidySingleCellExperiment)
```
# Create `tidySingleCellExperiment`, the best of both worlds!
This is a *SingleCellExperiment* object but it is evaluated as a tibble. So it is compatible both with SingleCellExperiment and tidyverse.
```{r}
pbmc_small_tidy <- tidySingleCellExperiment::pbmc_small
```
**It looks like a tibble**
```{r}
pbmc_small_tidy
```
**But it is a SingleCellExperiment object after all**
```{r}
assay(pbmc_small_tidy)
```
# Annotation polishing
We may have a column that contains the directory each run was taken from, such as the "file" column in `pbmc_small_tidy`.
```{r}
pbmc_small_tidy$file[1:5]
```
We may want to extract the run/sample name out of it into a separate column. Tidyverse `extract` can be used to convert a character column into multiple columns using regular expression groups.
```{r}
# Create sample column
pbmc_small_polished <-
pbmc_small_tidy %>%
extract(file, "sample", "../data/([a-z0-9]+)/outs.+", remove=FALSE)
# Reorder to have sample column up front
pbmc_small_polished %>%
select(sample, everything())
```
# Preliminary plots
Set colours and theme for plots.
```{r}
# Use colourblind-friendly colours
friendly_cols <- dittoSeq::dittoColors()
# Set theme
custom_theme <-
list(
scale_fill_manual(values=friendly_cols),
scale_color_manual(values=friendly_cols),
theme_bw() +
theme(
panel.border=element_blank(),
axis.line=element_line(),
panel.grid.major=element_line(size=0.2),
panel.grid.minor=element_line(size=0.1),
text=element_text(size=12),
legend.position="bottom",
aspect.ratio=1,
strip.background=element_blank(),
axis.title.x=element_text(margin=margin(t=10, r=10, b=10, l=10)),
axis.title.y=element_text(margin=margin(t=10, r=10, b=10, l=10))
)
)
```
We can treat `pbmc_small_polished` as a tibble for plotting.
Here we plot number of features per cell.
```{r plot1}
pbmc_small_polished %>%
tidySingleCellExperiment::ggplot(aes(nFeature_RNA, fill=groups)) +
geom_histogram() +
custom_theme
```
Here we plot total features per cell.
```{r plot2}
pbmc_small_polished %>%
tidySingleCellExperiment::ggplot(aes(groups, nCount_RNA, fill=groups)) +
geom_boxplot(outlier.shape=NA) +
geom_jitter(width=0.1) +
custom_theme
```
Here we plot abundance of two features for each group.
```{r}
pbmc_small_polished %>%
join_features(features=c("HLA-DRA", "LYZ")) %>%
ggplot(aes(groups, abundance_counts + 1, fill=groups)) +
geom_boxplot(outlier.shape=NA) +
geom_jitter(aes(size=nCount_RNA), alpha=0.5, width=0.2) +
scale_y_log10() +
custom_theme
```
# Preprocess the dataset
We can also treat `pbmc_small_polished` as a *SingleCellExperiment* object and proceed with data processing with Bioconductor packages, such as *scran* [@lun2016pooling] and *scater* [@mccarthy2017scater].
```{r preprocess}
# Identify variable genes with scran
variable_genes <-
pbmc_small_polished %>%
modelGeneVar() %>%
getTopHVGs(prop=0.1)
# Perform PCA with scater
pbmc_small_pca <-
pbmc_small_polished %>%
runPCA(subset_row=variable_genes)
pbmc_small_pca
```
If a tidyverse-compatible package is not included in the tidySingleCellExperiment collection, we can use `as_tibble` to permanently convert `tidySingleCellExperiment` into a tibble.
```{r pc_plot}
# Create pairs plot with GGally
pbmc_small_pca %>%
as_tibble() %>%
select(contains("PC"), everything()) %>%
GGally::ggpairs(columns=1:5, ggplot2::aes(colour=groups)) +
custom_theme
```
# Identify clusters
We can proceed with cluster identification with *scran*.
```{r cluster}
pbmc_small_cluster <- pbmc_small_pca
# Assign clusters to the 'colLabels' of the SingleCellExperiment object
colLabels(pbmc_small_cluster) <-
pbmc_small_pca %>%
buildSNNGraph(use.dimred="PCA") %>%
igraph::cluster_walktrap() %$%
membership %>%
as.factor()
# Reorder columns
pbmc_small_cluster %>% select(label, everything())
```
And interrogate the output as if it was a regular tibble.
```{r cluster count}
# Count number of cells for each cluster per group
pbmc_small_cluster %>%
tidySingleCellExperiment::count(groups, label)
```
We can identify and visualise cluster markers combining SingleCellExperiment, tidyverse functions and tidyHeatmap [@mangiola2020tidyheatmap]
```{r}
# Identify top 10 markers per cluster
marker_genes <-
pbmc_small_cluster %>%
findMarkers(groups=pbmc_small_cluster$label) %>%
as.list() %>%
map(~ .x %>%
head(10) %>%
rownames()) %>%
unlist()
# Plot heatmap
pbmc_small_cluster %>%
join_features(features=marker_genes) %>%
group_by(label) %>%
heatmap(feature, cell, abundance_counts, .scale="column")
```
# Reduce dimensions
We can calculate the first 3 UMAP dimensions using the SingleCellExperiment framework and *scater*.
```{r umap}
pbmc_small_UMAP <-
pbmc_small_cluster %>%
runUMAP(ncomponents=3)
```
And we can plot the result in 3D using plotly.
```{r umap plot, eval=FALSE}
pbmc_small_UMAP %>%
plot_ly(
x=~`UMAP1`,
y=~`UMAP2`,
z=~`UMAP3`,
color=~label,
colors=friendly_cols[1:4]
)
```
# Cell type prediction
We can infer cell type identities using *SingleR* [@aran2019reference] and manipulate the output using tidyverse.
```{r eval=FALSE}
# Get cell type reference data
blueprint <- celldex::BlueprintEncodeData()
# Infer cell identities
cell_type_df <-
assays(pbmc_small_UMAP)$logcounts %>%
Matrix::Matrix(sparse = TRUE) %>%
SingleR::SingleR(
ref = blueprint,
labels = blueprint$label.main,
method = "single"
) %>%
as.data.frame() %>%
as_tibble(rownames="cell") %>%
select(cell, first.labels)
```
```{r}
# Join UMAP and cell type info
pbmc_small_cell_type <-
pbmc_small_UMAP %>%
left_join(cell_type_df, by="cell")
# Reorder columns
pbmc_small_cell_type %>%
tidySingleCellExperiment::select(cell, first.labels, everything())
```
We can easily summarise the results. For example, we can see how cell type classification overlaps with cluster classification.
```{r}
# Count number of cells for each cell type per cluster
pbmc_small_cell_type %>%
count(label, first.labels)
```
We can easily reshape the data for building information-rich faceted plots.
```{r}
pbmc_small_cell_type %>%
# Reshape and add classifier column
pivot_longer(
cols=c(label, first.labels),
names_to="classifier", values_to="label"
) %>%
# UMAP plots for cell type and cluster
ggplot(aes(UMAP1, UMAP2, color=label)) +
geom_point() +
facet_wrap(~classifier) +
custom_theme
```
We can easily plot gene correlation per cell category, adding multi-layer annotations.
```{r}
pbmc_small_cell_type %>%
# Add some mitochondrial abundance values
mutate(mitochondrial=rnorm(dplyr::n())) %>%
# Plot correlation
join_features(features=c("CST3", "LYZ"), shape="wide") %>%
ggplot(aes(CST3 + 1, LYZ + 1, color=groups, size=mitochondrial)) +
geom_point() +
facet_wrap(~first.labels, scales="free") +
scale_x_log10() +
scale_y_log10() +
custom_theme
```
# Nested analyses
A powerful tool we can use with tidySingleCellExperiment is tidyverse `nest`. We can easily perform independent analyses on subsets of the dataset. First we classify cell types into lymphoid and myeloid, and then nest based on the new classification.
```{r}
pbmc_small_nested <-
pbmc_small_cell_type %>%
filter(first.labels != "Erythrocytes") %>%
mutate(cell_class=dplyr::if_else(`first.labels` %in% c("Macrophages", "Monocytes"), "myeloid", "lymphoid")) %>%
nest(data=-cell_class)
pbmc_small_nested
```
Now we can independently for the lymphoid and myeloid subsets (i) find variable features, (ii) reduce dimensions, and (iii) cluster using both tidyverse and SingleCellExperiment seamlessly.
```{r warning=FALSE}
pbmc_small_nested_reanalysed <-
pbmc_small_nested %>%
mutate(data=map(
data, ~ {
.x <- runPCA(.x, subset_row=variable_genes)
variable_genes <-
.x %>%
modelGeneVar() %>%
getTopHVGs(prop=0.3)
colLabels(.x) <-
.x %>%
buildSNNGraph(use.dimred="PCA") %>%
igraph::cluster_walktrap() %$%
membership %>%
as.factor()
.x %>% runUMAP(ncomponents=3)
}
))
pbmc_small_nested_reanalysed
```
We can then unnest and plot the new classification.
```{r}
pbmc_small_nested_reanalysed %>%
# Convert to tibble otherwise SingleCellExperiment drops reduced dimensions when unifying data sets.
mutate(data=map(data, ~ .x %>% as_tibble())) %>%
unnest(data) %>%
# Define unique clusters
unite("cluster", c(cell_class, label), remove=FALSE) %>%
# Plotting
ggplot(aes(UMAP1, UMAP2, color=cluster)) +
geom_point() +
facet_wrap(~cell_class) +
custom_theme
```
We can perform a large number of functional analyses on data subsets. For example, we can identify intra-sample cell-cell interactions using *SingleCellSignalR* [@cabello2020singlecellsignalr], and then compare whether interactions are stronger or weaker across conditions. The code below demonstrates how this analysis could be performed. It won't work with this small example dataset as we have just two samples (one for each condition). But some example output is shown below and you can imagine how you can use tidyverse on the output to perform t-tests and visualisation.
```{r, eval=FALSE}
pbmc_small_nested_interactions <-
pbmc_small_nested_reanalysed %>%
# Unnest based on cell category
unnest(data) %>%
# Create unambiguous clusters
mutate(integrated_clusters=first.labels %>% as.factor() %>% as.integer()) %>%
# Nest based on sample
tidySingleCellExperiment::nest(data=-sample) %>%
tidySingleCellExperiment::mutate(interactions=map(data, ~ {
# Produce variables. Yuck!
cluster <- colData(.x)$integrated_clusters
data <- data.frame(assays(.x) %>% as.list() %>% .[[1]] %>% as.matrix())
# Ligand/Receptor analysis using SingleCellSignalR
data %>%
cell_signaling(genes=rownames(data), cluster=cluster) %>%
inter_network(data=data, signal=., genes=rownames(data), cluster=cluster) %$%
`individual-networks` %>%
map_dfr(~ bind_rows(as_tibble(.x)))
}))
pbmc_small_nested_interactions %>%
select(-data) %>%
unnest(interactions)
```
If the dataset was not so small, and interactions could be identified, you would see something like below.
```{r}
tidySingleCellExperiment::pbmc_small_nested_interactions
```
# Session Info
```{r}
sessionInfo()
```
# References