Gene signature scoring with UCell
Introduction
In single-cell RNA-seq analysis, gene signature (or “module”) scoring constitutes a simple yet powerful approach to evaluate the strength of biological signals, typically associated to a specific cell type or biological process, in a transcriptome.
UCell is an R package for evaluating gene signatures in single-cell datasets. UCell signature scores, based on the Mann-Whitney U statistic, are robust to dataset size and heterogeneity, and their calculation demands less computing time and memory than other available methods, enabling the processing of large datasets in a few minutes even on machines with limited computing power. UCell can be applied to any single-cell data matrix, and includes functions to directly interact with Seurat objects.
Quick start
To test your installation, load a small sample dataset and run UCell:
library(UCell)
data(sample.matrix)
gene.sets <- list(Tcell_signature = c("CD2","CD3E","CD3D"),
Myeloid_signature = c("SPI1","FCER1G","CSF1R"))
scores <- ScoreSignatures_UCell(sample.matrix, features=gene.sets)
head(scores)## Tcell_signature_UCell Myeloid_signature_UCell
## L5_ATTTCTGAGGTCGTGA 0.8991989 0
## L4_TCACTATTCATCTCTA 0.6900312 0
## L1_TCCTTCTTCTTTACAC 0.5932354 0
## L5_AAAGTGAAGGCGCTCT 0.6090343 0
## E2L3_CCTCAGTAGTGCAGGT 0.8506898 0
## L5_CCCTCTCGTTCTAAGC 0.6557632 0Get some testing data
For this demo, we will download a single-cell dataset of lung cancer (Zilionis et al. (2019) Immunity) through the scRNA-seq package. This dataset contains >170,000 single cells; for the sake of simplicity, in this demo will we focus on immune cells, according to the annotations by the authors, and downsample to 5000 cells.
Define gene signatures
Here we define some simple gene sets based on the “Human Cell Landscape” signatures Han et al. (2020) Nature. You may edit existing signatures, or add new one as elements in a list.
Run UCell
Run ScoreSignatures_UCell and get directly signature
scores for all cells
## Tcell_UCell Myeloid_UCell NK_UCell Plasma_cell_UCell
## bcHTNA1 0 0.5227121 0 0.00000000
## bcHNVA2 0 0.5112892 0 0.00000000
## bcALZN3 0 0.3584502 0 0.07540874
## bcFWBP4 0 0.1546426 0 0.00000000
## bcBJYE5 0 0.4629927 0 0.00000000
## bcGSBJ6 0 0.5452238 0 0.00000000Show the distribution of predicted scores
library(reshape2)
library(ggplot2)
melted <- reshape2::melt(u.scores)
colnames(melted) <- c("Cell","Signature","UCell_score")
p <- ggplot(melted, aes(x=Signature, y=UCell_score)) +
geom_violin(aes(fill=Signature), scale = "width") +
geom_boxplot(width=0.1, outlier.size=0) +
theme_bw() + theme(axis.text.x=element_blank())
p
Pre-calculating gene rankings
The time- and memory-demanding step in UCell is the calculation of
gene rankings for each individual cell. If we plan to experiment with
signatures, editing them or adding new cell subtypes, it is possible to
pre-calculate the gene rankings once and for all and then apply new
signatures over these pre-calculated ranks. Run the
StoreRankings_UCell function to pre-calculate gene rankings
over a dataset:
## 5 x 5 sparse Matrix of class "dgCMatrix"
## bcHTNA1 bcHNVA2 bcALZN3 bcFWBP4 bcBJYE5
## 5S_rRNA . . . . .
## 5_8S_rRNA . . . . .
## 7SK . . . . .
## A1BG . . . . .
## A1BG-AS1 . . . . .Then, we can apply our signature set, or any other new signature to the pre-calculated ranks. The calculations will be considerably faster.
set.seed(123)
u.scores.2 <- ScoreSignatures_UCell(features=signatures,
precalc.ranks = ranks)
melted <- reshape2::melt(u.scores.2)
colnames(melted) <- c("Cell","Signature","UCell_score")
p <- ggplot(melted, aes(x=Signature, y=UCell_score)) +
geom_violin(aes(fill=Signature), scale = "width") +
geom_boxplot(width=0.1, outlier.size = 0) +
theme_bw() + theme(axis.text.x=element_blank())
p
new.signatures <- list(Mast.cell = c("TPSAB1","TPSB2","CPA3","MS4A2"),
Lymphoid = c("LCK"))
u.scores.3 <- ScoreSignatures_UCell(features=new.signatures,
precalc.ranks = ranks)
melted <- reshape2::melt(u.scores.3)
colnames(melted) <- c("Cell","Signature","UCell_score")
p <- ggplot(melted, aes(x=Signature, y=UCell_score)) +
geom_violin(aes(fill=Signature), scale = "width") +
geom_boxplot(width=0.1, outlier.size=0) +
theme_bw() + theme(axis.text.x=element_blank())
p
Multi-core processing
If your machine has multi-core capabilities and enough RAM, running
UCell in parallel can speed up considerably your analysis. The example
below runs on a single core - you may modify this behavior by setting
e.g. workers=4 to parallelize to 4 cores:
Interacting with SingleCellExperiment or Seurat
SingleCellExperiment and Seurat are popular environments for single-cell analysis. The UCell package implements functions to interact directly with these pipelines, as described in dedicated demos available on the Bioc landing page.
Resources
Please report any issues at the UCell GitHub repository.
More demos available on the Bioc landing page and at the UCell demo repository.
If you find UCell useful, you may also check out the scGate package, which relies on UCell scores to automatically purify populations of interest based on gene signatures.
See also SignatuR for easy storing and retrieval of gene signatures.
References
- Andreatta, M., Carmona, S. J. (2021) UCell: Robust and scalable single-cell gene signature scoring Computational and Structural Biotechnology Journal
- Zilionis, R., Engblom, C., …, Klein, A. M. (2019) Single-Cell Transcriptomics of Human and Mouse Lung Cancers Reveals Conserved Myeloid Populations across Individuals and Species Immunity
Session Info
## R version 4.4.2 (2024-10-31)
## Platform: x86_64-pc-linux-gnu
## Running under: Ubuntu 24.04.1 LTS
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## Matrix products: default
## BLAS: /usr/lib/x86_64-linux-gnu/openblas-pthread/libblas.so.3
## LAPACK: /usr/lib/x86_64-linux-gnu/openblas-pthread/libopenblasp-r0.3.26.so; LAPACK version 3.12.0
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## [11] LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
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## time zone: Etc/UTC
## tzcode source: system (glibc)
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## attached base packages:
## [1] stats4 stats graphics grDevices utils datasets methods
## [8] base
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## other attached packages:
## [1] reshape2_1.4.4 scater_1.35.0
## [3] scuttle_1.17.0 patchwork_1.3.0
## [5] ggplot2_3.5.1 Seurat_5.1.0
## [7] SeuratObject_5.0.2 sp_2.1-4
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