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Putative cell type discovery from single-cell gene expression data

Abstract

We present the Single-Cell Clustering Assessment Framework, a method for the automated identification of putative cell types from single-cell RNA sequencing (scRNA-seq) data. By iteratively applying a machine learning approach to a given set of cells, we simultaneously identify distinct cell groups and a weighted list of feature genes for each group. The differentially expressed feature genes discriminate the given cell group from other cells. Each such group of cells corresponds to a putative cell type or state, characterized by the feature genes as markers. Benchmarking using expert-annotated scRNA-seq datasets shows that our method automatically identifies the ‘ground truth’ cell assignments with high accuracy.

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Fig. 1: A self-projection approach.
Fig. 2: Using the connection graph to optimize clustering.
Fig. 3: Self-projection-based clustering optimization compared with ground truth.
Fig. 4: Self-projection accuracy indicates optimal clustering during clustering optimization.
Fig. 5: SCCAF captures the key stages in mouse hematopoiesis.

Data availability

The datasets together with the accession codes are as follows: pancreas12, accession no. GSE84133; cortex30, accession no. GSE60361; retinal bipolar neurons11, accession no. GSE81904; pancreatic islets13, accession no. E-MTAB-5061; visual cortex31, accession no. GSE102827; hematopoiesis35, GSE89754; hematopoiesis36, accession no. GSE92575; cortex51, accession no. GSE71585; cortex33, accession no. GSE115746; liver32, accession no. GSE124395; liver50, accession no. GSE115469. Source data for Figs. 1–5 are included with this paper.

Code availability

An open source implementation of SCCAF is available at GitHub (https://github.com/SCCAF/sccaf) and (https://doi.org/10.5281/zenodo.3695975) under the MIT license. The release includes tutorials and example vignettes for reproducing the analyses presented in this article, as well as all preprocessed datasets considered in this study. The software version used to generate the results presented in this article is also available as Supplementary Software. SCCAF is also accessible from the Python package index (https://pypi.org/project/SCCAF/) and it is implemented as a Galaxy tool in the Human Cell Atlas (https://humancellatlas.usegalaxy.eu/). The SCCAF Galaxy modules are available to install with a few clicks on any Galaxy instance through the main Galaxy Tool Shed at https://toolshed.g2.bx.psu.edu/view/ebi-gxa/suite_sccaf/.

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Acknowledgements

We thank all members of the Teichmann and Brazma labs for helpful discussions. We thank S. Aldridge for proofreading the text. Z.M. is supported by the Single Cell Gene Expression Atlas grant from the Wellcome Trust (no. 108437/Z/15/Z).

Author information

Affiliations

Authors

Contributions

Z.M. conceived the method, implemented the algorithm and website, conducted the analyses, created the figures and contributed to the manuscript. P.M., N.H. and I.P. packed the algorithm and implemented it as a Galaxy tool. A.B. and S.A.T. supervised the work and contributed to the manuscript.

Corresponding authors

Correspondence to Alvis Brazma or Sarah A. Teichmann.

Ethics declarations

Competing interests

In the last three years S.A.T. has consulted for Biogen, Genentech and Roche, and is a member of the Scientific Advisory Board of Foresite Labs and of the Functional Genomics & AI Scientific Advisory Board of GlaxoSmithKline.

Additional information

Peer review information Nicole Rusk and Lin Tang were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Self-projection accuracy comparison between the ground truth annotation and the clustering with under-clustering or over-clustering.

This test measures the self-projection accuracy on three conditions: 1) the “ground truth” clustering as annotated by human experts (marked as ‘correct-clustering’); 2) over-clustering and 3) under-clustering. The violin plots on the left column show the self-projection accuracy distributions (of both cross-validation as red and on the test set as green) for these three conditions in all the datasets by repeating the random sampling 100 times. These plots demonstrate that the “ground truth” clustering corresponds to the highest self-projection accuracy in almost all cases. According to the test results on the datasets: Hrvatin(48,266 cells), Tasic2018 (21,874 cells), Shekhar (26,830 cells), Segerstolpe (2,108 cells), Zeisel (3,005 cells), Baron (Mouse, 1,886 cells) and Baron (Human, 8,199 cells), it is possible to identify the best clustering using self-projection as the clustering consistency test. As for any classifier, it is always easier to perform well on fewer clusters. Thus if two clusterings show a similar level of self-projection accuracy, for example, Baron (Mouse), the clustering with more clusters should be chosen for consideration. Source data

Extended Data Fig. 2 Testing machine learning methods on ground truth datasets.

Five machine learning models were tested on the five ground truth datasets (Baron mouse cells (1,886 cells), Baron human cells (8,199 cells), Shekhar (26,830 cells), Segerstolpe (2,108 cells), Zeisel (3,005 cells)). The data were randomly split into a training set and a test set for self-projection, and this process was repeated 100 times. The distributions of the self-projection accuracies and the mean accuracy of cross-validation in the training were plotted as violin plots. Source data

Extended Data Fig. 3 Self-projection can assess over-clustering on real data.

The performance on 1000 BC1A cells from the mouse retina dataset (Shekhar et al.). When the data randomly assigned as two clusters, logistic regression cannot demonstrate any predictive ability in self-projection. When splitting the 1000 BC1A cells into two clusters based on the first principal component (PC), logistic regression shows certain but not ideal predictive ability in self-projection. The performance on 500 BC2 cells and 500 BC1A cells. Self-projection shows high predictive ability. When the 500 BC2 cells are over-clustered into two clusters based on PC1, the confusion always happens between the over-clustered clusters but hardly between BC1A cells and BC2 cells. Source data

Extended Data Fig. 4 SCCAF clustering optimization on mouse retina data.

The mouse retina data of Shekhar includes 26,830 cells. a, show the t-SNE plot of the initial clustering (Round0) and the clustering during the four Rounds (Round1 to Round4) of SCCAF optimization, (b) shows the self-projection results, while (c) shows the consistency (self-projection accuracy) between the clustering assignment and the self-projection results. Source data

Extended Data Fig. 5 The self-projection-based clustering optimization achieves clustering identical to human expert annotation.

The six expert-annotated datasets a Baron mouse cells (1,886 cells), b: Baron human cells (8,199 cells), c: Shekhar (26,830 cells), d, Segerstolpe (2,108 cells), e: Zeisel (3,005 cells), f: Hrvatin (48,266 cells), g: Aizarani (10,305 cells), h: Tasic2018 (21,874 cells) are used to compare the SCCAF clustering result and the human expert annotation. Source data

Extended Data Fig. 6 Adjusted Rand Index evaluation of the SCCAF results compared with Louvain clustering.

The Adjusted Rand Index (ARI) is calculated between the clustering results and the human expert annotation. The blue dots show the ARI of SCCAF, while the orange dots show the ARI of the initial Louvain clustering before SCCAF optimization (the initial clustering). Source data

Extended Data Fig. 7 SCCAF clustering compared with published annotation.

a, The clustering shows the result from the SCCAF clustering of the mouse hematopoiesis data (4,016 cells) from Tusi et al. b, The cell potential (of the 4,016 cells) to develop into different cell lineages (Er: Erythrocytes, Gr: Granulocytes, Ly: Lymphocytes, Mk: Megakaryocytes, Mo: Monocytes, Ba: Basophilic or mast cell) are colored as Viridis. Source data

Extended Data Fig. 8 Upregulated and downregulated genes in erythrocytes development.

The upregulated and downregulated genes in the erythrocytes’ development are colored on the SPRING plot and the UMAP plot of the Tusi dataset (4,016 cells) (a) and the Giladi dataset (20,202 cells) (b). Source data

Extended Data Fig. 9 SCCAF helps in annotating a new unannotated dataset of the human brain.

Human brain single nuclei-Seq data (http://celltypes.brain-map.org/rnaseq) from Middle Temporal Gyrus, Primary Visual Cortex, Anterior Cingulate Cortex and Lateral Geniculate (33,782 cells in total) were analyzed together. In the t-SNE plots, cells are colored according to the a) cortical area and the cell classes. Projection-based annotation approaches (logistic regression and CHETAH) were used to annotate the dataset using the mouse brain data from Tasic et al. were applied considering the ortholog genes between human and mouse. And the results are colored in the t-SNE plots in b. SCCAF was also used to identify the discriminative cell clusters and resulted in 38 clusters. c, Each cell cluster is annotated according to the top-ranked feature genes extracted from the SCCAF model. d, Self-projection accuracy and ROC curves are compared for these three annotation approaches. Source data

Extended Data Fig. 10 A hierarchical approach to cluster mouse visual cortex data.

a, The t-SNE plot shows the cell types in the Hrvatin dataset (48,266 cells). Three cell types, under the main cell type “Interneurons” (350 cells), cluster together in the red circle. The variances of these three cell types are not dominant when considering the whole dataset. b, The first round SCCAF clustering of the Hrvatin dataset (48,266 cells) cannot find such subpopulations, because (c) they are clustered together from the initial state. When looking at these three clusters (350 cells) (d), Louvain clustering (e) may achieve a similar clustering result as the manual annotation. Leiden clustering (f) can also identify the three cell types but shows a difference in the center cells. Source data

Supplementary information

Supplementary Information

Supplementary Discussion, Supplementary Figs. 1–18, Supplementary Table 1

Reporting Summary

Supplementary Software

Source data

Source Data Fig. 1

Raw data for the simulated one cell type and two cell types.

Source Data Fig. 2

Raw data for the simulated six cell types, number of recapitulated genes.

Source Data Fig. 3

Raw data for the t-SNE plots, river plot.

Source Data Fig. 4

Raw data for the simulated six cell types.

Source Data Fig. 5

Raw data for the violin plots and t-SNE plots.

Source Data Extended Data Fig. 1

Raw data for the violin plots and t-SNE plots.

Source Data Extended Data Fig. 2

Raw data for the violin plots.

Source Data Extended Data Fig. 3

Raw data for the PCA plots.

Source Data Extended Data Fig. 4

Raw data for the violin plots and t-SNE plots.

Source Data Extended Data Fig. 5

Raw data for the t-SNE plots.

Source Data Extended Data Fig. 6

Raw data for the dot plots.

Source Data Extended Data Fig. 7

Raw data for the SPRING plots.

Source Data Extended Data Fig. 8

Raw data for the SPRING plots and UMAP plots.

Source Data Extended Data Fig. 9

Raw data for the t-SNE plots.

Source Data Extended Data Fig. 10

Raw data for the UMAP plots and t-SNE plots.

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Miao, Z., Moreno, P., Huang, N. et al. Putative cell type discovery from single-cell gene expression data. Nat Methods 17, 621–628 (2020). https://doi.org/10.1038/s41592-020-0825-9

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