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Evaluating measures of association for single-cell transcriptomics

Abstract

Single-cell transcriptomics provides an opportunity to characterize cell-type-specific transcriptional networks, intercellular signaling pathways and cellular diversity with unprecedented resolution by profiling thousands of cells in a single experiment. However, owing to the unique statistical properties of scRNA-seq data, the optimal measures of association for identifying gene–gene and cell–cell relationships from single-cell transcriptomics remain unclear. Here, we conducted a large-scale evaluation of 17 measures of association for their ability to reconstruct cellular networks, cluster cells of the same type and link cell-type-specific transcriptional programs to disease. Measures of proportionality were consistently among the best-performing methods across datasets and tasks. Our analysis provides data-driven guidance for gene and cell network analysis in single-cell transcriptomics.

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Fig. 1: Single-cell transcriptomics compendium and functional coherence of single-cell gene coexpression networks.
Fig. 2: Overlap between single-cell gene coexpression networks and other biological networks.
Fig. 3: Reproducibility of single-cell gene coexpression networks of the human pancreas.
Fig. 4: Accuracy of measures of association for clustering single-cell transcriptomes of known cell types.
Fig. 5: Disease gene prioritization through single-cell gene coexpression analysis.

Data availability

The data that support the findings of this study are available from the following GitHub repository: https://github.com/skinnider/SCT-MoA. Raw data are available from the Gene Expression Omnibus, http://mousebrain.org, or https://support.10xgenomics.com, as detailed in the Methods; dataset identifiers are provided in Supplementary Data 1.

Code availability

The ‘dismay’ R package is available as Supplementary Software 1 and from the following GitHub repository: https://github.com/skinnider/dismay. R code used to reproduce the analysis and figures is available from the following GitHub repository: https://github.com/skinnider/SCT-MoA.

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Acknowledgements

We thank E. Su and M. Hirst for computational assistance and P. Pavlidis for comments on an earlier version of the manuscript. This work was supported by Genome Canada and Genome British Columbia (to L.J.F.; project 214PRO) and enabled in part by support provided by WestGrid and Compute Canada. M.A.S. is supported by a CIHR Vanier Canada Graduate Scholarship, an Izaak Walton Killam Memorial Pre-Doctoral Fellowship, a UBC Four Year Fellowship and a Vancouver Coastal Health–CIHR–UBC MD/PhD Studentship. J.W.S. is supported by an Izaak Walton Killam Memorial Pre-Doctoral Fellowship, a UBC Four Year Fellowship and a Vancouver Coastal Health–CIHR–UBC MD/PhD Studentship.

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M.A.S., J.W.S. and L.J.F. designed experiments. M.A.S. and J.W.S. performed experiments. M.A.S. wrote the first draft of the manuscript, which was edited by J.W.S. and L.J.F.

Corresponding authors

Correspondence to Michael A. Skinnider or Leonard J. Foster.

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The authors declare no competing interests.

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Integrated supplementary information

Supplementary Fig. 1 Functional coherence of scRNA-seq gene coexpression networks, considering all datasets from each publication (n = 213).

Functional coherence of single-cell gene coexpression networks. Known gene functions were randomly withheld and predicted from the coexpression network in threefold cross-validation, and the area under the receiver operating characteristic curve (AUC) was calculated to quantify the degree to which genes with similar functions are coexpressed in networks constructed with each measure of association. Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers).

Supplementary Fig. 2 Relationships between functional coherence and dropouts or number of cells in scRNA-seq gene coexpression networks.

a,b, Relationships between functional coherence and proportion of dropouts (a) or number of cells (b) in scRNA-seq gene coexpression networks.

Supplementary Fig. 3 Functional coherence of single-cell gene coexpression networks according to scRNA-seq protocol (including all datasets associated with each publication, n = 213).

Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers).

Supplementary Figure 4 Functional coherence of single-cell gene coexpression networks according to transcript coverage afforded by scRNA-seq protocol.

Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers), for the subset of n = 40 of the 43 datasets randomly sampled from each publication using scRNA-seq protocols discussed by Chen et al. (Annu. Rev. Biomed. Data Sci. 1, 29–51; 2018).

Supplementary Fig. 5 Adjusted r2 of experimental and analytical variables in univariate linear models of overall functional coherence (median AUC).

Univariate linear regression was performed using the subset of n = 35 of the 43 datasets randomly sampled from each publication (i) using scRNA-seq protocols discussed by Chen et al. (Annu. Rev. Biomed. Data Sci. 1, 29–51; 2018) and (ii) using protocols that were used in at least two different publications. Exact F test P values were 5.2 × 10–31 (measure of association); 1.3 × 10–13 (sequencing protocol); 3.6 × 10–12 (number of cells); 7.0 × 10–6 (cell isolation/capture); 1.3 × 10–3 (transcript coverage); and 0.13 (percentage of zeroes). Blue, significant at uncorrected P < 0.05; gray, not significant.

Supplementary Fig. 6 Overlap between sparse unweighted single-cell gene coexpression networks constructed with the top-ranked 20,000 or 100,000 edges and other biological networks (n = 43 datasets, one per publication).

a, Results with 20,000 edges. b, Results with 100,000 edges. Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers).

Supplementary Fig. 7

Dendrograms obtained from hierarchical clustering of 561 single-cell transcriptomes derived from seven cell lines27, with two lines sequenced in two batches, colored by cell line of origin and batch.

Supplementary Fig. 8

Adjusted Rand index of hierarchical clustering of single-cell transcriptomes from cell lines profiled in two different batches27 with each measure of association.

Supplementary Fig. 9 Normalized mutual information of hierarchical clustering of single-cell transcriptomes.

a,b, Normalized mutual information of hierarchical clustering of single-cell transcriptomes from seven different cell lines27 (a) or a subset of two cell lines profiled in two different batches (b) with each measure of association.

Supplementary Fig. 10 Adjusted Rand index and normalized mutual information of Louvain clustering applied to the shared nearest-neighbor graph of single-cell transcriptomes from seven different cell lines.

a, Adjusted Rand index. b, Normalized mutual information. Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers) for n = 5 values of k in nearest-neighbor graph construction (2, 5, 10, 20 and 50).

Supplementary Fig. 11 Computational time required to construct gene coexpression matrices for a random sample of 1,000 genes from the subset of scRNA-seq datasets obtained from the Gene Expression Omnibus (n = 162 datasets).

Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers). The median of 10 random samples from each dataset was retained for plotting.

Supplementary Fig. 12 Functional coherence of gene coexpression networks constructed with each measure of association at different thresholds for gene filtering, ranging from non-zero expression in less than 50% of cells (more stringent, less sparse) to non-zero expression in less than 95% of cells (less stringent, more sparse).

Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers) for n = 43 datasets, one per publication.

Supplementary Fig. 13 Trends in functional coherence of gene coexpression networks for each of 17 measures of association at different thresholds for gene filtering, ranging from non-zero expression in less than 50% of cells (more stringent, less sparse) to non-zero expression in less than 95% of cells (less stringent, more sparse).

Box plots show median (horizontal line), interquartile range (hinges) and the smallest and largest values no more than 1.5 times the interquartile range (whiskers) for n = 43 datasets, one per publication.

Supplementary information

Supplementary Information

Supplementary Figs. 1–13 and Supplementary Note

Reporting Summary

Supplementary Software

‘dismay’ R package providing a unified interface to calculate all 17 measures of association.

Supplementary Data 1

List of scRNA-seq datasets analyzed in this study.

Supplementary Data 2

Functional coherence of scRNA-seq gene coexpression networks.

Supplementary Data 3

Macromolecular interaction network overlap of scRNA-seq gene coexpression networks.

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Skinnider, M.A., Squair, J.W. & Foster, L.J. Evaluating measures of association for single-cell transcriptomics. Nat Methods 16, 381–386 (2019). https://doi.org/10.1038/s41592-019-0372-4

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