Benchmarking algorithms for gene regulatory network inference from single-cell transcriptomic data
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Abstract
We present a systematic evaluation of state-of-the-art algorithms for inferring gene regulatory networks from single-cell transcriptional data. As the ground truth for assessing accuracy, we use synthetic networks with predictable trajectories, literature-curated Boolean models and diverse transcriptional regulatory networks. We develop a strategy to simulate single-cell transcriptional data from synthetic and Boolean networks that avoids pitfalls of previously used methods. Furthermore, we collect networks from multiple experimental single-cell RNA-seq datasets. We develop an evaluation framework called BEELINE. We find that the area under the precision-recall curve and early precision of the algorithms are moderate. The methods are better in recovering interactions in synthetic networks than Boolean models. The algorithms with the best early precision values for Boolean models also perform well on experimental datasets. Techniques that do not require pseudotime-ordered cells are generally more accurate. Based on these results, we present recommendations to end users. BEELINE will aid the development of gene regulatory network inference algorithms.
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GRN inference accuracy has remained disappointingly low, marginally exceeding random predictions.
“Despite extensive efforts, GRN inference accuracy has remained disappointingly low, marginally exceeding random predictions39.”
From Inferring gene regulatory networks from single-cell multiome data using atlas-scale external data (2025) · cites this paper as [39]
Targeted perturbations can be modeled as affecting individual nodes of a regulatory network, which allows investigation of mechanisms and inference of regulatory interactions and their direction.
“Experiments with targeted perturbations can be modeled as affecting individual nodes of a regulatory network model, enabling investigation of mechanistic processes and inference of regulatory interactions and their directionality11.”
From scPerturb: harmonized single-cell perturbation data (2024) · cites this paper as [11]
Cell-type-specific ground-truth GRNs were generated in the same manner as in a previous benchmarking study.
“Cell-type-specific ground-truth GRNs were generated in the same manner as in a previous benchmarking study55.”
From Dissecting cell identity via network inference and in silico gene perturbation (2023) · cites this paper as [55]
GENIE3 emerged as one of the best-performing algorithms in a previous benchmarking study.
“GENIE3 emerged as one of the best-performing algorithms in a previous benchmarking study55.”
From Dissecting cell identity via network inference and in silico gene perturbation (2023) · cites this paper as [55]
GRN inference performance was evaluated by AUROC and EPR, following the evaluation method used in a previous benchmarking study.
“GRN inference performance was evaluated by the AUROC and the early precision ratio (EPR), following the evaluation method used in a previous benchmarking study55.”
From Dissecting cell identity via network inference and in silico gene perturbation (2023) · cites this paper as [55]
Existing GRN tools utilize scRNA-seq data with thousands of pre-selected genes and cells, because GRNs from full transcriptomes in large scRNA-seq datasets are often computationally intensive and intractable.
“Existing GRN tools utilize scRNA-seq data with thousands of pre-select genes and cells, because GRNs from full transcriptomes in large scRNA-seq datasets are often computationally intensive and intractable.13,15”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
Benchmarking studies have shown limited accuracy of existing methods on both synthetic and real data.
“In addition, benchmarking studies have shown limited accuracy of existing methods on both synthetic and real data.13”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
Pseudotime-based GRN inference approaches generally have inferior performance to those that rely solely on scRNA-seq data.
“Although there exist other GRN inference approaches based on pseudotime analysis,23,24,25 their performance is generally inferior to those that rely solely on the scRNA-seq data.13”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
Validation of GRN inference tools has remained challenging.
“Validation of GRN inference tools has remained challenging.13”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
Since ground truth networks are unknown for many conditions across cell types and tissues, current GRN accuracy evaluations rely on inferring synthetic ground truth networks that may not capture biological or curated interactions that forsake tissue and cell-type specificity.
“Since ground truth networks are unknown for many conditions across cell types and tissues, current GRN accuracy evaluations rely on inferring synthetic ground truth networks that may not capture biological or curated interactions that forsake tissue and cell-type specificity.13”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
To benchmark SCING, three existing methods (GRNBOOST2, ppcor, and PIDC) were selected with default parameters based on previous benchmarking studies where superior performance of these methods was supported.
“To benchmark the performance of SCING, we selected three existing methods, namely GRNBOOST2, ppcor, and PIDCm with default parameters. These methods were selected based on previous benchmarking studies where superior performance of these methods were supported.13”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
Single cell multiomics has become a powerful tool for identifying regulatory interactions between genes, but the performance of existing tools is limited in both accuracy and scalability.
“Single cell multiomics has become a powerful tool for identifying regulatory interactions between genes, but the performance of existing tools is limited in both accuracy and scalability.13”
From SCING: Inference of robust, interpretable gene regulatory networks from single cell and spatial transcriptomics (2023) · cites this paper as [13]
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- Authors
- Pratapa, A., Jalihal, A. P., Law, J. N., Bharadwaj, A., & Murali, T. M.
- Affiliations
- Virginia Tech (US)
- Corresponding author
- Murali, T. M.
- Venue
- Nature Methods
- Published
- 2020-01-06
- Volume
- 17
- Issue
- 2
- Pages
- 147-154
- ISSN
- 1548-7091 (ISSN-L)
- 1548-7105
- PMC
- PMC7098173
- OpenAlex
- W2997831639
- Cited by
- 792
- References
- 63
- Citations by year
- 2026: 135 · 2025: 146 · 2024: 135 · 2023: 142 · 2022: 101 · 2021: 94 · 2020: 36 · 2019: 3
- FWCI
- 31.76
- Type
- journal article
- Language
- en
- Open access
- green
- doi.org
- Locations
- Nature Methods (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Science Foundation (CCF-1617678, DBI-1759858)
- U.S. Department of Health & Human Services | NIH | National Cancer Institute (UH2CA203768)
- ODNI | Intelligence Advanced Research Projects Activity (W911NF-17-2-0105)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gene Regulatory Network Analysis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Single-cell and spatial transcriptomics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Microbial Metabolic Engineering and Bioproduction
- Keywords
- gene regulatory network inference, single-cell transcriptomics, single-cell RNA sequencing, algorithm benchmarking, synthetic gene regulatory networks, Boolean model, data simulation, pseudotime, precision and recall
- MeSH terms
- Algorithms, Sequence Analysis, RNA, Gene Regulatory Networks, Single-Cell Analysis, Transcriptome, Datasets as Topic
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