The shaky foundations of simulating single-cell RNA sequencing data

BACKGROUND: With the emergence of hundreds of single-cell RNA-sequencing (scRNA-seq) datasets, the number of computational tools to analyze aspects of the generated data has grown rapidly. As a result, there is a recurring need to demonstrate whether newly developed methods are truly performant—on their own as well as in comparison to existing tools. Benchmark studies aim to consolidate the space of available methods for a given task and often use simulated data that provide a ground truth for evaluations, thus demanding a high quality standard results credible and transferable to real data. RESULTS: Here, we evaluated methods for synthetic scRNA-seq data generation in their ability to mimic experimental data. Besides comparing gene- and cell-level quality control summaries in both one- and two-dimensional settings, we further quantified these at the batch- and cluster-level. Secondly, we investigate the effect of simulators on clustering and batch correction method comparisons, and, thirdly, which and to what extent quality control summaries can capture reference-simulation similarity. CONCLUSIONS: Our results suggest that most simulators are unable to accommodate complex designs without introducing artificial effects, they yield over-optimistic performance of integration and potentially unreliable ranking of clustering methods, and it is generally unknown which summaries are important to ensure effective simulation-based method comparisons. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13059-023-02904-1.