Benchmarking algorithms for RNA velocity inference

RNA velocity is a computational framework for single-cell RNA sequencing (scRNA-seq) that estimates the future transcriptional state of individual cells, thereby capturing the direction and rate of cell state transitions rather than providing a purely static snapshot. Since its introduction in 2018, multiple RNA velocity methods have been developed, differing in their modeling assumptions, required inputs, computational complexity, and robustness. However, there remains limited consensus on how best to evaluate these methods or on which tools are most reliable under specific biological and technical settings. Here, we perform a systematic comparison of 29 velocity inference algorithms across 114 simulated datasets with known ground-truth cell dynamics and 62 real scRNA-seq datasets, and we extend the evaluation to spatial and multi-omics levels where velocity is increasingly applied. We benchmark RNA velocity methods using a unified framework that decomposes performance into four practical dimensions: accuracy, scalability, stability, and usability. Version 1 provides an overview of the entire project. Version 2 contains the AnnData object constructed from real experimental data. Version 3 contains the AnnData object generated from simulated data.