Assessing RNA velocity stability across syntheticreplicates using count splitting

Abstract

MotivationRNA velocity is a computational framework that predicts future cell states from single-cell RNA sequencing data, offering valuable insights into dynamic biological processes. However, there is a lack of general methods to quantify the uncertainty and stability of these predictions from various RNA velocity methods. ResultsWe present a novel framework for evaluating RNA velocity stability using negative binomial count splitting to generate independent data replicates, a metric we call replicate coherence. Testing five RNA velocity methods across datasets for mouse erythroid, pancreatic, and human brain development, we identify significant performance differences and inconsistencies, such as reversed velocity flows. Our framework remains robust even when intermediary cell states are missing. Furthermore, we introduce a signal-to-random coherence metric to guide model selection. We demonstrate that selecting fits with high replicate coherence uncovers more biologically informative gene pathways. This broadly applicable approach provides a rigorous tool for assessing and comparing RNA velocity methods across diverse biological contexts. Availability and implementationThe code and analyses are available at https://github.com/linnykos/veloUncertainty.