We perform a thorough analysis of RNA velocity methods, with a view towards understanding the suitability of the various assumptions underlying popular implementations. In addition to providing a self-contained exposition of the underlying mathematics, we undertake simulations and perform controlled experiments on biological datasets to assess workflow sensitivity to parameter choices and underlying biology. Finally, we argue for a more rigorous approach to RNA velocity, and present a framework for Markovian analysis that points to directions for improvement and mitigation of current problems.

The simultaneous quantification of protein and RNA makes possible the inference of past, present, and future cell states from single experimental snapshots. To enable such temporal analysis from multimodal single-cell experiments, we introduce an extension of the RNA velocity method that leverages estimates of unprocessed transcript and protein abundances to extrapolate cell states. We apply the model to six datasets and demonstrate consistency among cell landscapes and phase portraits. The analysis software is available as the protaccel Python package.

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The question of how cell-to-cell differences in transcription rate affect RNA count distributions is fundamental for understanding biological processes underlying transcription. Answering this question requires quantitative models that are both interpretable (describing concrete biophysical phenomena) and tractable (amenable to mathematical analysis). This enables the identification of experiments which best discriminate between competing hypotheses. As a proof of principle, we introduce a simple but flexible class of models involving a continuous stochastic transcription rate driving a discrete RNA transcription and splicing process, and compare and contrast two biologically plausible hypotheses about transcription rate variation. One assumes variation is due to DNA experiencing mechanical strain, while the other assumes it is due to regulator number fluctuations. We introduce a framework for numerically and analytically studying such models, and apply Bayesian model selection to identify candidate genes that show signatures of each model in single-cell transcriptomic data from mouse glutamatergic neurons.