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scvelo

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RNA velocity analysis with scVelo. Estimate cell state transitions from unspliced/spliced mRNA dynamics, infer trajectory directions, compute latent time, and identify driver genes in single-cell RNA-seq data. Complements Scanpy/scVI-tools for trajectory inference.

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name: scvelo description: RNA velocity analysis with scVelo. Estimate cell state transitions from unspliced/spliced mRNA dynamics, infer trajectory directions, compute latent time, and identify driver genes in single-cell RNA-seq data. Complements Scanpy/scVI-tools for trajectory inference. license: BSD-3-Clause tags: [scientific-skills, scvelo, scanpy, bioinformatics] metadata: skill-author: Kuan-lin Huang -------|-----|-------------| | adata.layers | velocity | RNA velocity per gene per cell | | adata.layers | fit_t | Fitted latent time per gene per cell | | adata.obsm | velocity_umap | 2D velocity vectors on UMAP | | adata.obs | velocity_pseudotime | Pseudotime from velocity | | adata.obs | latent_time | Latent time from dynamical model | | adata.obs | velocity_length | Speed of each cell | | adata.obs | velocity_confidence | Confidence score per cell | | adata.var | fit_likelihood | Gene-level model fit quality | | adata.var | fit_alpha | Transcription rate | | adata.var | fit_beta | Splicing rate | | adata.var | fit_gamma | Degradation rate | | adata.uns | velocity_graph | Cell-cell transition probability matrix |

Velocity Models Comparison

ModelSpeedAccuracyWhen to Use
stochasticFastModerateExploratory; large datasets
deterministicMediumModerateSimple linear kinetics
dynamicalSlowHighPublication-quality; identifies driver genes

Best Practices

  • Start with stochastic mode for exploration; switch to dynamical for final analysis
  • Need good coverage of unspliced reads: Short reads (< 100 bp) may miss intron coverage
  • Minimum 2,000 cells: RNA velocity is noisy with fewer cells
  • Velocity should be coherent: Arrows should follow known biology; randomness indicates issues
  • k-NN bandwidth matters: Too few neighbors → noisy velocity; too many → oversmoothed
  • Sanity check: Root cells (progenitors) should have high unspliced/spliced ratios for marker genes
  • Dynamical model requires distinct kinetic states: Works best for clear differentiation processes

Troubleshooting

ProblemSolution
Missing unspliced layerRe-run velocyto or use STARsolo with --soloFeatures Gene Velocyto
Very few velocity genesLower min_shared_counts; check sequencing depth
Random-looking arrowsTry different n_neighbors or velocity model
Memory error with dynamicalSet n_jobs=1; reduce n_top_genes
Negative velocity everywhereCheck that spliced/unspliced layers are not swapped

Additional Resources