RNA velocity analysis with scVelo

scVelo — RNA Velocity Analysis

Overview

scVelo is the leading Python package for RNA velocity analysis in single-cell RNA-seq data. It infers cell state transitions by modeling the kinetics of mRNA splicing — using the ratio of unspliced (pre-mRNA) to spliced (mature mRNA) abundances to determine whether a gene is being upregulated or downregulated in each cell. This allows reconstruction of developmental trajectories and identification of cell fate decisions without requiring time-course data.

Installation: uv pip install scvelo

Key resources:

• Documentation: https://scvelo.readthedocs.io/
• GitHub: https://github.com/theislab/scvelo
• Paper: Bergen et al. (2020) Nature Biotechnology. PMID: 32747759

When to Use This Skill

Use scVelo when:

• Trajectory inference from snapshot data: Determine which direction cells are differentiating
• Cell fate prediction: Identify progenitor cells and their downstream fates
• Driver gene identification: Find genes whose dynamics best explain observed trajectories
• Developmental biology: Model hematopoiesis, neurogenesis, epithelial-to-mesenchymal transitions
• Latent time estimation: Order cells along a pseudotime derived from splicing dynamics
• Complement to Scanpy: Add directional information to UMAP embeddings

Prerequisites

scVelo requires count matrices for both unspliced and spliced RNA. These are generated by:

1. STARsolo or kallisto|bustools with lamanno mode
2. velocyto CLI: velocyto run10x / velocyto run
3. alevin-fry / simpleaf with spliced/unspliced output

Data is stored in an AnnData object with layers["spliced"] and layers["unspliced"].

Standard RNA Velocity Workflow

1. Setup and Data Loading

import scvelo as scv
import scanpy as sc
import numpy as np
import matplotlib.pyplot as plt

# Configure settings
scv.settings.verbosity = 3       # Show computation steps
scv.settings.presenter_view = True
scv.settings.set_figure_params('scvelo')

# Load data (AnnData with spliced/unspliced layers)
# Option A: Load from loom (velocyto output)
adata = scv.read("cellranger_output.loom", cache=True)

# Option B: Merge velocyto loom with Scanpy-processed AnnData
adata_processed = sc.read_h5ad("processed.h5ad")  # Has UMAP, clusters
adata_velocity = scv.read("velocyto.loom")
adata = scv.utils.merge(adata_processed, adata_velocity)

# Verify layers
print(adata)
# obs × var: N × G
# layers: 'spliced', 'unspliced' (required)
# obsm['X_umap'] (required for visualization)

2. Preprocessing

# Filter and normalize. As of scVelo 0.3, filter_and_normalize() only filters
# genes and normalizes per cell -- it no longer takes n_top_genes and no longer
# log-transforms, so the log step and HVG selection come from Scanpy.
scv.pp.filter_and_normalize(
    adata,
    min_shared_counts=20    # Minimum counts in spliced+unspliced
)
sc.pp.log1p(adata)
sc.pp.highly_variable_genes(adata, n_top_genes=2000, subset=True)

# Compute first and second order moments (means and variances)
# knn_connectivities must be computed first
sc.pp.neighbors(adata, n_neighbors=30, n_pcs=30)
scv.pp.moments(
    adata,
    n_pcs=30,
    n_neighbors=30
)

3. Velocity Estimation — Stochastic Model

The stochastic model is fast and suitable for exploratory analysis:

# Stochastic velocity (faster, less accurate)
scv.tl.velocity(adata, mode='stochastic')
scv.tl.velocity_graph(adata)

# Visualize
scv.pl.velocity_embedding_stream(
    adata,
    basis='umap',
    color='leiden',
    title="RNA Velocity (Stochastic)"
)

4. Velocity Estimation — Dynamical Model (Recommended)

The dynamical model fits the full splicing kinetics and is more accurate:

# Recover dynamics (computationally intensive; ~10-30 min for 10K cells)
scv.tl.recover_dynamics(adata, n_jobs=4)

# Compute velocity from dynamical model
scv.tl.velocity(adata, mode='dynamical')
scv.tl.velocity_graph(adata)

5. Latent Time

The dynamical model enables computation of a shared latent time (pseudotime):

# Compute latent time
scv.tl.latent_time(adata)

# Visualize latent time on UMAP
scv.pl.scatter(
    adata,
    color='latent_time',
    color_map='gnuplot',
    size=80,
    title='Latent time'
)

# Identify top genes ordered by latent time
top_genes = adata.var['fit_likelihood'].sort_values(ascending=False).index[:300]
scv.pl.heatmap(
    adata,
    var_names=top_genes,
    sortby='latent_time',
    col_color='leiden',
    n_convolve=100
)

6. Driver Gene Analysis

# Identify genes with highest velocity fit
scv.tl.rank_velocity_genes(adata, groupby='leiden', min_corr=0.3)
df = scv.DataFrame(adata.uns['rank_velocity_genes']['names'])
print(df.head(10))

# Speed and coherence
scv.tl.velocity_confidence(adata)
scv.pl.scatter(
    adata,
    c=['velocity_length', 'velocity_confidence'],
    cmap='coolwarm',
    perc=[5, 95]
)

# Phase portraits for specific genes
scv.pl.velocity(adata, ['Cpe', 'Gnao1', 'Ins2'],
               ncols=3, figsize=(16, 4))

7. Velocity Arrows and Pseudotime

# Arrow plot on UMAP
scv.pl.velocity_embedding(
    adata,
    arrow_length=3,
    arrow_size=2,
    color='leiden',
    basis='umap'
)

# Stream plot (cleaner visualization)
scv.pl.velocity_embedding_stream(
    adata,
    basis='umap',
    color='leiden',
    smooth=0.8,
    min_mass=4
)

# Velocity pseudotime (alternative to latent time)
scv.tl.velocity_pseudotime(adata)
scv.pl.scatter(adata, color='velocity_pseudotime', cmap='gnuplot')

8. PAGA Trajectory Graph

# PAGA graph with velocity-informed transitions
scv.tl.paga(adata, groups='leiden')
df = scv.get_df(adata, 'paga/transitions_confidence', precision=2).T
df.style.background_gradient(cmap='Blues').format('{:.2g}')

# Plot PAGA with velocity
scv.pl.paga(
    adata,
    basis='umap',
    size=50,
    alpha=0.1,
    min_edge_width=2,
    node_size_scale=1.5
)

Complete Workflow Script

import scvelo as scv
import scanpy as sc

def run_rna_velocity(adata, n_top_genes=2000, mode='dynamical', n_jobs=4):
    """
    Complete RNA velocity workflow.

    Args:
        adata: AnnData with 'spliced' and 'unspliced' layers, UMAP in obsm
        n_top_genes: Number of top HVGs for velocity
        mode: 'stochastic' (fast) or 'dynamical' (accurate)
        n_jobs: Parallel jobs for dynamical model

    Returns:
        Processed AnnData with velocity information
    """
    scv.settings.verbosity = 2

    # 1. Preprocessing (scVelo 0.3 dropped log/HVG from filter_and_normalize)
    scv.pp.filter_and_normalize(adata, min_shared_counts=20)
    sc.pp.log1p(adata)
    sc.pp.highly_variable_genes(adata, n_top_genes=n_top_genes, subset=True)

    if 'neighbors' not in adata.uns:
        sc.pp.neighbors(adata, n_neighbors=30)

    scv.pp.moments(adata, n_pcs=30, n_neighbors=30)

    # 2. Velocity estimation
    if mode == 'dynamical':
        scv.tl.recover_dynamics(adata, n_jobs=n_jobs)

    scv.tl.velocity(adata, mode=mode)
    scv.tl.velocity_graph(adata)

    # 3. Downstream analyses
    if mode == 'dynamical':
        scv.tl.latent_time(adata)
        scv.tl.rank_velocity_genes(adata, groupby='leiden', min_corr=0.3)

    scv.tl.velocity_confidence(adata)
    scv.tl.velocity_pseudotime(adata)

    return adata

Key Output Fields in AnnData

After running the workflow, the following fields are added:

Location	Key	Description
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

Model	Speed	Accuracy	When to Use
stochastic	Fast	Moderate	Exploratory; large datasets
deterministic	Medium	Moderate	Simple linear kinetics
dynamical	Slow	High	Publication-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

Problem	Solution
Missing unspliced layer	Re-run velocyto or use STARsolo with --soloFeatures Gene Velocyto
Very few velocity genes	Lower min_shared_counts; check sequencing depth
Random-looking arrows	Try different n_neighbors or velocity model
Memory error with dynamical	Set n_jobs=1; reduce n_top_genes
Negative velocity everywhere	Check that spliced/unspliced layers are not swapped

Additional Resources

• scVelo documentation: https://scvelo.readthedocs.io/
• Tutorial notebooks: https://scvelo.readthedocs.io/tutorials/
• GitHub: https://github.com/theislab/scvelo
• Paper: Bergen V et al. (2020) Nature Biotechnology. PMID: 32747759
• velocyto (preprocessing): http://velocyto.org/
• CellRank (fate prediction, extends scVelo): https://cellrank.readthedocs.io/
• dynamo (metabolic labeling alternative): https://dynamo-release.readthedocs.io/