Single Trajectory Analysis

Velocity coupling (VIA + scVelo)

• Use scv.pp.filter_and_normalize, scv.pp.moments, and scv.tl.velocity to generate velocity layers.
• Provide VIA with adata.layers['velocity'] to refine lineage directionality (via.VIA(..., velocity_weight=...)).
• Compare VIA pseudotime with scVelo latent time (scv.tl.latent_time) to validate directionality and root selection.

Advanced RNA Velocity Backends (ov.single.Velo)

OmicVerse provides a unified Velo class wrapping 4 velocity backends. Use this when you need more than basic scVelo:

Backend selection guide

Unified Velo pipeline

import omicverse as ov

velo = ov.single.Velo(adata)

# 1. Filter (scvelo backend) or preprocess (dynamo backend)
velo.filter_genes(min_shared_counts=20)     # For scvelo
# velo.preprocess(recipe='monocle', n_neighbors=30, n_pcs=30)  # For dynamo

# 2. Compute moments
velo.moments(backend='scvelo', n_pcs=30, n_neighbors=30)
# backend: 'scvelo' or 'dynamo'

# 3. Fit kinetic parameters
velo.dynamics(backend='scvelo')

# 4. Calculate velocity
velo.cal_velocity(method='scvelo')
# method: 'scvelo', 'dynamo', 'latentvelo', 'graphvelo'

# 5. Build velocity graph and project to embedding
velo.velocity_graph(basis='umap')
velo.velocity_embedding(basis='umap')

latentvelo specifics (deep learning velocity)

latentvelo uses a VAE + neural ODE to learn latent dynamics. It handles batch effects and complex trajectories better than classical scVelo:

velo.cal_velocity(
    method='latentvelo',
    celltype_key='cell_type',    # Optional: AnnotVAE uses cell type info
    batch_key='batch',           # Optional: batch correction
    velocity_key='velocity_S',
    n_top_genes=2000,
    latentvelo_VAE_kwargs={},    # Pass custom VAE hyperparameters
)
# Requires: pip install torchdiffeq
# Uses GPU if available, falls back to CPU

graphvelo specifics (refinement layer)

GraphVelo refines velocity estimates from any base method by leveraging the cell graph structure. Run it after scvelo or dynamo:

# First: compute base velocity with scvelo or dynamo
velo.cal_velocity(method='scvelo')

# Then: refine with graphvelo
velo.graphvelo(
    xkey='Ms',                          # Spliced moments key
    vkey='velocity_S',                  # Base velocity key to refine
    basis_keys=['X_umap', 'X_pca'],    # Project to multiple embeddings
    gene_subset=None,                   # Optional: restrict to gene subset
)

Downstream fate scoring notebooks

• CellFateGenie: For pseudotime-associated gene discovery, use search_skills('CellFateGenie fate genes') to load the dedicated CellFateGenie skill.
• t_metacells.ipynb: Aggregate metacell trajectories for robustness checks and meta-state differential expression.
• t_cytotrace.ipynb: Integrate CytoTRACE differentiation potential with velocity-informed lineages for maturation scoring.

Required preprocessing

1. Quality control: remove low-quality cells/genes, apply doublet filtering.
2. Normalization & log transformation (sc.pp.normalize_total, sc.pp.log1p).
3. Highly variable gene selection tailored to immune datasets (sc.pp.highly_variable_genes).
4. Batch correction if necessary (e.g., scvi-tools, bbknn).
5. Compute PCA, neighbor graph, and embedding (UMAP/FA) used by all trajectory methods.
6. For velocity: compute moments on the same neighbor graph before running VIA coupling.

Parameter tuning

• Neighbor graph n_neighbors and n_pcs should be harmonized across PAGA, VIA, and Palantir to maintain consistency.
• In VIA, adjust knn, too_big_factor, and root_user for datasets with uneven sampling.
• Palantir requires careful start cell selection; use marker genes and velocity arrows to confirm.
• For PAGA, tweak threshold to control edge sparsity; ensure connected components reflect biological branches.
• Velocity estimation: compare mode='stochastic' vs mode='dynamical' in scVelo; recalibrate if terminal states disagree with VIA.

Visualization and export

1. Overlay PAGA edges on UMAP (scv.pl.paga) and annotate branch labels.
2. Plot Palantir pseudotime and branch probabilities on embeddings.
3. Visualize VIA trajectories using via.plot_fates and via.plot_scatter.
4. Export pseudotime tables and fate probabilities to CSV for downstream notebooks.
5. Save high-resolution figures (PNG/SVG) and notebook artifacts for reproducibility.
6. Update notebooks with consistent color schemes and metadata columns before sharing.

Defensive Validation Patterns

# Before PAGA: verify neighbor graph exists
assert 'neighbors' in adata.uns, "Neighbor graph required. Run sc.pp.neighbors(adata) first."

# Before VIA velocity coupling: verify velocity layers exist
if 'velocity' not in adata.layers:
    print("WARNING: velocity layer missing. Run scv.tl.velocity(adata) first for VIA coupling.")
assert 'spliced' in adata.layers and 'unspliced' in adata.layers, \
    "Missing spliced/unspliced layers. Check loom/H5AD import preserved velocity layers."

# Before Palantir: verify PCA/diffusion components
assert 'X_pca' in adata.obsm, "PCA required. Run ov.pp.pca(adata) first."

Troubleshooting tips

• Missing velocity layers: re-run scv.pp.moments and scv.tl.velocity ensuring adata.layers['spliced']/['unspliced'] exist; verify loom/H5AD import preserved layers.
• Disconnected PAGA graph: inspect neighbor graph or adjust n_neighbors; confirm batch correction didn’t fragment the manifold.
• Palantir convergence issues: reduce diffusion components or reinitialize start cells; ensure no NaN values in data matrix.
• VIA terminal states unstable: increase iterations (cluster_graph_pruning_iter), or provide manual terminal state hints based on marker expression.
• Notebook kernel memory errors: downsample cells or precompute summaries (metacells) before rerunning.
• latentvelo ImportError: torchdiffeq: Install with pip install torchdiffeq. Required for neural ODE backend.
• graphvelo returns NaN velocities: Ensure base velocity (scvelo/dynamo) was computed first. graphvelo refines — it doesn't compute from scratch.
• dynamo preprocess fails: dynamo expects spliced/unspliced layers. Verify with 'spliced' in adata.layers.