opencode-knowledge-work 0.0.0-stage → 0.1.1

package/content/bio-research/skills/bio-research-scvi-tools/references/troubleshooting.md

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+# Troubleshooting Guide for scvi-tools
+
+This reference provides a consolidated guide for diagnosing and resolving common issues across all scvi-tools models.
+
+## Quick Diagnosis
+
+| Symptom | Likely Cause | Quick Fix |
+|---------|--------------|-----------|
+| "X should contain integers" | Normalized data in X | Use `layer="counts"` in setup |
+| CUDA out of memory | GPU memory exhausted | Reduce `batch_size`, use smaller model |
+| Training loss is NaN | Bad data or learning rate | Check for all-zero cells/genes |
+| Batches not mixing | Too few shared features | Increase HVGs, check gene overlap |
+| Over-correction | Too aggressive integration | Use scANVI with labels |
+| Import error | Missing dependencies | `pip install scvi-tools[all]` |
+
+## Data Format Issues
+
+### Issue: CITE-seq protein data from Seurat is CLR-normalized
+
+**Cause**: Seurat's `NormalizeData(normalization.method = "CLR")` transforms raw ADT counts. totalVI requires raw integer counts for protein data.
+
+**Symptoms**:
+- Protein values are not integers
+- Protein values contain negative numbers
+- Model training produces poor results
+
+**Solution**:
+```python
+# Check if protein data is normalized
+protein = adata.obsm["protein_expression"]
+print(f"Min value: {protein.min()}")  # Should be 0 if raw counts
+print(f"Contains integers: {np.allclose(protein, protein.astype(int))}")
+
+# If importing from Seurat, use the raw counts assay, not the normalized one
+# In R/Seurat, export the RNA assay's counts slot, not the data slot
+# GetAssayData(seurat_obj, assay = "ADT", slot = "counts")
+```
+
+### Issue: "layer not found" or "X should contain integers"
+
+**Cause**: scvi-tools requires raw integer counts, not normalized data.
+
+**Solution**:
+```python
+# Check if X contains integers
+import numpy as np
+print(f"X max: {adata.X.max()}")
+print(f"Contains integers: {np.allclose(adata.X.data, adata.X.data.astype(int))}")
+
+# If normalized, recover from raw
+if hasattr(adata, 'raw') and adata.raw is not None:
+    adata = adata.raw.to_adata()
+
+# Or use existing counts layer
+adata.layers["counts"] = adata.X.copy()
+scvi.model.SCVI.setup_anndata(adata, layer="counts")
+```
+
+### Issue: Sparse matrix errors
+
+**Cause**: Incompatible sparse format or dense array expected.
+
+**Solution**:
+```python
+from scipy.sparse import csr_matrix
+
+# Convert to CSR format (most compatible)
+if hasattr(adata.X, 'toarray'):
+    adata.X = csr_matrix(adata.X)
+
+# Or convert to dense if small enough
+if adata.n_obs * adata.n_vars < 1e8:
+    adata.X = adata.X.toarray()
+```
+
+### Issue: NaN or Inf values in data
+
+**Cause**: Missing values or corrupted data.
+
+**Solution**:
+```python
+import numpy as np
+
+# Check for issues
+X = adata.X.toarray() if hasattr(adata.X, 'toarray') else adata.X
+print(f"NaN count: {np.isnan(X).sum()}")
+print(f"Inf count: {np.isinf(X).sum()}")
+print(f"Negative count: {(X < 0).sum()}")
+
+# Replace NaN/Inf with 0
+X = np.nan_to_num(X, nan=0, posinf=0, neginf=0)
+X = np.clip(X, 0, None)  # Ensure non-negative
+adata.X = csr_matrix(X)
+```
+
+### Issue: batch_key or labels_key not found
+
+**Cause**: Column name mismatch in adata.obs.
+
+**Solution**:
+```python
+# List available columns
+print(adata.obs.columns.tolist())
+
+# Check for similar names
+for col in adata.obs.columns:
+    if 'batch' in col.lower() or 'sample' in col.lower():
+        print(f"Potential batch column: {col}")
+```
+
+## GPU and Memory Issues
+
+### Issue: CUDA out of memory
+
+**Cause**: Model or batch doesn't fit in GPU memory.
+
+**Solutions** (try in order):
+
+```python
+# 1. Reduce batch size
+model.train(batch_size=64)  # Default is 128
+
+# 2. Use smaller model architecture
+model = scvi.model.SCVI(
+    adata,
+    n_latent=10,   # Default is 10-30
+    n_layers=1     # Default is 1-2
+)
+
+# 3. Subset to fewer genes
+sc.pp.highly_variable_genes(adata, n_top_genes=1500)
+adata = adata[:, adata.var['highly_variable']].copy()
+
+# 4. Clear GPU cache between models
+import torch
+torch.cuda.empty_cache()
+
+# 5. Use CPU if GPU is too small
+model.train(accelerator="cpu")
+```
+
+### Issue: No GPU detected
+
+**Cause**: CUDA not installed or version mismatch.
+
+**Diagnosis**:
+```python
+import torch
+print(f"CUDA available: {torch.cuda.is_available()}")
+print(f"PyTorch version: {torch.__version__}")
+print(f"CUDA version: {torch.version.cuda}")
+```
+
+**Solution**:
+```bash
+# Check system CUDA
+nvidia-smi
+nvcc --version
+
+# Reinstall PyTorch with matching CUDA
+pip install torch --index-url https://download.pytorch.org/whl/cu118  # For CUDA 11.8
+# Or
+pip install torch --index-url https://download.pytorch.org/whl/cu121  # For CUDA 12.1
+```
+
+### Issue: Memory error with large datasets
+
+**Cause**: Dataset too large for system RAM.
+
+**Solutions**:
+```python
+# 1. Process in chunks (for very large data)
+# Subsample for initial exploration
+adata_sample = adata[np.random.choice(adata.n_obs, 50000, replace=False)].copy()
+
+# 2. Use backed mode for AnnData
+adata = sc.read_h5ad("large_data.h5ad", backed='r')
+
+# 3. Reduce gene count aggressively
+adata = adata[:, adata.var['highly_variable']].copy()
+```
+
+## Training Issues
+
+### Issue: Training loss is NaN
+
+**Cause**: Numerical instability, bad data, or learning rate issues.
+
+**Solutions**:
+```python
+# 1. Check for problematic cells/genes
+sc.pp.filter_cells(adata, min_genes=200)
+sc.pp.filter_genes(adata, min_cells=3)
+
+# 2. Remove cells with zero counts
+adata = adata[adata.X.sum(axis=1) > 0].copy()
+
+# 3. Use gradient clipping (built into scvi-tools)
+model.train(max_epochs=200, early_stopping=True)
+```
+
+### Issue: Training doesn't converge
+
+**Cause**: Insufficient epochs, poor hyperparameters, or data issues.
+
+**Solutions**:
+```python
+# 1. Train longer
+model.train(max_epochs=400)
+
+# 2. Check training curves
+import matplotlib.pyplot as plt
+plt.plot(model.history['elbo_train'])
+plt.plot(model.history['elbo_validation'])
+plt.xlabel('Epoch')
+plt.ylabel('ELBO')
+plt.legend(['Train', 'Validation'])
+
+# 3. Adjust model size for data size
+# Small data (<10k cells): smaller model
+model = scvi.model.SCVI(adata, n_latent=10, n_layers=1, dropout_rate=0.2)
+
+# Large data (>100k cells): can use larger model
+model = scvi.model.SCVI(adata, n_latent=30, n_layers=2)
+```
+
+### Issue: Overfitting (validation loss increases)
+
+**Cause**: Model too complex or trained too long.
+
+**Solutions**:
+```python
+# 1. Enable early stopping
+model.train(early_stopping=True, early_stopping_patience=10)
+
+# 2. Add regularization
+model = scvi.model.SCVI(adata, dropout_rate=0.2)
+
+# 3. Reduce model complexity
+model = scvi.model.SCVI(adata, n_layers=1)
+```
+
+## Integration Issues
+
+### Issue: Batches don't mix
+
+**Cause**: Too few shared features, strong biological differences, or technical issues.
+
+**Solutions**:
+```python
+# 1. Check gene overlap between batches
+for batch in adata.obs['batch'].unique():
+    batch_genes = adata[adata.obs['batch'] == batch].var_names
+    print(f"{batch}: {len(batch_genes)} genes")
+
+# 2. Use more HVGs
+sc.pp.highly_variable_genes(adata, n_top_genes=4000, batch_key="batch")
+
+# 3. Train longer
+model.train(max_epochs=400)
+
+# 4. Increase latent dimensions
+model = scvi.model.SCVI(adata, n_latent=50)
+```
+
+### Issue: Over-correction (biological signal lost)
+
+**Cause**: Model removes too much variation.
+
+**Solutions**:
+```python
+# 1. Use scANVI with cell type labels
+scvi.model.SCANVI.from_scvi_model(scvi_model, labels_key="cell_type")
+
+# 2. Reduce model capacity
+model = scvi.model.SCVI(adata, n_latent=10)
+
+# 3. Use categorical covariates instead of batch_key
+scvi.model.SCVI.setup_anndata(
+    adata,
+    layer="counts",
+    categorical_covariate_keys=["batch"]  # Less aggressive than batch_key
+)
+```
+
+### Issue: One batch dominates clusters
+
+**Cause**: Unbalanced batch sizes or incomplete integration.
+
+**Solutions**:
+```python
+# 1. Check batch distribution
+print(adata.obs['batch'].value_counts())
+
+# 2. Subsample to balance
+from sklearn.utils import resample
+balanced = []
+min_size = adata.obs['batch'].value_counts().min()
+for batch in adata.obs['batch'].unique():
+    batch_data = adata[adata.obs['batch'] == batch]
+    balanced.append(batch_data[np.random.choice(len(batch_data), min_size, replace=False)])
+adata_balanced = sc.concat(balanced)
+```
+
+## Model-Specific Issues
+
+### scANVI: Poor label transfer
+
+**Solutions**:
+```python
+# 1. Check label distribution
+print(adata.obs['cell_type'].value_counts())
+
+# 2. Use Unknown for low-confidence cells
+adata.obs.loc[adata.obs['prediction_score'] < 0.5, 'cell_type'] = 'Unknown'
+
+# 3. Train scVI longer before scANVI
+scvi_model.train(max_epochs=300)
+scanvi_model = scvi.model.SCANVI.from_scvi_model(scvi_model, labels_key="cell_type")
+scanvi_model.train(max_epochs=100)
+```
+
+### totalVI: Noisy protein signal
+
+**Solutions**:
+```python
+# 1. Use denoised protein values
+_, protein_denoised = model.get_normalized_expression(return_mean=True)
+
+# 2. Check isotype controls
+# Isotype controls should have low expression
+for i, name in enumerate(adata.uns["protein_names"]):
+    if 'isotype' in name.lower():
+        print(f"{name}: mean={adata.obsm['protein_expression'][:, i].mean():.1f}")
+```
+
+### PeakVI: Poor clustering
+
+**Solutions**:
+```python
+# 1. Use more variable peaks
+from sklearn.feature_selection import VarianceThreshold
+selector = VarianceThreshold(threshold=0.05)
+adata = adata[:, selector.fit(adata.X).get_support()].copy()
+
+# 2. Binarize data
+adata.X = (adata.X > 0).astype(np.float32)
+```
+
+### MultiVI: Different cell counts between modalities
+
+**Solutions**:
+```python
+# Ensure same cells in same order
+common_cells = adata_rna.obs_names.intersection(adata_atac.obs_names)
+adata_rna = adata_rna[common_cells].copy()
+adata_atac = adata_atac[common_cells].copy()
+```
+
+### DestVI: Poor deconvolution
+
+**Solutions**:
+```python
+# 1. Check gene overlap
+common_genes = adata_ref.var_names.intersection(adata_spatial.var_names)
+print(f"Common genes: {len(common_genes)}")  # Should be >1000
+
+# 2. Use tissue-matched reference
+# Reference should contain all cell types expected in spatial data
+
+# 3. Check reference quality
+print(adata_ref.obs['cell_type'].value_counts())
+```
+
+## Version Compatibility
+
+### scvi-tools 1.x vs 0.x API changes
+
+Key differences:
+```python
+# 0.x API
+scvi.data.setup_anndata(adata, ...)
+
+# 1.x API (current)
+scvi.model.SCVI.setup_anndata(adata, ...)
+```
+
+### Check versions
+```python
+import scvi
+import scanpy as sc
+import anndata
+import torch
+
+print(f"scvi-tools: {scvi.__version__}")
+print(f"scanpy: {sc.__version__}")
+print(f"anndata: {anndata.__version__}")
+print(f"torch: {torch.__version__}")
+```
+
+### Recommended versions (as of late 2024)
+```
+scvi-tools>=1.0.0
+scanpy>=1.9.0
+anndata>=0.9.0
+torch>=2.0.0
+```
+
+## Getting Help
+
+1. **Check documentation**: https://docs.scvi-tools.org/
+2. **GitHub issues**: https://github.com/scverse/scvi-tools/issues
+3. **Discourse forum**: https://discourse.scverse.org/
+4. **Tutorials**: https://docs.scvi-tools.org/en/stable/tutorials/index.html
+
+When reporting issues, include:
+- scvi-tools version (`scvi.__version__`)
+- Python version
+- Full error traceback
+- Minimal reproducible example

package/content/bio-research/skills/bio-research-scvi-tools/scripts/cluster_embed.py

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+#!/usr/bin/env python3
+"""
+Cluster and embed data using scvi-tools latent representation.
+
+Computes neighbors, UMAP, and Leiden clustering on the latent space.
+Input should have latent representation from train_model.py.
+
+Usage:
+    python cluster_embed.py input.h5ad output_dir/
+    python cluster_embed.py input.h5ad output_dir/ --resolution 0.5 --use-rep X_scVI
+"""
+
+import argparse
+import os
+import sys
+
+
+def cluster_and_embed(
+    adata,
+    use_rep=None,
+    n_neighbors=15,
+    resolution=1.0,
+    min_dist=0.3
+):
+    """
+    Cluster and compute UMAP embedding.
+
+    Parameters
+    ----------
+    adata : AnnData
+        Data with latent representation in obsm
+    use_rep : str, optional
+        Key in obsm to use (auto-detects if None)
+    n_neighbors : int
+        Number of neighbors for graph
+    resolution : float
+        Leiden clustering resolution
+    min_dist : float
+        UMAP min_dist parameter
+
+    Returns
+    -------
+    AnnData with neighbors, UMAP, and leiden clustering
+    """
+    import scanpy as sc
+
+    # Auto-detect representation
+    if use_rep is None:
+        candidates = ["X_scANVI", "X_scVI", "X_totalVI", "X_PeakVI", "X_MultiVI"]
+        for key in candidates:
+            if key in adata.obsm:
+                use_rep = key
+                break
+
+        if use_rep is None:
+            # Fall back to PCA
+            if "X_pca" not in adata.obsm:
+                print("No scvi-tools embedding found, computing PCA...")
+                sc.pp.pca(adata)
+            use_rep = "X_pca"
+
+    print(f"Using representation: {use_rep}")
+    print(f"Embedding shape: {adata.obsm[use_rep].shape}")
+
+    # Compute neighbors
+    print(f"Computing neighbors (n={n_neighbors})...")
+    sc.pp.neighbors(adata, use_rep=use_rep, n_neighbors=n_neighbors)
+
+    # UMAP
+    print(f"Computing UMAP (min_dist={min_dist})...")
+    sc.tl.umap(adata, min_dist=min_dist)
+
+    # Leiden clustering
+    print(f"Computing Leiden clustering (resolution={resolution})...")
+    sc.tl.leiden(adata, resolution=resolution)
+
+    n_clusters = adata.obs['leiden'].nunique()
+    print(f"Found {n_clusters} clusters")
+
+    return adata
+
+
+def plot_results(adata, output_dir, batch_key=None, labels_key=None):
+    """Generate and save visualization plots."""
+    import scanpy as sc
+    import matplotlib.pyplot as plt
+
+    plots = []
+
+    # Always plot clusters
+    plots.append(("leiden", "Clusters"))
+
+    # Plot batch if available
+    if batch_key is not None and batch_key in adata.obs.columns:
+        plots.append((batch_key, f"Batch ({batch_key})"))
+
+    # Plot labels if available
+    if labels_key is not None and labels_key in adata.obs.columns:
+        plots.append((labels_key, f"Labels ({labels_key})"))
+
+    # Check for common columns
+    for col in adata.obs.columns:
+        if col not in [p[0] for p in plots]:
+            if 'cell' in col.lower() and 'type' in col.lower():
+                plots.append((col, col))
+            elif 'predict' in col.lower():
+                plots.append((col, col))
+
+    # Limit to 6 plots
+    plots = plots[:6]
+
+    # Create figure
+    n_plots = len(plots)
+    n_cols = min(3, n_plots)
+    n_rows = (n_plots + n_cols - 1) // n_cols
+
+    fig, axes = plt.subplots(n_rows, n_cols, figsize=(5 * n_cols, 4 * n_rows))
+    if n_plots == 1:
+        axes = [axes]
+    else:
+        axes = axes.flatten()
+
+    for i, (color, title) in enumerate(plots):
+        try:
+            sc.pl.umap(adata, color=color, ax=axes[i], show=False, title=title)
+        except Exception as e:
+            axes[i].set_title(f"Could not plot {color}: {e}")
+
+    # Hide unused axes
+    for i in range(len(plots), len(axes)):
+        axes[i].set_visible(False)
+
+    plt.tight_layout()
+
+    plot_path = os.path.join(output_dir, "umap_clusters.png")
+    plt.savefig(plot_path, dpi=150, bbox_inches="tight")
+    plt.close()
+    print(f"UMAP plot saved to {plot_path}")
+
+    # Save cluster counts
+    cluster_counts = adata.obs['leiden'].value_counts().sort_index()
+    counts_path = os.path.join(output_dir, "cluster_counts.csv")
+    cluster_counts.to_csv(counts_path)
+    print(f"Cluster counts saved to {counts_path}")
+
+
+def main():
+    parser = argparse.ArgumentParser(
+        description="Cluster and embed using scvi-tools latent space",
+        formatter_class=argparse.RawDescriptionHelpFormatter,
+        epilog="""
+Examples:
+    # Basic clustering
+    python cluster_embed.py adata_trained.h5ad results/
+
+    # Custom resolution
+    python cluster_embed.py adata_trained.h5ad results/ --resolution 0.5
+
+    # Specify representation
+    python cluster_embed.py adata_trained.h5ad results/ --use-rep X_scANVI
+
+    # Include batch and label columns in plots
+    python cluster_embed.py adata_trained.h5ad results/ --batch-key batch --labels-key cell_type
+        """
+    )
+    parser.add_argument("input", help="Input h5ad file with latent representation")
+    parser.add_argument("output_dir", help="Output directory")
+    parser.add_argument("--use-rep", help="Representation key in obsm (auto-detects)")
+    parser.add_argument("--n-neighbors", type=int, default=15, help="Neighbors for graph (default: 15)")
+    parser.add_argument("--resolution", type=float, default=1.0, help="Leiden resolution (default: 1.0)")
+    parser.add_argument("--min-dist", type=float, default=0.3, help="UMAP min_dist (default: 0.3)")
+    parser.add_argument("--batch-key", help="Batch column for plotting")
+    parser.add_argument("--labels-key", help="Labels column for plotting")
+
+    args = parser.parse_args()
+
+    try:
+        import scanpy as sc
+    except ImportError:
+        print("Error: scanpy required. Install with: pip install scanpy")
+        sys.exit(1)
+
+    # Create output directory
+    os.makedirs(args.output_dir, exist_ok=True)
+
+    # Load data
+    print(f"Loading {args.input}...")
+    adata = sc.read_h5ad(args.input)
+    print(f"Data: {adata.shape}")
+
+    # Cluster and embed
+    adata = cluster_and_embed(
+        adata,
+        use_rep=args.use_rep,
+        n_neighbors=args.n_neighbors,
+        resolution=args.resolution,
+        min_dist=args.min_dist
+    )
+
+    # Save results
+    adata_path = os.path.join(args.output_dir, "adata_clustered.h5ad")
+    adata.write_h5ad(adata_path)
+    print(f"AnnData saved to {adata_path}")
+
+    # Plot
+    plot_results(adata, args.output_dir, args.batch_key, args.labels_key)
+
+    print("\nDone!")
+
+
+if __name__ == "__main__":
+    main()