# Spatial Transcriptomics Tutorials With Omicverse > Spatial transcriptomics: Visium/HD, Stereo-seq, Slide-seq preprocessing (crop, rotate, cellpose), deconvolution (Tangram, cell2location, Starfysh), clustering (GraphST, STAGATE), integration, trajectory, communication. - Skill: `omicverse/spatial-transcriptomics-tutorials-with-omicverse-3` (Agent Skill, multi-file: 2 files) - Install (CLI): `npx skillmds@latest add omicverse/spatial-transcriptomics-tutorials-with-omicverse-3` - Raw SKILL.md: https://api.skillmd.com/api/skills/omicverse/spatial-transcriptomics-tutorials-with-omicverse-3/raw - Safety review: pending - Works with: Claude Code, Claude.ai, OpenAI Codex - Category: Docs & Writing - Author: omicverse (https://skillmd.com/u/omicverse) - Updated: 2026-09-22 - Page: https://skillmd.com/skills/omicverse/spatial-transcriptomics-tutorials-with-omicverse-3 --- # Spatial Transcriptomics with OmicVerse This skill covers spatial analysis workflows organized into three stages: Preprocessing, Deconvolution, and Downstream Analysis. Each stage includes the critical function calls, parameter guidance, and common pitfalls. ## Defensive Validation: Always Check Spatial Coordinates First Before ANY spatial operation, verify that spatial coordinates exist and are numeric: ```python # Required check before spatial analysis assert 'spatial' in adata.obsm, \ "Missing adata.obsm['spatial']. Load with ov.io.spatial.read_visium() or set manually." # Cast to float64 to prevent coordinate precision issues during rotation/cropping adata.obsm['spatial'] = adata.obsm['spatial'].astype('float64') ``` ## Stage 1: Preprocessing ### Crop, Rotate, and Align Coordinates Load Visium data and manipulate spatial coordinates for region selection and alignment: ```python import scanpy as sc, omicverse as ov ov.plot_set() adata = sc.datasets.visium_sge(sample_id="V1_Breast_Cancer_Block_A_Section_1") library_id = list(adata.uns['spatial'].keys())[0] # Cast coordinates before manipulation adata.obsm['spatial'] = adata.obsm['spatial'].astype('float64') # Crop to region of interest adata_crop = ov.space.crop_space_visium(adata, crop_loc=(0, 0), crop_area=(1000, 1000), library_id=library_id, scale=1) # Rotate and auto-align adata_rot = ov.space.rotate_space_visium(adata, angle=45, library_id=library_id) ov.space.map_spatial_auto(adata_rot, method='phase') # For manual refinement: ov.space.map_spatial_manual(adata_rot, ...) ``` ### Visium HD Cell Segmentation Segment Visium HD bins into cells using cellpose: ```python adata = ov.space.read_visium_10x(path="binned_outputs/square_002um/", source_image_path="tissue_image.btf") ov.pp.filter_genes(adata, min_cells=3) ov.pp.filter_cells(adata, min_counts=1) # H&E-based segmentation adata = ov.space.visium_10x_hd_cellpose_he(adata, mpp=0.3, gpu=True, buffer=150) # Expand labels to neighboring bins ov.space.visium_10x_hd_cellpose_expand(adata, labels_key='labels_he', expanded_labels_key='labels_he_expanded', max_bin_distance=4) # Gene-expression-driven seeds ov.space.visium_10x_hd_cellpose_gex(adata, obs_key="n_counts_adjusted", mpp=0.3, sigma=5) # Merge labels and aggregate to cell-level ov.space.salvage_secondary_labels(adata, primary_label='labels_he_expanded', secondary_label='labels_gex', labels_key='labels_joint') cdata = ov.space.bin2cell(adata, labels_key='labels_joint') ``` ## Stage 2: Deconvolution ### Critical API Reference: Method Selection ```python # CORRECT — Tangram: method passed to deconvolution() call decov_obj = ov.space.Deconvolution(adata_sc=sc_adata, adata_sp=sp_adata, celltype_key='Subset', result_dir='result/tangram') decov_obj.preprocess_sc(max_cells=5000) decov_obj.preprocess_sp() decov_obj.deconvolution(method='tangram', num_epochs=1000) # CORRECT — cell2location: method passed at INIT time cell2_obj = ov.space.Deconvolution(adata_sc=sc_adata, adata_sp=sp_adata, celltype_key='Subset', result_dir='result/c2l', method='cell2location') cell2_obj.deconvolution(max_epochs=30000) cell2_obj.save_model('result/c2l/model') ``` Note: For cell2location, the `method` parameter is set at initialization, not at the `deconvolution()` call. For Tangram, it's passed to `deconvolution()`. ### Starfysh Archetypal Deconvolution ```python from omicverse.external.starfysh import AA, utils, plot_utils visium_args = utils.prepare_data(adata_path="data/counts.h5ad", signature_path="data/signatures.csv", min_cells=10, filter_hvg=True, n_top_genes=3000) adata, adata_normed = visium_args.get_adata() aa_model = AA.ArchetypalAnalysis(adata_orig=adata_normed) aa_model.fit(k=12, n_init=10) visium_args = utils.refine_anchors(visium_args, aa_model, add_marker=True) model, history = utils.run_starfysh(visium_args, poe=False, n_repeat=5, lr=5e-3, max_epochs=500) ``` ## Stage 3: Downstream Analysis ### Spatial Clustering ```python # GraphST + mclust ov.utils.cluster(adata, use_rep='graphst|original|X_pca', method='mclust', n_components=7) # STAGATE ov.utils.cluster(adata, use_rep='STAGATE', method='mclust', n_components=7) # Merge small clusters ov.space.merge_cluster(adata, groupby='mclust', resolution=0.5) ``` Algorithm choice: GraphST and STAGATE require precalculated latent spaces. For standard clustering without spatial-aware embeddings, use Leiden/Louvain directly. ### Multi-Slice Integration (STAligner) ```python import anndata as ad Batch_list = [ov.read(p) for p in slice_paths] adata_concat = ad.concat(Batch_list, label='slice_name', keys=section_ids) STAligner_obj = ov.space.pySTAligner(adata=adata_concat, batch_key='slice_name', hidden_dims=[256, 64], use_gpu=True) STAligner_obj.train_STAligner_subgraph(nepochs=800, lr=1e-3) STAligner_obj.train() adata_aligned = STAligner_obj.predicted() sc.pp.neighbors(adata_aligned, use_rep='STAligner') ``` ### Spatial Trajectories **SpaceFlow** — pseudo-spatial maps: ```python sf_obj = ov.space.pySpaceFlow(adata) sf_obj.train(spatial_regularization_strength=0.1, num_epochs=300, patience=50) sf_obj.cal_pSM(n_neighbors=20, resolution=1.0) ``` **STT** — transition dynamics: ```python STT_obj = ov.space.STT(adata, spatial_loc='xy_loc', region='Region', n_neighbors=20) STT_obj.stage_estimate() STT_obj.train(n_states=9, n_iter=15, weight_connectivities=0.5) ``` ### Cell Communication (COMMOT + FlowSig) ```python df_cellchat = ov.external.commot.pp.ligand_receptor_database(species='human', database='cellchat') df_cellchat = ov.external.commot.pp.filter_lr_database(df_cellchat, adata, min_expr_frac=0.05) ov.external.commot.tl.spatial_communication(adata, lr_database=df_cellchat, distance_threshold=500, result_prefix='cellchat') # FlowSig network adata.layers['normalized'] = adata.X.copy() ov.external.flowsig.tl.construct_intercellular_flow_network( adata, commot_output_key='commot-cellchat', flowsig_output_key='flowsig-cellchat', edge_threshold=0.7) ``` ### Structural Layers (GASTON) and Slice Alignment (SLAT) **GASTON** — iso-depth estimation: ```python gas_obj = ov.space.GASTON(adata) A = gas_obj.prepare_inputs(n_pcs=50) gas_obj.load_rescale(A) gas_obj.train(hidden_dims=[64, 32], dropout=0.1, max_epochs=2000) gaston_isodepth, gaston_labels = gas_obj.cal_iso_depth(n_layers=5) ``` **SLAT** — cross-slice alignment: ```python from omicverse.external.scSLAT.model import Cal_Spatial_Net, load_anndatas, run_SLAT, spatial_match Cal_Spatial_Net(adata1, k_cutoff=20, model='KNN') Cal_Spatial_Net(adata2, k_cutoff=20, model='KNN') edges, features = load_anndatas([adata1, adata2], feature='DPCA', check_order=False) embeddings, *_ = run_SLAT(features, edges, LGCN_layer=5) best, index, distance = spatial_match(embeddings, adatas=[adata1, adata2]) ``` ## Troubleshooting - **`ValueError: spatial coordinates out of bounds` after rotation**: Cast `adata.obsm['spatial']` to `float64` BEFORE calling `rotate_space_visium`. Integer coordinates lose precision during trigonometric rotation. - **Cellpose segmentation fails with memory error**: For large `.btf` images, use `backend='tifffile'` to memory-map the image. Reduce `buffer` parameter if GPU memory is insufficient. - **Gene ID overlap failure in Tangram/cell2location**: Harmonise identifiers (ENSEMBL vs gene symbols) between `adata_sc` and `adata_sp` before calling `preprocess_sc`/`preprocess_sp`. Drop non-overlapping genes. - **`mclust` clustering error**: Requires `rpy2` and the R `mclust` package. If R bindings are unavailable, switch to `method='louvain'` or `method='leiden'`. - **STAligner/SpaceFlow embeddings collapse to a single point**: Verify `adata.obsm['spatial']` exists and coordinates are scaled appropriately. Tune learning rate (try `lr=5e-4`) and regularisation strength. - **FlowSig returns empty network**: Build spatial neighbor graphs before Moran's I filtering. Increase bootstraps or lower `edge_threshold` (try 0.5) if the network is too sparse. - **GASTON `RuntimeError` in training**: Provide a writable `out_dir` path. PyTorch nondeterminism may cause variation between runs—set `torch.manual_seed()` for reproducibility. - **SLAT alignment has many low-quality matches**: Regenerate spatial graphs with a higher `k_cutoff` value. Inspect `low_quality_index` flags and filter cells with high distance scores. - **STT pathway enrichment fails**: `gseapy` needs network access for gene set downloads. Cache gene sets locally with `ov.utils.geneset_prepare()` and pass the dictionary directly. ## Dependencies - Core: `omicverse`, `scanpy`, `anndata`, `squidpy`, `numpy`, `matplotlib` - Segmentation: `cellpose`, `opencv-python`/`tifffile`, optional GPU PyTorch - Deconvolution: `tangram-sc`, `cell2location`, `pytorch-lightning`; Starfysh needs `torch`, `scikit-learn` - Downstream: `scikit-learn`, `commot`, `flowsig`, `gseapy`, torch-backed modules (STAligner, SpaceFlow, GASTON, SLAT) ## Examples - "Crop and rotate my Visium slide, then run cellpose segmentation on the HD data and aggregate to cell-level AnnData." - "Deconvolve my lymph node spatial data with Tangram and cell2location, compare proportions, and plot cell-type maps." - "Integrate three DLPFC slices with STAligner, cluster with STAGATE, and infer communication with COMMOT+FlowSig." ## References - Quick copy/paste commands: [`reference.md`](reference.md)