--- license: mit pretty_name: spot Stage 1 — CD4 continuous transcriptional-program scores tags: - single-cell - immunology - perturb-seq - CD4-T-cells - transcriptomics - reproducibility size_categories: - 100K - Zhu, Dann, et al., *Genome-scale perturb-seq in primary human CD4+ T cells maps context-specific regulators of T cell programs and human immune traits*, bioRxiv 2025.12.23.696273, . - Authors' analysis repository: . `ntc_clustered.h5ad` is a **spot-derived pinned input object**, not an original authors' release file. It contains 396,000 NTC cells: 33,000 cells from each of four coded donors in each of `Rest`, `Stim8hr`, and `Stim48hr`. This equal quota is a spot sampling decision and does not preserve the source population's condition proportions. The object is 396,000 × 18,130. Its `.X` matrix is already normalized to a per-cell target total of approximately 9,819 and `log1p` transformed. No raw counts layer is retained. Cluster metadata are retained, but latent and UMAP matrices are not stored in the H5AD (`obsm`, `obsp`, `layers`, and `uns` are empty). Frozen display coordinates are supplied separately. ## Scoring method For cell `c` and program `p`: ```text score(c, p) = mean(.X[c, measured panel genes]) - mean(.X[c, frozen expression-matched control genes]) ``` The deterministic control construction is fully materialized in the release: - 25 expression bins computed over the pinned 396,000-cell matrix; - eligible pool = detected genes minus every program marker and activation predictor marker; - program-order-invariant keyed SHA-256 selection; - 50 controls per occupied marker bin, without replacement; - master seed `12345`. There are 11 primary axes: Th1-like, Th2-like, Th17-like, Tfh-like, Treg-like, CD4 CTL-like, Th9-like, Naive, Activated, Memory, and Checkpoint. The activation-adjusted CD4 CTL-like residual is a sensitivity/display lane and is not a primary pole. Marker panels are curated canonical-marker panels with per-gene primary-source provenance in the registry. Masopust et al., , is used as a nomenclature framework, not as the source of the marker panels. ## Files | Path | Purpose | |---|---| | `ntc_clustered.h5ad` | Pinned 396,000-cell normalized NTC scoring source object. | | `stage1-continuous-v3.0.1/data/stage01_scores_full.parquet` | Authoritative 396,000 × 15 table: barcode, donor, condition, 11 primary scores, and one activation-adjusted sensitivity score. | | `stage1-continuous-v3.0.1/data/stage01_summary_v3.json` | Full-table program summaries by condition. | | `stage1-continuous-v3.0.1/data/stage01_umap_coordinates.json` | Frozen coordinates for the 40,000-cell display sample. | | `stage1-continuous-v3.0.1/data/stage01_umap_overlay_v3_hf.json` | The same 40,000 barcodes with v3 scores; display-only. | | `stage1-continuous-v3.0.1/method/` | Input identity, registry and marker provenance, frozen bins, controls, eligible pool, activation-association diagnostic, and Stage-2 scorer projection. | | `stage1-continuous-v3.0.1/validation/` | Frozen gate specification, descriptive validation, semantic amendment, constituent evidence, and non-gating diagnostics. | | `stage1-continuous-v3.0.1/contracts/` | Generic Stage-1 selection-contract schema. | | `stage1-continuous-v3.0.1/receipts/` | Independent reconstruction receipt. | | `MANIFEST.json` | Exact size and SHA-256 ledger for every current release file. | `stage01_stage2_registry_view.json` is only a Stage-1 scorer projection for downstream binding; it is not a Stage-2 result. ## Validation status - Full-score raw SHA-256: `de63b496e8121c77babe380e0c3b5ddfd66f9ce67d0d4e80f55645d177e27e5f`. - Canonical full-score content SHA-256: `43c4296d5166740c334441a69df23bb440a073382bbe79628a3bb89e43d51316`. - The independent reconstruction receipt reports all checks passing, including exact overlay-to-full score equality for all 40,000 display barcodes. - The 40,000-cell overlay is not the scientific analysis universe. An archived representativeness diagnostic contains one failed overlay-distribution check; use the full 396,000-cell table for analysis and summaries. - The frozen validation archive records that 0/33 program-condition pairs cleared its prospective small-panel leave-one-marker robustness gate. That result is retained for auditability, has `active_gate:false` in the current selection contract, and is not a claim that the programs are biologically invalid. Null/PENDING selectability fields inside the scorer registry are the pre-validation snapshot; `stage01_selectability_v3.json` records the later historical disposition. - `stage01_activation_association_v1.json` reports descriptive activation association for every primary axis and the pooled activation-adjusted CTL residual. Activation/timepoint association is not claimed to be removed. External-dataset confirmation and protein, lineage, and functional validation remain separate biological work; they are not implied by this scoring release. The HF overlay differs from the repository-generated overlay only in its top-level explanatory `note`. Cell records, coordinates, score fields, canonical score hash, and coordinate hash are unchanged. Both source and emitted raw hashes are recorded in `MANIFEST.json`; raw-byte reproduction is not claimed for that note-only packaging transform. ## Reproducibility The pinned input identity is: - source HF revision: `e5fcf98b56a9302921d402e97fc5a190bd88f9a6`; - `ntc_clustered.h5ad` raw SHA-256: `2edc6d318415c8b0ee779d707ab86e26ddb6f0274db51ab4a12f21ebfda50e43`. Reproduction and verification code is in the public [`spot` repository](https://github.com/Kirit-Singh/spot/tree/29bd367b6e0ed674868041a4fe85d1f6fafd4cb8/01_programs/analysis). The input manifest, control algorithm, materialized controls/bins, validation constituents, and independent receipt are included here so the release can be audited without relying on moving branch names. ## Privacy The H5AD contains coded donor, guide, lane, and library identifiers but no donor demographics. In particular, it does not include age, sex, ethnicity, weight, height, smoking status, blood type, or collection date. Coded identifiers may be joinable to upstream public metadata and therefore should not be described as anonymous. Release files were scanned for machine-local paths and credentials. ## License and release history The official CZI dataset page declares the upstream dataset **MIT License**. The exact upstream copyright-holder notice is not supplied on that page, so this repository does not invent one. Spot-authored transformations and metadata are MIT licensed; see `LICENSE` and `NOTICE`. The historical v2 release is preserved at immutable commit `e5fcf98b56a9302921d402e97fc5a190bd88f9a6` and release tag `stage1-continuous-v2`. Its root `stage01_umap_seed.json` is intentionally absent from the current tree. v3.0.1 supersedes the earlier categorical-call, permutation-FDR, prevalence, paper-exact-embedding, and CP10k-equivalence claims; none of those claims is made by this release. Current release tag: **`stage1-continuous-v3.0.1`**.