README.md

---
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<n<1M
source_datasets:
  - CZI Virtual Cells Platform — Primary Human CD4+ T Cell Perturb-seq
language:
  - en
annotations_creators:
  - machine-generated
---

# spot Stage 1 — CD4 continuous transcriptional-program scores

This repository contains the public data release for `spot` Stage 1,
method version **`stage1-continuous-v3.0.1`**. It provides continuous RNA-program
scores for a quota-balanced non-targeting-control (NTC) subset of the Marson
GWCD4i primary-human-CD4 Perturb-seq dataset.

The scores are measurements of specified transcriptional modules. They are not
categorical cell-type, lineage, protein, cytotoxicity, or suppressive-function
calls. The Stage-1 scoring and validation outputs contain no inferential p-values,
q-values, or FDR estimates and do not report cell-type prevalence.

This dataset repository deliberately contains **Stage 1 only**. Stage 2–4 result
artifacts use additional sources and are not bundled here.

## Source and subset

Upstream public source:

- **Primary Human CD4+ T Cell Perturb-seq**, CZI Virtual Cells Platform, v1.0,
  released 22 December 2025:
  <https://virtualcellmodels.cziscience.com/dataset/genome-scale-tcell-perturb-seq>
- 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, <https://doi.org/10.64898/2025.12.23.696273>.
- Authors' analysis repository:
  <https://github.com/emdann/GWT_perturbseq_analysis_2025>.

`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.,
<https://doi.org/10.1038/s41577-025-01238-2>, 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`**.