# HTO DND - Demultiplex Hashtag Data
[](https://badge.fury.io/py/hto-dnd)
[](https://github.com/sail-mskcc/hto_dnd/actions/workflows/test.yml)
`hto` is a Python package designed for efficient and accurate demultiplexing of hash-tagged oligonucleotides (HTOs) in single-cell data.
It normalises based on observed background signal and denoises the data to remove batch effects and noise:
- **Normalization**: Normalize HTO data using background signal, inspired by the DSB method (see citation below).
- **Denoising**: Remove batch effects and noise from the data by regressing out cell by cell variation.
- **Demultiplexing**: Cluster and classify cells into singlets, doublets, or negatives using clustering methods like k-means or Gaussian Mixture Models (GMM).
The package supports command-line interface (CLI) usage and Python imports.
## Installation
Using `pip`:
```bash
pip install hto
```
From source:
```bash
git clone https://github.com/sail-mskcc/hto_dnd.git
cd hto_dnd
pip install .
```
## Usage
### Python API
The python API is built around AnnData. it is highly recommended two work with three AnnData objects:
* `adata_hto`: Filtered AnnData object with HTO data, containing only actual cells.
* `adata_hto_raw`: Raw AnnData object with HTO data, containing actual cells and background signal.
* `adata_gex`: Raw AnnData object with gene expression data. This is optional and can be used to construct a more informative background signal.
```python
import hto
# get mockdata
mockdata = hto.data.generate_hto(n_cells=1000, n_htos=3, seed=10)
adata_hto = mockdata["filtered"]
adata_hto_raw = mockdata["raw"]
adata_gex = mockdata["gex"]
# denoise, normalize, and demultiplex
adata_demux = hto.demultiplex(
adata_hto,
adata_hto_raw,
adata_gex=adata_gex,
inplace=False,
)
# see results
adata_demux.obs[["hash_id", "doublet_info"]].head()
```
### Command-Line Interface (CLI)
The CLI provides an API for the `hto demultiplex` scripts. Make sure to define `--adata-out` to save the output.
```
hto demultiplex \
--adata-hto /path/to/adata_hto.h5ad \
--adata-hto-raw /path/to/adata_hto_raw.h5ad \
--adata-gex /path/to/adata_gex.h5ad \
--adata-out /path/to/output.h5ad
```
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"description": "# HTO DND - Demultiplex Hashtag Data\n\n[](https://badge.fury.io/py/hto-dnd)\n[](https://github.com/sail-mskcc/hto_dnd/actions/workflows/test.yml)\n\n`hto` is a Python package designed for efficient and accurate demultiplexing of hash-tagged oligonucleotides (HTOs) in single-cell data.\nIt normalises based on observed background signal and denoises the data to remove batch effects and noise:\n\n- **Normalization**: Normalize HTO data using background signal, inspired by the DSB method (see citation below).\n- **Denoising**: Remove batch effects and noise from the data by regressing out cell by cell variation.\n- **Demultiplexing**: Cluster and classify cells into singlets, doublets, or negatives using clustering methods like k-means or Gaussian Mixture Models (GMM).\n\nThe package supports command-line interface (CLI) usage and Python imports.\n\n\n\n## Installation\n\nUsing `pip`:\n\n```bash\npip install hto\n```\n\nFrom source:\n\n```bash\ngit clone https://github.com/sail-mskcc/hto_dnd.git\ncd hto_dnd\npip install .\n```\n\n## Usage\n\n### Python API\n\nThe python API is built around AnnData. it is highly recommended two work with three AnnData objects:\n\n* `adata_hto`: Filtered AnnData object with HTO data, containing only actual cells.\n* `adata_hto_raw`: Raw AnnData object with HTO data, containing actual cells and background signal.\n* `adata_gex`: Raw AnnData object with gene expression data. This is optional and can be used to construct a more informative background signal.\n\n```python\nimport hto\n\n# get mockdata\nmockdata = hto.data.generate_hto(n_cells=1000, n_htos=3, seed=10)\nadata_hto = mockdata[\"filtered\"]\nadata_hto_raw = mockdata[\"raw\"]\nadata_gex = mockdata[\"gex\"]\n\n# denoise, normalize, and demultiplex\nadata_demux = hto.demultiplex(\n adata_hto,\n adata_hto_raw,\n adata_gex=adata_gex,\n inplace=False,\n)\n\n# see results\nadata_demux.obs[[\"hash_id\", \"doublet_info\"]].head()\n```\n\n### Command-Line Interface (CLI)\n\nThe CLI provides an API for the `hto demultiplex` scripts. Make sure to define `--adata-out` to save the output.\n\n```\nhto demultiplex \\\n --adata-hto /path/to/adata_hto.h5ad \\\n --adata-hto-raw /path/to/adata_hto_raw.h5ad \\\n --adata-gex /path/to/adata_gex.h5ad \\\n --adata-out /path/to/output.h5ad\n```\n",
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