PREPS

The microenvironment of glioma is heterogeneous, including tumor cells, neurons, and immune cells, making it difficult to develop an effective treatment. Our previous study also demonstrated neuronal behaviors of glioma tumor cells, especially firing an action potential, highlighting the importance of characterizing the electrophysiological properties of each single cell. The electrophysiology data is achieved through Patch-sequencing experiments. However, the available data size is limited due to the experimental difficulty. Here, we introduce PREPS (Predicting Electrophysiological Properties of Single-cell RNA-seq), a machine-learning-based computational framework that employs the state-of-the-art GPT (Generative Pre-trained Transformer) models to predict electrophysiological features of glioma samples by single-cell RNA-sequencing.

Methodology

In the first step of PREPS, leveraging the foundational GPT model, Geneformer, which has captured the complexity within human gene networks based on a broad range of healthy tissues, we fine-tuned the model into a series of brain-specific cell type classifiers using the transcriptomes of various developing brain and glioma datasets. Besides clustering and annotating glioma cells, we extracted and concatenated embeddings from the intermediate layers of these classifiers to represent the comprehensive transcriptomic features of each cell. Next, we built a group of predictive Elastic Nets (i.e., PREPS models) that map the electrophysiological features of glioma cells to their embeddings, with models optimized through a systematic grid search of all parameter combinations. Finally, we applied PREPS models to predict electrophysiological features of a larger amount of glioma data, where conducting many Patch-seq experiments is time-consuming and labor-intensive.

We also developed a single-cell gene set enrichment-like method (scoring.py) to assign cell types using gene attention scores derived from our fine-tuned transformer models. For each cell, we averaged multi-head attention weights from the final transformer layer and ranked genes based on the [CLS] token’s attention vector. Gene identifiers were converted to symbols, producing ranked gene lists per cell. To define marker sets, we automatically extracted and weighted marker genes for each cell type using PubMed abstracts (2021-2024) and GPT-4.1, prioritizing genes frequently cited or included in canonical brain cell markers. Using these weighted marker lists, we calculated enrichment scores per cell via a modified ssGSEA approach, assigning each cell to the highest scoring type. Final cell type labels were determined by consensus across multiple ranked gene inputs, and both enrichment scores and final annotations were exported.

Installation & Setup

To ensure all dependencies run correctly, it is highly recommended to install this project within a Python virtual environment.

1. Clone the repository

git clone https://github.com/akdess/preps.git  
cd preps  

2. Create and activate a virtual environment

• For macOS and Linux:

  python3 -m venv venv  
  source venv/bin/activate  
  

• For Windows:

  python -m venv venv  
  venv\Scripts\activate  
  

3. Install the required dependencies
Make sure your virtual environment is active, then run:

pip install -r requirements.txt  

Quickstart

PREPS/
| ----- preps.sh <-- The master wrapper script
| ----- scripts/ <-- Core pipeline scripts
| ----- | ----- tokenize_data.py
| ----- | ----- finetune.py
| ----- | ----- annotate.py
| ----- | ----- patchseq_glm.py
| ----- | ----- select_best_models.py
| ----- | ----- patchseq_predict.py
| ----- data/
| ----- paper_reproducibility/
| ----- | ----- mouse_model_pipeline/
| ----- | ----- | ----- models/
| ----- | ----- | ----- scripts/
| ----- | ----- human_model_pipeline/
| ----- | ----- | ----- models/
| ----- | ----- | ----- scripts/
| ----- requirements.txt
| ----- README.md

PREPS is fully automated through a single master Bash script (preps.sh). All data tokenization, model training, annotation, and prediction steps are handled automatically based on the workflow you select.

Command Structure

./preps.sh [workflow] [dataset_name] [species]  

• workflow: Choose between finetune, train, or apply.
• dataset_name: The prefix of your dataset (e.g., if your file is glioma.h5ad, use glioma).
• species: human or mouse (defaults to human if left blank).

Prerequisites

Before running for the first time, make sure the master script is executable:

chmod +x preps.sh  

Ensure your input dataset is in .h5ad format and placed in the main directory (e.g., my_dataset.h5ad).

Example 1: Fine-tuning the Foundation Model

Use the finetune workflow to adapt the Geneformer foundation model to your specific biological context.

./preps.sh finetune allen_cortex mouse  

What it does: Tokenizes allen_cortex.h5ad using Mouse-Geneformer, fine-tunes the transformer for cell-type classification, and saves the output to a predictable trained_model/ directory.

Example 2: Training Predictive Models (Patch-seq)

Use the train workflow on your paired Patch-seq dataset to build the electrophysiological and cell-type prediction models. Make sure you have your metadata (_meta_data.txt) and ephys features (_ephys_features.csv) in the directory.

./preps.sh train m1_patchseq mouse  

What it does: Tokenizes the Patch-seq data, extracts latent embeddings using the fine-tuned model, trains Elastic Net regressors and Logistic Regression classifiers, and automatically scans and saves the lowest MAE / highest Accuracy models as best__*.joblib.

Example 3: Atlas-Scale Application (Inference)

Use the apply workflow to project your trained electrophysiological models onto a massive, unimodal scRNA-seq atlas.

./preps.sh apply glioma_patients human  

What it does: Tokenizes the unimodal glioma_patients.h5ad data, extracts embeddings using the human foundation model, dynamically loads your best__ predictive models, and outputs clean Excel files with the predicted continuous electrophysiological features and cell-type probabilities for every single cell.

Workflow Tip: The Complete End-to-End Pipeline

If you are starting from scratch with a new species or brain region, a complete end-to-end pipeline simply looks like this:

# 1. Fine-tune on a large reference atlas  
./preps.sh finetune brain_reference human  
  
# 2. Train predictive models on a paired Patch-seq dataset  
./preps.sh train glioma_patchseq human  
  
# 3. Predict electrophysiology on a massive new unimodal dataset  
./preps.sh apply glioma_unimodal human  

Step-wise Application

R Data conversion

If the scRNA-seq dataset adata.h5ad is available, skip this step and proceed to (2) Tokenization. Otherwise, suppose [seuratObj].rda is in the directory ./mouse/. In R, we convert seuratObj into meta.tsv, matrix.mtx, genes.tsv, and barcodes.tsv, saving them in the same directory.

library(Matrix)
library(Seurat)

load("mouse/malcolm_rao_cx3cr1_mouse_032525_seuratObj_ann.rda")
write.table(seuratObj@meta.data, file = "mouse/meta.tsv", 
            sep = "\t", row.names = T, col.names = T, quote = F)
writeMM(seuratObj@assays$RNA@layers$counts, file = "mouse/matrix.mtx")
write.table(rownames(seuratObj), file = "mouse/genes.tsv", 
            sep = "\t", row.names = F, col.names = F, quote = F)
write.table(colnames(seuratObj), file = "mouse/barcodes.tsv", 
            sep = "\t", row.names = F, col.names = F, quote = F)

Notes

• In meta.tsv, the colname of cell IDs (i.e., barcodes) should be CellID.
• In matrix.mtx, raw read counts should be saved instead of processed or scaled data.

Tokenization

tokenize_data.py

• This script loads the scRNA-seq data adata.h5ad or the equivalent set {meta.tsv, matrix.mtx, genes.tsv, barcodes.tsv} from the directory ./[test_name]/, converts them into an intermediate [test_name].loom, and tokenizes [test_name].loom, saving the results in a new folder ./[test_name]/[test_name].dataset/.
• Human (the default [species]) or mouse gene symbols will be mapped to human Ensembl IDs through the GProfiler online search.

Usage

python tokenize_data.py [test_name] --species [species]

Examples

python tokenize_data.py mouse -s mouse  

python tokenize_data.py glioma -s human  

Notes

• adata.h5ad or matrix.mtx should contain raw read counts.
• Keep all genes and do not filter.

Fine-tuning

finetune.py

• This script fine-tunes the fundamental GPT model loaded from the directory ./Geneformer/ for a more specific context using a single reference dataset ./[ref_name]/[ref_name].dataset.
• The fine-tuned model will be saved in the folder ./[ref_name]/finetune.

Usage

python finetune.py [ref_name] --gpu_name [gpu_name]

Examples

python finetune.py aldinger_2000perCellType  

python finetune.py bhaduri_3000perCellType -g 2  

Notes

• The reference dataset should have been tokenized using tokenize_data.py and saved as ./[ref_name]/[ref_name].dataset.
• Run nvidia-smi to select an idle [gpu_name] with low Memory-Usage and GPU-Utility, default 0.

With the GPT models fine-tuned and the predictive PREPS models trained, it is easy to predict the electrophysiological features of a new scRNA-seq dataset (either human or mouse). Users can choose to run either the single script with the whole workflow integrated or separate scripts for flexible adjustment. Starting from an input [seuratObj].rda or adata.h5ad, the workflow consists of Tokenization, Annotation, and Electrophysiological feature/celltype prediction.

Annotation

annotate.py

• This script loads the tokenized data [test_name].dataset from the directory ./[test_name]/, extracts cell embeddings, and annotates cell types using fine-tuned GPT models, saving the results in a new folder ./[test_name]_preds/.
• Loading [test_name].dataset generates many temporary files within the folder. This script creates and works with ./[test_name]_preds/tokenized_copy.dataset to keep [test_name].dataset clean for future use, similar to finetune.py.

Usage

python annotate.py [test_name] --gpu_name [gpu_name]

Examples

python annotate.py mouse  

python annotate.py glioma -g 1  

Notes

• Each fine-tuned GPT model's folder should have been saved in the current directory (e.g., ./aldinger_2000perCellType, ./bhaduri_3000perCellType).
• ./[test_name]_preds/tokenized_copy.dataset can be deleted afterwards.

Electrophysiological feature/celltype prediction

patchseq_predict.py

• This script loads the pre-trained PREPS [models] (patchseq or celltype, default patchseq) to predict the electrophysiological features or cell types of the dataset [test_name] based on its cell embeddings loaded from the directory ./[test_name]_preds/.
• The predicted features or cell types are saved in the directory ./[test_name]_[models]/.

Usage

python patchseq_predict.py [test_name] --models [models]

Examples

python patchseq_predict.py mouse -m patchseq  

python patchseq_predict.py glioma -m celltype  

Notes

• The PREPS models with parameters grid-searched have been saved in the directory ./combined_patchseq_all_preds/. Do not change the folder or file names that contain keys to identify the optimal model for each feature or cell type prediction.

Attention-based DEEPS celltype scoring

scoring.py

• This script serves as the attention-based cell type scoring framework that combines transformer-derived gene attention values with literature-curated marker gene sets.
• It integrates multi-head attention patterns from fine-tuned Geneformer models and GPT-extracted marker weights from PubMed (queries of [tumor] OR [tissue]).
• Each cell in the testing dataset [test_name] is assigned an enrichment-based score across candidate cell types.

Usage

python scoring.py [tumor] [test_name] --species [species] --tissue [tissue] --gpu_name [gpu_name]

Examples

python scoring.py DIPG mouse -s mouse -t brain -g 0  

python scoring.py glioma glioma -s human -t brain -g 1  

Notes

• The testing dataset [test_name] should have been tokenized before running scoring.py.