scE2G

This project is licensed under the terms of the MIT license.

Input: Single-cell ATAC-seq or paired ATAC and RNA-seq (mulitome) data per cell cluster

Output: Genomewide enhancer-gene regulatory link predictions per cell cluster

The pipeline consists of the following components:

1. Compute ABC model predictions for each cell cluster
2. Generate E2G features from ABC predictions
3. If running scE2G (Multiome): compute Kendall correlation and/or ARC-E2G score for each cell cluster
4. Combine 2 & 3 to construct a feature file to be used as input to predictive model
5. (optional) Train predictive model using CRISPR-validated E-G pairs from K562 dataset
6. Apply trained model to make predictions by assigning a score to each E-G pair

Set up

Clone the repo and set it up for submdoule usage

git clone --recurse-submodules https://github.com/EngreitzLab/scE2G.git
git config --global submodule.recurse true

When running for the first time, the conda environments have to be setup. For speed, it's recommended that your current environment has mamba installed

conda config --set channel_priority flexible  # Make sure your conda config uses flexible channel packaging to prevent unsatisfiable errors
conda create -n mamba -c conda-forge mamba "python<=3.11"
conda activate mamba
mamba install -c conda-forge -c bioconda snakemake=7

Apply model

Before running this workflow, users should perform clustering to define cell clusters through regular single-cell analysis (such as Seurat & Signac). Required input data includes (refer to the example data in the resources/example_chr22_multiome_cluster folder):

1. Pseudobulk fragment files and their corresponding *.tbi index files in the same directory for each cell cluster
   • Must be sorted by coordinates and gzipped. If it is not sorted, you can use the sortBed tool from bedtools: sortBed atac_fragments.unsorted.tsv > atac_fragments.tsv.
   • Must have 5 columns (no header) corresponding to chr, start, end, cell_name, read_count (usually just 1)
   • The cell_name column should correspond to the cell names in the RNA count matrix. All cells in the RNA matrix must be represented in the fragment file.
   • To create a .tbi index, use bgzip from HTSlib instead of gzip to compress the fragment file: bgzip atac_fragments.tsv, then generate the corresponding .tbi index file using tabix -p bed atac_fragments.tsv.gz.
2. For scE2G (Multiome): RNA count matrix (gene x cell) for each cell cluster
   • Use unnormalized (raw) counts
   • Must be in either .csv.gz or .h5ad format
   • Ensure there are not duplicated gene names

To configure the pipeline:

• Modify config/config.yaml to specify paths for results_dir.
• Modify config/config_cell_clusters.tsv to specify the RNA matrix path, fragment file path, Hi-C file path, Hi-C data type, Hi-C resolution, TSS coordinates, and gene coordinates for each cell cluster. If running scE2G (ATAC), leave the RNA matrix path blank.
• Specify model directory from those in models/ to be applied

Running the pipeline:

snakemake -j1 --use-conda

This command make take a while the first time you run it, as it needs to build the conda environments. But if it takes more than 1 hour, that's usually a bad sign, meaning that you're not using mamba and/or need more memory to build the environment.

Output will show up in the results/ directory by default, with the structure results/cell_cluster/model_name/encode_e2g_predictions.tsv.gz. The score column to use is E2G.Score.qnorm.

Train model

Important: Only train models for biosamples matching the corresponding CRISPR data (in this case, K562)

Modify config/config_training.yaml with your model and cell_cluster configs

• model_config has columns: model, dataset, ABC_directory, feature_table, polynomial (do you want to use polynomial features?) Note that trained models generated using polynomial features cannot directly be used in the Apply model workflow
• cell_cluster_config has rows representing each "dataset" in model_config, where each "dataset" must correspond to a "cluster" in cell_cluster_config
  • If an ABC_directory is not specified for a dataset, its entry in cell_cluster_config must also contain the required ABC biosample parameters
  • TO DO: specify how to generate and formats for Kendall parameters
• To apply a trained model, it must contain the following files: 1) model.pkl, 2) feature_table.tsv, 3) score_threshold_.XX , 5) tpm_threshold_YY (YY=0 if ATAC-only model), 4) qnorm_reference.tsv.gz (single column with header E2G.Score that contains raw scores for genomewide predictions)

Running the pipeline:

snakemake -s workflow/Snakefile_training -j1 --use-conda

Output will show up in the results_training/ directory by default