TCRclub

a tool to clustering T cells by integrating sc-RNA seq and sc-TCR seq on local harmony

Introduction

TCRclub is a novel approach that identifies the functional relevance of T cells at single-cell resolution. TCRclub receives scRNA expression and the numeric embeddings of the CDR3β sequences as inputs. It aims to bridge the gap between scRNA expression and TCRs by focusing on the inner relationship between T cells with similar functions. To achieve this, TCRclub models the relationship between pair-wise TCR embedding and pair-wise expression distances according to the local harmony. Local harmony means the nearby homogeneity existing in the local neighbours of any cell, since the neighbouring cells in the distance space are more likely to have similar characteristics and belong to the same category. By emphasizing local harmony, TCRclub reduces noise and increases the robustness of integration. Considering the built-in cell structure, TCRclub builds the T-cell hierarchy based on the distances updated by the integration and extracts the T-cell clubs. Finally, TCRclub repeats multiple times to obtain the consensus results of the clubs as the final output.

Prerequisite

TCRclub is implemented in Python and requires a GPU for the acceleration at default. we recommend using CUDA versions 11.2 or 11.6 along with cudnn8.1.0 for the users who have access to GPU. If you intend to utilize CUDA with different versions, please ensure compatibility of your CUDA environment with TensorFlow versions 2.6 to 2.9.

If you do not have a GPU, TCRclub can still be utilized with a CPU, but it may not meet speed expectations. For your convenience, you can also try our webpage interface TIMEDB-TCRclub ( Please note that the web-sever is not equipped with GPU. Due to high demand in the sever, it may take additional time to receive the output file. We are continuously working to update the server.)

Environment Setup

To avoid conflicts with your existing environment, we suggest creating a new Anaconda environment. There are several ways to set up the environment:

1. Use Provided Conda Environment （Recommended)

You can directly utilize the provided conda environment TCRclub or TCRclub on google drive using the following instructions. If you encounter any download problems, please open an issue and let me know.

# Navigate to your anaconda3 directory 
cd /home/XX/anaconda3/envs/
 
mkdir -p TCRclub
tar zxvf /<the path you store the provided .tar.gz>/TCRclubConda.tar.gz -C TCRclub
source /home/XX/anaconda3/envs/TCRclub/bin/activate
conda-unpack

# Check the new environment
conda env list
## Output
conda environments:
base    /home/XX/anaconda3
TCRclub */home/XX/anaconda3/envs/TCRclub

2. Create Conda Environment

• Clone the repository.
• Create a conda environment with python3.8 or python3.9, for example conda create -n <Environment Name> python=3.8
• Activate the conda environment you just created. Navigate to the TCRclub directory and execute install.sh. Please note that in install.sh, the default version of PyTorch is 1.13.0 + CUDA 11.6, and the version of TensorFlow is 2.9.0. You can reinstall the proper version of PyTorch and TensorFlow that are suitable for your CUDA version.

3. Use Your Existing Environment

You have the option to install the required Python packages manually in your existing environment. However, for the successful execution of TCRclub, we recommend installing the following Python packages in sequential order along with their respective versions:

• pyseat (version 0.0.1.4)
• tensorflow (version 2.6.0~2.9.0)
• torch (version 1.13.0)
• numba (version 0.59.0)
• networkx (version 2.8.8)

pySEAT can be installed by the following command:

pip install pyseat

Running TCRclub

TCRclub includes a VAE model for converting CDR3β sequences into embeddings. To ensure the proper functioning of TCRclub, please download the "autoencoder" folder and the Python script TCRclub.py if you do not want to clone the whole repository.

Input data

TCRclub requires two files to identify functional-similar clubs for T cells. The first column of both files should be named "barcode", with each element being unique. Each row represents a single T cell, and the order of T cells should be the same in both files.

1. sc-TCR sequences file

For the sc-TCR sequences file, two columns are necessary: "barcode" and "cdr3". If the file contains cells from multiple samples, you should include a column labelled "sample" to indicate the source of each T cell. Because of the setting of our autoencoder, CDR3 longer than 30 should be removed before using TCRclub to avoid insufficient embedding.

Fig.1 An example of required scTCR file in .csv format.

2. sc-RNA expression file

For the sc-RNA expression file, each row corresponds to a T cell, and the columns (except the first column) correspond to genes. The first column should be named as "barcode". We suggest using the top 10% of highly variable genes extracted from the original sc-RNA expression file. You can select the input genes according to your own criteria. Normalization and log-transformation are recommended. Be cautious with selecting too many genes, as it may cause GPU memory issues.

Fig.2 An example of a required scRNA expression file in .csv format.

Parameters

TCRclub accepts several parameters, as listed in the table below:

Parameters	Description
tcr_file	(Required) The path of the .csv file contains the scTCR sequences (see Input data).
rna_file	(Required) The path of the .csv file contains the scRNA expression (see Input data).
k	Number of selected neareast neighbours. Default: 10.
repeat_times	Repeat times for obtaining the consensus results. Default: 50. Set to 1 if using fixed_initialization.
beta	L2 regularization parameter. Default: 1e-7.
single_cutoff	Cut-off parameter to split the cell hierarchy in a single run. Default: 1e-4.
con_cutoff	Cut-off parameter to split the cell hierarchy based on the consensus matrix. Default: 5e-4.
con_topk	Parameter to choose the number of results with the smallest loss from the repeat_times results to produce the consensus matrix. Default: 15.
out	Output directory name.
epoch	Iterations for each execution to convergence. Default: 1000.
multiple_sample	A binary value indicating whether the input T cells are derived from different samples. If this parameter is selected, the input TCR file should contain a column specified as "sample". Default: False
fixed_initialization	A binary value indicating whether the initialization of TCRclub starts in the default way (randomness). If this parameter is selected, the initialization of matrix C in TCRclub will be fixed. In this case, we suggest the parameter repeat_times should be set as 1. Default: False.
CPU	TCRclub runs on GPU by default. If you do not have access to a GPU, please select this parameter. Default: False

TCRclub has several hyper-parameters such as k, beta, single_cutoff and con_cutoff. All of them can influence the clustering performance and can be adjusted. For users seeking to fine-tune clustering coverage and its associated purity as immediately as possible, adjusting the con_cutoff parameter is advised. This approach offers the quickest adjustment, allowing users to increase (or decrease) con_cutoff for higher (or lower) clustering coverage in the consensus result.

Identifying T-cell clubs

TCRclub runs on GPU by default.

If you do not have access to a GPU, simply append --CPU to the end of the command.

To identify T-cell clubs, follow the instructions below based on your specific scenario:

1. If the cells come from a single sample/patient, run the Python script TCRclub.py using the following command:

python3 TCRclub.py --tcr_file ./example_data/processed_tcr.csv --rna_file ./example_data/processed_rna.csv

CPU command: python3 TCRclub.py --tcr_file ./example_data/processed_tcr.csv --rna_file ./example_data/processed_rna.csv --CPU

2. If the cells come from multiple samples/patients, run python script TCRclub.py with the additional --multiple_sample parameter:

python3 TCRclub.py --tcr_file ./example_data/processed_tcr.csv --rna_file ./example_data/processed_rna.csv --multiple_sample

CPU command: python3 TCRclub.py --tcr_file ./example_data/processed_tcr.csv --rna_file ./example_data/processed_rna.csv --multiple_sample --CPU

3. If you choose the fixed_initialization option, run Python script TCRclub.py with the following command.

python3 TCRclub.py --tcr_file ./example_data/processed_tcr.csv --rna_file ./example_data/processed_rna.csv --fixed_initialization --repeat_times 1

CPU command: python3 TCRclub.py --tcr_file ./example_data/processed_tcr.csv --rna_file ./example_data/processed_rna.csv --fixed_initialization --repeat_times 1 --CPU

The T-cell clubs will be saved in the "consensus_result.csv" file within the output directory specified by the out parameter. The output file will include a new column named "club" in the input TCR file, where T cells with the same club ID are considered to belong to the same club. Fig.3 An example of the produced result in .csv format.

Tutorial (onGPU) is available to guide you through the process step by step.