Cell Ranger ATAC Docker Pipeline

This repository contains a Dockerfile "recipe" of 10x Genomics Cell Ranger ATAC pipeline for processing ATAC-seq data.

Quick Start

1. Build Docker Image

docker build -t cellranger-atac:latest .

To log all the build process you can run

docker build -t cellranger-atac:latest . > >(tee -a log.txt) 2> >(tee -a log.txt >&2)

• > >(tee -a log.txt) captures standard output (stdout) and appends it to log.txt
• 2> >(tee -a log.txt >&2) captures standard error (stderr) and appends it to log.txt

2. Prepare Data Directories

Create required directories for data organization:

mkdir -p ~/cellranger_data/{fastqs,reference,output}

3. Data Preparation

• Place your FASTQ files in ~/cellranger_data/fastqs/
• Download reference data to ~/cellranger_data/reference/
• Output will be generated in ~/cellranger_data/output/

4. Run Analysis

Non-interactive docker container run

docker run -v ~/cellranger_data:/data cellranger-atac \
    cellranger-atac count \
    --id=run1 \
    --reference=/data/reference/refdata-cellranger-arc-GRCh38-2020-A-2.0.0 \
    --fastqs=/data/fastqs \
    --sample=SAMPLE_NAME

Interactive docker container run

docker run -it -v ~/cellranger_data:/data cellranger-atac bash

This command:

• -it enables interactive terminal
• -v mounts your local data directory
• bash starts an interactive bash shell

Once inside the container, you can:

• Navigate directories: cd /data
• Check Cell Ranger ATAC version: cellranger-atac --version
• Run commands directly:

cellranger-atac count \
    --id=run1 \
    --reference=/data/reference/refdata-cellranger-arc-GRCh38-2020-A-2.0.0 \
    --fastqs=/data/fastqs \
    --sample=SAMPLE_NAME

• Explore outputs in real-time
• Test different parameters
• Exit container when done: exit

The interactive session gives flexibility in monitoring the analysis within the container environment.

Input Requirements

FASTQ Files

• R1: Forward genomic reads
• R2: Cell barcode and sample index
• R3: Reverse genomic reads Example naming convention:

SAMPLE_S1_L002_R1_001.fastq.gz
SAMPLE_S1_L002_R2_001.fastq.gz
SAMPLE_S1_L002_R3_001.fastq.gz

Reference Genome

Download from 10x Genomics:

• Mouse genome (mm10)

wget https://cf.10xgenomics.com/supp/cell-atac/refdata-cellranger-arc-mm10-2020-A-2.0.0.tar.gz

• Human genome (GRCh38)

wget https://cf.10xgenomics.com/supp/cell-atac/refdata-cellranger-arc-GRCh38-2020-A-2.0.0.tar.gz

Expected Outputs

The pipeline generates several key outputs in the ~/cellranger_data/output/run1/ directory:

1. web_summary.html: A summary report of the analysis results.
2. cloupe.cloupe: A file for visualization in Loupe Browser.
3. filtered_peak_bc_matrix/: Directory containing filtered peak-barcode matrices.
4. filtered_tf_bc_matrix/: Directory containing filtered transcription factor matrices.
5. analysis/: Directory with various analysis results (clustering, dimensionality reduction, etc.).
6. possorted_bam.bam: Aligned reads in BAM format.
7. peaks.bed: Called peaks in BED format.
8. fragments.tsv.gz: Fragment file for custom analyses.

Key Metrics

• Number of cells detected
• Median fragments per cell
• TSS enrichment score
• Fraction of reads in peaks
• Fragment size distribution

Analysis Results

• Dimension reduction plots (UMAP/tSNE)
• Clustering information
• Peak annotations
• Cell-type assignments (if reference is provided)

Resource Requirements

Recommended specifications:

RAM: 32GB minimum CPU: 8 cores minimum Storage: 500GB minimum

Troubleshooting

If you encounter issues:

1. Ensure Docker has sufficient resources allocated.
2. Verify input data integrity and format.
3. Check for sufficient disk space.
4. Review logs in the output directory for specific error messages.

Additional Resources

• Cell Ranger ATAC Documentation
• 10x Genomics Support

License

10x Cell Ranger belongs to 10x. Licence is available here. No changes or any kind of licence infringement, or violations have been introduced to the software

Contact

For questions or support, please open an issue in this repository.