SCRATCH-QC Subworkflows

Introduction

This repository contains three Nextflow subworkflows for alignment (GEX and TCR), quality control (sample and cell filtering) and clustering analysis in single-cell RNA sequencing (scRNA-seq) data. The subworkflows are designed to handle and analyze provided input files, ensuring reproducibility and scalability.

Disclaimer: Subworkflows are chained modules providing a high-level functionality (e.g., Alignment, QC, Differential expression) within a pipeline context. These subworkflows should ideally be bundled with the pipeline implementation and shared among different pipelines as needed.

Prerequisites

Before running any of the subworkflows, ensure you have the following installed:

• Nextflow (version 21.04.0 or higher)
• Java (version 8 or higher)
• Docker or Singularity for containerized execution
• Git

Installation

Clone the repository to your local machine:

git clone https://github.com/WangLab-ComputationalBiology/SCRATCH-QC.git
cd SCRATCH-QC

Subworkflows

1. scratch_align_entry.nf

This subworkflow runs the alignment process for scRNA-seq data.

Usage

nextflow run scratch_align_entry.nf -profile [docker/singularity] --samplesheet <path/to/samplesheet> --modality <GEX|TCR|GEX+TCR> --genome <GRCh38|GRCm39>

Parameters

• --samplesheet: Path to the samplesheet file (default: assets/test_sample_table.csv).
• --modality: Modality type, either GEX, TCR, or GEX+TCR (default: GEX).
• --genome: Genome reference, either GRCh38 or GRCm39 (default: GRCh38).

Example

nextflow run scratch_align_entry.nf -profile docker --samplesheet data/samplesheet.csv --modality GEX --genome GRCh38

2. scratch_qc_entry.nf

This subworkflow performs QC checks on the GEX matrices.

Usage

nextflow run scratch_qc_entry.nf -profile [docker/singularity] --input_gex_matrices_path <path/to/gex_matrices> --input_exp_table <path/to/exp_table>

Parameters

• --input_gex_matrices_path: Path to the GEX matrices (default: data/SCRATCH_ALIGN:CELLRANGER_COUNT/**/outs/*).
• --input_exp_table: Path to the experiment table (default: data/pipeline_info/samplesheet.valid.csv).
• --expected_cells: Expected number of cells (default: 5000).
• --total_droplets: Total number of droplets (default: 15000).
• --fpr: False positive rate (default: 0.01).
• --epochs: Number of epochs (default: 150).
• --skip_cellbender: Skip Cellbender step (default: true).
• --thr_estimate_n_cells: Threshold for estimated number of cells (default: 300).
• --thr_mean_reads_per_cells: Threshold for mean reads per cell (default: 25000).
• --thr_median_genes_per_cell: Threshold for median genes per cell (default: 900).
• --thr_median_umi_per_cell: Threshold for median UMI per cell (default: 1000).
• --thr_n_feature_rna_min: Minimum threshold for RNA features (default: 300).
• --thr_n_feature_rna_max: Maximum threshold for RNA features (default: 7500).
• --thr_percent_mito: Threshold for mitochondrial gene percentage (default: 25).
• --thr_n_observed_cells: Threshold for observed cells (default: 300).
• --skip_scdblfinder: Skip scDblFinder step (default: false).
• -profile: Execution profile. Use docker or singularity depending on your containerization preference. Alternatively, you can create an HPC-aware profile for your institution.

Example

nextflow run scratch_qc_entry.nf -profile docker --input_gex_matrices_path data/gex_matrices/ --input_exp_table data/exp_table.csv

3. scratch_cluster_entry.nf

This subworkflow performs clustering on the merged Seurat object.

Usage

nextflow run scratch_cluster_entry.nf -profile [docker/singularity] --input_merged_object <path/to/seurat_object.RDS>

Parameters

• --input_merged_object: Path to the merged Seurat object file (default: data/SCRATCH_QC:SEURAT_MERGE/*_merged_object.RDS).
• --thr_n_features: Threshold for the number of features (default: 2000).
• --thr_n_dimensions: Threshold for the number of dimensions (default: 100).
• --input_integration_dimension: Integration dimension (default: auto).
• --input_group_plot: Group plot input (default: patient_id;timepoint).
• --thr_resolution: Clustering resolution threshold (default: 0.5).
• --thr_proportion: Clustering proportion threshold (default: 0.25).

Example

nextflow run scratch_cluster_entry.nf -profile docker --input_merged_object data/seurat_object.RDS

Configuration

The subworkflow can be configured using the nextflow.config file. Modify this file to set default parameters, profiles, and other settings. An institution profile should be created whenever running the pipeline in an HPC environment, please refer to Step-by-step guide to writing an institutional profile

Output

Upon successful completion, the results will be available in the specified output directory. You can open the reports in your browser to review the QC metrics and other outputs.

Documentation

For more detailed documentation and advanced usage, refer to the Nextflow documentation and the comments within the subworkflow scripts.

Contributing

Contributions are welcome! Please submit a pull request or open an issue to discuss any changes.

License

This project is available under the GNU General Public License v3.0. See the LICENSE file for more details.

Contact

For questions or issues, please contact:

• [email protected]
• [email protected]
• [email protected]