Spatial and single-cell Transcriptomics Integration Tool for CHaracterization (STITCH)

Introduction

This Nextflow pipeline provides a comprehensive framework for tertiary analysis of Visium spatial transcriptomics data and single-cell RNA-sequencing (scRNA-seq). The pipeline includes the following modules:

1. Quality Control (QC): Evaluate the quality of the input data, and identify thresholds for QC metrics.
2. Normalization: Apply data normalization to ensure comparability between samples.
3. Data Integration and Merging: Combine multiple samples using integration-based analysis (high batch-effect) or merge-based analysis (minimal batch-effect or different sample-to-sample compositions).
4. Clustering: Identify clusters of cells or regions based on gene expression and/or spatial context.
5. Differential Expression Analysis: Detect differentially expressed genes across conditions or clusters.
6. Reporting: Generate summary reports for QC and a final analysis report.

This pipeline is designed to provide reproducible and efficient analysis workflows, generating both intermediate and final outputs. The pipeline is largely built on Seurat framework.

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Setup Instructions

1. Clone the Repository

Clone the pipeline repository to your local machine using the following command:

git clone https://github.com/Liuy12/STITCH.git
cd STITCH
## optional, specify .cache directory for renv
mkdir .cache/
export RENV_PATHS_CACHE="$PWD/.cache/"

2. Install R Dependencies

Ensure you have R version 4.4.1 installed. Open a new R session, then use renv to restore the required R packages:

renv::restore()

This will install all the necessary R packages specified in the repository, and might take a while.

3. Prepare the Sample Information Sheet

Create a sample information sheet in tab-delimited format with at least the following first three columns. Ensure that the first three column names are "sampleid", "condition", "secondary_output".

• sampleid: Unique identifier for each sample. Ensure the sample ids do not contain space or special characters.
• condition: Experimental condition for the sample, e.g. Control or Case.
• secondary_output: Path to the secondary output directory from Cell Ranger (scRNA-seq) or Space Ranger (Visium).

An example samplesheet.tsv:

sampleid    condition   secondary_output
sample1 control /path/to/sample1/outs
sample2 treatment   /path/to/sample2/outs

4. Modify the Configuration File

Adjust the provided configuration file (e.g., nextflow.config.scRNAseq) to suit your analysis. Some key parameters to examine/modify include:

• feature_list: Path to genes of interest, one gene per line; Final report will generate visualizations of expression levels for those genes.
• output_dir: Path to output directory.
• qc_only: Whether to perform stop after QC. THis could be helpful to identify cutoffs for various QC metrics.
• adaptive_cutoff_flag: Whether to apply adaptive cutoff idenfication (based on IQR). Rathern than selecting the same cutoffs across all samples, this will identify cutoffs based on distribtion of QC metrics within each sample to create sample-specific cutoffs.
• norm_dimreduc/norm_diff: Normalization method for dimension reduction/differential testing, either SCT or LogNormalize
• cellcycle_correction_flag: Whether to estimate and correct for cell-cycle effect.
• merge_analysis/integration_analysis: whether to perform merge-based/integration-based analysis.
• merge_only/integration_only: Whether to stop after merge-based/integration-based analysis.
• integration_method: Integration strategy.
• sketch_flag: Whether to perform sketch-based workflow.
• resolution: Resolution parameter used to identify number of clusters.
• spatial_cluster: Method for spatial clustering.
• control_var/case_var: control/case group for differential expression analysis.
• test: Statistical test.

5. Run the Pipeline

Execute the pipeline with the following command:

nextflow run main.nf --samplesheet samplesheet.tsv -c nextflow.config.scRNAseq -work-dir ./work 

• --samplesheet: Path to the prepared sample sheet.
• -c: Specifies the configuration file.
• -work-dir: Specifies processing directory.

The pipeline currenlty supports local(default, -profile local) or slurm (-profile slurm). You can modify the config file based on your own needs.

You can add -resume option to the command if you want to resume a pipeline.

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Notes

• Ensure all required dependencies (Nextflow, R, and other tools) are installed and configured.
• Customize the pipeline to suit your specific data and experimental design.
• For further assistance, consult the documentation or open an issue in the repository.