SPC Genome Pipelines

Pipelines for processing single-cell whole genome sequencing (CapWGS) and genome-transcriptome co-assay (CapGTA) data. Includes tools for alignment, cell detection, variant calling, and AnnData generation.

Installation

git clone https://github.com/SrivatsanLab/SPC_genome
cd SPC_genome

# Create the environment
micromamba create -n spc_genome -f environment.yml
micromamba activate spc_genome

Pipelines

CapWGS_PP.sh - Whole Genome Sequencing with Variant Calling

Complete pipeline for single-cell whole genome sequencing data:

• BWA-MEM alignment
• Cell detection from barcode counts
• Single-cell BAM extraction
• GATK variant calling (HaplotypeCaller + GenotypeGVCFs)
• AnnData generation with variant calls

Output: data/{sample}/, data/{sample}/sc_outputs/, results/{sample}/variants.h5ad

CapWGS_PP_QC_only.sh - Coverage QC Pipeline

QC-only pipeline for coverage benchmarking (no variant calling):

• BWA-MEM alignment
• Cell detection
• Single-cell BAM extraction
• BigWig generation
• Lorenz curve analysis for coverage uniformity

Output: data/{sample}/sc_outputs/, results/{sample}/ (QC metrics, Lorenz curves)

CapGTA_PP.sh - Genome-Transcriptome Co-Assay

Pipeline for simultaneous genome and transcriptome profiling:

• STAR alignment (separates DNA/RNA by splice junctions)
• Dual-modality cell detection
• Single-cell DNA and RNA BAM extraction
• BCFtools variant calling on DNA
• RNA count matrix generation
• Dual AnnData output (variants + gene expression)

Output: data/{sample}/sc_outputs/, results/{sample}/variants.h5ad, results/{sample}/rna_counts.h5ad

Usage Example

./CapWGS_PP.sh \
  -o sample_name \
  -1 /path/to/read1.fastq.gz \
  -2 /path/to/read2.fastq.gz \
  -r 1000000000 \
  -g /shared/biodata/reference/GATK/hg38/

Required arguments:

• -o Sample name (creates data/{sample}/ and results/{sample}/)
• -1 Read 1 FASTQ file(s)
• -2 Read 2 FASTQ file(s)
• -r Total read count
• -g Reference genome directory

Optional arguments:

• -s Scripts directory (default: .)
• -n Number of chunks for parallelization (default: 500)
• -t Temporary directory (default: /hpc/temp/srivatsan_s/SPC_genome_preprocessing/{sample}/)
• -h Show help message

All pipelines support the same argument structure and can optionally use config.yaml for default values.

Configuration (Optional)

Create config.yaml to set defaults:

processing:
  n_chunks: 500
  tmp_dir: /hpc/temp/srivatsan_s/SPC_genome_preprocessing

reference:
  genome_dir: /shared/biodata/reference/GATK/hg38/

data:
  read1: /path/to/read1.fastq.gz
  read2: /path/to/read2.fastq.gz
  read_count: 1000000000

output:
  sample_name: my_sample

Command-line arguments override config values.

Directory Structure

SPC_genome/
├── CapWGS_PP.sh              # Main WGS pipeline
├── CapWGS_PP_QC_only.sh      # QC-only pipeline
├── CapGTA_PP.sh              # Genome-transcriptome pipeline
├── scripts/                  # Pipeline scripts
│   ├── CapWGS/              # WGS + GATK scripts
│   ├── CapWGS_QC/           # Coverage QC scripts
│   ├── CapGTA/              # Genome-transcriptome scripts
│   ├── bulk/                # Bulk processing utilities
│   └── utils/               # Shared utilities
├── bin/                      # Experiment metadata and one-off scripts
├── data/                     # Alignments and single-cell outputs (gitignored)
│   └── {sample}/
│       ├── {sample}.bam     # Bulk alignment
│       └── sc_outputs/      # Single-cell BAMs, VCFs, bigwigs
├── results/                  # Final outputs (gitignored)
│   └── {sample}/
│       ├── variants.h5ad    # Variant AnnData
│       ├── rna_counts.h5ad  # RNA AnnData (CapGTA only)
│       └── *_qc_summary.csv # QC metrics
├── notebooks/                # Analysis notebooks
│   ├── K562_tree/           # K562 tree experiment analysis
│   ├── benchmarking/        # CapWGS benchmarking analysis
│   └── PolE_worm_pilot/     # C. elegans CapGTA analysis
└── environment.yml           # Micromamba environment

Analysis Notebooks

• K562_tree/ - K562 lineage tree experiment
  • sc_analysis.ipynb - Single-cell variant analysis
  • bulk_spectra_analysis.ipynb - Mutational spectra
• benchmarking/ - CapWGS validation
  • benchmarking.ipynb - Comparison with public scWGS datasets
• PolE_worm_pilot/ - C. elegans CapGTA analysis
  • PolE_worm_pilot_analysis.ipynb - Multi-modal analysis

Citation

Capsule-based Whole Genome Sequencing and lineage tracing - bioRxiv