NuCSOI

Nuclease Cleavage Site and Overhang Identification

Description

NuCSOI processes paired-end sequencing data to identify nuclease cleavage sites on plasmid references. The pipeline performs quality control, mapping, coverage analysis, and statistical analysis to identify cleavage sites with single base-pair resolution.

Installation

pip install nucsoi

External dependencies (install separately):

• fastp (quality control)
• bwa (read mapping)
• samtools (BAM processing)

Inputs

• FASTQ files: Paired-end sequencing files (R1 and R2). Must be an even number of files.
• Plasmid reference: FASTA file containing the circular plasmid sequence.

Usage

nucsoi -f R1.fastq.gz R2.fastq.gz -p plasmid.fasta -o output_dir/

Use nucsoi --help for all available options.

Pipeline Stages

1. Quality Control: Filters reads using fastp with configurable quality threshold (default: Q30).
2. Plasmid Mapping: Maps quality-filtered reads to the plasmid reference using BWA. Handles circular references.
3. Coverage Analysis: Calculates coverage at each position. Identifies regions with coverage drop-offs.
4. Position Analysis: Statistical analysis of mapping positions. Applies multiple testing correction (Bonferroni and Benjamini-Hochberg).

Outputs

Results are written to the specified output directory:

output_dir/
├── inputs/
│   ├── raw_fastqgzs/          # Input FASTQ files
│   ├── qc_reads/              # Quality-controlled reads
│   └── plasmid/               # Plasmid reference
├── results/
│   └── plasmid_mapping_*/
│       ├── coverage_analysis.png
│       ├── coverage_data.csv
│       ├── coverage_zoomed_data.csv
│       ├── coverage_data.txt
│       ├── comprehensive_summary_plot.png
│       └── comprehensive_position_analysis.txt
├── scripts/                   # Analysis scripts
├── configs.yaml              # Configuration file
└── Makefile                  # Pipeline makefile

• coverage_analysis.png: Coverage plots for entire plasmid and zoomed regions
• coverage_data.csv: Coverage data for all positions
• coverage_zoomed_data.csv: Coverage data for zoomed regions
• coverage_data.txt: Coverage statistics
• comprehensive_summary_plot.png: Statistical analysis plots
• comprehensive_position_analysis.txt: Position statistics with multiple testing corrections

Options

• -f, --fastq-files: Paired FASTQ files (required)
• -p, --plasmid: Plasmid reference FASTA file (required)
• -o, --output-dir: Output directory (required)
• -q, --quality-cutoff: Quality cutoff for fastp (default: 30)
• --run-pipeline: Automatically run pipeline after setup
• --version: Show version number
• -h, --help: Show help message

License

MIT License