Create Additional Probes on Unknown Mixed Samples

Overview

RiboZAP is designed to generate custom RNaseH probes for metatranscriptomics samples without prior knowledge of sample compostion. Addition of these probes to RiboZero Plus Microbiome kit will significantly improve depletion of additional ribosomal RNA (rRNA) regions.

Depleting rRNA, which typically constitutes 80–90% of total RNA, before sequencing significantly reduces sequencing costs and increases the coverage of mRNA.

Installation

Prerequisites

• Docker
• Python3

Clone and install:

git clone https://github.com/ilmn-mouse-cecal/RiboZAP.git
cd RiboZAP
pip install .
docker build -t ribozap:latest .

Sample Sheet Format

Your input must be a CSV with three columns:

sample_id,read1,read2

An example sample sheet is included here

Run the App:

Probe design:

ribozap \
  --sample-sheet samplesheet.csv \
  --output-dir ./my_out/ \
  --analysis-name my_analysis \
  --cpus 4 \
  --memory 16 \
  --image ribozap \
  --image-tag 'latest' \
  --resume

Validation: For validation, --probes-fasta and --probes-summary are required. These are produced by the probe design step (see outputs under your --output-dir). Example:

ribozap \
  --sample-sheet samplesheet.csv \
  --output-dir ./my_out/ \
  --analysis-name my_analysis \
  --probes-fasta ./my_out/reports/probes.fasta \
  --probes-summary ./my_out/probes/probes.summary.csv \
  --cpus 4 \
  --memory 16 \
  --image ribozap \
  --image-tag 'latest' \
  --resume

Code Style

The codebase follows PEP 8 conventions and uses Black for formatting.

Citation

If you use ribozap in your research, please cite our publication:

Bunga, S., Tan, A., Roos, M., & Kuersten, S. (2025). RiboZAP: Custom probe design for rRNA depletion in complex metatranscriptomes. bioRxiv, 2025-11.