usage-guide.md

Ribo-seq Pipeline - Usage Guide

Overview

Complete workflow from Ribo-seq FASTQ to translation efficiency and ORF detection.

Prerequisites

# CLI tools
conda install -c bioconda cutadapt bowtie2 star sortmerna

# Python
pip install plastid ribocode

# R
BiocManager::install('riborex')

Quick Start

• "Analyze my Ribo-seq data from FASTQ to translation efficiency"
• "Run the ribosome profiling pipeline"
• "Detect translated ORFs from my Ribo-seq"

Example Prompts

Full Pipeline

"Run the complete Ribo-seq pipeline"

"Calculate translation efficiency from my ribosome profiling data"

Specific Steps

"Just run P-site calibration"

"Detect novel ORFs from my Ribo-seq data"

What the Agent Will Do

1. Trim adapters and filter by size
2. Remove rRNA contamination
3. Align to transcriptome
4. Calibrate P-site offsets
5. Calculate translation efficiency
6. Call translated ORFs
7. Generate QC reports

Tips

• Read length - 28-30nt typical for ribosome-protected fragments
• 3-nt periodicity - Key QC metric; should see strong triplet periodicity
• P-site offset - Usually 12-13nt from 5' end for 28-30nt reads
• rRNA removal - Critical; >80% can be rRNA in raw data
• Paired RNA-seq - Required for translation efficiency calculation