Ribo-seq Pipeline - Usage Guide

Overview

Complete workflow from Ribo-seq FASTQ through periodicity QC, P-site calibration, ORF detection, and differential translation efficiency. The pipeline gates each downstream analysis on library quality and records the two upstream decisions (harvest/drug and UMIs) that determine which conclusions are valid.

Prerequisites

conda install -c bioconda cutadapt umi_tools bowtie2 star plastid samtools ribocode

BiocManager::install(c("riboWaltz", "riborex", "DESeq2"))

Quick Start

• "Analyze my Ribo-seq data from FASTQ to translation efficiency"
• "Run the ribosome profiling pipeline with periodicity QC"
• "Detect translated ORFs and differential TE from my Ribo-seq"

Example Prompts

Full Pipeline

"Run the complete Ribo-seq pipeline from FASTQ"

"Process my footprints and paired RNA-seq through to differential TE"

Specific Steps

"Just calibrate P-site offsets with riboWaltz"

"Gate ORF detection on the periodicity QC result"

"My library has UMIs - include extraction and deduplication"

What the Agent Will Do

1. Extract UMIs, trim, deplete rRNA, align end-to-end, and deduplicate (UMIs only)
2. Run periodicity QC and calibrate per-length P-site offsets
3. Gate downstream analyses on the frame-0 fraction
4. Detect ORFs with RiboCode
5. Test differential TE with a count-based GLM
6. Optionally run stalling or initiation-site mapping

Tips

• Harvest gates dwell-time - CHX pre-treatment invalidates stalling analysis
• UMIs gate dedup - deduplicate only with UMIs
• End-to-end alignment - soft-clipping corrupts P-site offsets
• Periodicity is a hard gate - no periodicity means gene-level counts only
• RiboCode read lengths come from metaplots, not -l
• Differential TE uses count GLMs (riborex/Xtail/anota2seq), not ratio tests

Related Skills

• ribo-seq/riboseq-preprocessing - Preprocessing decisions
• ribo-seq/ribosome-periodicity - Periodicity QC and offsets
• ribo-seq/orf-detection - ORF calling
• ribo-seq/translation-efficiency - Differential TE
• ribo-seq/initiation-site-mapping - Start-codon mapping
• differential-expression/deseq2-basics - Count-based testing