name: bio-ribo-seq-initiation-site-mapping description: Map translation initiation sites, including non-AUG and alternative starts, from initiation-drug ribosome profiling (TI-seq). Use when locating start codons, detecting near-cognate or upstream initiation, or analyzing harringtonine, lactimidomycin (GTI-seq/QTI-seq), or retapamulin (Ribo-RET) data. origin: openai4s category: bioskills/ribo-seq metadata: tool_type: mixed primary_tool: Ribo-TISH third_party: name: GPTomics/bioSkills repository: https://github.com/GPTomics/bioSkills commit: d91ed3d563019e649dc854c56ccd62551359488a license: MIT
Reference examples tested with: Ribo-TISH 0.2.7+, PRICE/GEDI 1.0.5+, samtools 1.19+
Before using code patterns, verify installed versions match. If versions differ:
- CLI: <tool> --version then <tool> --help to confirm flags
- Python: pip show <package> then help(module.function) to check signatures
If code throws ImportError, AttributeError, or TypeError, introspect the installed package and adapt the example to match the actual API rather than retrying.
"Map where translation starts in my Ribo-seq data" -> Locate translation initiation sites (TIS) at single-nucleotide resolution, including non-AUG and upstream starts, from initiation-drug profiling experiments.
- CLI: Ribo-TISH for TIS detection from harringtonine/LTM data; PRICE for EM-based cryptic-start detection
This is a distinct analysis from elongation ORF detection: it asks WHERE initiation occurs (which start codon), not which ORF bodies are translated. It typically requires a dedicated initiation-drug library paired with a standard elongation library.
| Method | Drug(s) | Signal | Citation |
|---|---|---|---|
| Harringtonine TIS | harringtonine | binds free 60S, blocks the first peptide bond; broad start peak | Ingolia 2011 |
| GTI-seq | lactimidomycin (LTM) + CHX in parallel | LTM blocks translocation at the assembled 80S; sharp start peak | Lee 2012 |
| QTI-seq | LTM then puromycin (sequential) | puromycin strips elongating ribosomes; quantitative, low background | Gao 2015 |
| Ribo-RET (bacteria) | retapamulin | arrests initiating 70S at start codons | Meydan 2019 |
All three drugs CREATE the initiation signal by halting or removing elongation; the data is a deliberate artifact read out at the start codon. LTM gives sharper peaks than harringtonine because it cannot act on elongating ribosomes whose E-site is occupied. QTI-seq (LTM then puromycin) is analyzed on the same LTM path below; the puromycin step only strips elongating ribosomes to lower the background, so the TIS library is still passed as the LTM-type -t input. Without an initiation-drug library, start codons can only be inferred indirectly from elongation periodicity (see orf-detection).
Initiation occurs at AUG and near-cognate codons differing by one base; the biologically used set is CUG, GUG, ACG, UUG, AUU, AUC, AUA (AAG/AGG also differ by one base but initiate negligibly). CUG is the dominant near-cognate start (~16% of mapped sites in GTI-seq; AUG remains >50%). uORFs especially use near-cognate starts, so initiation mapping must enable alternative start codons to recover them; an AUG-only search misses most upstream initiation.
| Situation | Tool | Why |
|---|---|---|
| TIS from harringtonine/LTM data, with QC | Ribo-TISH | quality + predict modes; near-cognate via --alt; differential TIS |
| Cryptic/near-cognate starts, EM model | PRICE | per-codon EM; handles near-cognate; designed for cryptic events |
| Bacterial initiation (Ribo-RET) | dedicated retapamulin analysis | prokaryote initiation; eukaryote periodicity tools fit poorly |
Goal: Confirm the drug enriched start-codon signal and pick P-site offsets before predicting.
Approach: Run Ribo-TISH quality, which reports the metagene profile and writes a per-length offset parameter file.
# Writes a <ribo.bam>.para.py offset file and a QC figure
ribotish quality -b ribo_elongation.bam -g annotation.gtf -o ribo_quality.txt -f ribo_qc.pdf
ribotish quality -b ribo_tis.bam -g annotation.gtf -o tis_quality.txt -f tis_qc.pdf
Goal: Call TIS, including non-AUG starts, using the initiation-drug library.
Approach: Run ribotish predict with the elongation BAM (-b) and the TIS/harringtonine/LTM BAM (-t), enabling alternative start codons.
# --harr marks the TIS library as harringtonine-type; --alt enables near-cognate starts
ribotish predict \
-b ribo_elongation.bam \
-t ribo_tis.bam \
-g annotation.gtf \
-f genome.fa \
--harr --harrwidth 15 --alt \
-o tis_predictions.txt
The output lists initiation sites with the start codon, ORF type, and significance. For differential initiation across conditions, ribotish tisdiff compares two TIS libraries.
Goal: Detect cryptic and near-cognate initiation with an EM model.
Approach: Prepare the genome and run the Price tool in GEDI on the Ribo-seq reads.
gedi -e Price -reads ribo_elongation.bam -genomic prepared_genome -prefix price_out
PRICE reports a per-ORF p-value from a generalized binomial model (not multiple-testing corrected); codon-level activity is written to price_out.codons.cit.
A called TIS is strongest when it shows a sharp drug-induced start peak, a downstream in-frame elongation signal in the standard library, and (for novel sites) conservation or peptide support. Alternative N-terminal starts and uORF starts frequently use near-cognate codons; report the start codon identity, not just the position. Initiation at a uORF does not guarantee a stable protein product (see orf-detection validation).
| Symptom | Cause | Fix |
|---|---|---|
| Only AUG starts found | Alternative starts not enabled | Add --alt (Ribo-TISH) or use PRICE for near-cognate |
| Broad, smeared start peaks | Harringtonine data treated as sharp LTM data | Use --harr; expect broader peaks than LTM |
predict gives weak calls |
Missing the paired elongation BAM (-b) | Provide both -b (elongation) and -t (TIS) libraries |
| TIS analysis on elongation-only data | No initiation-drug library present | Initiation mapping needs harringtonine/LTM/RET data; otherwise infer from periodicity |
| Bacterial data mis-called | Eukaryote TIS tool on Ribo-RET data | Use a retapamulin/prokaryote initiation workflow |