| 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"}} |
Version Compatibility
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.
Translation Initiation Site Mapping
"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.
Initiation-drug data types (which experiment produced the data)
| 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).
Near-cognate and alternative starts
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.
Tool selection
| 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 |
QC the initiation library
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.
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
Predict initiation sites with Ribo-TISH
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.
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.
Alternative: cryptic starts with PRICE
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.
Interpreting initiation sites
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).
Common Errors
| 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 |
Related Skills
- orf-detection - Call and validate the ORF bodies downstream of mapped starts
- ribosome-periodicity - Calibrate P-site offsets for both libraries
- riboseq-preprocessing - Align the elongation and initiation-drug libraries
- ribosome-stalling - Initiation drugs are not for elongation pausing
References
- Ingolia NT, Lareau LF, Weissman JS. 2011. Ribosome profiling of mouse embryonic stem cells reveals the complexity and dynamics of mammalian proteomes. Cell 147(4):789-802. doi:10.1016/j.cell.2011.10.002
- Lee S, Liu B, Lee S, Huang SX, Shen B, Qian SB. 2012. Global mapping of translation initiation sites in mammalian cells at single-nucleotide resolution. Proc Natl Acad Sci USA 109(37):E2424-E2432. doi:10.1073/pnas.1207846109
- Gao X, Wan J, Liu B, Ma M, Shen B, Qian SB. 2015. Quantitative profiling of initiating ribosomes in vivo. Nat Methods 12(2):147-153. doi:10.1038/nmeth.3208
- Zhang P, He D, Xu Y, et al. 2017. Genome-wide identification and differential analysis of translational initiation. Nat Commun 8:1749. doi:10.1038/s41467-017-01981-8
- Erhard F, Halenius A, Zimmermann C, et al. 2018. Improved Ribo-seq enables identification of cryptic translation events. Nat Methods 15(5):363-366. doi:10.1038/nmeth.4631
- Meydan S, Marks J, Klepacki D, et al. 2019. Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome. Mol Cell 74(3):481-493. doi:10.1016/j.molcel.2019.02.017
