Bio Ribo Seq Initiation Site Mapping

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)

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

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.

# 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

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.

# --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.

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

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