amber-codon-scanner

A Python library for scanning archaeal DNA sequences for UAG (amber) codons and classifying each as a likely pyrrolysine-coding codon or a true translation stop.

Built to support research from the Nayak lab:

Shalvarjian, Chadwick et al. (2025). Methanogenic archaea encoding pyrrolysine maintain ambiguous amber codon usage. PNAS 122(45):e2517473122.

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Background

Pyrrolysine (Pyl) is the 22nd genetically encoded amino acid, incorporated at UAG codons in certain methanogenic archaea including Methanosarcina acetivorans, M. mazei, and M. barkeri. These organisms use UAG ambiguously — the same codon serves as both a stop signal and a pyrrolysine codon depending on context.

This tool uses two evidence sources to classify each UAG:

Evidence	Description	Source
Mid-ORF context	UAG flanked by in-frame coding sequence with a downstream in-frame stop	Heuristic
PYLIS element	GC-rich stem-loop downstream that promotes Pyl-tRNA read-through	Heuristic pattern (see PYLIS section below)

⚠ Important: Classification is heuristic only. No validated sensitivity/specificity data exist for this classifier across all archaeal lineages. For definitive results, check for the pylTSBCD gene cluster in the genome (see below) and/or use experimental data.

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Installation

# From source
git clone https://github.com/CameronPiepkorn/amber-codon-scanner
cd amber-codon-scanner
pip install -e ".[dev]"

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Quick Start

from amber_codon_scanner import AmberCodonScanner, Classification, report

scanner = AmberCodonScanner(
    min_orf_length=10,          # codons upstream required to call mid-ORF
    downstream_stop_window=50,  # codons downstream to search for in-frame stop
    check_pylis=True,           # search for PYLIS-like element
)

codons = scanner.scan_sequence(my_dna_string, seq_id="MA0859_mttB")
print(report.summary(codons))

# Export
report.to_tsv(codons, "results.tsv")
report.to_json(codons, "results.json")

# Filter to candidates only
candidates = [c for c in codons if c.classification == Classification.PYL_CANDIDATE]

Scan a whole FASTA file

results = scanner.scan_fasta("my_genome.fasta")
for seq_id, codons in results.items():
    print(f"\n=== {seq_id} ===")
    print(report.summary(codons))

---

Classification Rules

mid-ORF	PYLIS detected	Classification
✅	✅	pyrrolysine_candidate
✅	❌	ambiguous
❌	✅	ambiguous
❌	❌	stop_likely

---

Confirming Results — pylTSBCD Gene Cluster

The most reliable confirmation of pyrrolysine encoding is the presence of the pyl biosynthesis gene cluster in the same genome:

Gene	Function
pylS	Pyrrolysyl-tRNA synthetase (charges tRNA with pyrrolysine)
pylT	Pyrrolysine tRNA (anticodon CUA, decodes UAG)
pylB/C/D	Pyrrolysine biosynthesis enzymes

To check for these genes using HMMER against your genome:

# Download pyl HMM profiles from Pfam / TIGRFAM
# Then:
hmmsearch --tblout pyl_hits.txt Pyl_synthetase.hmm my_genome.faa

Known pyrrolysine-encoding genes in M. acetivorans C2A (GenBank AE010299.1):

Locus	Gene	Contains UAG
MA0859	mttB	Yes (trimethylamine methyltransferase)
MA4384	mtmB	Yes (monomethylamine methyltransferase)

For the full list of pyrrolysine-encoding methyltransferases and their locus tags, query GenBank AE010299.1 directly or see the NCBI gene pages linked in the example FASTA file header.

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PYLIS Element Detection

The PYLIS (Pyrrolysine Insertion Sequence) element is a GC-rich stem-loop found downstream of UAG codons in pyrrolysine-encoding genes. It promotes read-through of UAG by the pyrrolysyl-tRNA.

This tool uses a relaxed heuristic pattern (GC-rich stem ≥5 bp + loop 4-8 nt + GC-rich continuation, within an 80 nt window downstream of UAG) to flag PYLIS-like structures. The detection parameters are heuristic choices, not calibrated from a published benchmark. This is intentionally permissive and will produce false positives in GC-rich genomes. Results are labelled pylis_detected: True only, not "PYLIS confirmed".

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Repository Structure

amber-codon-scanner/
├── amber_codon_scanner/
│   ├── __init__.py
│   ├── scanner.py      ← AmberCodonScanner, AmberCodon, Classification
│   ├── report.py       ← TSV / JSON / text export
│   └── utils.py        ← FASTA parser, codon table, reverse complement
├── tests/
│   └── test_scanner.py
├── examples/
│   ├── example_sequences.fasta   ← synthetic placeholders + NCBI links
│   └── basic_usage.py
├── .github/workflows/ci.yml
├── pyproject.toml
└── README.md

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Running Tests

pytest
pytest --cov=amber_codon_scanner

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Citation

If you use this tool in published research, please cite:

@article{shalvarjian2025,
  title   = {Methanogenic archaea encoding pyrrolysine maintain
             ambiguous amber codon usage},
  author  = {Shalvarjian, Katherine E and Chadwick, Garrett L and
             P{\'e}rez, Paloma I and Woods, Patrick H and Orphan, Victoria J
             and Nayak, Dipti D},
  journal = {Proceedings of the National Academy of Sciences},
  volume  = {122},
  number  = {45},
  pages   = {e2517473122},
  year    = {2025}
}

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License

MIT © 2024. See LICENSE.