This repository contains a small comparative genomics project on snake limb loss. The project asks whether snake limb reduction is more likely explained by changes in the SHH protein-coding sequence or by changes in a non-coding regulatory enhancer, especially the ZRS limb enhancer near SHH.
The Kaggle version provides a browser-based notebook and a public dataset page for viewing the analysis outputs more easily.
- Kaggle Dataset: https://www.kaggle.com/datasets/binivin/snake-shh-and-zrs-analysis
- Kaggle Notebook: https://www.kaggle.com/code/binivin/exploring-snake-shh-and-zrs
Use the Kaggle notebook to view the tables, heatmaps, bar plots, and written interpretation directly in a browser.
Do snakes show major sequence-level divergence in the Sonic hedgehog protein, or is the stronger signal found in the ZRS enhancer that regulates limb-specific SHH expression?
The analysis is built around a two-step hypothesis test.
- Protein-level test: compare SHH protein sequences across vertebrates.
- Regulatory-level test: compare a human ZRS enhancer window across vertebrates and scan ETS-core motif candidates.
If the SHH protein remains conserved in snakes but ZRS enhancer motifs are disrupted, the result supports a regulatory-change hypothesis rather than a simple protein-loss hypothesis.
The SHH protein FASTA file contains six vertebrate sequences retrieved from NCBI.
| Species | Group | Accession | Length |
|---|---|---|---|
| Homo sapiens | Mammal | NP_000184.1 | 462 aa |
| Mus musculus | Mammal | NP_033196.1 | 437 aa |
| Gallus gallus | Bird | NP_990152.1 | 425 aa |
| Anolis carolinensis | Lizard | XP_003221976.1 | 427 aa |
| Python bivittatus | Snake | XP_007433256.1 | 426 aa |
| Protobothrops mucrosquamatus | Snake | XP_015688035.1 | 426 aa |
The ZRS enhancer analysis uses a human reference window near SHH:
homo_sapiens 7:156791102-156791874
The Ensembl PECAN amniote alignment contains five species:
homo_sapiens
mus_musculus
gallus_gallus
anolis_carolinensis
pseudonaja_textilis
snake-limb-loss-shh-zrs-analysis/
├── README.md
├── requirements.txt
├── data/
│ ├── shh/
│ │ ├── accessions.csv
│ │ └── shh_selected.fasta
│ └── zrs/
│ ├── zrs_5species_aligned.fasta
│ └── zrs_5species_ungapped.fasta
├── results/
│ ├── shh_pairwise_similarity.csv
│ ├── zrs_pairwise_stats.csv
│ ├── zrs_sequence_lengths.csv
│ ├── zrs_ETS_motif_scan.csv
│ └── zrs_ETS_motif_summary.csv
├── figures/
│ ├── shh_similarity_heatmap.png
│ ├── zrs_pairwise_identity_heatmap.png
│ └── zrs_ets_motif_status.png
├── src/
│ ├── fetch_shh_sequences.py
│ ├── summarize_shh_sequences.py
│ ├── calculate_shh_similarity.py
│ ├── download_zrs_alignment.py
│ ├── scan_zrs_ets_motifs.py
│ └── visualize_results.py
├── notebooks/
│ └── snake_analysis.ipynb
└── docs/
└── project_summary_ko.md
pip install -r requirements.txtTo reproduce the SHH protein analysis:
python src/fetch_shh_sequences.py --email your_email@example.com
python src/summarize_shh_sequences.py
python src/calculate_shh_similarity.pyTo reproduce the ZRS enhancer analysis:
python src/download_zrs_alignment.py
python src/scan_zrs_ets_motifs.py
python src/visualize_results.pyFor a quick browser-based reproduction, open the Kaggle notebook instead:
https://www.kaggle.com/code/binivin/exploring-snake-shh-and-zrs
The SHH protein comparison shows high similarity between the two snake species and moderate-to-high similarity between snakes and other vertebrates.
Key examples from the pairwise SHH protein comparison:
| Species 1 | Species 2 | Similarity |
|---|---|---|
| Python bivittatus | Protobothrops mucrosquamatus | 95.31% |
| Anolis carolinensis | Python bivittatus | 87.35% |
| Homo sapiens | Python bivittatus | 76.41% |
| Homo sapiens | Protobothrops mucrosquamatus | 75.97% |
This result weakens the idea that snake limb loss was mainly caused by a complete loss of SHH protein function.
The ZRS alignment shows a stronger divergence signal in Pseudonaja textilis than in mouse, chicken, or anole when compared with human.
| Comparison with human | Ungapped identity |
|---|---|
| human vs mouse | 89.37% |
| human vs chicken | 88.17% |
| human vs anole | 82.32% |
| human vs Pseudonaja textilis | 69.46% |
The ETS-core motif scan identified eight human reference ETS candidate sites. In Pseudonaja textilis, only one of eight was conserved, one was mutated, and six were gap-disrupted.
| Species | Conserved | Mutated | Gap-disrupted | Total |
|---|---|---|---|---|
| mus_musculus | 7 | 0 | 1 | 8 |
| gallus_gallus | 7 | 0 | 1 | 8 |
| anolis_carolinensis | 5 | 2 | 1 | 8 |
| pseudonaja_textilis | 1 | 1 | 6 | 8 |
The combined result suggests the following pattern:
SHH protein sequence: relatively conserved
ZRS enhancer sequence: more disrupted in snake
ETS-core motif candidates: strongly disrupted in Pseudonaja textilis
Therefore, this project supports the hypothesis that snake limb reduction is more plausibly connected to changes in SHH regulation than to loss of the SHH protein itself.
This project is a comparative sequence analysis, not a functional experiment. Motif disruption does not automatically prove loss of enhancer activity. Also, only a small number of species and a limited set of ETS-core motifs were analyzed. Experimental validation would require reporter assays, gene expression data, or broader comparative genomics.
This project shows how bioinformatics can connect protein conservation, non-coding regulatory sequence evolution, and evolutionary developmental biology. It also demonstrates why coding sequences and enhancers should be analyzed separately when studying morphological evolution.