S. mutans Variant and Phylogeny Analysis

Overview

This repository contains a reproducible bioinformatics pipeline developed to investigate the evolutionary relationships and genetic variations of Streptococcus mutans genomes using ancient DNA (aDNA) sequencing data. The workflow covers all computational steps, from processing raw sequencing reads to constructing and annotating phylogenetic trees.

Dataset

The study analyzes 25 ancient S. mutans samples with known archaeological contexts and age estimates.

• Reference Genome: GCF_009738105.1
• Detailed sample information, including estimated age, geographical origin, average read depth, and damage profiles (metaDMG Bayesian Z score), is documented in metadata/s_mutans_metadata.tsv.

Bioinformatics Pipeline

The analysis architecture is designed to run computationally intensive steps on a High-Performance Computing (HPC) cluster, while data visualization is handled in a local R environment.

1. Quality Control & Trimming: Read quality assessment using FastQC and adapter trimming.
2. Alignment: Reference mapping, SAM/BAM format conversions, and PCR duplicate marking.
3. Variant Calling: SNP identification using bcftools with parameters optimized for aDNA characteristics, followed by strict variant filtering (min4 filter).
4. Phylogeny: Maximum Likelihood (ML) phylogenetic tree construction using IQ-TREE 2 (featuring automatic model selection via ModelFinder and 1000 iterations of Ultrafast Bootstrap).
5. Annotation & Visualization: Integrating the phylogenetic tree with archaeological metadata and visualizing evolutionary distances using R (ggtree, tidyverse).

Repository Structure

S_mutans_Variant_and_Phylogeny_Analysis/
├── metadata/           # Sample metadata and environmental data (TSV)
├── results/            # Intermediate outputs (Alignment BAMs, VCFs, Logs)
│   └── phylogeny/      # Final tree files (.treefile) and visualization outputs
├── scripts/            # Pipeline scripts
│   ├── 01_qc/          # Quality control automation
│   ├── 02_alignment/   # Mapping scripts
│   ├── 03_variant_calling/ # bcftools SNP filtering
│   └── 04_phylogeny/   # Python (vcf2phylip.py) and RMarkdown annotation scripts
└── README.md           # Project documentation

Dependencies

The following software dependencies are required to execute the scripts in this repository:

• HPC / Linux Environment: bcftools, IQ-TREE 2
• R Environment (for Annotation): R (>= 4.0), ggtree, treeio, tidyverse, rmarkdown

Researchers

• Oğuzhan Işılay - Mersin University, Biotechnology
• Advisor: Assoc. Prof. Emrah Kırdök

Acknowledgment: The computational steps of this research were performed using the high-performance computing resources of TRUBA (Turkish National Science e-Infrastructure).

References

This pipeline builds upon essential methodologies and computational tools in ancient DNA analysis. If you adapt this workflow, please consider citing the original authors:

• aDNA Damage Estimation (metaDMG): Thygesen, C., et al. (2026). Genome Biology. DOI: 10.1186/s13059-026-04018-w
• Phylogenetic Inference (IQ-TREE 2): Minh, B. Q., et al. (2020). IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in the Genomic Era. Molecular Biology and Evolution.
• Tree Visualization (ggtree): Yu, G., et al. (2017). ggtree: an R package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution.