This repository contains a reproducible workflow for the analysis of Illumina paired-end 16S rRNA sequencing data using Mothur.
The pipeline was developed during my MSc thesis and demonstrates a complete microbiome preprocessing workflow from raw FASTQ files to OTU distance matrix generation.
This workflow performs:
- Paired-end read assembly
- Quality filtering
- Dereplication
- Alignment to SILVA reference database
- Chimera detection and removal
- Pre-clustering to reduce sequencing noise
- Abundance filtering
- Distance matrix generation for downstream analysis
Microbiome-16S-mothur-pipeline
│
├── workflow/
│ └── mothur_workflow.md
│
├── scripts/
│ └── script-16S-microbiome-analysis.txt
│
├── example_logs/
│ └── mothur_log_snippet.txt
│
└── README.md
- Linux/Windows environment
- Mothur (v1.44+)
- SILVA reference database
Launch mothur and execute the commands from:
scripts/script-16S-microbiome-analysis.txt
The workflow produces:
- Cleaned and filtered 16S sequences
- Chimera-free dataset
- OTU distance matrix (0.03 cutoff)
These outputs was used for:
- Alpha diversity
- Taxonomy assignment
- Functional annotation
Example heatmap showing the relative abundance of dominant bacterial taxonomic groups across samples. Each cell’s color indicates the abundance of the OTU that contributed to the dissimilarity of the two clusters, based on the Simper analysis. The OTUs are ordered from those with the highest contribution to dissimilarity to those with the lowest.
Raw sequencing data are not included due to size and privacy constraints.
This repository focuses on workflow reproducibility and documentation.
Eleni Terpsidou
MSc Bioinformatics & Computational Biology