🌺 Cacao Flower Microbiome Project Overview

This repository contains the bioinformatics analysis pipeline for investigating the effects of pollinators on flower-associated microbial communities in cacao (Theobroma cacao L.) across deforestation gradients in Ghana.

🔬 Research Questions

1. Microbial Community Characterization: Describe bacterial and fungal T. cacao flower communities for the first time with a focus on pathogens and mutualistic symbionts;
2. Management Effects: Investigate how farm management type affects flower microbiomes along the deforestation gradient;
3. Pollinator Footprint: Identify the microbial signature of flower-visitng animals;
4. Functional Correlations: Examine relationships between microbiome structure and the success of the early step of fertilisation.

🏞️ Study Design

Experimental Framework & Sampling Strategy

• Visitint insect exclusion experiment: 3 bagged + 3 openly pollinated flowers per tree
• Deforestation gradient: 4 management types across 7 farms in Ghana at different stages of extensive farming:
  • Full sun (2 farms)
  • Agroforest (2 farms)
  • Near forest (2 farms)
  • Inside tropical forest (1 farm)
• Biological samples: 294 flowers (7 trees/farm × 6 flowers/tree)
• Controls: 42 total (14 extraction + 14 PCR negative + 14 mock communities)

Molecular Methods

• Marker genes: 16S rRNA V4 (515f/806r, bacteria) + ITS1 (ITS1f/ITS2, fungi)
• Sequencing platform: Illumina NovaSeq 2×250bp
• Multiplexing and indexing: Two-level approach enabling high sample throughput with combinatorial dual-indexing (48 sublibraries, 336 samples per marker)

📁 Repository Structure

cacao_flower_microbiome/
├── docs/                          # Analysis documentation
│   └── analysis_log.md            # Detailed progress tracking
├── data/                          # Metadata and small data files
├── qiime2/                        # QIIME2 analysis pipeline
│   ├── scripts/                   # Analysis scripts
│   ├── import/                    # Raw data preprocessing and import into QIIME2
│   ├── denoise/                   # DADA2 ASV calling
│   ├── taxonomy/                  # Taxonomic classification
│   ├── filtered/                  # Quality- and taxonomy-filtered datasets
│   └── rarefaction/               # Alpha rarefaction analysis
├── logs/                          # SLURM job outputs
└── README.md                      # This file

🔄 Current Status

⚠️ This is an ongoing analysis repository

The analysis pipeline is under active development. Raw sequencing data will be deposited in public repositories upon publication.

🤝 Open Science & Reproducibility

All analysis scripts are documented for reproducibility. Development includes AI assistance for code optimisation. Methodology and results will be made fully available upon publication.

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Keywords: microbiome, anthosphere, pollination ecology, Theobroma cacao, deforestation, 16S rRNA, ITS1, QIIME2, metabarcoding, Ghana, pollen-pistil interaction