QIIME2 Microbiome Analysis Workflow

This repository contains a complete QIIME2 workflow for analyzing microbiome data from paired-end sequencing.

Prerequisites

• QIIME2 installed (recommended version: 2023.5 or later)
• Install DADA2 using conda(https://anaconda.org/qiime2/q2-dada2)
• Paired-end fastq.gz files in the data/ directory
• Properly formatted metadata file (metadata.tsv)

Setup Instructions

1. Activate QIIME2 environment:

   conda activate qiime2-2023.5  # or your QIIME2 environment name

2. Prepare your data:

   • Place all your fastq.gz files in the data/ directory
   • Files should be named with the pattern: SAMPLEID_1.fastq.gz and SAMPLEID_2.fastq.gz
   • Update the metadata.tsv file with your sample information

3. Run the workflow:

   bash qiime2_workflow.sh

Workflow Steps

1. Data Import: Imports paired-end fastq.gz files using a manifest file
2. Quality Control: Uses DADA2 for denoising, chimera removal, and ASV identification
3. Phylogenetic Analysis: Builds a phylogenetic tree for diversity analyses
4. Taxonomic Classification: Assigns taxonomy using the Silva 138 classifier
5. Diversity Analysis: Calculates alpha and beta diversity metrics
6. Statistical Tests: Performs statistical tests on diversity metrics based on metadata categories

Output Files

All output files are stored in the qiime2_output/ directory:

• qiime2_output/table.qza: Feature table (ASV counts)
• qiime2_output/rep-seqs.qza: Representative sequences
• qiime2_output/taxonomy.qza: Taxonomic classifications
• qiime2_output/rooted-tree.qza: Phylogenetic tree
• qiime2_output/diversity/: Directory containing diversity metrics

Visualizations are stored in qiime2_output/visualizations/:

• demux-paired-end.qzv: Quality plots for raw sequences
• table.qzv: Feature table summary
• rep-seqs.qzv: Representative sequences
• taxonomy.qzv: Taxonomy summary
• taxa-bar-plots.qzv: Taxonomic bar plots
• alpha-rarefaction.qzv: Rarefaction curves
• Various alpha and beta diversity visualizations

Interpreting Results

1. Rarefaction Curves: Check if curves plateau to ensure sufficient sequencing depth
2. Alpha Diversity: Compare diversity metrics across groups using statistical tests
3. Beta Diversity: Examine community composition differences between groups using PERMANOVA tests

Troubleshooting

• If the script fails at any step, check the error message for details
• Ensure QIIME2 is properly installed and activated
• Verify that your metadata file is properly formatted
• Check that your fastq.gz files follow the expected naming pattern

Customization

You can modify the following parameters in the script:

• DADA2 trimming parameters (trim-left-f, trim-left-r, trunc-len-f, trunc-len-r)
• Sampling depth for diversity analyses
• Taxonomic classifier