Microbiome Analysis Pipeline

This repository contains scripts and workflows for conducting microbiome analyses using QIIME2 and R. The workflows are designed to handle metagenomic and taxonomic data, performing tasks such as sequence processing, differential abundance analysis, and visualization.

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Table of Contents

1. Overview
2. Prerequisites
3. Pipeline Details
   • QIIME2 Workflow
   • R Workflow
4. Usage
5. Outputs

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Overview

This repository supports two primary analysis pipelines:

1. QIIME2 Workflow: Focused on sequence processing, taxonomic classification, and phylogenetic diversity analysis.
2. R Workflow: Performs differential abundance analysis using ANCOMBC2 and creates detailed taxonomic visualizations.

These workflows are complementary and provide end-to-end support for microbiome data analysis.

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Prerequisites

Software and Dependencies

1. QIIME2 (v2021.8 or later)
   • Ensure all plugins like dada2, feature-classifier, phylogeny, and taxa are installed.
2. R (v4.0 or later)
   • Required R packages: tidyverse, phyloseq, qiime2R, microbiome, ANCOMBC, microViz, ggplot2, lme4.

Input Files

• QIIME2 Workflow:
  • Raw paired-end sequences in CasavaOneEightSingleLanePerSampleDirFmt format.
  • A pre-trained classifier for taxonomic classification (e.g., Silva database).
  • Metadata file in .tsv format.
• R Workflow:
  • Exported .qza and .qzv files from QIIME2.
  • Metadata file with relevant grouping columns.

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Pipeline Details

QIIME2 Workflow

The QIIME2 pipeline includes the following steps:

1. Data Import: Converts raw paired-end sequence data into QIIME2's SampleData[PairedEndSequencesWithQuality] format.
2. Denoising with DADA2: Removes noise and generates a feature table and representative sequences.
3. Taxonomic Classification: Classifies sequences using a pre-trained classifier (e.g., Silva database).
4. Diversity Analysis: Computes alpha and beta diversity metrics based on phylogenetic trees.
5. Visualization: Produces bar plots, tables, and diversity summaries.

R Workflow

The R pipeline is designed for downstream statistical and visualization tasks:

1. Import Data: Converts exported QIIME2 .qza files into phyloseq objects.
2. Differential Abundance Analysis:
   • Performs ANCOMBC2 at various taxonomic levels (e.g., Phylum, Genus).
   • Identifies significant taxa with adjusted p-values and log fold changes.
3. Visualization:
   • Generates bar plots with error bars for log fold changes.
   • Outputs results as .tsv files and high-resolution images.

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Usage

Running the QIIME2 Workflow

1. Update the QIIME2 paths in the Bash script (qiime2_workflow.sh).
2. Execute the script:

   bash qiime2_workflow.sh
   
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