Metagenomics - iffatAGheyas/bioinformatics-tutorial-wiki GitHub Wiki

6.2 Metagenomics

In this module we will cover:

1. Overview & Experimental Design
   – Marker-gene (16S/ITS) vs. shotgun approaches
   – Study design considerations (replicates, depth, controls)

2. Data Types & Sequencing
   – 16S rRNA amplicon (V4, V3–V4 regions)
   – Shotgun metagenomics (short‐ and long‐reads)

3. Raw Read Quality Control
   – Demultiplexing & adapter trimming
   – Quality filtering (FastQC, MultiQC, cutadapt)

4. 16S/ITS Amplicon Analysis
   – DADA2 (error correction & ASV inference)
   – QIIME2 pipeline (plugin ecosystem)
   – Taxonomy assignment (SILVA, Greengenes)

5. Shotgun Taxonomic Profiling
   – Kraken2 / Bracken
   – MetaPhlAn3
   – Kaiju / Centrifuge

6. Metagenomic Assembly & Binning
   – Assemblers: MEGAHIT, metaSPAdes
   – Binning: MetaBAT2, MaxBin2, CONCOCT
   – Refinement & dereplication (DASTool, dRep)

7. Functional Profiling
   – HUMAnN3 (pathway abundance)
   – eggNOG-mapper (orthology annotation)
   – MG-RAST overview

8. Diversity & Statistical Analysis
   – Alpha diversity (Shannon, Simpson)
   – Beta diversity (Bray–Curtis, UniFrac; PCoA, NMDS)
   – Differential abundance (ANCOM, DESeq2 on counts)

9. MAG Quality & Annotation
   – CheckM completeness/contamination
   – BUSCO for single-copy orthologs
   – Prokka for genome annotation

10. Visualization & Reporting
    – Krona interactive plots
    – phyloseq (R) heat trees & barplots
    – Combined dashboards (MicrobiomeAnalyst)

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