Metagenomics & Microbiome Data Analysis
From environmental and clinical samples to uncultured microbial genomes. Master 16S amplicon denoising with DADA2 and QIIME 2, shotgun host decontamination, ultra-fast taxonomic profiling (Kraken2 & MetaPhlAn 4), metabolic pathway quantification (HUMAnN3), MAG binning (MetaBAT2), CheckM2 quality benchmarking, and antimicrobial resistance surveillance.
Curriculum Modules
Click on any chapter below to explore complete code, command lines, and ecological statistics.
1: The Microbiome Landscape: Amplicon vs Shotgun Metagenomics
16S/18S/ITS rRNA hypervariable regions vs whole metagenome shotgun (WMS), primer selection biases (V3-V4 vs V4), mock communities, and contamination controls.
2: Amplicon Read Processing, Error Modeling & ASVs with DADA2
OTUs at 97% identity vs exact Amplicon Sequence Variants (ASVs), DADA2 parametric error modeling, dereplication, paired-end merging, and chimera removal.
3: Taxonomic Classification & QIIME 2 Architecture
Reference databases (SILVA 138, Greengenes2, UNITE), naive Bayes classifiers, QIIME 2 artifacts (.qza, .qzv), provenance tracking, and taxonomic barplots.
4: Alpha and Beta Diversity Statistics & Ordination
Within-sample richness (Shannon, Simpson, Faith's PD), between-sample distance (Bray-Curtis, UniFrac), PCoA ordination, and PERMANOVA hypothesis testing.
5: Differential Abundance Testing: ANCOM-BC & MaAsLin2
The compositionality problem in microbiome sequencing, centered log-ratio (CLR) transformations, ANCOM-BC2 bias correction, ALDEx2, and multivariable linear modeling.
6: Shotgun Metagenomics QC & Host DNA Decontamination
Quality filtration with Fastp, aligning against host reference genomes (human/mouse), removing host contamination with kneaddata, and estimating microbial DNA proportions.
7: Ultra-Fast Taxonomic Profiling: Kraken2 & Bracken
Exact k-mer matching against Lowest Common Ancestor (LCA) trees with Kraken2, Bayesian reallocation of reads to species level with Bracken, and Krona charts.
8: Clade-Specific Marker Profiling with MetaPhlAn 4
Single-copy clade-specific marker gene catalogs, direct relative abundance estimation, viral and eukaryotic microbial profiling, and heatmaps with hclust2.
9: Functional Profiling & Metabolic Pathway Reconstruction with HUMAnN3
Tiered functional search (ChocoPhlAn pangenomes & UniRef90), MetaCyc metabolic pathways, pathway completeness and abundance, and MinPath parsimony.
10: De Novo Metagenome Assembly: metaSPAdes & MEGAHIT
Multi-species de Bruijn graphs with uneven sequencing depth, memory-efficient assembly with MEGAHIT, metaSPAdes for complex samples, and MetaQUAST validation.
11: Metagenome-Assembled Genomes (MAGs): Binning with MetaBAT2 & MaxBin
Tetranucleotide frequency (TNF) and co-abundance coverage profiles across samples, contig binning with MetaBAT2 and MaxBin2, and multi-binner refinement with DASTool.
12: MAG Quality Assessment (CheckM) & Taxonomic Placement (GTDB-Tk)
Single-copy marker gene completeness and contamination metrics with CheckM2, MIMAG standards, and phylogenomic taxonomic placement with GTDB-Tk.
13: Antimicrobial Resistance (AMR) & Viral Metagenomics
Antimicrobial resistance profiling with CARD RGI and ResFinder, virulence factors (VFDB), bacteriophage identification with VirSorter2, and viral quality with CheckV.