Metagenomics

1. Amplicon Profiling vs Shotgun Metagenomics

Traditional microbiology relies on laboratory culturing; however, over 99% of environmental and human gut microorganisms cannot be cultured using standard techniques. Metagenomics circumvents this limitation by sequencing microbial DNA extracted directly from environmental or host samples:

• Marker Gene / Amplicon Sequencing (16S rRNA / ITS): PCR amplifies hypervariable regions (such as V3–V4 of the 16S ribosomal RNA gene in bacteria or ITS in fungi). Advantage: Low cost ($20–$50/sample), tolerates high host DNA contamination. Limitation: Genus-level resolution only; no direct information on gene function or antimicrobial resistance.
• Whole Metagenome Shotgun (WMS): Unbiased sequencing of all extracted DNA (bacterial, viral, fungal, archaeal). Advantage: Species and strain-level resolution, enables de novo assembly of uncultured genomes (MAGs), direct profiling of metabolic pathways (HUMAnN 3), and antibiotic resistance genes (AMR).

2. Hands-on Shotgun Profiling & MAG Binning Commands

# Step 1: Remove host contamination (e.g. human/mouse genome)
bowtie2 -x GRCh38_bowtie2_idx -1 sample_R1.fastq.gz -2 sample_R2.fastq.gz \
        --un-conc-gz decontaminated_reads.fastq.gz -p 16 > /dev/null

# Step 2: Ultra-fast taxonomic profiling with Kraken 2 & Bracken
kraken2 --db standard_db/ --paired decontaminated_reads.1.fastq.gz decontaminated_reads.2.fastq.gz \
        --output kraken.out --report kraken.report --threads 16
bracken -d standard_db/ -i kraken.report -o bracken_species.txt -l S -t 10

# Step 3: Metagenome assembly and contig binning into MAGs
megahit -1 decontaminated_reads.1.fastq.gz -2 decontaminated_reads.2.fastq.gz \
        -o megahit_out/ -t 16
metabat2 -i megahit_out/final.contigs.fa -a contig_depth.txt -o bins/bin

# Step 4: Validate MAG quality with CheckM 2
checkm2 predict --threads 16 --input bins/ --output-directory checkm2_out/ -x fa