Shotgun Metagenomic Analysis: Taxonomic Profiling, Functional Pathways & Genome Binning

Conducted every alternate Sunday (10:00 AM – 5:00 PM IST · Live Online). Kindly register below and our team will get back to you with the upcoming cohort schedule and onboarding details.

Workshop Overview

Unlike 16S rRNA amplicon sequencing, whole-metagenome shotgun sequencing captures the entire genetic repertoire of complex microbial communities — resolving species and strains, unearthing metabolic pathways, profiling antimicrobial resistance (AMR), and reconstructing draft genomes of uncultured microbes. This hands-on workshop teaches the complete computational pipeline from raw sequencing reads to publication-ready biological insights.

1. Raw Read Quality Control & Host DNA Decontamination

• Understanding shotgun metagenomic study design, sequencing depth requirements, and paired-end FASTQ structure.

• Adapter trimming and quality filtering using FastQC, MultiQC, and fastp / Trimmomatic.

• Removing host (human, mouse, plant) contamination using Bowtie2 / KneadData before downstream profiling.

2. Read-Based Taxonomic Profiling at Species & Strain Resolution

• Ultra-fast k-mer classification with Kraken2 and Bayesian abundance re-estimation with Bracken.

• Clade-specific marker gene profiling with MetaPhlAn 4 and strain-level tracking.

• Merging taxonomic profiles into unified abundance tables and controlling false-positive low-abundance calls.

3. Functional Pathway, CAZyme & Resistome (AMR) Profiling

• Reconstructing microbial metabolic pathways and gene families using HUMAnN 3, UniRef, MetaCyc, and KEGG Orthology (KO).

• Screening for Antimicrobial Resistance Genes (ARGs) using CARD/RGI and AMRFinderPlus, virulence factors (VFDB), and carbohydrate-active enzymes (dbCAN / CAZy).

4. Metagenome Assembly & Metagenome-Assembled Genomes (MAGs)

• De novo metagenomic contig assembly using MEGAHIT and metaSPAdes, and evaluating assembly metrics with QUAST.

• Contig binning into Metagenome-Assembled Genomes (MAGs) using MetaBAT2, MaxBin2, and consensus refinement with DAS Tool.

• Assessing MAG quality (completeness and contamination) with CheckM2, dereplicating genomes with dRep, taxonomic classification via GTDB-Tk, and functional annotation with Prokka/Bakta & DRAM.