Metagenomics Principles and Workflow

Introduction to Metagenomics

Definition: Genomic analysis of microbial communities through direct extraction and sequencing or cloning of their DNA.
Purpose: Allows the study of microbial communities directly in their natural environment.

Definition: Genome of many individual microorganisms present in an environmental sample.
Importance:
- Microbial life exists in almost every environment, including extreme conditions (e.g., sea bottom, arctic ice, salt deserts).
- Microbes inhabit familiar environments (e.g., garden soil, plant leaves and roots, human skin, and gut).
- Metagenomics offers cultural, independent information about environmental microorganisms since most cannot grow in pure culture.

Isolation of Metagenomic DNA:
- Obtained from a sample of a defined environment encompassing genomes of the entire microbial population.
Analysis Approaches:
- Functional Metagenomics:
  - Construction of a metagenomics library.
  - DNA fragments are generated and ligated to a vector, creating a library of recombinant vectors.
  - Library used to transform a bacterial host for laboratory manipulation.
  - Screening the library to find genes expressing specific functions (e.g., UV radiation resistance).
- Metagenomic DNA Sequencing:
  - Provides insights into microbial and functional diversity.
  - Involves sequencing the 16S RNA gene or directly sequencing the metagenomic DNA.
  - Aligns sequences to generate a phylogenetic tree to reveal microbial diversity.
  - Reassembly of overlapping DNA fragments into longer sequences (contigs).
  - Genes are predicted, functionally and taxonomically annotated by comparison with reference databases.

Observational Power: Provides a broad lens for observing microbial communities.
Applications: Addresses fundamental questions of microbial diversity, evolution, and ecology.