Metagenomics principles and workflow. Metagenomics is the genomic analysis of microbial communities by direct extraction and sequencing or cloning of their DNA that allows studying communities of organisms directly in their natural environment. What is the metagenome? Microbial life exists in almost every environment from the most familiar environments such as garden soil the leaves of green plants as well as their roots, or the pipe underneath our sink. But these microbial habitats also include environments considered hard to survive due to extreme conditions, for example at the bottom of the sea, in the arctic ice, or in salt deserts.
Microbes also populate our own body and live for example on our skin or inside the gut. Almost every environment on earth is colonized by different types of microorganisms. The metagenome consists of the genome of many individual microorganisms, present in an environmental sample. We study the metagenome because most microorganisms cannot grow in a pure culture and culturing can never capture the full spectrum of microbial diversity.
Metagenomics provides cultural, independent information about environmental microorganisms. How do we study the metagenome? The first step is the isolation of metagenomic DNA from a sample of a defined environment that encompasses genomes of the entire microbial population.
The resulting metagenomic DNA can then be analyzed by two distinct approaches. A functional metagenomics to search for specific enzymatic activities or proteins or b metagenomic DNA sequencing to study microbial and functional diversity. Functional metagenomics starts with the construction of a metagenomics library. DNA fragments are generated by restriction enzyme digestion or mechanical methods and then ligated to a vector resulting in a library of recombinant vectors.
This library is used to transform a bacterial host that can be manipulated in the laboratory. The resulting metagenomic library can later be screened in order to find genes that are expressing a particular function. For example, UV radiation resistance. Metagenomic DNA sequencing provides insights into the microbial diversity as well as the functional potential of microorganisms of a sample. One option is the sequencing of the highly conserved 16S RNA gene amplified from the metagenomic DNA.
The sequences obtained can be aligned to generate a phylogenetic tree, in which the diversity of microorganisms present in the sample is revealed. Another option to obtain information about the diversity of the genes and their function in the microorganisms of a sample is to sequence directly the metagenomic DNA. Smaller DNA fragments that overlap are reassembled into a longer sequence or continuous sequence, a condic.
Through this procedure, very comprehensive sequences of the microbial genomes of the sample can be obtained. These genes are then predicted and functionally and taxonomically annotated by comparison with other known genes from a reference database. Metagenomics offers a powerful lens for observing microbial communities on a broader scale. and addressing fundamental questions of microbial diversity, evolution and ecology.