In metagenomics, information about micro-organisms in an environment can be extracted with two main techniques:
- Amplicon sequencing, which sequences only the rRNA or ribosomal DNA of organisms
- Shotgun sequencing, which sequences full genomes of the micro-organisms in the environment
Metagenomics, the sequencing of DNA directly from a sample without first culturing and isolating the organisms, has become the principal tool of “meta-omic” analysis. It can be used to;
- Explore the diversity, function
- Ecology of microbial communities.
- Apply appropriate statistics to undertake rigorous data analysis
Understanding the role of the microbiome in human health and how it can be modulated is becoming increasingly relevant for preventive medicine and for the medical management of chronic diseases. The development of high-throughput sequencing technologies has boosted microbiome research through the study of microbial genomes and allowing a more precise quantification of microbiome abundances and function. Microbiome data analysis is challenging because it involves high-dimensional structured multivariate sparse data and because of its compositional nature. The terms microbiome and microbiota are used indistinctly to describe the community of microorganisms that live in a given environment. High-throughput DNA sequencing technologies have powered microbiome research by enabling the study of the genomes of all microorganisms of a given environment and a more precise quantification of microbiome abundances and function. The main steps of a microbiome study:
- microbial DNA extraction and sequencing according to two main approaches, amplicon sequencing and shotgun sequencing
- bioinformatics sequence processing
- statistical analysis.
Shotgun metagenomics sequencing involves sequencing the total microbial DNA of a sample, instead of just a particular marker gene. With this technique, we can find out the relative abundance of every microbial gene and quantify specific metabolic pathways to predict the potential functionality of the entire community. This is achieved by mapping the obtained sequences against a database such as Kyoto Encyclopedia of Genes and Genomes. A gene pathway table resulting from this type of functional study provides the number of sequences associated to a particular function for each sample.
Metagenomic approaches are a growing branch of science and have many applications in different fields. Metagenomics seems to be the ideal culture-independent technique for unraveling the biodiversity of soils and to study how this biodiversity is affected with continuously changing conditions. In addition, its application in clinical and diagnostic approaches was reported. The emergence of several next-generation sequencing (NGS) strategies enriched metagenomics. The combination between NGS and metagenomic approaches helped the investigators resolve several issues regarding the microbial diversity and the functions and relationships among different microbial flora. A number of NGS approaches were developed including;
- Roche/454 pyrosequencing
- Illumina/Solexa sequencing
- Applied Biosystems/SOLiD sequencing
Metagenomic analysis is a sophisticated process and involves several steps. Of these steps, the sampling process is very crucial for the downstream applications. Sample collection, preparation, and storage should be handled carefully to prevent lysis and decomposition of the sample compositions. Multiple freezing–thawing cycles may cause changes in the microbial community profile under investigation. As well, a suitable DNA extraction protocol should be adopted to cope with the different chemical and physical characteristics of each sample. For instance, soils contain many substances that are co-extracted with the genomic DNA and may have inhibitory effects on the downstream experiments.
Metagenomics applies a suite of genomic technologies and bioinformatics tools to directly access the genetic content of entire communities of organisms. The field of metagenomics has been responsible for substantial advances in microbial ecology, evolution, and diversity over the past 5 to 10 years, and many research laboratories are actively engaged in it now. With the growing numbers of activities also comes a plethora of methodological knowledge and expertise that should guide future developments in the field.