Functional metagenomics is a strong experimental approach in studying gene function, beginning from the extracted DNA of mixed microbial populations. A functional approach is vested on the building and screening of metagenomic libraries, which are basically, physical libraries that contain DNA cloned from environmental metagenomes.
The information gotten from functional metagenomics can be used in future annotations of gene function and could serve as a supplement to sequence-based metagenomics. In this perspective, we examine functional metagenomics and its applications by summarizing the technical challenges of building metagenomic libraries and focusing on their value as resources.
Functional metagenomics entails restricting DNA from microbial communities to examine the uses of encoded proteins. It includes cloning DNA fragments, expressing genes in a surrogate host, and screening for enzymatic activities.
Applying this function-based approach brings about the discovery of new enzymes whose functions would not be ascertained based on DNA sequence alone. The information from function-based analyses can then be applied to annotate genomes and metagenomes derived entirely from sequence-based analyses.
Therefore, the process complements sequence-based metagenomics, equivalent to how molecular genetics of model organisms has proffered knowledge of gene application that is widely applicable in genomics.
This is one of two functional metagenomics and its applications. The underlying process starts with the building of a metagenomics library. Cosmid or fosmid-based libraries are usually preferred due to their wide and consistent insert size and high cloning capabilities. DNA is first taken from the environmental sample of interest, then size-selected, end-repaired, and ligated to a cos-based vector, allowing packaging by lambda phage for upcoming transduction of Escherichia coli.
The library that comes from this retains relatively large insert DNA, typically 25–40 kb for cos-based vectors. It has been implied above that functional metagenomics analyses can be applied on metagenomics libraries through the isolation and purification of DNA from an environmental sample, cloning of the DNA into a suitable vector, heterologous expression of the insert vector containing environmental DNA fragments in a suitable surrogate host (usually Escherichia coli), and analysis of subsequent transformants by either sequencing-or phenotypic based approaches or both.
However, screening of metagenomics libraries via phenotypic-based approaches is undergone to view, if possible, the expression of a specific phenotype bestowed on the host by inserted DNA. The process of screening is commonly performed on many clones on a fixed matrix in which the entire group is assayed with an indicator to display the presence of a phenotypically relevant clone. Such assays inspire the functional protein to be secreted from the host cell to allow for extracellular detection.
These clones may also be grown on particular indicator media, to enable visual identification of a dominant clone. For example, hemolytic activity on blood agar, lipolytic activity, etc. In other cases, the appearance of zones of inhibition in soft agar overlay assays using indicator microorganisms can display residual or antimicrobial agents provided by a dominant clone.
Libraries may also be examined based on selection approaches. In these cases, only the clones on which the activity of interest has been bestowed by the metagenomic DNA insert will survive. Selections might entail, for instance, the ability to metabolize a specific substrate as a clone’s sole carbon source, the capacity to be immune to a potent antimicrobial agent, or the capacity to grow in the appearance of a lethal concentration of heavy metal.
Metagenomics analyses start with the restriction of microbial DNA from an environmental sample. The acquired metagenomic DNA specimen should be as pure and of as high quality as possible, and should accurately personify all species available both qualitatively and quantitatively.
Direct sequencing of extracted metagenomic DNA, followed with careful bioinformatics analyses, can encourage the elucidation of the functional traits of microorganisms colonizing particular environments. In the beginning, the break from culture-dependent to culture-independent outlooks for the microbiological analysis of an environmental sample included the sequencing of genes encoding microbial ribosomal RNAs (rRNAs).
Highly conserved primer binding sites within the bacterial 16S rRNA gene encourage the amplification and sequencing of hypervariable regions that can provide species-specific signature sequences useful for bacterial identification in an environmental sample.
This innovation permits microbiologists to determine phylogenetic relationships between unculturable bacteria and view and measure the microbial consistency of a sample. In addition, through the 16S rRNA gene sequencing of a metagenomic sample, a functional profile of the bacteria present in a given environment can also be gotten.
Information, as regards the functional duties of already studied bacterial species, is available in database archives, and includes both cultured bacteria whose functional proteins have been extensively characterized as well as functions directed to bacterial proteins produced by uncultured bacteria through previous metagenomic studies.
Once a member of a previously described bacterial family has been profiled in an environmental sample, or an appropriate closest known relative has been appointed, the phylogenetic analysis may assign forecasted functions to an identified bacterial species by defining the functional information available regarding that particular taxonomic group.
This process can be used to potentially most, if not all, of the different bacterial species seen in a sample and hence, community roles can be predicted for the microbes residing in the sampled niche without the need for what is referred to as the shotgun sequencing.
Finally, asides functional metagenomics and its applications, there are other means to predict the functional properties of microorganisms in a metagenomic sample. One of these is PICRUSt. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) is defined as a computational approach that can be used to forecast the functional properties of microorganisms in a metagenomic sample from characterized relatives in available databases using 16S rRNA sequencing data.
By measuring the individual species dominance in a sample and, in doing so, measuring the function(s) assigned to that family, PICRUSt can predict the overall functional composition of the community. This concept is explored via a combinatorial approach of 16S rDNA metabarcoding and single genomics for assessing the compositional and functional diversity of a microbial community.
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