The human microbiota consists of microorganisms that live inside as well as on humans and they outnumber the human germ cells and somatic cells ten times (Turnbaugh et al., 2007). The collection of genes of these microbial organisms (the microbiome) provides the characteristics that human beings lack (Zimmer 2010). There are two major techniques that are currently used to characterize the microbiome, namely; the (rRNA) small-subunit ribosomal RNA, and the metagenomic study, wherein shotgun sequencing is used to characterize community DNA of these microorganisms.
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Shotgun Sequencing of the Human Microbiome
Shotgun sequencing, also called shotgun cloning in genetics, is a process used for sequencing long strands of DNA. Shotgun sequencing was referred so by resemblance with the quasi-random shotgun firing pattern. The shotgun sequencing method was developed by Fredrick Sanger in the 1970s. The chain termination sequencing process is only be used to sequence relatively short DNA strands. Longer sequences of DNA must be sub-divided into shorter fragments, and then subsequently reassembled to give the entire sequence (Venter et al., 2004). Shotgun sequencing process, which uses random fragments, is used to sequence shorter fragments of longer DNA strands. The process is faster but more complex process.
The DNA strand is randomly broken up into many small fragments, in shotgun sequencing, which are then sequenced using the process of chain termination to obtain reads. The multiple target DNA overlapping reads are obtained through performing many rounds of DNA fragmentation and sequencing. The overlapping ends of many reads are then used by the computer programs to assemble the reads into a continuous DNA sequence.
Through metagenomics and the use of shotgun sequencing, much progress has occurred in the characterization of the human microbiome plus the role it plays in human health and disease. Recent technological advances, especially the high-throughput sequencing has been required for revealing large-scale trends relating the micro-biomes of most individuals. To begin with, direct human microbiome sequencing has revealed community differences (HMC, 2012: Turnbaugh et al., 2007). Shotgun metagenomic information is available so far only for gut microbiome. One robust finding reveals that there seems to be a larger degree of difference in the composition of the microbial community amongst individuals than it differs within the same person over time. The variation in the composition of the microbial community between individuals has been established to be right in different research and within a broad range of body habitations. For example, the composition of the gut microbial community is fairly stable within the same person over some time when the diet is consistent and even to a certain extent when the diet is altered (Costello et al., 2009). Likewise, the composition of the skin microbial community is more analogous within an individual than between individuals for months, as are nasal, external auditory canal, and oral communities. These findings show that people are likely to be much similar to themselves for some months than to their friends at the present when it comes to the bacteria they harbor.
Another thing that shotgun sequencing has contributed to the human microbiome is the uncovering of the changes in the microbial community associated with human disease (Turnbaugh et al., 2007: Morgan & Huttenhower, 2012). While measures of community multiplicity have dominated historical research, modern high-throughput techniques have also been developed for a multitude of other meta assays from un-cultured microorganisms. The body of human beings has been found to be composed of innumerable complex environments that incline colonization by many different communities of microbes. These microbial communities have been found to play essential roles in the human health and disease (HMC, 2012: Turnbaugh et al., 2007: Morgan & Huttenhower, 2012). For example, the microbial community of the gut has been found to have an impact upon both the natural and the adaptive immune systems, and it causes immune disorders evident outside and within the gut. The inflammatory bowel patients, for instance, have been found to have a dysregulated gut microbial community immune response (Turnbaugh et al., 2007). In addition, the human microbiota has been revealed to contain the capacity of altering the expression of the host genes encoding antimicrobial compounds.
The interpersonal variations in the overall composition of the microbial community and their correlations with diseases have also been uncovered through shotgun sequencing of the human microbiome. The mechanisms through which these microbial communities contribute to the ailment have been discovered. Metagenomics is a term used to refer more specifically, to WMS (whole-metagenome shotgun) sequencing of fragments of genomic DNA from the communitys meta-genome. Verberkmoes et al. (2009) argues that shotgun sequencing has also enabled the sequencing of reverse-transcribed transcripts of RNA (metatranscriptomics), the quantification of the peptide or protein levels (meta-proteomics), and the investigation of small molecule metabolites (community metabolomics). Through whole-metagenome shotgun sequencing, the potential usefulness of microbial community metabolomics research in understanding the human microbiome and the role it plays in obesity, metabolic disorders and energy harvest is now clear.
In addition to the diversity and composition of the human microbial communities, significant efforts have been put forward to sequence reference human microbial genomes from numerous body sites. Shotgun metagenomics has allowed the sequencing of the microbial genomes from five major body sites and eighteen sub-sites; skin, gastrointestinal tract, oral, airway, and vagina/urogenital tract, (Xie, Lo C-C, Scholz, & Chain, 2014). Between 13 percent and 92 percent, that is 62.3 percent on average, of the shotgun reads have been aligned to a 2780 complete list of reference genomes, together with 1583 human microbiome references (Qin et al., 2010: Xie, Lo C-C, Scholz, & Chain, 2014). The study also unveiled that there is no human microbial reference genome that is universal in all body habitats. It has also been found that, for any given meta-genome, the body habitat-specific reference genomes accounts for, on average, 58.8 percent of the shotgun reads that have been mapped. The Human Microbiome Projects metagenomic study has uncovered over 3.5 T base-pairs of shotgun sequences of DNA isolated from seven body sites (Xie, Lo C-C, Scholz, & Chain, 2014).
The high-throughput shotgun sequencing of the human microbiome has also uncovered the metabolic and the broader bio-molecular functions associated with the community (Turnbaugh et al., 2007: Zimmer, 2010). Additionally, the manners in which they provide specialization within different body sites microbial niches have also been revealed (HMC, 2012). For example, the gut microbiota has been revealed to play an imperative role in the immune response and metabolism. A scalable methodology, the Network of the Human Microbiome Project Metabolic Analysis, has been developed and used accurately to deduce community functions from the short DNA community functions from the short DNA reads directly. The use of the HUMAnN to the HMP metagenomic shotgun data has shown that, unlike personal microbial species, several metabolic processes exist among all body sites (Turnbaugh et al., 2007). The incidences of these metabolic processes, however, vary dramatically, and some are highly enriched in different body sites to give niche specialization, for example, the gut is rich in food matter, however, low in oxygen.
Other community functions have been connected specifically to the human host properties, such as the biochemical processes present in the vaginal habitats only with particular low or high pH. Functional characterization of the human microbial metagenomes has provided for the determination of the metabolism plus other biological activities that might be occurring (HMC, 2012: Turnbaugh et al., 2007: Zimmer, 2010). Current metagenomic techniques for characterizing the microbial community roles rely upon a best-BLAST-hit approach. In this best-BLAST-hit method, the sequenced communitys short reads are individually searched against a fully characterized reference database with translated BLAST.
Shotgun sequencing of the human microbiome has provided for the full characterization of the genes encoding the microbial communities and their habitats within human beings. Shotgun sequencing of these microbial communities has provided information about their composition and diversity within the body habitats. Shotgun sequencing has also provided information about the biochemical and metabolic functions of these microbial communities, in addition to uncovering the mechanisms through which they contribute to disease.
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