J Bacteriol Virol.  2012 Dec;42(4):263-275. 10.4167/jbv.2012.42.4.263.

Application of Metagenomic Techniques: Understanding the Unrevealed Human Microbiota and Explaining the in Clinical Infectious Diseases

• Kim W

• KMID: 2168661
• DOI: http://doi.org/10.4167/jbv.2012.42.4.263

Abstract

Uncultured microorganisms comprise the majority of the planet's biological diversity. In many environments, as many as 99% of the microorganisms cannot be cultured by standard techniques, and the uncultured fraction includes diverse organisms that are only distantly related to the cultured ones. Therefore, culture-independent methods are essential to understand the genetic diversity, population structure, and ecological roles of the majority of microorganisms. Recently, new techniques for studying microbial communities, collectively called metagenomics, have been developed to overcome the limitations of culturing. This review assesses the potential of metagenomic techniques to analyze the relative abundance of microbial species under varying human environmental conditions and to discover infectious causes of unexplained human diseases.

Keyword

Metagenomics; Sanger sequencing; Next-generation DNA sequencing

MeSH Terms

Biodiversity
Communicable Diseases
Genetic Variation
Humans
Metagenome
Metagenomics

Figure

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  Figure 1 In high-throughput shotgun Sanger sequencing, genomic DNA is fragmented, then cloned into a plasmid vector and transformed into E. coli. For each sequencing reaction, a single bacterial colony is picked and plasmid DNA isolated. Each cycle sequencing reaction takes place within a microliter-scale volume, generating a ladder of ddNTP-terminated, dye-labeled products that are subjected to high-resolution electrophoretic separation within one of 96 or 384 capillaries in one run of a sequencing instrument. As fluorescently labeled fragments of discrete sizes pass a detector, the four-channel emission spectrum is used to generate a sequencing trace (26).

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  Figure 2 Clonal amplification of sequencing features. The 454, Polonator, and SOLiD platforms rely on emulsion PCR20 to amplify clonal sequencing features. In brief, an in vitro-constructed adaptor flanked shotgun library (shown as gold and turquoise adaptors flanking unique inserts) is PCR amplified (that is, multi-template PCR, not multiplex PCR, as only a single primer pair is used, corresponding to the gold and turquoise adaptors) in the context of a water-in-oil emulsion. One of the PCR primers is tethered to the surface (5'-attached) of micron-scale beads that are also included in the reaction. A low template concentration results in most bead-containing compartments having either zero or one template molecule present. In productive emulsion compartments (where both a bead and template molecule are present), PCR amplicons are captured on the surface of the bead. After breaking the emulsion, beads bearing amplification products are selectively enriched. Each clonally amplified bead has PCR products on its surface corresponding to amplification of a single molecule from the template library (26).

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  Figure 3 Comparison of the microbial diversity in the oropharynx of bronchial asthma versus non-asthmatic persons.

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  Figure 4 UniFrac analysis of V3 16S sequences of the microbial community of oropharynx samples from bronchial asthma and non-asthmatic persons. (A) A jackknifed clustering of the environments in the UniFrac dataset. The numbers next to the nodes represent the number of times that particular node was observed in a random sampling from the whole dataset. (B) Principal component analysis scatter plot of the microbiota of individuals with bronchial asthma and healthy individuals (non-asthmatic persons, red; bronchial asthma, blue).

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  Figure 5 Diagram of mycobiome communities at the phylum level. (A) Distribution of mycobiomes between normal scalps and dandruff-afflicted scalps. (B) Distribution of genera belonging to the phylum Ascomycota in healthy and dandruff-afflicted scalps. (C) Distribution of genera belonging to the phylum Basidiomycota in healthy and dandruff-afflicted scalps.

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  Figure 6 Diagram of the fungal communities at the generic level. Comparison of genus frequencies in the mycobiomes of normal and dandruff-afflicted scalps.

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