Metagenomics

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Definition of Metagenomics

Metagenomics is the culture-independent genomics analysis of microbial communities. "Metagenomics" describes the functional and sequence-based analysis of the collective microbial genomes contained in an environmental sample. The term is derived from the statistical concept of meta-analysis ( the process of statistically combining separate analyses) and genomics (the comprehensive analysis of an organism's genetic material)


Ecologial Inference From Metagenomics

1. Symbiosis(^^*)
2. Biogeochemical Cycles(*^^)
3. Population Genetics and Microheterogeneity(-..-;)


Measures of microbial diversity

1. Nucleotide diversity
2. Gene diversity
3. Genotype diversity
4. Species diversity
5. Phylogenetic diversity
6. Evolutionary diversity
7. Ecological niche diversity
8. Functional diversity
9. Morphological diversity
10. Structural diversity
11. Metabolic diversity
12. Metabolite diversity
13. Protein diveristy


Metagenomic Library Constructing Method

1. Genomic methods in microbial ecology research DNA sequencing
The most significant technical advance in genomic is the development of efficient, high throughput DNA-sequencing techniques and instruments. While the basic principle for DNA sequencing was established in the mid-1970s, it was not until the mid-1990s when efficient automated DNA sequencers and fluorescent dyes to tag the dideoxyribonucleotides (with one colour for each of the four types of nucleotides) were developed. At present, high throughput DNA sequencing facilities are found in most academic institutions and many molecular biology laboratories. Furthermore, faster and cheaper sequencing methods and equipment are continuously developed.

2. Polyerase chain reaction
The second tool is the polymerase chain reaction (PCR) that allows the analysis of minute amount of DNA from laboratory and environmental sources. In combination with appropriate DNA extraction protocols, PCR allows highly selective amplification of target DNA. Indeed, the PCR technique is permeating almost every aspect of biological research, including many other DNA-based genomics techniques.

3. DNA cloning systems(plasmind, lamda-phase, cosmid, bacterial artificial chromosome or 
BAC, yeast artificial chromosoems or YAC)
The third highly useful genomics tool for microbial ecological studies is the availability of efficient in vivo cloning systems (including cloning vectors and hosts). These systems allow the separation and amplification of individual DNA sequences from often unknown but heterogeneous gene pools. A large variety of such systems is now available to accommodate different types and sizes of DNA fragments.

4. Fluorescent in situ hybridization(FISH)
Several other traditional DNA analytical techniques have also been widely used in microbial ecological studies. These include DNA re-association kinetic analysis and fluorescent in situ hybridization (FISH). Using fluorescently tagged specific probes, FISH allows the direct observation and estimation of micro-organisms from specific species, genera, families or phyla in a given environmental sample. In contrast, the analyses of DNA re-association kinetics can be used to provide estimates on the diversity of microbial genomes in environmental DNA samples.

5. Micro-array technology

6. Representational difference analysis(RDA)

7. 2-D gel electrophoresis

8. DNA re-association

9. Denaturing gradient gel electrophoresis(DGGE)

Microbial community Shotgun Sequencing Project

95 shotgun sequening projects of various communities have been sequenced to date(http://www.genomesonline.org/). The biological insights from these studies have been well-reviewd elsewhere.

1. Soil

Soil is an important reservoir for organic carbon, and prokaryotes are an essential component of the soil decomposition system. Despite the high concentration of organic matter in most soil types, only low concentrations of organic carbon are readily available to microorganisms. Reasons for this include the transformation of most of the organic matter that is derived from plants, animals and microorganisms into humus by a combination of microbiological and abiotic processes, and the uneven distribution of microorganisms and organic compounds in the soil matrix. Humic substances are stable and recalcitrant to microbial decomposition processes ? the half-life of these stable organic matter complexes with respect to biological degradation is approximately 2,000 years.

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2. Marine

Ocean covered 70% all of earth microbial life. The world's oceans are teeming with microscopic life forms. Nominal cell counts of >105 cells per ml in surface sea water  predict that the oceans harbor 3.6 x 1029 microbial cells with a total cellular carbon content of {approx}3 x 1017 g. Communities of bacteria, archaea, protists, and unicellular fungi account for most of the oceanic biomass. These microscopic factories are responsible for 98% of primary production and mediate all biogeochemical cycles in the ocean. Given the enormous number of microbes and their vast metabolic diversity, the accumulation of mutations during the past 3.5 billion years should have led to very high levels of genetic and phenotypic variation.

3. Feces
The human gut is colonized by an abundant, active, and diverse microbiota. This microbiota has been studied extensively using culture-based assays and, more recently, by a variety of molecular methods including fluorescent in situ hybridization, terminal restriction fragment length polymorphism, membrane assays, microarrays, and direct sequencing of 16S libraries. A lot of sediment of feces have shown that there are 400 to 500 human intestinal microbial species, with 30 to 40 species accounting for 99% of the total population.

4. Human mouth
The mouth is awash in microbes, but scientists so far have merely scratched the surface in identifying and studying the hundreds of bacteria that live in biofilm communities that stick to the teeth and gums. The mouth's microbial communities tend to congregate in biofilms - sticky, mat-like films that often include hundreds of distinct organisms that cooperate with each other to adapt to changes in their environment.

5. Drinking water
6. Ancient DNA
7. River biofilm
8. Beetle symbionts
9. Sponge symbionts
10. Polychaete symbionts
11. Anaerobic digester

High-quality Automated Annotation Tools for Metagenomics


1. ERGO(http://ergo.integratedgenomics.com/ERGO/)
The current version of the ERGOTM database contains 618 complete or nearly complete genomes, of which 319 are Bacteria, 116 Eukarya, 34 Archaea and 149 Viruses (Figure 1). In total, these genomes contain over 1,300,000 Open Reading Frames (ORFs), more than 60% of which have a functional annotation. This percentage of annotated genes is actually much higher for the bacterial genomes, reaching an average of 70%. Every genome that goes into the ERGO system, is annotated from scratch whether it has been sequenced at Integrated Genomics, or at another sequencing center. More than 450 of the genomes are available for subscription or as part of a stand-alone ERGO server package from Integrated Genomics.


2. GenDB(The system is open source)
The GenDB genome annotation system has been developed as an extensible and user friendly framework for both bioinformatics researchers and biologists to use in their genome projects. From a user's and from a programmer's point of view, several key features can be identified that a genome annotation system has to provide: From a user's perspective, the quality of the automatic annotation, the range of functions available for manual annotation, and the quality of the user interface play the key role. From a programmer's perspective, the application programmer's interface, the internal data representation, and the extensibility of any system are the most important features. While the implemented core functionality for annotating a genome showed its usability in more than 10 microbial genome projects, the current work on the GenDB system is directed towards building a platform for systems biology. We present the latest developments of the open source GenDB software for both comparative and functional genomics analyses. We have also implemented a novel web interface that can be used for remote genome annotation. For the automated annotation of coding regions (CDS) we enhanced the gene prediction based on a strategy that was evaluated for 113 microbial genomes. Furthermore, the automatic functional assignment was improved by combining the results of different bioinformatics tools.


3. PRIAM(http://www.renabi.fr/article250.html)
PRIAM is a method for automated enzyme detection in a fully sequenced genome, based on all sequences available in the ENZYME database. PRIAM relies on sets of position-specific score matrices (PSSMs) automatically tailored for each ENZYME entry. The whole Swiss-Prot database has been used to parameterise and to assess the method. As an example, PRIAM was applied to predict metabolic pathways from the complete genome of the nitrogen fixing bacterium Sinorhizobium meliloti and on the plant pathogen Ralstonia solanacearum.



Approaches To Metagenomic Analysis

Metagenomic analysis involves isolating DNA from an environmental sample, cloning the DNA into a suitable vector, transforming the clones into a host bacterium, and screening the resulting transformants. The clones can be screened for phylogenetic markers or "anchors", such as 16S rRNA and recA, or for othedr conserved genes by hybridization or multiplex PCR or for expression of specific traits, such as enzyme activity or antibiotic production, or they can be sequenced randomly

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