1. Metagenomics and beyond: current approaches and integration with complementary technologies

Tracy L. Meiring, Rolene Bauer, Ilana Scheepers, Colin Ohlhoff, Marla I. Tuffin and Donald A. Cowan

Metagenomics is the cultivation independent analysis of the collective genomes of microbes within a given environment, using sequence- and function-based approaches. Early metagenomic studies, aimed at cataloguing the phylogenetic diversity of different habitats, revealed the vast size and richness of the microbial and viral realms. Access to previously unexplored volumes of sequence space from uncultured organisms has since opened up many new avenues of research, with the number of projects and potential applications ever expanding. The rapid growth of the research area is furthermore a direct reflection of the advances in the throughput, as well as the reduction in cost, of sequencing and screening technologies. In this chapter we present a comprehensive introduction to the current methodologies, applications and challenges of metagenomics. We review the major improvements in the technologies that form the metagenomic toolkit, as well as the limitations they currently place on research. We assess the achievements made thus far in the exploration of novel sequence space. Finally, we consider future research trends in light of recent novel metagenomic approaches and the incorporation of complementary technologies.

2. Bacterial genealogy: not dead

Robert L. Dorit and Margaret A. Riley

The importance of horizontal gene transfer (HGT) in bacterial evolution has led many bacteriologists to question the very existence of bacterial species. If gene transfer is as rampant as comparative genomic and metagenomic studies suggest, how could bacterial species survive such genomic fluidity? Indeed, some go so far as to propose the metagenome as the appropriate unit of evolutionary distinction. The coherence and continuity of the metagenome remains, for us, an open question. Whatever the ultimate fate of metagenome-based phylogenetic reconstructions, we contend that genetic information in the bacterial world still comes in discrete, discernible and discontinuous packages that retain their integrity over evolutionary time. Despite the many fascinating instances of horizontal gene transfer that have been documented, the genetic coherence of bacterial species has not devolved into a continuous smear of promiscuously shared genetic information. The distinctions that we see and name in the microbial world are not arbitrary slicings of a continuous distribution, but natural breakpoints and boundaries that reflect the operation of time, history and selection on bacterial populations.

3. Viral metagenomics and the regulation of prokaryotic communities

Fernando Santos and Josefa Antón

Since the publication of the first genomic analysis of a marine uncultured viral community in 2002, the analysis of the viral metagenomes from a wide variety of natural environments has provided a wealth of information on the diversity, abundance and metabolic capabilities of viruses. Here, we summarize how such studies have shed light into the control than viruses exert on prokaryotic communities. We have focused on three main aspects of this control: (i) what metagenomes tell about the dynamics of virus-host interaction and how the well established 'kill-the-winner' theory fits into new metagenomic data; (ii) which picture the analysis of viral metagenomes provides on the extend of lysogeny in the microbial communities; and (iii) which are the new findings on the role of viruses as gene transfer agents. Finally, we give some hints on the analysis of expression of virome genes ("metavirotranscriptomes") and which are, in our opinion, the likely future trends in the "omics" analysis of viruses in nature.

4. Functional metagenomics and systems biology: understanding the human organismal complexity in disease and health

Liping Zhao and Jian Shen

A devastating epidemic of chronic diseases is threatening the public health worldwide. Preventive healthcare systems require novel types of health assessment technologies which focus on the early warning biomarkers before the clinical onset of chronic diseases. In light of the systems theory, emergent functions of the human body should be measured for health evaluation. Humans are superorganisms harbouring two integrated genomes, the human genome and the microbiome which is the collective genomes of all symbiotic microorganisms, particularly those inhabiting the gut. The gut microbiota and the host interact intimately. The structure and functions of the gut microbiota, together with the host metabolism as reflected in urine metabolite profiles, are the emergent functions of the human body. Metagenomics and metabonomics can be used to monitor the dynamics of gut microbiota and host metabolism. Large scale cohort studies in which urine and faecal samples are analyzed by the whole body systems approaches may lead to the discovery of patterns of gut microbiota and host metabotypes which can in turn be used as a biomarker for diagnosis or target for developing new therapeutics for chronic diseases. The application of these systems approaches in traditional Chinese medicine and nutritional studies may lead to a significant paradigm shift in modern medicine and nutritional sciences.

5. Next-generation sequencing approaches to metagenomics

John Walshaw, Graham J. Etherington and Dan MacLean

Next-generation sequencing approaches enable us to gather many more times sequence data than was possible a few years ago. Next-generation sequencers from the main vendors, Illumina, 454 and ABI SOLiD are distinct and varied technologies with unique approaches to sequencing that produce sequence reads with different strengths and weaknesses. We describe these technologies in detail and also discuss the applicability of the co-evolving new approaches for manipulation of reads and assembly and alignment of the high volume of reads that have also been developed to tackle the particular challenges of the shorter read format. By using next-generation sequence data in metagenomics experiments a wide range of new analyses are possible and we discuss the scope, goals, utility and practicality of these in modern laboratories including analysing environmental communities with partial assembly, clustering of taxonomically related reads to identify community structure, phylogenetic classification of unclustered reads and functional analyses that rely on identification of specific pathways or gene family members in samples. Metagenomic study has an increasingly powerful partner in the next-generation sequence technology and this partnership is likely to get more productive as software and hardware mature.

6. Stable isotope probing: uses in metagenomics

Ondrej Uhlik, Lucie Musilova, Katerina Demnerova, Tomas Macek and Martina Mackova

Until recently, investigating functions of microbial populations was restricted to thorough studying of pure cultures. Molecular biology tools enabled scientists to take a much deeper insight into the phylogenetic as well as metabolic diversity but hardly allowed for linking the phylogenetic identity with the metabolic activity which the microbes disposed of. Stable isotope probing (SIP) was one of the first microbial ecology tools to enable such a linkage. SIP consists of providing the community with a stable isotope labelled substrate and subsequent extraction and analysis of the labelled biomarkers. This text summarizes why SIP is a technique of an outstanding importance for microbial ecology and related fields and further focuses on SIP using DNA as a biomarker since DNA is one of the few molecules that bears both phylogenetic and functional information. Much deeper insight into stable isotope labelled DNA has been allowed especially due to current advances in high-throughput sequencing technologies. Metagenomic analyses, however, profit from stable isotope probing as well by reduced complexity of studied DNA (only populations actively performing a particular process). Therefore, when DNA-SIP is used in metagenomics, it is much easier to reconstruct individual genomes of key uncultured microbes as well as detect genes of interest.

7. DNA sequencing of uncultured microbes from single cells

Roger S. Lasken, Mary-Jane Lombardo, Mark Novotny, Joyclyn Yee-Greenbaum and Rashel V. Grindberg

Development of a method to sequence DNA from a single cell has enabled new strategies to investigate the microbial world. Only a few years ago, sequencing from one cell was not feasible. A bacterium only contains a few femtograms of DNA which is insufficient for current sequencing technologies. This limitation was overcome with the development of a method to amplify DNA called multiple displacement amplification (MDA) which can generate micrograms of genomic sequence from one cell. Improvements have also been made in our ability to isolate cells by flow cytometry, micromanipulation and microfluidics and to lyse the cells to release the single genome copy as a template for MDA. Large portions of the genome can be obtained from each cell and this has opened up a new front in the effort to sequence uncultivated species. Cells can be isolated from an environment or clinical specimen and directly sequenced with no need to develop culture methods. This chapter will review the current methodologies, the strengths and limitations of the single cell approach and its application to microbial genomics.

8. A Community cyberinfrastructure resource for metagenomics research: CAMERA 2.0

Jing Chen, Shulei Sun, Weizhong Li and John C. Wooley

The sustained deluge of complex experimental data in metagenomics opens extraordinary opportunities for this new science, but also requires an advanced, cyberinfrastructure-based knowledge resource that provides a platform for rigorous analysis of the data sets and in turn, of the microbial communities. In response, we established the Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis or CAMERA and recently released a very major extension to this resource, termed CAMERA 2.0. In this chapter, we describe the essential attributes of CAMERA 2.0 and their relationship to expectations of the experimental and computational researchers in metagenomics. An analysis portal provides access to a semantically aware database along with computational infrastructure. Meticulous attention is paid to annotating the sequence data with metadata, the associated contextual information (environmental parameters). Using the standards established by the Genomics Standard Consortium, CAMERA 2.0 enables ready depositing, locating, analyzing, visualizing and sharing data and the use of a range of actual analytical processes or workflows. These workflows, most notably, are user-selected, allowing a flexible approach in which an investigator can set their own requirements for a given sequential analysis. The analytical and computational history is archived, allowing accurate reproducibility, detailed further analysis, and validation.

9. Metagenomics for the identification of novel viruses

Vincent Montoya, Eunice C. Chen, Charles Y. Chiu and Patrick Tang

Viruses are the most abundant and genetically diverse biological entities on Earth and the vast majority is yet to be discovered. Therefore, systematic surveillance for viruses requires techniques that have both broad specificity and high sensitivity. Conventional laboratory techniques in virology often fail to detect a specific etiology in many syndromes that are thought be caused by viruses. Metagenomics-based tools such as pan-viral microarrays and ultra-high-throughput sequencing have significantly improved our ability to detect and characterize divergent as well as novel viruses. Some of these methods rely on the fact that any one virus will possess some degree of conservation within its genomic sequence with other members of the same family. Thus, nucleic acid amplification tests targeting conserved regions in the viral families associated with a particular disease can often lead to a successful diagnosis. However, metagenomics-based techniques such as pan-viral microarrays are able to transcend our predetermined lists of viruses associated with each syndrome and allow for the simultaneous interrogation of thousands of conserved and specific genetic regions within all taxa of known virus families. Second generation high-throughput sequencing offers the unique opportunity to discover novel pathogens with no a priori sequence information with sensitivities comparable to that of PCR. As the costs for these techniques continue to decrease and the technology becomes more widely available, they will have the potential to revolutionize our approach to detecting viruses and diagnosing viral diseases.

10. Metagenomics applied to arbuscular mycorrhizal fungal communities

Valeria Bianciotto, Erica Lumini, Alberto Orgiazzi, Roberto Borriello and Paola Bonfante

Metagenomics studies have recently offered new approaches that shed light on microbial communities in a variety of environments. In this context, DNA pyrosequencing is being used more and more to investigate prokaryotic assemblages in soil environment. Fungi, which are crucial components of soil microbial communities, functioning as decomposers, pathogens and mycorrhizal symbionts, have instead been largely neglected. However, the last year has been characterized by an explosion of metagenomic studies applied to fungal communities, based on the pyrosequencing technology. The aim of this chapter is to focus on Arbuscular Mycorrhizal Fungi (AMF), the most widespread symbionts in many ecosystems, and to demonstrate how metagenomics may help us to understand the composition and dynamics of AMF communities. At the moment, only two studies have investigated AMF biodiversity using the pyrosequencing approach and SSU rDNA as the target gene. Although both studies targeted the same group of fungi, they focused on different habitats. Compared to similar studies, carried out using a cloning-sequencing approach or DNA barcoding, the main outcome that emerged from pyrosequencing analyses applied to AMF and to other fungal communities is the unexpected fungal biodiversity observed in the analyzed environments. Interestingly, the pyrosequencing approach applied to isolated spores of AMF has demonstrated that they are a niche for highly polymorphic endobacterial communities. The data confirm the powerfulness of the pyrosequencing approach, which represents a promising new tool to better understand the natural distribution of an essential group of soil microorganisms, such as fungi. The large number of reads that have been obtained increases the likelihood of capturing sequences from rare organisms, which would instead remain undetected with the cloning- sequencing approach.

11. The human microbiome: exploring and manipulating our microbial selves

Corinne F. Maurice and Peter J. Turnbaugh

The human body is home to roughly ten times more microbial cells than human cells, containing a vast array of genes and metabolic activities referred to in aggregate as the human microbiome. Metagenomics has recently enabled an initial map of the microbial diversity found in different body habitats, individuals, and populations. These developments include an extensive catalog of genes and genomes, characterization of the human gut viriome, a description of the patterns of succession of the gut microbiota during development, and links between obesity and the gut microbiome. The application of principles from macro-ecology, in addition to studies of defined communities in model organisms, have begun to reveal the basic operating principles that govern community assembly, stability, and function. These studies are beginning to move beyond simple characterizations of the organisms and genes found in a given habitat at a single timepoint, to a systems biology approach that allows the characterization of this complex microbial community at a variety of spatial and temporal scales. In the foreseeable future, studies of the human microbiome promise to reveal new biomarkers for disease, novel strategies for manipulation, and a more comprehensive view of human physiology.

12. Metagenomics and integrative omics technologies in microbial bioremediation: current trends and potential applications

Varun Shah, Kunal Jain, Chirayu Desai and Datta Madamwar

Implementation of efficacious bioremediation strategies relies heavily on intrinsic microbial community dynamics, structure and function. Any one particular microorganism is incapable of processing all the metabolic reactions to degrade environmental pollutants, however a group of diverse organisms form a community and collectively process all the metabolic reactions for bioremediation. Therefore, metagenomics based analyses of entire microbial community genomes becomes imperative to delineate the metabolic pathways responsible for biodegradation. The essential genes for bioremediation may be present, however to ascertain how many of them are involved in bioremediation we need high throughput metatranscriptomics and metaproteomics, transcriptome and proteome analyses of entire community respectively. Metametabolomics, analyses of the entire repertoire of microbial community metabolites and fluxomics, real time flux analysis of molecules/metabolites over a time period provide the missing links about regulation of metabolism of anthropogenic/xenobiotic compounds. Interactive studies between metagenomics, metatranscriptomics, metaproteomics and metametabolomics have become a trend in microbial bioremediation. In this chapter, we discuss the potential of recent innovative breakthroughs in molecular and '-omics' technologies such as molecular profiling, ultrafast pyro-sequencing, microarrays, mass spectrometry and other novel techniques and applications along with bioinformatics tools to gain insights of indigenous microbial communities and their mechanism in bioremediation of environmental pollutants.

13. Escherichia coli host engineering for efficient metagenomic enzyme discovery

Reia Hosokawa-Okamoto and Kentaro Miyazaki

Enzymes are environmentally friendly biocatalysts that are widely used in modern life, e.g., in food processing, laundry detergent, and production of medicinal compounds. An increasing demand to shift focus from petrochemicals to biotechnology-based industries has expanded the use of enzymes. To date, most industrially relevant enzymes are of microbial origin. Therefore, mining for microbial enzymes is key to the development of the biotechnology industry; however, less than 1% of environmental bacteria can be cultured in the laboratory at present. To accelerate the discovery of industrially relevant enzymes, it would therefore be advantageous to employ a metagenomic approach to extend the available microbial sources to presently 'unculturable' taxa. However, such an approach also risks a low hit rate; typically, only a few positives are obtained from the hundreds of thousands of library clones screened. This is largely because of the discrepancy between the host's transcriptional/translational machineries and the genetic signals present in the metagenomes. Escherichia coli has long been used as a generic host for cloning and production purposes and is, in most instances, suitable for this purpose. However, several modifications are necessary to overcome the problems of heterologous gene expression. In this chapter, we discuss several approaches that may be useful for developing the utility of E. coli as a host for efficient functional screening of metagenomic libraries.

14. Recent contributions of metagenomics to studies on quorum-sensing and plant-pathogen interactions

Denis Faure, Mélanie Tannières, Samuel Mondy and Yves Dessaux

Though metagenomics is a novel tool in the field of plant-microbe interaction, the technique has already led to remarkable advances. Among these, the identification of yet-uncultivable phytopathogens or the description of the plant and rhizosphere microflora, are two features that may lead to a better description of the quality of agricultural lands, for instance in the case of disease suppressive soils. At a more molecular level, the identification of novel density-dependent regulatory functions (quorum sensing) and antagonizing elements (quorum quenching) that may be used to develop sustainable, biological control strategies directed at plant pathogen, are examples of valuable outcomes of metagenomics in the plant-microbe interaction field.

15. Metagenomics analysis of belowground microbial communities using microarrays

Joy D. Van Nostrand, Zhili He and Jizhong Zhou

The use of microarrays has revolutionized the field of microbiology. While many types of microarrays are available, the functional gene arrays (FGAs) afford a way to link environmental processes with microbial communities. FGAs probe for a wide range of genes involved in functional activities of interest to microbial ecology (e.g. carbon degradation, N₂-fixation, metal resistance) from many different microorganisms, cultured and uncultured. The most comprehensive FGA reported to date are the GeoChip arrays. GeoChip 3.0 targets tens of thousands of genes involved in the geochemical cycling of carbon, nitrogen, phosphorus, and sulphur, metal resistance and reduction, energy processing, antibiotic resistance and contaminant degradation as well as phylogenetic information (gyrB). This technology has been used successfully to examine the effects of global climate change on microbial communities. This chapter provides an overview of FGA development with a focus on the GeoChip. Several GeoChip studies, which have established the GeoChip's worth as a rapid, sensitive and specific tool for the examination of microbial communities, will be highlighted.