Metagenomics: Perspectives, Methods, and Applications

Nagarajan

Chapter 1

Metagenomics: A Paradigm Shift in Microbiology

Andrey V. Mardanov; Vitaly V. Kadnikov; Nikolai V. Ravin    Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

Abstract

Uncultured microorganisms are the ones mostly responsible for natural biodiversity on Earth. Typically, more than 99% of microorganisms from natural ecosystems cannot be cultured under laboratory conditions. Therefore, there is a demand for culture-independent approaches for identification and characterization of such microorganisms and uncovering their ecological roles in the biosphere. Metagenomics is culture-independent, sequencing-based and/or function-based analysis of the collective genome of a microbial community, which enables collection of essential information about community structure and genetic and metabolic potential of the members, thus providing insights into the biology of these microorganisms. Another culture-independent method is single-cell genomics, which obtains information about microbial population based on the isolation and genome sequencing of a single cell. This chapter presents an overview of the history of metagenomics and describes the main approaches used for analysis of microbial communities, including the synergy between metagenomics and single-cell genomics, and outlines further directions in this field.

Keywords

Metagenomics; Uncultured microorganism; Sequencing; Single-cell genomics; Microbial ecology

1.1 The Birth of Metagenomics

Traditional objects of microbiology are pure microbial cultures composed of genetically identical cells obtained by clonal propagation of an isolated single cell. All the knowledge provided by microbiology in the 20th century regarding the diversity of metabolic pathways and biochemical processes in microorganisms was obtained through analysis of pure cultures. Development of cultivation techniques and establishment of pure cultures for different microbes, followed by their subsequent characterization using microbiological, biochemical, and genetic methods, resulted in the creation of representative collections of microorganisms and ultimately in a deeper understanding of the roles of microorganisms in the biosphere.

The seminal discovery of DNA as a carrier of hereditary information, indicating that all properties of the organism are encrypted in the genome, and the rise of molecular biology in the middle of the 20th century gave rise to genomic microbiology. The first hallmark application of molecular methods in microbiology dates back to the end of the 1970s, when Carl Woese suggested a universal phylogenetic system for prokaryotes based on comparison of 16S ribosomal (r)RNA [1]. The development of genome sequencing techniques, especially next-generation sequencing (NGS) methods in the middle of the 2000s, resulted in the so-called genomic revolution in microbiology, making complete genome sequencing routine and feasible not only for a few well-studied model organisms such as Escherichia coli, but also for multiple microbial species. Thus, genome sequencing has become the leading approach in the analysis of new microorganisms isolated as pure cultures.

Since the very beginning, environmental microbiology was technically based on the segregation of microbial communities into pure cultures of individual microbial species, followed by their physiological and biochemical characterization. However, in spite of more than a hundred-year history of microbial cultivation, the majority of microorganisms inhabiting natural environments have not been isolated as pure laboratory cultures. As early as 1933, Arthur Henrichi noted that, It has long been known that the standard methods of bacteriology—pure culture isolation and observation upon artificial media—often yield only an incomplete knowledge of a particular microbial flora. [2] Particularly, he found different bacteria with atypical morphology in freshwater lakes, which had not been isolated as pure cultures at that time [3].

Microbial ecology reached a new stage when sequencing of rRNA and the corresponding genes obtained directly from the environment without prior cultivation of microorganisms started to be used for the characterization of microbial diversity. The first example was sequencing of fragments cloned in a 5S rRNA cDNA library obtained from the microbial community of a giant tubeworm Riftia pachyptila [4]. However, another approach, based on the isolation of total community DNA from an environmental sample followed by PCR amplification, cloning, and sequencing of 16S rRNA gene fragments, appeared to be easier and more informative. These studies have led to the recognition that usually less than 1% of microorganisms from natural sources could be cultivated under laboratory conditions [5] and that uncultured species not only constitute the major part of microbial communities, but could also perform key functions in ecological processes.

Extreme environments such as the hot springs of Yellowstone National Park became one of the first focuses in rRNA survey studies [6]. Analysis of the microbial community in only one hot spring, Obsidian Pool, revealed novel phylogenetic lineages of Archaea belonging to a high taxonomic rank, including the candidate archaeal phylum Korarchaeota [7,8], and 11 novel candidate phyla of bacteria, OP1–OP11 [9,10]. Considering that at the time when Woese first proposed the phylogenetic system of prokaryotes, only 12 bacterial and archaeal phyla were recognized and all of them have cultured representatives, the results of these early studies clearly indicate that we are very far from understanding the true phylogenetic diversity of the microbial world.

Molecular studies of different natural ecosystems revealed a number of novel phylogenetic lineages of Bacteria and Archaea, and only some of them were subsequently cultured. According to recent estimates, cultured microorganisms account for less than 20% of real phylogenetic diversity of prokaryotes [11]. At present, the uncultured majority of prokaryotes include about 50 bacterial phyla and about the same number of major archaeal groups lacking cultured representatives [12,13]. However, although uncultured microorganisms have a major role in important biogeochemical processes on Earth and are highly abundant in the ocean, soils, sediments, animal microbiomes, and other ecosystems, they remained poorly characterized [14–16]. To emphasize the scope and significance of uncultured microorganisms that dominate life, the term microbial dark matter has been proposed [11,14,17,18], by analogy with the dark matter substance in astrophysics.

A description of phylogenetic diversity among microorganisms answering the question who is here? is only the first step in the study of microbial communities. A much more difficult task is to assess the functional activities and ecological roles of particular microorganisms, i.e., to answer the question what can they do? Similar to genomic sequencing of cultivated microorganisms, which allows prediction of their metabolic potential, analysis of a collective genome of a microbial community, a metagenome, provides information about functional characteristics of the community as a whole as well as about each of its members, including uncultured species. The idea of isolating and cloning DNA directly from the environment was first proposed by Norman Pace in 1985 [19] and realized in 1991, when a library of total DNA isolated from sea water was cloned in the λ phage vector [20]. This approach became more common with the advances in genome sequencing technologies. Thus, in 1996, the DeLong group cloned and sequenced a 40-kbp fragment from the genome of an uncultured marine archaeon [21]. In 2004, Tyson et al. [22] sequenced about 76 Mbp of DNA extracted from acid mine drainage and determined near complete genomes of several uncultured microorganisms, while in the same year Venter et al. [23] reported large-scale sequencing of the Sargasso Sea water metagenome, which generated more than 1 Gbp of nonredundant sequences. All these studies were conducted using traditional genome sequencing strategies, including cloning of environmental DNA in plasmid vectors and sequencing by the Sanger method.

The development of high-throughput next generation sequencing (NGS) technologies has revolutionized genomic research as they enabled massive parallel sequencing in a short time, which resulted in the generation of orders of magnitude more nucleotide sequences at a much lower cost, making metagenomic studies of complex environmental communities affordable even to small labs. As a result, it was possible to perform comprehensive characterization of such complex microbial populations as animal and human microbiomes [24], cow rumen [25], soil, seawater, and sediments (reviewed in Refs. [16,26]).

The term metagenomics, first coined in 1998 by Jo Handelsman [27], refers to investigation of collective genetic material obtained from a certain environmental sample, which is, in principle, is similar to that of individual genomes from cultured microorganisms. In the strict sense of this definition, the metagenomics concept excludes analysis of only a particular gene (e.g., 16S rRNA) present in the environment, since it does not provide information about the whole genetic potential of a microbial community [28]. However, these community profiling methods are often used together with true metagenomic analysis and complement it.

1.2 Function-Based and Sequencing-Based Metagenomics

Metagenomic analysis targeting total DNA isolated from the environment could be performed using two conceptually different strategies (Fig. 1.1). The first is the so-called functional metagenomics, which evaluates biochemical and metabolic activities of interest [29] and is based on cloning random fragments of community DNA in large insert-holding vectors (cosmids, fosmids, etc.) to generate an expression library, which is then screened for a target reaction with a specific substrate [30]. In this case, the choice of the ecosystem for analysis allows identification enzymes with desired characteristics [31]; for example, it could be expected that the metagenome of a thermal spring would contain specific genes encoding thermostable enzymes. Functional metagenomics was successfully applied to identify different antibiotics, hydrolytic enzymes, antibiotic resistance genes, and many other functions [29]; moreover, it allowed characterization of genes encoding enzymes with a particular activity, which represent completely novel sequence types carrying no similarity to the ones already known [31].

Fig. 1.1 Overview of metagenomic analysis of microbial communities.

However, function-based metagenomics has several limitations. First, the range of functional activities is limited to those identifiable by available screening systems, such as used for characterization of antibiotics or hydrolytic enzymes. Second, not all genes, especially those from phylogenetically distant uncultured organisms, could be efficiently expressed in standard hosts such as E. coli. Finally, there is a size limitation for metagenomic libraries; thus, a fosmid library containing 50,000 clones with an average insert size of 40 kbp is equivalent to about 500 bacterial genomes with the size of 4 Mbp, while even 1 g of soil could contain several thousand different microbial species [32].

An alternative to functional metagenomics is sequencing-based metagenomics, which involves large-scale sequencing of the total environmental DNA, followed by gene search and functional annotation by bioinformatics methods, usually through homology to known sequences. This strategy is more productive since it provides information about all potential activities of microorganisms represented by the metagenome rather than of a limited set of those targeted by functional screening, offering such advantages as scalability and independence from availability of high-throughput screening systems. However, similar to genomic sequencing of cultured microorganisms, this approach depends on existing gene annotations, which limits its potential to identify fundamentally novel genes encoding desired functions [33]. Nevertheless, the accumulation of large new data in genetic databases and development of more sophisticated bioinformatics methods promotes the use of sequencing-based metagenomics as the method of choice for the search of functional activities of interest.

1.3 An Overview of Methods Used in Sequencing-Based Metagenomics

The ultimate goal of a metagenomic project is the characterization of a microbial community in terms of taxonomic composition and actual or potential metabolic and/or biogeochemical contribution to the environment, which could be further analyzed by means of metatranscriptomics and metaproteomics.

Currently, community taxonomic structure is determined by genetic profiling based on PCR amplification and sequencing of 16S rRNA genes. NGS methods, especially pyrosequencing, can generate relatively long reads (400–700 nt) sufficient for 16S rRNA-based taxonomic identification of microorganisms, providing deep quantitative characterization of microbial community structure through analysis of dozens to hundreds of thousands of 16S rRNA gene sequences [34]. However, PCR amplification of 16S rRNA gene fragments may introduce a bias due to unequal amplification rates of different 16S rRNA genes and generation of chimeric sequences. Primers which are supposed to be universal may not match their target regions in 16S rRNA genes of some taxa, especially in phylogenetically distant uncultured lineages. Precise taxonomic assignment of determined 16S rRNA sequences is the most important aspect in the analysis of microbial community structure. Taxonomic identification is typically performed based on similarity searches against reference 16S rRNA databases, such as Greengenes [35], SILVA [36], and RDP [37], which comprise 16S rRNA sequences of taxonomically identified cultured microorganisms, but could also contain sequences from uncultured microbial lineages. The accuracy of such a classification depends on the presence of closely related reference sequences in the database and could be problematic for microbial communities including novel groups phylogenetically distant from the characterized species. Alternatively, reference-free methods, such as UCLUST [38], which perform clustering of 16S rRNA sequences into operational taxonomic units (OTUs) based on sequence similarity, can be used. The following taxonomic identification of the obtained OTUs could be done by comparison with reference 16S rRNA databases or using different methods of phylogenetic analysis.

A methodologically similar approach is employed for functional profiling of microbial community. This strategy is based on identification and characterization of sequence diversity among representatives of a particular gene family through sequencing of fragments amplified using conservative primers specific for the target gene family [39]. Taxonomic placement of the obtained sequences reveals phylogenetic groups in the microbial community that carry the gene of interest and, thus, could perform the corresponding function.

Reconstruction of metabolic pathways of microorganisms comprising the community and prediction of their functional roles in the ecosystem are the main goals of sequencing-based metagenomics. This requires determination of genome sequences of microorganisms present in the community. Assembly of contigs from individual metagenomic sequencing reads is performed using the same algorithms and bioinformatic software as for the genomes of cultured organisms, although some of these tools have versions optimized for the assembly of metagenomes (e.g., MetaVelvet [40] and Meta-IDBA [41]). However, two principal differences between microbial cultures and environmental microbial communities should be considered. First, in pure cultures the cells represent a clonal population and have identical genomes, while in the natural sample even a single species may actually comprise closely related but nonidentical strains with a wide range of genetic differences: from small sequence variations (such as single nucleotide polymorphisms) to the presence or absence of genes or genomic regions. Such heterogeneity complicates contig assembly and could result in chimeric contigs due to sequence similarity between genomes of closely related but distinct organisms. Therefore, genomes reconstructed by metagenomics as a set of contigs actually represent composite genomes of a group comprising closely related microorganisms present in the community. The second important difference between an isolate and an environmental sample is a diversity of the community at different taxonomic levels and huge variations in the relative abundance of particular groups. Therefore, even in the case of high metagenome sequence coverage, sequencing reads representing minor groups could not be assembled into contigs. For example, in a metagenomic study of a soil community comprising about 3000 species of which none dominated, about 99% of reads were not assembled into contigs [42].

Nevertheless, metagenome sequencing of simple communities or diverse communities containing a small number of dominant lineages allows assembling relatively long contigs (in the range from 10³ to 10⁶ nucleotides), and continuous progress in sequencing technology enables such results to be obtained for more complex microbial communities. To construct a composite genome, the assembled contigs should be distributed among the microorganisms present in the community. Binning of metagenomic sequences (reads or contigs) is based on their nucleotide composition, including GC content, tetra-nucleotide frequency, and codon usage, as well as on sequence similarity with reference genomes, sequence coverage, and other parameters [43–47]. The accuracy of such clustering strongly depends on the contig length and is typically efficient for contigs longer than several thousand nucleotides. Taxonomic assignment of the obtained contig bins could be performed by sequence similarity search and phylogenetic analysis of conserved vertically inherited marker genes such as those encoding ribosomal RNA and proteins. Completeness of the obtained composite genome could be quantitatively assessed by the presence of an entire set of single-copy conserved marker genes in the corresponding bin [48], while the detection of two or more copies of such genes would indicate incorrect binning and contamination of the analyzed bin by contigs representing other microorganisms.

Extreme ecosystems usually have low biodiversity and thus represent a convenient object for metagenomic studies aiming to assemble composite genomes of individual microorganisms. A good example is the acidic mine drainage environment formed as a result of oxidation of sulfide ores and characterized by very low pH and high concentration of dissolved metal ions. In one of the first large-scale metagenomic studies, Tyson et al. [22] analyzed the microbial community structure of a biofilm grown in acid mine drainage of the Richmond Mine (California, United States) and determined near complete composite genomes of Leptospirillum group II bacterium and Ferroplasma type II archaeum, as well as partial genomes of Leptospirillum group III, Ferroplasma type I, and G-plasma archaeon. Metagenomic analysis of various extreme ecosystems allowed reconstruction of complete or nearly complete genome sequences for representatives of several uncultured candidate phyla: archaea of the ARMAN group [49] subsequently assigned to the novel phylum Parvarchaeota [14], Nanohaloarchaeota [50], Hot Water Crenarchaeal Group I [51], anaerobic methane-oxidizing ANME-1 archaea [52], and bacteria of the candidate phyla OP1 [53], OP9 [54] and others.

Despite technical advances in DNA sequencing and metagenomic data analysis, it appears difficult or even impossible to assemble long contigs and, consequently, to determine hogh-quality composite genomes of individual species for very complex microbial communities or those containing multiple closely related species. In such cases, nucleotide sequences of individual reads could be used for functional and taxonomic classification of the metagenome, although read-based analysis is more frequently employed for the identification of known organisms in the metagenome, which is achieved by mapping reads to reference genomes. Analysis of individual sequencing reads rather than contigs addresses the problem of excluding minor members of the community whose genomes could not be assembled into contigs because of insufficient coverage. However, taxonomical and functional predictions are usually effective only for a small fraction of reads because of their much shorter length compared to contigs.

Recently developed sequencing platforms such as PacBio RS II (Pacific Biosciences, Menlo Park, United States) and nanopore-based sequencers (Oxford Nanopore Technologies, Oxford, UK) enable sequencing of individual DNA molecules without amplification, generating several thousands of nucleotide-long reads, and are expected to greatly facilitate reconstruction of composite genomes from metagenomic sequences. However, at present these technologies have several limitations, the most important being a significant error rate requiring high sequence coverage (more than 50-fold) to achieve high accuracy of genome assembly [55].

1.4 Interplay of Single-Cell Genomics and Metagenomics

Contrary to metagenomics, which analyzes the total community DNA with subsequent bioinformatics-based binning of obtained sequences among individual microbial species, single-cell genomics studies genomes of individual cells recovered from the environment [56–58]. Single cells isolated using fluorescence-activated cell sorting or optofluidics are lysed, and whole-genome DNA obtained in femtogram amounts is subjected to a multiple displacement amplification [59] with subsequent generation of micrograms of DNA sufficient for convenient sequencing. The resultant single amplified genome (SAG) is sequenced as an ordinary microbial genome, assembled into contigs, and analyzed (Fig. 1.2). The limitations of this approach are associated with the isolation and lysis of single cells, sensitivity to contamination, and incomplete and uneven amplification of single-cell genomes [16,56]. As a result, sequenced SAGs are usually represented by a set of contigs covering only a part of the whole genome [14]. These problems could be solved in part by combining the data obtained from sequencing of several SAGs from cells of a single species [14]. An example of the successful application of single-cell genomics is a study of Rinke et al. [14], where they sequenced 201 SAGs representing 29 uncultured lineages of microbial dark matter [14].

Fig. 1.2 Synergy between metagenomics and single-cell genomics.

Genomes of uncultured microorganisms obtained by metagenomics and single-cell genomics are fundamentally different, as metagenomic bins represent composite genomes of a genetically heterogeneous population, while SAGs are genomes of individual cells; the latter are often incomplete and do not cover the whole genetic potential of different strains of the same species occurring in the population [16].

Methodological platforms used in metagenomics and single-cell genomics are mutually complementary, as metagenomics do not face the problems associated with the isolation of single cells and incomplete SAG amplification, while single-cell genome sequencing enables unambiguous phylogenetic assignment of the obtained genomic sequences [16,59,60]. Therefore, SAG sequencing could enhance the efficiency of metagenome binning and, in certain cases, enables the reconstruction of complete composite genomes of microorganisms belonging to novel phylogenetic lineages [14,18]. In turn, metagenomic contigs could be used to fill the gaps in SAG assembly [18,57].

1.5 Concluding Remarks

Application of metagenomics and single-cell genomics for analysis of natural ecosystems has revealed a high genomic and metabolic diversity of microorganisms that was not anticipated. Multiple uncultured lineages of Bacteria and Archaea characterized at the genomic level have already exceeded the cultured lineages in number, indicating that metagenomics together with single-cell genomics have become the main instruments in environmental microbiology. However, our understanding of taxonomic diversity in the microbial world is far from being complete, and, according to the estimation of Rinke et al. [14], sequencing of at least 16,000 genomes from different ecosystems will be required to cover 50% of phylogenetic diversity known from 16S rRNA profiling. In most cases, the reconstructed genomes of uncultured microorganisms remain incomplete and have low sequence coverage, in reality representing composite genomes of closely related strains. The development of sequencing technologies, especially single-molecule sequencing, which allows obtaining significantly longer reads, would play a key role in assembling complete genomes from metagenomic datasets.

With the growing amount of genomic information and continuous filling of the gaps in the tree of life, approaches to verify genome-based functional predictions will be brought to the forefront of microbial ecology. Although metagenomics can provide information about community structure and functional genetic potential, it cannot reveal what the members of the microbial community are actually doing in particular environmental conditions. To address these aspects, metagenomics should be complemented with metatranscriptomic and metaproteomic analyses and isotope labeling approaches.

Although the success of applying cultivation-independent methods to genetic profiling of the microbial world is obvious, one of the most valuable outcomes of sequencing genomes from uncultured microorganisms is so-called genome-enabled cultivation. In the future, scientists would employ a combination of culture-independent approaches and characterization of pure cultures by the whole arsenal of microbiological, biochemical, and genetic methods to obtain knowledge about yet uncultured microbial lineages.

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