Steps in Metagenomics: Let’s Avoid Garbage in and Garbage Out

Is metagenomics a revolution or a new fad? Metagenomics is tightly associated with the availability of next-generation sequencing in all its implementations. The key feature of these new technologies, moving beyond the Sanger-based DNA sequencing approach, is the depth of nucleotide sequencing per sample.1 Knowing much more about a sample changes the traditional paradigms of “What is the most abundant?” or “What is the most significant?” to “What is present and potentially sig­nificant that might influence the situation and outcome?” Let’s take the case of identifying proper biomarkers of disease state in the context of chronic disease prevention. Prevention has been deemed as a viable option to avert human chronic diseases and to curb health­care management costs.2 The actual implementation of any effective preventive measures has proven to be rather difficult. In addition to the typically poor compliance of the general public, the vagueness of the successful validation of habit modification on the long-term risk, points to the need of defining new biomarkers of disease state. Scientists and the public are accepting the fact that humans are super-organisms, harboring both a human genome and a microbial genome, the latter being much bigger in size and diversity, and key for the health of individuals.3,4 It is time to investigate the intricate relationship between humans and their associated microbiota and how this relationship mod­ulates or affects both partners.5 These remarks can be expanded to the animal and plant kingdoms, and holistically to the Earth’s biome. By its nature, the evolution and function of all the Earth’s biomes are influenced by a myriad of interactions between and among microbes (planktonic, in biofilms or host associated) and the surrounding physical environment. The general definition of metagenomics is the cultivation-indepen­dent analysis of the genetic information of the collective genomes of the microbes within a given environment based on its sampling. It focuses on the collection of genetic information through sequencing that can target DNA, RNA, or both. The subsequent analyses can be solely fo­cused on sequence conservation, phylogenetic, phylogenomic, function, or genetic diversity representation including yet-to-be annotated genes. The diversity of hypotheses, questions, and goals to be accomplished is endless. The primary design is based on the nature of the material to be analyzed and its primary function

Structure, function and diversity of the healthy human microbiome

Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome

Metagenomics for Bacteriology

The study of bacteria, or bacteriology, has gone through transformative waves since its inception in the 1600s. It all started by the visualization of bacteria using light microscopy by Antonie van Leeuwenhoek, when he first described “animalcules.” Direct cellular observation then evolved into utilizing different wavelengths on novel platforms such as electron, fluorescence, and even near-infrared microscopy. Understanding the link between microbes and disease (pathogenicity) began with the ability to isolate and cultivate organisms through aseptic methodologies starting in the 1700s. These techniques became more prevalent in the following centuries with the work of famous scientists such as Louis Pasteur and Robert Koch, and many others since then. The relationship between bacteria and the host’s immune system was first inferred in the 1800s, and to date is continuing to unveil its mysteries. During the last century, researchers initiated the era of molecular genetics. The discovery of the first-generation sequencing technology, the Sanger method, and, later, the polymerase chain reaction technology propelled the molecular genetics field by exponentially expanding the knowledge of relationship between gene structure and function. The rise of commercially available next-generation sequencing methodologies, in the beginning of this century, is drastically allowing larger amount of information to be acquired, in a manner open to the democratization of the approach

Microbiota, Oral Microbiome, and Pancreatic Cancer

Only 30% of patients diagnosed with pancreatic cancer survive one year post-diagnosis. Progress in understanding the causes of pancreatic cancer has been made, including solidifying the associations with obesity and diabetes, and a proportion of cases should be preventable through lifestyle modifications. Unfortunately, identifying reliable biomarkers of early pancreatic cancer has been extremely challenging, and no effective screening modality is currently available for this devastating form of cancer. Recent data suggest the microbiota may play a role in the disease process, but many questions remain. Future studies focusing on the human microbiome, both etiologically and as a marker of disease susceptibility, should shed light on how to better tackle prevention, early detection, and treatment of this highly fatal disease

Promises and Prospects of Microbiome Studies

Since Anthony van Leeuwenhoek, first microscopic observations of the unseen microbiota and the more recent realization that little of the microbes in the biosphere are known, humans have developed a deep curiosity to fully understand the inner workings of the microbial realm. Our ability to characterize the complexity of microbial communities in their natural habitats has dramatically improved over the past decade thanks to advances in high-throughput methodologies. By eliminating the need to isolate and culture individual species, metagenomics approaches have removed many of the obstacles that hindered research in the ecology of mixed microbial consortia, providing valuable information about the diversity, composition, function, and metabolic capability of the community. Microbes are the unseen majority with the capability to colonize every environment, including our bodies. The establishment and composition of a stable human microbiome is determined by the host genetics, immunocompetence, and life-style choices. Our life-style choices determine our exposure to many external and internal environmental factors that permanently or temporarily can influence our microbiome composition. Figure 1 illustrates some of the life-style-related factors that might influence the microbiota of the skin, mouth, and gut. It is not limited to what we carry, touch, breath, and eat. Other dispersal vectors include secretion, excretions, aerosols, air flow, animals, moving surfaces, water, beverages, food, contact, wind, tools, toiletry, and others. These influence the microbiome membership, who are present, and they have the ability to participate in the microbiome dynamic within an environment. The establishment of a microbial community is dependent on many environmental factors, including pH, temperature, altitude, weather, soil type, nutrient availability, relative humidity, air quality, pollutants, microbial competitors, and others. In other words, we are superorganisms interconnected with other living forms on this Earth

A framework for human microbiome research

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies

The unseen world: environmental microbial sequencing and identification methods for ecologists

Archaea, bacteria, microeukaryotes, and the viruses that infect them (collectively “microorganisms”) are foundational components of all ecosystems, inhabiting almost every imaginable environment and comprising the majority of the planet’s organismal and evolutionary diversity. Microorganisms play integral roles in ecosystem functioning; are important in the biogeochemical cycling of carbon (C), nitrogen (N), sulfur (S), phosphorus (P), and various metals (eg Barnard et al. 2005); and may be vital to ecosystem responses to large-scale climatic change (Mackelprang et al. 2011). Rarely found alone, microorganisms often form complex communities that are dynamic in space and time (Martiny et al. 2006). For these and other reasons, ecologists and environmental scientists have become increasingly interested in understanding microbial dynamics in ecosystems. Ecological studies of microbes in the environment generally focus on determining which organisms are present and what functional roles they are playing or could play. Rapid advances in molecular and bioinformatic approaches over the past decade have dramatically reduced the difficulty and cost of addressing such questions (Figure 1; WebTable 1). Yet the range of methodologies currently in use and the rapid pace of their ongoing development can be daunting for researchers unaccustomed to these technologies

Genetic and Structural Analyses of Cytoplasmic Filaments of Wild-Type Treponema phagedenis and a Flagellar Filament-Deficient Mutant

Unique cytoplasmic filaments are found in the treponeme genus of spirochete bacteria. Their function is unknown, but their location underneath the periplasmic flagellar filaments (PFF) suggests a role in motility and/or cell structure. To better understand these unique structures, the gene coding for the cytoplasmic filaments, cfpA, was identified in various treponemal species. Treponema phagedenis cfpA was 2,037 nucleotides long, and the encoded polypeptide showed 78 to 100% amino acid sequence identity with the partial sequence of CfpA from T. denticola, T. vincentii, and T. pallidum subsp. pertenue. Wild-type T. phagedenis and a PFF-deficient isolate were analyzed by electron microscopy to assess the structural relationship of the cytoplasmic filaments and the PFF. The number of cytoplasmic filaments per cell of T. phagedenis (mean, 5.7) was compared with the number of PFF at each end of the cell (mean, 4.7); the results suggest that there is no direct one-to-one correlation at the cell end. Moreover, a structural link between these structures could not be demonstrated. The cytoplasmic filaments were also analyzed by electron microscopy at different stages of cell growth; this analysis revealed that they are cleaved before or during septum formation and before the nascent formation of PFF. A PFF-deficient mutant of T. phagedenis possessed cytoplasmic filaments similar to those of the wild type, suggesting that intact PFF are not required for their assembly and regulation. The extensive conservation of CfpA among pathogenic spirochetes suggests an important function, and structural analysis suggests that it is unlikely that the cytoplasmic filaments and the flagellar apparatus are physically linked

\u3ci\u3eMetagenomics for Microbiology:\u3c/i\u3e Preface

It is well known that only a small fraction of extant microbial life has been identified. Metagenomics, the direct sequencing and characterization of genes and genomes present in complex microbial ecosystems (e.g., metagenomes), has revolutionized the practice of microbiology by bypassing the hurdle of pure culture isolation. Metagenomics shows promise of advancing our understanding of the diversity, function, and evolution of the uncultivated majority. Metagenomics as a field arose in the 1990s after the application of molecular biology techniques to genomic material directly extracted from microbial assemblages present in diverse habitats, including the human body. The application of metagenomic approaches allows for the acquisition of genetic/genomic information from the viruses, bacteria, archaea, fungi, and protists forming complex assemblages. The field of metagenomics addresses the fundamental questions of which microbes are present and what their genes are potentially doing. In the mid-2000s, the availability of high-throughput or next-generation sequencing technologies propelled the field by lowering the monetary and time constraints imposed by traditional DNA sequencing technologies. These advances have allowed the scientific community to examine the microbiome of diverse environments/habitats, follow spatial and temporal changes in community structure, and study the response of the communities to treatment or environmental modifications. In 2012, the publication of the large-scale characterization of the microbiome of healthy adults created high expectations about the influence of the microbiota in human health and disease. With the publication of the results of the Human Microbiome Project, metagenomics has emerged as a major research area in microbiology, particularly, when it comes to the characterization of the role of microbiota in complex disorders, such as obesity. With contributions by leading researchers in the field, we provide a series of chapters describing best practices for the collection and analysis of metagenomic data, as well as the promises and challenges of the field. The chapters have been dedicated to different aspects of metagenomics

\u3ci\u3eMetagenomics for Microbiology:\u3c/i\u3e Preface

It is well known that only a small fraction of extant microbial life has been identified. Metagenomics, the direct sequencing and characterization of genes and genomes present in complex microbial ecosystems (e.g., metagenomes), has revolutionized the practice of microbiology by bypassing the hurdle of pure culture isolation. Metagenomics shows promise of advancing our understanding of the diversity, function, and evolution of the uncultivated majority. Metagenomics as a field arose in the 1990s after the application of molecular biology techniques to genomic material directly extracted from microbial assemblages present in diverse habitats, including the human body. The application of metagenomic approaches allows for the acquisition of genetic/genomic information from the viruses, bacteria, archaea, fungi, and protists forming complex assemblages. The field of metagenomics addresses the fundamental questions of which microbes are present and what their genes are potentially doing. In the mid-2000s, the availability of high-throughput or next-generation sequencing technologies propelled the field by lowering the monetary and time constraints imposed by traditional DNA sequencing technologies. These advances have allowed the scientific community to examine the microbiome of diverse environments/habitats, follow spatial and temporal changes in community structure, and study the response of the communities to treatment or environmental modifications. In 2012, the publication of the large-scale characterization of the microbiome of healthy adults created high expectations about the influence of the microbiota in human health and disease. With the publication of the results of the Human Microbiome Project, metagenomics has emerged as a major research area in microbiology, particularly, when it comes to the characterization of the role of microbiota in complex disorders, such as obesity. With contributions by leading researchers in the field, we provide a series of chapters describing best practices for the collection and analysis of metagenomic data, as well as the promises and challenges of the field. The chapters have been dedicated to different aspects of metagenomics