Metagenomics

Metagenomics is the study of all genetic material from all organisms in a particular environment, providing insights into their composition, diversity, and functional potential. Metagenomics has allowed researchers to profile the microbial composition of environmental and clinical samples without the need for time-consuming culture of individual species.
Metagenomics has transformed microbial ecology and evolutionary biology by uncovering previously hidden biodiversity and metabolic capabilities. As the cost of DNA sequencing continues to decline, metagenomic studies now routinely profile hundreds to thousands of samples, enabling large-scale exploration of microbial communities and their roles in health and global ecosystems.
Metagenomic studies most commonly employ shotgun sequencing though long-read sequencing is being increasingly utilised as technologies advance. The field is also referred to as environmental genomics, ecogenomics, community genomics, or microbiomics and has significantly expanded the understanding of microbial life beyond what traditional cultivation-based methods can reveal.
Metagenomics is distinct from Amplicon sequencing, also referred to as Metabarcoding or PCR-based sequencing. The main difference is the underlying methodology, since metagenomics targets all DNA in a sample, while Amplicon sequencing amplifies and sequences one or multiple specific genes. Data utilisation also differs between these two approaches. Amplicon sequencing provides mainly community profiles detailing which taxa are present in a sample, whereas metagenomics also recovers encoded enzymes and pathways. Amplicon sequencing was frequently used in early environmental gene sequencing focused on assessing specific highly conserved marker genes, such as the 16S rRNA gene, to profile microbial diversity. These studies demonstrated that the vast majority of microbial biodiversity had been missed by cultivation-based methods.

Etymology

The term "metagenomics" was first used by Jo Handelsman, Robert M. Goodman, Michelle R. Rondon, Jon Clardy, and Sean F. Brady, and first appeared in publication in 1998. The term metagenome referenced the idea that a collection of genes sequenced from the environment could be analyzed in a way analogous to the study of a single genome. In 2005, Kevin Chen and Lior Pachter defined metagenomics as "the application of modern genomics technique without the need for isolation and lab cultivation of individual species".

History

Conventional sequencing begins with a culture of identical cells as a source of DNA. However, early metagenomic studies revealed that there are probably large groups of microorganisms in many environments that cannot be cultured and thus cannot be sequenced. These early studies focused on 16S ribosomal RNA sequences which are relatively short, often conserved within a species, and generally different between species. Many 16S rRNA sequences have been found which do not belong to any known cultured species, indicating that there are numerous non-isolated organisms. These surveys of ribosomal RNA genes taken directly from the environment revealed that cultivation based methods find less than 1% of the bacterial and archaeal species in a sample. Much of the interest in metagenomics comes from these discoveries that showed that the vast majority of microorganisms had previously gone unnoticed.
In the 1980s early molecular work in the field was conducted by Norman R. Pace and colleagues, who used PCR to explore the diversity of ribosomal RNA sequences. The insights gained from these breakthrough studies led Pace to propose the idea of cloning DNA directly from environmental samples as early as 1985. This led to the first report of isolating and cloning bulk DNA from an environmental sample, published by Pace and colleagues in 1991 while Pace was in the Department of Biology at Indiana University. Considerable efforts ensured that these were not PCR false positives and supported the existence of a complex community of unexplored species. Although this methodology was limited to exploring highly conserved, non-protein coding genes, it did support early microbial morphology-based observations that diversity was far more complex than was known by culturing methods. Soon after that in 1995, Healy reported the metagenomic isolation of functional genes from "zoolibraries" constructed from a complex culture of environmental organisms grown in the laboratory on dried grasses. After leaving the Pace laboratory, Edward DeLong continued in the field and has published work that has largely laid the groundwork for environmental phylogenies based on signature 16S sequences, beginning with his group's construction of libraries from marine samples.
In 2002, Mya Breitbart, Forest Rohwer, and colleagues used environmental shotgun sequencing to show that 200 liters of seawater contains over 5000 different viruses. Subsequent studies showed that there are more than a thousand viral species in human stool and possibly a million different viruses per kilogram of marine sediment, including many bacteriophages. Essentially all of the viruses in these studies were new species. In 2004, Gene Tyson, Jill Banfield, and colleagues at the University of California, Berkeley and the Joint Genome Institute sequenced DNA extracted from an acid mine drainage system. This effort resulted in the complete, or nearly complete, genomes for a handful of bacteria and archaea that had previously resisted attempts to culture them.
Beginning in 2003, Craig Venter, leader of the privately funded parallel of the Human Genome Project, has led the Global Ocean Sampling Expedition, circumnavigating the globe and collecting metagenomic samples throughout the journey. All of these samples were sequenced using shotgun sequencing, in hopes that new genomes would be identified. The pilot project, conducted in the Sargasso Sea, found DNA from nearly 2000 different species, including 148 types of bacteria never before seen. Venter thoroughly explored the West Coast of the United States, and completed a two-year expedition in 2006 to explore the Baltic, Mediterranean, and Black Seas. Analysis of the metagenomic data collected during this journey revealed two groups of organisms, one composed of taxa adapted to environmental conditions of 'feast or famine', and a second composed of relatively fewer but more abundantly and widely distributed taxa primarily composed of plankton.
In 2005 Stephan C. Schuster at Penn State University and colleagues published the first sequences of an environmental sample generated with high-throughput sequencing, in this case massively parallel pyrosequencing developed by 454 Life Sciences. Another early paper in this area appeared in 2006 by Robert Edwards, Forest Rohwer, and colleagues at San Diego State University.
there are few metagenomics laboratories in the world; for example, in the UK only the metagenomics labs at Great Ormond Street Hospital is recognised to carry out these tests. The cost is high, but this is expected to drop as the technology is developed.

Sequencing

Recovery of DNA sequences longer than a few thousand base pairs from environmental samples was very difficult until recent advances in molecular biological techniques allowed the construction of libraries in bacterial artificial chromosomes, which provided better vectors for molecular cloning.

Shotgun metagenomics

Advances in bioinformatics, refinements of DNA amplification, and the proliferation of computational power have greatly aided the analysis of DNA sequences recovered from environmental samples, allowing the adaptation of shotgun sequencing to metagenomic samples. The approach, used to sequence many cultured microorganisms and the human genome, randomly shears DNA, sequences many short sequences, and reconstructs them into a consensus sequence. Shotgun sequencing reveals genes present in environmental samples. Historically, clone libraries were used to facilitate this sequencing. However, with advances in high throughput sequencing technologies, the cloning step is no longer necessary and greater yields of sequencing data can be obtained without this labour-intensive bottleneck step. Shotgun metagenomics provides information both about which organisms are present and what metabolic processes are possible in the community. Because the collection of DNA from an environment is largely uncontrolled, the most abundant organisms in an environmental sample are most highly represented in the resulting sequence data. To achieve the high coverage needed to fully resolve the genomes of under-represented community members, large samples, often prohibitively so, are needed. On the other hand, the random nature of shotgun sequencing ensures that many of these organisms, which would otherwise go unnoticed using traditional culturing techniques, will be represented by at least some small sequence segments.

High-throughput sequencing

An advantage to high throughput sequencing is that this technique does not require cloning the DNA before sequencing, removing one of the main biases and bottlenecks in environmental sampling. The first metagenomic studies conducted using high-throughput sequencing used massively parallel 454 pyrosequencing. Three other technologies commonly applied to environmental sampling are the Ion Torrent Personal Genome Machine, the Illumina MiSeq or HiSeq and the Applied Biosystems SOLiD system. These techniques for sequencing DNA generate shorter fragments than Sanger sequencing; Ion Torrent PGM System and 454 pyrosequencing typically produces ~400 bp reads, Illumina MiSeq produces 400-700bp reads, and SOLiD produce 25–75 bp reads. Historically, these read lengths were significantly shorter than the typical Sanger sequencing read length of ~750 bp, however the Illumina technology is quickly coming close to this benchmark. However, this limitation is compensated for by the much larger number of sequence reads. In 2009, pyrosequenced metagenomes generate 200–500 megabases, and Illumina platforms generate around 20–50 gigabases, but these outputs have increased by orders of magnitude in recent years.
To achieve higher resolution and more complete assemblies from complex environmental samples, the field has adopted several advanced techniques. One approach combines shotgun sequencing and chromosome conformation capture, which measures the proximity of any two DNA sequences within the same cell, to guide microbial genome assembly. Another technique is single-cell metagenomic sequencing, which resolves the heterogeneity present within the community. Additionally, long read sequencing technologies, such as those from Pacific Biosciences and Oxford Nanopore Technologies, generate significantly longer reads that simplify the assembly process, particularly in repetitive or structurally complex regions.