Metagenomics is the study of a collection of genetic material (genomes) from a mixed community of organisms. Metagenomics usually refers to the study of microbial communities.

"Metagenomics" is the two words "meta" and "genomics". So genomics is obtaining the DNA sequence, but meta implies that we're doing it of many organisms together. And metagenomics is usually used when we are studying microbial communities where we can't separate one microbe from another. Like there may be two bacteria that grow together, and so when you take the DNA sequence, you're getting the DNA sequence of two bacteria together. Now, as an example of this, you can imagine that I could go in and take the DNA sequence of a person who lives in New York City. But if I were to come in and take the DNA from everyone who lives in New York City and sequence it together, that would be the equivalent of what we're doing when were sequencing the DNA of all of the bacteria that live in one place on your skin or your intestine together. So we're not just looking at one organism, we're looking at the DNA sequence of all of the organisms together. Because we could imagine sequencing the DNA of an individual in New York, but imagine if our technology was limited and we couldn't separate these people in New York. If we need to take the DNA sequence of every person in New York together, and then later we try to figure out which DNA belonged to which person, that's often what we are doing when we're studying bacterial and fungal communities together.

— Julie A. Segre, Ph.D.

Did you know that the new methods for DNA sequencing have been used to read the genomes of tens of thousands of organisms, some alive and some extinct, allowing us to redefine the "tree of life?" Darwin could never have imagined the full complexity of the tree of life, but genomic technologies have been used to illuminate the natural world in amazing ways. We are all connected to every living thing on earth, from plants to animals to microbes, through our genomes.

Genomics helps us understand the biology of organisms across the world: why are some faster or smarter than others? Why have some gone extinct, while others are resilient to environmental changes? What do their genomes teach us about our own?

Have you ever taken a close look at a platypus? It has a bill instead of a nose and it lays eggs, just like a bird. But it also carries deadly venom like a reptile, and it has fur and nurses its young like a mammal. In fact, when the first body of a platypus was shipped from Australia to a museum in England, the curator thought it was a hoax.

In 2008, scientists determined the genome sequence of the platypus. That study showed that the platypus is truly a unique mix of reptiles and mammals, with genes for venom and milk being retained in the same animal lineage over many years of evolution! Similar genomic studies involving thousands of other life forms have yielded amazing new insights into about every branch on the tree of life.

Recent genomic discoveries have forced us to rethink many long-standing, basic assumptions. For example, we have learned that many seemingly "simpler" species have more genes than humans, including the octopus and the water flea. Other species have much larger genomes than humans, including the loblolly pine (7-times larger than human) and a salamander called the axolotl (10-times larger than human!). We have also had to change the placement of some organisms on the "Tree of Life" - like when we learned that turtles are more closely related to birds than to lizards and snakes, or when DNA sequencing showed how much DNA had been exchanged between different types of bacteria over time [see Microbes and Microbiomes]. We've even been able to sequence the genomes of extinct animals using preserved specimens, like a museum sample of a Tasmanian tiger and the pelt from a woolly mammoth. Although sequencing their genomes cannot bring back extinct species, these studies reveal what these animals had in common with modern life forms on earth - and this, in turn, teaches us important things about evolution.

DNA Barcoding

Scientists can now use information about short stretches of DNA that are unique to each species to establish DNA "barcodes." These barcodes distinguish one species from another. DNA barcoding can be used to make complete catalogs of organisms in a certain ecosystem, like on the island of Mo'orea in French Polynesia. Then researchers can more precisely track how the organisms change over time and in response to environmental changes. DNA barcodes also provide insights about the diets of animals through the DNA in their feces, so we can determine where animals migrate and can help to protect their habitats. In a different application, DNA barcoding has enabled food safety experts to monitor the food supply and prevent fraud. For example, they showed that the large side wings of fish called skates or rays were being cut into circles and improperly sold as scallops in multiple countries. Stopping this illegal activity is critical for skate and ray conservation.

Metagenomics

Have you ever wondered what organisms live in the air in the New York subway system or in extreme environments like hot springs, or how bacterial communities respond to environmental disasters like the Deepwater Horizon oil spill?

Thanks to advances in DNA sequencing, there is a new and exciting scientific field called "metagenomics," which involves studying all the DNA in an environmental sample. Following sequencing of mixed samples of DNA, scientists perform computer analyses to catalog all the living things that are present based on matches to known genome sequences. Metagenomics is important because researchers cannot always grow an organism in the laboratory to study its DNA by traditional methods. Now such organisms can be studied while existing in their natural community, like the microbial mats in Yellowstone National Park. From nine environmental samples taken in the Times Square subway station in New York City, researchers identified traces of 114 unique bacteria, in addition to other organisms like plants and humans. Metagenomic studies are allowing us to appreciate the complex interactions of our world as never before.

Did you know that your body contains many more microbes (including bacteria and viruses) than human cells? And, even though some microbes can cause dangerous infections, you can't live without others? Viral and bacterial genomes might be small, but they are packed with information and some surprises about evolution and human health.

When you think of bacteria or viruses, what comes to mind? If you've been sick recently, you might not have good thoughts about them.

In 2018, infections from the influenza virus have been particularly rough. A big difference between 2018 and 15 years ago when the Human Genome Project was completed, is that genomics is now used routinely to track influenza virus infections, in real time, as the virus spreads across the country. These data are used to help design the vaccine used for our annual "flu shot." Even so, just one mutation in the virus's genome can make a big difference in the severity of someone's infection. Beyond influenza, the most advanced genomics technologies are being used to track infections caused by the Ebola and Zika viruses as they spread and mutate.

You may have also heard of situations where the overuse of antibiotics in both the medical and agricultural fields has contributed to bacteria becoming resistant to antibiotics, because with more exposure, bacteria that spontaneously develop resistance survive and reproduce more in this new environment, spreading this trait to the next generation. Now that we can track various microbes' genome sequences very accurately, we can see that antibiotic resistance most often happens when bacteria trade their DNA back and forth transferring the resistance trait. DNA sequencing of many, many bacterial species has now shown us that there has been rampant transfer of genes throughout history from early species of bacteria to those in existence today. These findings should make people think about the overuse of antibiotics in our world, since in the most extreme cases of infection with antibiotic-resistant bacteria can lead to serious illness and even death.

Microbes Make You Who You Are

Besides the microbes that make you ill, there are also "good" microbes. We contain multitudes of microbes (bacteria, yeast, and viruses), called our "microbiome." We would not be able to properly digest food without them living in our gut, or to have a working immune system without regular exposure to these microbes. However, things can also go wrong with your gut microbiome, such as when infection with the bacterium H. pylori leads to ulcers - this discovery led to the Nobel Prize in 2005. But, for the most part, our gut bacteria are huge helpers; they even train our own immune systems on what is "good" and what is "bad" in the microbial world. In another story related to our microbiome, we acquire millions of microbes from our mother at birth, including ones that live in our throats and quickly spread to keep out other, more harmful species. Genome sequencing has shown us how diverse our microbial communities are, and we will learn even more as the NIH's Human Microbiome Project finishes its work in 2018.

We Can Engineer New Microbes

Now that we have learned so much about microbial genomics, what else can we do? The field of synthetic biology has emerged directly from advances in DNA sequencing technologies, allowing for the design and construction of new biological systems. Teams from around the world are manipulating microbial genomes for applications in energy production and medicine. There is even the International Genetically Engineered Machine Competition (or iGEM) in which thousands of students compete each year to build new systems using standard parts called BioBricks. In 2016, a winning high school team from Taiwan constructed a new strain of E. coli, a famous bacterium that lives in your gut, that can detect levels of poisonous metals in Chinese medicines.

While the genomic revolution rolls on, a new one has been quietly fomenting over the last decade or so, only to take over the science of microbiology in the last couple of years.

The name of this new game is metagenomics.

Metagenomics is concerned with the complex communities of microbes.

Traditionally, microbes have been studied in isolation, but to do that, a microbe or virus has to be grown in a laboratory. While that might sound easy, only 0.1% of the world’s microbes will grow in artificial media, with the success rate for viruses even lower.

Furthermore, studying microbes in isolation can be somewhat misleading because they commonly thrive in nature as tightly knit communities.

Metagenomics addresses both problems by exhaustively sequencing all the microbial DNA or RNA from a given environment. This powerful, direct approach immensely expands the scope of biological diversity accessible to researchers.

But the impact of metagenomics is not just quantitative. Over and again, metagenomic studies—because they look at microbes in their natural communities and are not restricted by the necessity to grow them in culture—result in discoveries with major biological implications and open up fresh experimental directions.

In virology, metagenomics has already become the primary route to new virus discovery. In fact, in a dramatic break from tradition, such discoveries are now formally recognized by the International Committee on Taxonomy of Viruses. This decision all but officially ushers in a new era, I think.

Here is just one striking example that highlights the growing power of metagenomics.

In 2014, Rob Edwards and colleagues at San Diego State University achieved a remarkable metagenomic feat. By sequencing multiple human gut microbiomes, they managed to assemble the genome of a novel bacteriophage, named crAssphage (for cross-Assembly). They then went on to show that crAssphage is, by a wide margin, the most abundant virus associated with humans.

This discovery was both a sensation and a shock. We had been completely blind to one of the key inhabitants of our own bodies—apparently because the bacterial host of the crAssphage would not grow in culture. Thus, some of the most common microbes in our intestines, and their equally ubiquitous viruses, represent “dark matter” that presently can be studied only by metagenomics.

But the crAssphage genome was dark in more than one way.

Once sequenced, it looked like nothing in the world. For most of its genes, researchers found no homologs in sequence databases, and even those few homologs identified shed little light on the biology of the phage. Furthermore, we had been unable to establish any links to other phages, nor could we tell which proteins formed the crAssphage particle.

Such results understandably frustrate experimenters, but computational biologists see opportunity.

A few days after the crAssphage genome was published, Mart Krupovic of Institut Pasteur visited my lab, where we attempted to decipher the genealogies and functions of the crAssphage proteins using all computational tools available to us at the time. The result was sheer disappointment. We detected some additional homologies but could not shed much light on the phage evolutionary relationships or reproduction strategy.

We moved on. With so many other genomes to analyze, crAssphage dropped from our radar.

Then, in April 2017, Anca Segall, a sabbatical visitor in my lab, invited Rob Edwards to give a seminar at NCBI about crAssphage. After listening to Rob’s stimulating talk—and realizing that the genome of this remarkable virus remains a terra incognita—we could not resist going back to the crAssphage genome armed with some new computational approaches and, more importantly, vastly expanded genomic and metagenomic sequence databases.

This time we got better results.

After about eight weeks of intensive computational analysis by Natalya Yutin, Kira Makarova, and myself, we had fairly complete genomic maps for a vast new family of crAssphage-related bacteriophages. For all these phages, we predicted with good confidence the main structural proteins, along with those involved in genome replication and expression. Our work led to a paper we recently published in the journal Nature Microbiology. We hope and believe our findings provide a roadmap for experimental study of these undoubtedly important viruses.

Apart from the immediate importance of the crAss-like phages, this story delivers a broader lesson. Thanks to the explosive growth of metagenomic databases, the discovery of a new virus or microbe does not stop there. It brings with it an excellent chance to discover a new viral or microbial family. In addition, analyzing the gene sequences can yield interesting and tractable predictions of new biology. However, to take advantage of the metagenomic treasure trove, we must creatively apply the most powerful sequence analysis methods available, and novel ones may be required.

Put another way, if you know where and how to look, you have an excellent chance to see wonders.

As a result, I cannot help being unabashedly optimistic about the future of metagenomics. Fueled by the synergy between increasingly high-quality, low-cost sequencing, improved computational methods, and emerging high-throughput experimental approaches, the prospects appear boundless. There is a realistic chance we will know the true extent of the diversity of life on earth and get unprecedented insights into its ecology and evolution within our lifetimes. This is something to work for.

Eugene Koonin, PhD, has served as a senior investigator at NLM’s National Center for Biotechnology Information since 1996, after working for five years as a visiting scientist. He has focused on the fields of computational biology and evolutionary genomics since 1984.

Metagenomics, or identifying the organisms present in a sample through DNA-based analysis, is a revolutionary technology made possible through advances in sequencing technologies and computing power. Like all sample analyses, standards are needed to benchmark performance and enable translation into applications such as clinical diagnostics, microbiome therapeutics, environment, and bio-surveillance. Given the complexities of the analyses, an entire suite of NIST Reference Materials are under development to provide measurement assurance.

DESCRIPTION

Metagenomics is a powerful technology combining high-throughput (a.k.a. “next-generation”) sequencing with advanced bioinformatics to identify and quantify mixtures of organisms in a sample. Metagenomics workflows consist of 4 main steps:

• Sample collection: location, amount, storage, etc.
• Extraction: method, amount
• Sequencing: preparation, platform, chemistry
• Bioinformatics Analysis: algorithm, database, pre- and post-filtering

Each step contributes some error, or bias, to the overall measurement, which in some cases can result in wildly different results from what is known or expected. Error propagation is linear, which offers an opportunity to characterize it by working backwards using well-characterized materials suited for benchmarking each critical step.