Metagenomics is a very broad field that can often be summarized down to the analysis of a microbiome. These microbiomes are all around and inside us. They’re in the air we breathe, the water we drink and the food we eat. They’re high up in the atmosphere, at the bottom of the ocean, in pools of boiling acid - they exist everywhere that hasn’t been carefully sterilized. These microbiomes represent diverse ecosystems with countless species of prokaryotes, fungi, viruses, protozoa, plants, and more. The main challenges when studying them are how to isolate an individual species for study, and perhaps now more importantly, how can we possibly investigate all the complex interactions between them.

The traditional approach to studying microbes has been to isolate and then culture a specific cell line1. This homogeneous population can then be tested against any number of chemicals or pathogens, for example. This has proven impossible in microbiome analysis - a big reason being that most species do not lend themselves well to being cultured. Luckily, the more traditional approach is going out the window with the advent of massively-parallel shotgun sequencing. We no longer need to isolate and culture a microbe to investigate it. Researchers can now prepare entire heterogeneous samples and sequence them to determine the species present using phylotyping2.

Phylotyping involves amplifying, sequencing, and comparing ribosomal RNA (rRNA)3 against pre-defined phylogenies. These studies use rRNA because it is highly conserved by nature, meaning it varies very little between different species compared to other regions. This quality is important because primers used for amplification are likely to be on-target for a broader collection of species. Moreover, the little variance that does exist between species is enough to differentiate them. Different types of rRNA are looked at depending on the organism under study. Within prokaryotes, 16s rRNA is usually looked at. In practice, sequencing uses the complementary rDNA and has been very effective in identifying novel bacteria, especially within rare or difficult to culture species4. Studies involving eukaryotes typically make use of 18s rDNA.

Microeukaryotes play important roles in diverse ecosystems, such as waterways, and can be incredibly difficult to culture in a lab. Fortunately, studies investigating them can employ a sequencing approach that only requires sample extraction and amplification5. Finally, the ITS region within fungal species is of clinical importance for microbiome analysis. It represents a region similar enough to capture most fungal species, but different enough that individual species can be told apart6.

A lot of these advances may appear to have fully solved the problem of microbiome analysis. However, challenges do of course still exist. The most pressing issue is that of bias introduced by PCR amplification. Although designing primers for the highly-conserved regions discussed above will work for most species, some will always get missedsup>7. The catch when trying to identify novel species is that it’s hard to find something you aren’t looking for. The spotlight for human microbiome analysis rests on gut microbiome, and how it relates to the brainsup>8. However, studies are investigating other areas such as the surface of our skinsup>9. Other studies are not only looking at the broader soil microbiomesup>10, but also how these communities interact synergistically with plantssup>11. All in all, we’re well on our way to having a much better understanding of the tiny communitiessup>12 all around us and how they interact.

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2Thomas, Torsten, Jack Gilbert, and Folker Meyer. "Metagenomics-a guide from sampling to data analysis." Microbial informatics and experimentation 2.1 (2012): 3.


4Woo, P. C. Y., et al. "Then and now: use of 16S rDNA gene sequencing for bacterial identification and discovery of novel bacteria in clinical microbiology laboratories." Clinical Microbiology and Infection 14.10 (2008): 908-934.

5Searle, Daniel, et al. "18S rDNA dataset profiling microeukaryotic populations within Chicago area nearshore waters." Data in brief 6 (2016): 526-529.

6Schoch, Conrad L., et al. "Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi." Proceedings of the National Academy of Sciences 109.16 (2012): 6241-6246.

7Anderson, Ian C., Colin D. Campbell, and James I. Prosser. "Potential bias of fungal 18S rDNA and internal transcribed spacer polymerase chain reaction primers for estimating fungal biodiversity in soil." Environmental Microbiology 5.1 (2003): 36-47.

8O’Mahony, S. M., et al. "Serotonin, tryptophan metabolism and the brain-gut-microbiome axis." Behavioural brain research 277 (2015): 32-48.

9Kong, Heidi H., and Julia A. Segre. "The molecular revolution in cutaneous biology: investigating the skin microbiome." Journal of Investigative Dermatology 137.5 (2017): e119-e122.

10Schöler, Anne, et al. "Analysis of soil microbial communities based on amplicon sequencing of marker genes." (2017): 485-489.

11Lundberg, Derek S., et al. "Defining the core Arabidopsis thaliana root microbiome." Nature 488.7409 (2012): 86.

12Gopal, Murali, and Alka Gupta. "Microbiome selection could spur next-generation plant breeding strategies." Frontiers in microbiology 7 (2016): 1971.