The typical adult human is home to about as many microbial cells as there are cells in his own body [1]. This collection of up to 100 trillion individual cells is made up of the bacteria, archaea, protozoans, and fungi [2] that reside on and within our bodies [3]. Together with the viruses that also inhabit us, this array of microorganisms is called the human microbiota [4]. 

This “hidden half” interacts with us from childbirth [3], immediately becoming critical to life and development [5]. As we mature, our microbiota settles into a dynamic equilibrium sensitive to environmental factors as well as external interferences, such as the use of antibiotics [2]. If this balance is disturbed, the change in microbial biodiversity could lead to life-threatening illness [3].

Until recently, the limitations of traditional microbiology made it impossible to observe the full range of microorganisms in any given habitat [6]. Metagenomics has since emerged as a powerful approach that can capture genomic information from entire microbial communities [7]. The collection of these genomes, called the microbiome, depicts ‘true’ biodiversity [8].

What is Metagenomics?

In 1674, a Dutch cloth merchant wrote to London’s Royal Society about “animalcules”: impossibly tiny creatures, each a thousand times smaller than a mite, that he had found in the water from a nearby lake [9]. This letter kick-started Antonie van Leeuwenhoek’s ascent into scientific fame; today, we know that his animalcules were the first microorganisms ever observed [10]. 

The unlikely scientist would go on to discover microorganisms in seemingly everything – even people. In another letter to the Royal Society, Leeuwenhoek describes the microbes that he found in an old man’s dental plaque [11]: “an unbelievably great company of living animalcules, a-swimming more nimbly than any I had ever seen up to this time.” With this, Leeuwenhoek had made some of the earliest observations on living bacteria…after he had already found algae (Spirogyra) and protists (Vorticella) in various bodies of water [11].

Leeuwenhoek would go even further and compare microbes between samples: he noted that not only were microbes different between body sites (i.e. mouth vs. stool), they were also different at the same site between healthy and diseased individuals [12]. By pioneering the study of what would later be called the human microbiota, Leeuwenhoek had cemented his legacy as the “Father of Microbiology.” 

Still, two centuries would pass before techniques that could explore Leeuwenhoek’s findings were developed. And, even after Robert Koch devised techniques for the isolation of pure bacterial cultures in the late 1800s [13], the study of human microbial diversity was held back by two factors: the labor-intensive processes that were necessary (each microbial species had to be cultivated and isolated before it could be characterized), and what would later be called the ‘the great plate count anomaly’ [14].

What Staley and Konopka merely coined in 1985 had been known since Koch’s era: as little as 1% of the cells directly observed under a microscope will actually form colonies on culture plates [15]. The remaining 99% was effectively microbial “dark matter”, unknown in both identity and diversity. 

This changed in the late 1970s, when Carl Woese and George Fox pioneered the use of the 16S ribosomal RNA (rRNA) gene as a phylogenetic marker [16]. The ability to differentiate between organisms at the genus level without the need for cultivation made it possible, in a landmark 1996 study, for Edward DeLong to recover a 40-kbp genome fragment from an unculturable archaeon. By amplifying the 16S rRNA gene, DeLong was able to identify which clones, from a library constructed from environmental DNA, contained archaeal genes [17].

This first attempt at sampling what would later be called the “metagenome” ushered in a new way to study microorganisms. Today, metagenomics is a powerful approach that can capture entire microbial communities [18]: it uses molecular biology to retrieve genomes directly from natural samples, bypassing the need for pure cultures [19]. The collective genomes of all the microorganisms in a particular environment is called the microbiome. 

The Microbiome and Human Health

Humans have coexisted with microorganisms since the very beginning, and we’ve evolved to depend on the microbiome as a “virtual organ,” responsible for metabolic functions beyond our own physiology [20]. An emerging notion is that each of us, together with our microbiota, forms a superorganism: a community of organisms all working for the benefit of the collective [20]. Where, then, do we draw the boundaries that define us?

The numbers don’t lie: the microbiome of the gastrointestinal (GI) tract, alone, includes at least 3.3 million non-redundant genes – already 150 times greater than the entire human genome [21]. And, while the GI tract (or the “gut”) is the most densely populated site [22], it is only one of five main body niches together with the oral cavity, the skin, the respiratory tract, and the urogenital tract [22]. 

In total, the human microbiome is estimated to contain 200- to 300-fold more genes than its host [23], a “second genome” that complements our own [24]. The gut microbiome, for instance, encodes the proteins that help break down certain complex carbohydrates and dietary fibers [25]. These enzymes are found nowhere in the human genome, but their byproducts – short-chain fatty acids, or SCFAs – fuel the cells that help digest food, absorb nutrients, and shield the interior of the body from pathogenic invasion [26][27].

While the gut microbiome is considered especially important [28], the microbiota of the other niches have their roles. The oral cavity, in particular, is home to several genera of bacteria that can reduce dietary nitrates to nitrite, a precursor of nitric oxide [29]. A vasodilator, nitric oxide allows blood vessels to relax and expand, making it critical to cardiovascular health [30].  

More complex and dynamic than even the gut microbiota [31], the oral microbiota contributes positively to human health…but only when in balance. Any disruption in the finely tuned equilibrium of the oral ecosystem can diminish the population of “good” microbes and allow pathogens to flourish, leading to disease [30][32]. For example, if acid-producing species (e.g., Mutans streptococci, Bifidobacterium) proliferate, dental caries (or “tooth decay”) can form as the byproducts of fermentation break down tooth enamel [33][34]. This overgrowth is a type of dysbiosis, or imbalance in microbial composition [35].

Dysbiosis can occur in three different ways, although two or more of these usually occur at the same time [35]. First, potentally harmful microorganisms can proliferate, as in the case of tooth decay [35]. 

Second, the population of “good” microorganisms can diminish [35]. Acid-producing Lactobacillus bacteria, for example, help keep the female genital tract inhospitable to opportunistic pathogens: when they decline, infection becomes more likely [36]. 

Lastly, overall microbial diversity in a particular body niche may simply be lost. In the gut, a decrease in the populations of Bacteroides and Firmicutes–the two most abundant groups of bacteria–is characteristic of inflammatory bowel disease (IBD) [35]. Firmicutes, in particular, exhibit anti-inflammatory effects [37]. 

A metagenomic approach can reveal the species composition of a microbiota [38] (i.e., identify the dominant taxa, of “good” or “bad” microbes alike), and/or detect a change in overall biodiversity [39]: because metagenomics captures entire microbial communities, it can be used to study all three types of dysbiosis. Today, metagenomics is the most powerful tool for the exploration of the human microbiome. 

Metagenomics and Microbiome Research

The two types of metagenomics approaches are targeted sequencing and shotgun sequencing:

• Targeted sequencing: Only specific genetic markers or conserved regions (e.g., 16s rRNA, 18s rRNA, or internal transcribed spacer [ITS] regions) are amplified (via PCR) and sequenced. These segments have variable regions that can be used to identify different groups of organisms; however, this method typically can’t provide strain-level resolution [40][41]. 
• Shotgun sequencing: Involves random sequencing of all genetic material in a sample. Unlike targeted sequencing, this method doesn’t focus on specific regions, but is instead non-discriminant. Viable today because of next generation sequencing (NGS) technologies, shotgun metagenomics can capture the taxonomic composition of a microbial community (down to the species level) and map biological information to specific gene sequences (called functional annotation) [40][41]. 

As sequencing technologies improve and NGS becomes cheaper, the older “targeted” approach – which provides no information on function, and generally can’t be used to identify individual species – is becoming phased out [41].

Any NGS-based metagenomics experiment needs five fundamental steps [42]:

1. Sample collection and processing: The biological (or environmental) samples are collected. What sample, specifically, depends on what you’re trying to study: for example, stool samples are often used as proxies for gut microbiota [43], while saliva is used to investigate the oral microbiota [44].
2. Isolation of DNA: Various physical and chemical methods can be used to isolate DNA from the samples, but they need to be appropriate for the sample type. 
3. Library preparation: This step can vary depending on the sequencing platform, but because none of these can sequence intact DNA yet [45], it always involves fragmentation of the sample DNA. Only after the DNA has been broken into smaller pieces can they be converted into libraries, which in all cases consist of the fragments of the unknown DNA, flanked by pieces of known DNA (called adapters) [45]. 
4. Sequencing: Popular NGS platforms include Illumina, Pacific Biosciences (PacBio), and Ion Torrent.  
5. Data analysis: The “raw reads” are processed through a bioinformatics pipeline, which includes the trimming of poor-quality (or unreliable) reads. 

“Garbage in, garbage out”: this common saying in the field of ‘omics simply emphasizes that sample preparation is the basis of good results. That is, biased or poor quality (“garbage”) input will always produce flawed (“garbage”) output. A good sampling protocol is consistent, and minimizes contamination (such as from external sources – the air, the ground, or even the DNA isolation kits themselves) while maximizing DNA recovery [40][45].   

Once the samples have been collected, it is necessary to preserve them until they can be processed. Sample degradation is always a concern, but microbiome samples are particularly hard to maintain: at the wrong storage conditions, for example, some microorganisms may continue to grow at the expense of others. When this happens, the sample is no longer representative of the original microbial community [45][46]

Microbiome samples are traditionally frozen before shipping and long-term storage [46], but this isn’t always possible – cold chains are difficult to maintain, and freezers can fail [45]. Next-generation solutions involve microbiome DNA collection kits with reagents that stabilize samples upon contact, enabling transport and storage at ambient temperatures.

Our partners at Isohelix have further optimized this technology for a range of sample types, such as stool and saliva [45]. Isohelix collection devices and buffers are also completely free from even traces of human or bacterial DNA: because a potential source of external contamination is eliminated from the get-go, you can be more confident in the accuracy of your results. 

To support your entire metagenomics workflow, Isohelix also offers DNA isolation kits specifically designed to work with their microbiome DNA collectors to maximize yields of high-quality DNA. Isohelix’s saliva DNA isolation kit, for example, extracts twice as much DNA as other commonly available kits while keeping the material intact and therefore suitable for all downstream applications [45]. 

Conclusion

Metagenomics finally allowed us to capture entire microbial communities, and when we applied this powerful approach to the study of the human microbiota, we found that the distinction between “us” and “them” isn’t as clear-cut as we’d thought: our microbiome is responsible for functions beyond our own genome that are nonetheless essential to human physiology. Today, some consider the human microbiome a “virtual organ”, and the community formed by a human and their resident microbiota a “superorganism”. 

By investigating the complex interplay between our bodies and the trillions of microorganisms that inhabit them, researchers are uncovering new insights into the mechanisms of disease and paving the way for novel diagnostic tools, treatments, and personalized medicine strategies. As the field of metagenomics continues to advance, with the help of innovators like Isohelix, we can expect to see even more exciting discoveries and applications in the years to come.

References:

[1] Sender, R., Fuchs, S. and Milo, R. (2016) ‘Revised estimates for the number of human and bacteria cells in the body’, PLOS Biology, 14(8). doi:10.1371/journal.pbio.1002533.

[2] Martín, R., Miquel, S., Langella, P., & Bermúdez-Humarán, L. G. (2014). The role of metagenomics in understanding the human microbiome in health and disease. Virulence, 5(3), 413–423. https://doi.org/10.4161/viru.27864

[3] Ogunrinola, G. A., Oyewale, J. O., Oshamika, O. O., & Olasehinde, G. I. (2020). The human microbiome and its impacts on health. International Journal of Microbiology, 2020, 1–7. https://doi.org/10.1155/2020/8045646

[4] Rogers, K. (2024, February 29). Human microbiome. Encyclopædia Britannica. https://www.britannica.com/science/human-microbiome 

[5] Reynoso-García, J., Miranda-Santiago, A. E., Meléndez-Vázquez, N. M., Acosta-Pagán, K., Sánchez-Rosado, M., Díaz-Rivera, J., Rosado-Quiñones, A. M., Acevedo-Márquez, L., Cruz-Roldán, L., Tosado-Rodríguez, E. L., Figueroa-Gispert, M. D., & Godoy-Vitorino, F. (2022). A complete guide to human microbiomes: Body niches, transmission, development, dysbiosis, and restoration. Frontiers in Systems Biology, 2. https://doi.org/10.3389/fsysb.2022.951403

[6] Stewart, E. J. (2012). Growing unculturable bacteria. Journal of Bacteriology, 194(16), 4151–4160. https://doi.org/10.1128/jb.00345-12

[7] Nwachukwu, B. C., & Babalola, O. O. (2022). Metagenomics: A tool for exploring key microbiome with the potentials for improving sustainable agriculture. Frontiers in Sustainable Food Systems, 6. https://doi.org/10.3389/fsufs.2022.886987

[8] Choudhari, J. K., Choubey, J., Verma, M. K., Chatterjee, T., & Sahariah, B. P. (2022). Metagenomics: The boon for microbial world knowledge and current challenges. Bioinformatics, 159–175. https://doi.org/10.1016/b978-0-323-89775-4.00022-5

[9] Antoni van Leeuwenhoek. Micropia. (n.d.) https://www.micropia.nl/en/discover/stories/antoni-van-leeuwenhoek/

[10] Cassidy, C. (2022, September 27). The Secret Microscope that sparked a Scientific Revolution. Wired. https://www.wired.com/story/secret-microscope-sparked-scientific-revolution/

[11] Antony van Leeuwenhoek (1632-1723). Antony van Leeuwenhoek. (n.d.). https://ucmp.berkeley.edu/history/leeuwenhoek.html

[12] Ursell, L. K., Metcalf, J. L., Parfrey, L. W., & Knight, R. (2012). Defining the human microbiome. Nutrition Reviews, 70. https://doi.org/10.1111/j.1753-4887.2012.00493.x 

[13] 1.1D: Koch and pure culture – biology libretexts. (n.d.). https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Boundless)/01:_Introduction_to_Microbiology/1.01:_Introduction_to_Microbiology/1.1D:_Koch_and_Pure_Culture

[14] Staley, J. T., & Konopka, A. (1985). Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annual Review of Microbiology, 39(1), 321–346. https://doi.org/10.1146/annurev.mi.39.100185.001541 

[15] Harwani, D. (2012). The Great Plate Count Anomaly and the unculturable bacteria. International Journal of Scientific Research, 2(9), 350–351. https://doi.org/10.15373/22778179/sep2013/122

[16] Woese, C. R., & Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proceedings of the National Academy of Sciences, 74(11), 5088–5090. https://doi.org/10.1073/pnas.74.11.5088

[17] Stein, J. L., Marsh, T. L., Wu, K. Y., Shizuya, H., & DeLong, E. F. (1996). Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon. Journal of Bacteriology, 178(3), 591–599. https://doi.org/10.1128/jb.178.3.591-599.1996\

[18] Kumari, N., & Rai, L. C. (2020). Cyanobacterial diversity: molecular insights under multifarious environmental conditions. In Elsevier eBooks (pp. 17–33). https://doi.org/10.1016/b978-0-12-819311-2.00002-4

[19] Ghosh, A., Mehta, A., & Khan, A. M. (2019). Metagenomic Analysis and its Applications. In Elsevier eBooks (pp. 184–193). https://doi.org/10.1016/b978-0-12-809633-8.20178-7

[20] Sleator, R. D. (2010). The human superorganism – Of microbes and men. Medical Hypotheses, 74(2), 214–215. https://doi.org/10.1016/j.mehy.2009.08.047

[21] Qin, J., Li, R., Raes, J., Arumugam, M., Burgdorf, K. S., Manichanh, C., Nielsen, T., Pons, N., Levenez, F., Yamada, T., Mende, D. R., Li, J., Xu, J., Li, S., Li, D., Cao, J., Wang, B., Liang, H., Zheng, H., . . . Wang, J. (2010). A human gut microbial gene catalogue established by metagenomic sequencing. Nature, 464(7285), 59–65. https://doi.org/10.1038/nature08821

[22] Ma, Z., Zuo, T., Frey, N., & Rangrez, A. Y. (2024). A systematic framework for understanding the microbiome in human health and disease: from basic principles to clinical translation. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01946-6

[23] Shivaji, S. (2017). We are not alone: a case for the human microbiome in extra intestinal diseases. Gut Pathogens, 9(1). https://doi.org/10.1186/s13099-017-0163-3

[24] Grice, E. A., & Segre, J. A. (2012). The human microbiome: our second genome. Annual Review of Genomics and Human Genetics, 13(1), 151–170. https://doi.org/10.1146/annurev-genom-090711-163814

[25] Fujisaka, S., Watanabe, Y., & Tobe, K. (2022). The gut microbiome: a core regulator of metabolism. Journal of Endocrinology, 256(3). https://doi.org/10.1530/joe-22-0111

[26] Venegas, D. P., De La Fuente, M. K., Landskron, G., González, M. J., Quera, R., Dijkstra, G., Harmsen, H. J. M., Faber, K. N., & Hermoso, M. A. (2019). Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases. Frontiers in Immunology, 10. https://doi.org/10.3389/fimmu.2019.00277

[27] Rios-Arce, N. D., Collins, F. L., Schepper, J. D., Steury, M. D., Raehtz, S., Mallin, H., Schoenherr, D. T., Parameswaran, N., & McCabe, L. R. (2017). Epithelial Barrier Function in Gut-Bone Signaling. Advances in Experimental Medicine and Biology, 151–183. https://doi.org/10.1007/978-3-319-66653-2_8

[28] Hou, K., Wu, Z., Chen, X., Wang, J., Zhang, D., Xiao, C., Zhu, D., Koya, J. B., Wei, L., Li, J., & Chen, Z. (2022). Microbiota in health and diseases. Signal Transduction and Targeted Therapy, 7(1). https://doi.org/10.1038/s41392-022-00974-4

[29] Rosier, B. T., Johnston, W., Carda-Diéguez, M., Simpson, A., Cabello-Yeves, E., Piela, K., Reilly, R., Artacho, A., Easton, C., Burleigh, M., Culshaw, S., & Mira, A. (2024). Nitrate reduction capacity of the oral microbiota is impaired in periodontitis: potential implications for systemic nitric oxide availability. International Journal of Oral Science, 16(1). https://doi.org/10.1038/s41368-023-00266-9

[30] Kilian, M., Chapple, I. L. C., Hannig, M., Marsh, P. D., Meuric, V., Pedersen, A. M. L., Tonetti, M. S., Wade, W. G., & Zaura, E. (2016). The oral microbiome – an update for oral healthcare professionals. BDJ, 221(10), 657–666. https://doi.org/10.1038/sj.bdj.2016.865

[31] Maki, K. A., Kazmi, N., Barb, J. J., & Ames, N. (2020). The Oral and Gut Bacterial Microbiomes: Similarities, Differences, and Connections. Biological Research for Nursing, 23(1), 7–20. https://doi.org/10.1177/1099800420941606

[32] Maier, T. (2023). Oral Microbiome in Health and Disease: Maintaining a Healthy, Balanced Ecosystem and Reversing Dysbiosis. Microorganisms, 11(6), 1453. https://doi.org/10.3390/microorganisms11061453

[33] Yama, K., Nishimoto, Y., Kumagai, K., Jo, R., Harada, M., Maruyama, Y., Aita, Y., Fujii, N., Inokuchi, T., Kawamata, R., Sako, M., Ichiba, Y., Tsutsumi, K., Kimura, M., Murakami, S., Kakizawa, Y., Kumagai, T., Yamada, T., & Fukuda, S. (2023). Dysbiosis of oral microbiome persists after dental treatment-induced remission of periodontal disease and dental caries. mSystems, 8(5). https://doi.org/10.1128/msystems.00683-23

[34] Zhan, L. (2018). Rebalancing the Caries Microbiome Dysbiosis: Targeted Treatment and Sugar Alcohols. Advances in Dental Research, 29(1), 110–116. https://doi.org/10.1177/0022034517736498

[35] DeGruttola, A. K., Low, D., Mizoguchi, A., & Mizoguchi, E. (2016). Current Understanding of Dysbiosis in Disease in Human and Animal Models. Inflammatory Bowel Diseases, 22(5), 1137–1150. https://doi.org/10.1097/mib.0000000000000750

[36] Valeriano, V. D., Lahtinen, E., Hwang, I., Zhang, Y., Du, J., & Schuppe-Koistinen, I. (2024). Vaginal dysbiosis and the potential of vaginal microbiome-directed therapeutics. Frontiers in Microbiomes, 3. https://doi.org/10.3389/frmbi.2024.1363089

[37] Tsai, Y., Tai, W., Liang, C., Wu, C., Tsai, M., Hu, W., Huang, P., Chen, C., Kuo, Y., Yao, C., & Chuah, S. (2024). Alternations of the gut microbiota and the Firmicutes/Bacteroidetes ratio after biologic treatment in inflammatory bowel disease. Journal of Microbiology Immunology and Infection. https://doi.org/10.1016/j.jmii.2024.09.006

[38] Masenya, K., Manganyi, M. C., & Dikobe, T. B. (2024). Exploring Cereal Metagenomics: Unravelling Microbial Communities for Improved Food Security. Microorganisms, 12(3), 510. https://doi.org/10.3390/microorganisms12030510

[39] Pal, A., & Chakravarty, A. (2019). Advanced genomic techniques for studying immune-response genes. In Elsevier eBooks (pp. 209–234). https://doi.org/10.1016/b978-0-12-816406-8.00014-0

[40] Tamang, S. (2024, December 13). Metagenomics: Principle, Types, Steps, Uses, Examples, Diagram. Microbe Notes. https://microbenotes.com/metagenomics/

[41] Metagenomics. (2018, January 25). NGS Analysis. https://learn.gencore.bio.nyu.edu/metgenomics/

[42] Navgire, G. S., Goel, N., Sawhney, G., Sharma, M., Kaushik, P., Mohanta, Y. K., Mohanta, T. K., & Al-Harrasi, A. (2022). Analysis and Interpretation of metagenomics data: an approach. Biological Procedures Online, 24(1). https://doi.org/10.1186/s12575-022-00179-7

[43] Tang, Q., Jin, G., Wang, G., Liu, T., Liu, X., Wang, B., & Cao, H. (2020). Current Sampling Methods for Gut Microbiota: A Call for More Precise Devices. Frontiers in Cellular and Infection Microbiology, 10. https://doi.org/10.3389/fcimb.2020.00151

[44] Roca, C., Alkhateeb, A. A., Deanhardt, B. K., Macdonald, J. K., Chi, D. L., Wang, J. R., & Wolfgang, M. C. (2024). Saliva sampling method influences oral microbiome composition and taxa distribution associated with oral diseases. PLoS ONE, 19(3), e0301016. https://doi.org/10.1371/journal.pone.0301016

[45] Apone, L., Dimalanta, E, & Stewart, F. (2017). Improving Enzymatic DNA Fragmentation for Next Generation Sequencing Library Construction. New England Biolabs, Inc. https://www.neb.com/en/tools-and-resources/feature-articles/improving-enzymatic-dna-fragmentation-for-next-generation-sequencing-library-construction

[45] Seaton, A. (2023, December 15). Microbiome DNA sampling and analysis. Isohelix. https://isohelix.com/microbiome-dna-sampling-and-analysis/

[46] Izard, J. (2014). Steps in Metagenomics: Let’s Avoid Garbage in and Garbage Out. In Elsevier eBooks (pp. 1–23). https://doi.org/10.1016/b978-0-12-410472-3.00001-4