A comparison of 16S rRNA and Shotgun techniques

The medical microbiology of the intestinal tract, microbiome research, examines the microbial colonization of the intestinal tract, which plays a vital role in health. This microbial community—comprising bacteria, viruses, fungi, and other microorganisms—interacts with the host organism and is connected to virtually all organ systems, influencing metabolism, hormones, the immune system, and even the brain.

In recent years, an increasing number of people have turned to microbiome testing for various reasons, including digestive complaints, skin problems, chronic fatigue, or even personalized nutritional guidance. Those interested in undergoing such testing will quickly find that not only has the number of providers grown, but the testing technologies also vary significantly.

Traditional stool testing methods include macroscopic examination: observing consistency, color, fat content, mucus, blood, undigested food particles, and determining pH, which may indicate dysbiosis. Additionally, immunological tests are employed, such as the detection of occult blood or markers indicative of intestinal cancer. Inflammatory and mucosal integrity markers like calprotectin, lactoferrin, alpha-1-antitrypsin, and histamine can also be measured. The level of pancreatic elastase gives insights into the exocrine function of the pancreas. Other classic tests include microscopic microbiological and parasitological analyses and stool cultures. Clostridium toxins or the presence of Salmonella can also be detected.

Modern testing methods focus not on the bacterial species themselves, but on their metabolic products—i.e., their metabolites. These include tryptophan, serotonin, GABA, indole and its derivatives (e.g., tryptamine, kynurenic acid, p-cresol), and various bile acids (free, conjugated, protective, or cell-damaging). These substances can provide critical insights into the patient’s physical and mental condition and the possible causes of symptoms. A limitation of this method is that the specific bacteria involved in these processes are not always known.

With technological advancement, genetic-based stool microbiota testing methods have emerged. The most common are 16S rRNA-based sequencing and shotgun metagenome sequencing.

It is important to distinguish these microbiome tests from classic stool tests routinely performed in hospitals, which serve an entirely different purpose. Hospital stool tests typically cover only a few dozen known microorganisms and are not designed to give a comprehensive view of the gut microbiome’s composition or function.

16S rRNA-based sequencing: simple but limited

16S rRNA-based analysis ^1–2 is one of the most widely used methods for profiling the gut microbiome. This method examines a genetically conserved but species-specific gene segment of bacteria—the 16S ribosomal RNA. The analysis generally identifies bacteria at the genus level, as the gene sequences can be highly similar among closely related species. Therefore, while the genus—roughly analogous to a surname—can usually be identified, the species—comparable to a given name—is often unclear.

The major advantage of this method is that it is fast, cost-effective, and technically easy to standardize, making it widely adopted in both scientific research and commercial laboratory settings. Its popularity has grown since the early 2000s alongside advances in DNA sequencing technologies. Several international initiatives, such as the American Gut Project and Atlas Biomed, also use this technique.

However, the method is limited to analyzing bacteria and does not identify other microorganisms such as viruses. Moreover, it has limited capacity for species-level identification and provides no functional data—i.e., whether the microbes produce toxins, metabolites, or show antibiotic resistance. Typically, the method distinguishes a few thousand species.

Main advantages:

• Easy to perform, economical, and fast
• Suitable for general mapping of bacterial communities
• Widely validated

Limitations:

• Only bacteria are analyzed; fungi and viruses are not detected
• Accuracy is mainly at the genus level; species-level identification is limited or not possible
• No information is provided on microbial metabolism or function

Shotgun metagenome sequencing: function and accuracy

Shotgun metagenomics ^3–4 is a state-of-the-art, high-resolution analytical method that reveals not only the composition of the microbiome but also its functional activity. The essence of the method is that it analyzes not just a single gene segment, but the entire DNA pool of the microbial community, offering a much more detailed and accurate view of the organisms present and the biological processes they are involved in.

This technique allows for the identification of bacteria, viruses, fungi, and parasites. It also detects functional genes involved in metabolism, vitamin and metabolite production, toxin formation, and antibiotic resistance. Importantly, large volumes of genetic data alone are not enough; advanced bioinformatics tools are essential for processing and interpreting the data meaningfully. Leading international research institutes and diagnostic providers such as CosmosID, Microba, and Onegevity use this method.

Main advantages:

• Comprehensive genome analysis and species-level identification
• Detects viruses, fungi, and archaea
• Can potentially distinguish tens of thousands of bacterial species
• Also maps functions: what microbes produce and their capabilities, such as SCFA production, methane production, toxin generation, and antibiotic resistance
• It can be used for clinical purposes and personalized nutritional interventions, enabling targeted and specific adjustments based on the composition of the gut microbiota. In such cases, the desired physiological effect can be achieved by introducing, removing, or deliberately modifying the proportions of certain dietary components, such as macronutrients, specific types of fiber, or plant compounds.

Limitations:

• More expensive than simpler methods
• Requires complex data processing and interpretation

Comparison of shotgun and 16S rRNA-based sequencing

16S rRNA sequencing targets a single conserved bacterial gene and is mainly suitable for genus-level identification, with limited species-level accuracy. In contrast, shotgun metagenomics sequences all microbial DNA in the sample, enabling precise species-level identification and functional mapping. This method can detect all microorganisms comprising more than 0.01% of the sample, including previously unknown species. It also identifies viruses, fungi, and archaea, offering a more comprehensive view of the microbial community.

While 16S is quicker, cheaper, and simpler, shotgun metagenomics delivers more detailed and functional data—albeit with higher costs and computational demands.

Comparative overview

Table 1: Comparative overview of 16S rRNA sequencing and shotgun metagenomics

Why did we choose shotgun metagenomics?

The shotgun metagenomics test we use detects all bacterial species identified by science (above a 0.01% threshold) and all fungal species above 0.001%. It reveals bacterial toxins, metabolite production, and resistance genes, which can guide antibiotic selection for planned treatments. The test also includes a "human background" marker, which reflects the ratio of human to bacterial DNA in the sample. Human DNA in stool may originate from dead intestinal cells, immune cells (pus), or blood; elevated levels can indicate intestinal damage, inflammation, or even tumors. Knowledge of the functional characteristics of bacteria is also crucial from a clinical perspective. For example, a toxin-producing Escherichia coli requires a completely different treatment than Escherichia fergusonii, which is typically found in the urinary tract. However, it is not only well-known pathogens that can cause problems. Certain bacteria that are generally considered beneficial can also become harmful if they proliferate excessively. This overgrowth can disrupt the microbial balance in the intestine, displace other beneficial species, and lead to the overproduction of metabolic byproducts that, in high concentrations, can be corrosive (e.g., butyric acid, succinate) or otherwise harmful, potentially causing metabolic issues (e.g., lactic acid). It is also important to determine whether a particular bacterial species is beneficial or whether it exists in a toxin-producing variant (e.g., Bacteroides fragilis). Many bacterial species are capable of producing mild or severe toxins, and this knowledge can inform the appropriate therapeutic strategy and the selection of suitable active ingredients. Based on the data obtained during testing, we develop a personalized, step-by-step plan tailored to your individual microbiome profile, lifestyle, and health goals. This plan typically lasts 1 to 3 months, depending on the specific issue.

Other related tests

To obtain a comprehensive understanding of the body’s current state, microbiome analysis can be complemented by symptom scales, blood tests, inflammatory markers, allergy and food intolerance testing, and hormone profiles.