Metatranscriptomic Analysis — The Next Frontier in Microbiome Technology is Available Today

Naveen Jain

Image: Pixabay
Summary
DNA-based gut microbiome analyses have been available for years. They include 16S and metagenomic sequencing technologies, which have identified strong connections between the gut microbiome and human health. However, these technologies cannot provide accurate food and supplement recommendations that can improve many aspects of human health. To do this comprehensively, it is critical to measure what the microbes are doing for you, the host — which can only be done with the technology called “metatranscriptomics,” which measures microbial activity (which microbes are present and how active they are), and also their biochemical functions (what they are doing).

Recently, gut microbiome testing has received well-deserved criticism due to the widespread use of the 16S gut microbiome analysis method. [i],[ii] The 16S technology identifies bacteria by sequencing a portion of their 16S ribosomal RNA genes.[iii] As a result, this technology cannot detect other living microorganisms in the gut, such as viruses, phages, yeast, fungi and mold. Moreover, this technology cannot even identify all bacteria. 16S sequencing is also limited by the level of detail it can produce; bacteria can mostly be identified at the taxonomic levels of genus or higher. Despite these serious limitations, 16S sequencing continues to be used by many companies because it is inexpensive.

In some recent scientific publications, the 16S technology has been shown to produce lots of false results. A peer-reviewed study by Edgar determined that 16S sequencing of known bacterial communities resulted in a 56% to 88% false positive rate of predicted genus names.[iv] In other words, more than 50% of the genera reported in the results are not actually present in the sample. Ultimately, the study concludes that “biological inferences obtained using these methods are…not reliable”. Since many bacterial genera (plural of genus) comprise various types of bacteria, predicting responses to potential interventions is difficult when the microbiome is examined at the genus level. This problem is amplified when the technology produces false positives at such alarming rates.

Here at Viome, we are so convinced that 16S sequencing is worthless that we actually remove 16S ribosomal RNAs from our samples. We call this step Removal of Non-informative RNAs (RNR), because 16S RNAs are not informative.

After sending the same stool sample to American Gut and uBiome, a journalist found that her results from the two companies were very different, with Firmicutes-to-Bacteroidetes ratio being the opposite. Retrieved from Retrieved from: https://www.sciencenews.org/blog/gory-details/here%E2%80%99s-poop-getting-your-gut-microbiome-analyzed

Fortunately, sequencing technologies have progressed beyond the commonly used 16S method. Some companies have implemented metagenomic analysis of gut samples. This is a relatively simple technology, but significantly more expensive than 16S. While metagenomic analysis can identify most microbes (except for RNA viruses and phages) at much higher resolution than 16S, it provides no insight into the functions of these organisms. Both 16S and metagenomic methods have another drawback: they analyze DNA, not live microorganisms. DNA is very stable, so even DNA from the food we consume and from dead microorganisms finds its way into stool samples, thus wasting sequencing data and confounding the analyses.

The introduction of metatranscriptomic analysis has transcended the limitations of all other gut microbiome methods and demonstrated the gut microbiome’s promising potential as a target for personalized nutrition. Metatranscriptomic analysis allows for the detection of all living organisms (bacteria, archaea, viruses, phages, yeast, fungi, mold, parasites, etc.) at the highest resolution (strain level), precise quantification of the activity of these organisms, and most importantly, analysis of their functions that directly impact our own metabolism and health. Major differences between other gut microbiome analysis methods and Viome’s state-of-the-art metatranscriptomic technology are outlined in the table below.

Metatranscriptomic analysis allows microbiome researchers to measure gene expression of all living organisms, identifying how gut microorganisms interact with their human environment and elucidating functional changes that contribute to health and disease.[i] Metatranscriptomic technology has allowed Viome to generate personalized, actionable nutritional recommendations. Automated analyses of individual and collective data allow for unprecedented insights into the metabolic activities and functional capacity of an individual’s microbiome. These insights are used to provide a personalized action plan for optimizing wellness through specific diet, supplement and lifestyle modifications.

Research has established that the gut microbiome influences and interacts with a wide range of human physiological functions, especially those related to metabolism and the digestive, immune, and neuroendocrine systems.[ii],[iii],[iv],[v],[vi],[vii],[viii],[ix],[x] Specific microbiome functions have been identified that can influence specific human physiological functions. For example, research has shown that an important microbiome metabolic product called butyrate — produced from bacterial fermentation of dietary fiber — plays an important role in immune balance by stimulating the production of T regulatory cells, which help to suppress inflammation. Butyrate also helps to support the energy needs of colon cells, and contributes to metabolic and neurological health. Traditional ways of assessing butyrate production via 16S or metagenomic analyses are poor indicators, since these methods can only tell which organisms are present, but not what they are doing. Viome’s metatranscriptomic technology determines the activity of butyrate production and related pathways to obtain a much more accurate assessment. If Viome’s analysis demonstrates low levels of butyrate production in an individual, then — depending on the overall assessment of an individual’s microbiome and wellness — Viome can recommend higher intake of foods rich in certain dietary fibers that are known to stimulate butyrate production.

Why is it so important to tell if and how butyrate is actively made? Because the proprietary pathway analysis methods and functional scoring logic that Viome uses can suggest different recommendations based on the levels of specific butyrate production pathways and specific butyrate-producing organisms. This means you can get more personalized recommendations suggesting you focus on “feeding the good guys” (giving those butyrate producers more fibers they love), “adding the good guys” (taking probiotic-rich foods and supplements with butyrate producers themselves), “balancing out the bad guys” (other species which may suppress butyrate producers), or supplementing with butyrate, and/or different combinations of the above-listed actions for various lengths of times and with particular amounts/ doses that suit your gut microbiome and your functional profile best.

While butyrate is known to be produced by specific microbes, some pathway insights gained from metatranscriptomic data analyses also point to unexpected findings, not previously tied to any specific organisms. For instance, microbial organisms in your gut are often seen actively transporting and metabolizing a number of known environmental toxins, such as methylnaphthalene/naphthalene (could come from smoke, overly-fried foods or moth balls), toluene, acrylamide/ acrylate, atrazine, and many of the known persistent organic pollutants known to be present in the environment. They can come from plastics, rubber kitchen utensils, gels and emulsifiers, aerosols, shampoos, and many personal care products, as well as from unfiltered contaminated water sources. Seeing unusually high toxin-processing pathway activities can warrant some additional recommendations, which include more antioxidants, as well as liver support, in the food or supplement form.

There are thousands of other molecular pathway insights that allow Viome to understand which functions are actively carried out by the gut microbiome, allowing food and supplement recommendations specifically tailored to individual functional profiles. For example, if enzymes that are known to degrade proteins and membranes are overly active, they may lead to damage of mucosal lining and contribute to “leaky gut”. If such a trend is observed, Viome may suggest eating specific fiber-rich foods, bone broth (for collagen), adding specific probiotics, and taking supplements, like glutamine or zinc carnosine. Viome’s technology can also determine the energy sources of individual microbes based on active enzymes and functional pathways. Based on this functional profile, Viome offers recommendations for foods in relative amounts that would most benefit individual gut health.

In addition to energy production, we can determine which byproducts the microbes are synthesizing in the process — such as ammonia, cyanide, peroxide, some ketones, or other compounds. If microbes are creating these byproducts, they can be mitigated with specific nutrition and supplement plans. The synthesis of fats, as well as the types of fats (long- or short-chain, saturated or unsaturated) can be determined based on specific enzyme levels. If beneficial short-chain fatty acids (SCFAs) are being produced by microbiota, Viome’s technology can predict how this will affect human metabolism, mood, and even eating behavior — and make subsequent recommendations to correct the imbalances. If the biosynthesis pathway of a key SCFA is low, but breakdown enzymes are high, Viome can determine probiotic recommendations that would replenish the gut with beneficial butyrate producers.

Only at Viome is it possible to pinpoint the microbial pathway activities specific to production of particular bacterial products, such as indoles, lipopolysaccharides (LPS), reactive oxygen species (ROS)-like molecules, hydrogen sulfide or sulfites, putrescine, or cadaverine, and/or a number of neurotransmitters. The quantity of these molecules suggest if individuals will benefit more from foods like turmeric and pineapple or green tea, or supplements, such as curcumin, bromelain, resveratrol, or glutathione. Viome’s metatranscriptomic analysis can determine if glutathione is actively being produced by gut microbes, which is indicative of microbiota mitigating a hostile gut environment.

Viome’s technology extracts information from the human microbiome that has never been available before. As described in this article with a few examples, it generates many clear, scientifically-backed recommendations based on the high-resolution microbiome data. This technology allows Viome’s scientific and AI experts to develop the unique analytical methods that give you personalized guidance with foods and supplements that really address what is going on in your gut.

Do you know if you are getting what you need from your microbiome? Do you know if you are a “good host” to your microbes and the functions they perform for you? Viome can help you find out — and guide you on your path to ultimate wellness.

Naveen Jain is the CEO of Viome, Inc.

[i] Ranjan et al., “Analysis of the Microbiome.”

[ii] Qin et al., “A Human Gut Microbial Gene Catalogue Established by Metagenomic Sequencing.”

[iii] Sussman and Tang, Molecular Medical Microbiology Three-Volume-Set.

[iv] Edgar, “Accuracy of Microbial Community Diversity Estimated by Closed- and Open-Reference OTUs.”

[i] Bashiardes, Zilberman-Schapira, and Elinav, “Use of Metatranscriptomics in Microbiome Research.”

[ii] Chervonsky, “Microbiota and Autoimmunity.”

[iii] Karlsson et al., “Symptomatic Atherosclerosis Is Associated with an Altered Gut Metagenome.”

[iv] Rogers et al., “From Gut Dysbiosis to Altered Brain Function and Mental Illness.”

[v] Zhang and Zhang, “Microbiota Associated with Type 2 Diabetes and Its Related Complications.”

[vi] Harach et al., “Reduction of Abeta Amyloid Pathology in APPPS1 Transgenic Mice in the Absence of Gut Microbiota.”

[vii] David et al., “Diet Rapidly and Reproducibly Alters the Human Gut Microbiome.”

[viii] Scheperjans et al., “Gut Microbiota Are Related to Parkinson’s Disease and Clinical Phenotype.”

[ix] Baxter et al., “Microbiota-Based Model Improves the Sensitivity of Fecal Immunochemical Test for Detecting Colonic Lesions.”

[x] Parekh, Balart, and Johnson, “The Influence of the Gut Microbiome on Obesity, Metabolic Syndrome and Gastrointestinal Disease.”