Metatranscriptomic analyses of stool and transcriptomic analyses of capillary blood samples
The molecular analyses for the studies reported here focus on sequencing messenger RNAs (mRNAs) isolated from human stool and blood samples. Stool samples were collected and analyzed as previously reported [44]. Briefly, stool samples were collected by the study participants using the Viome commercial kits that included ambient temperature preservation solution and pre-paid return mailers. Stool metatranscriptomic analyses (RNA sequencing, RNAseq) were performed using an automated, clinically-validated laboratory and bioinformatics methods. Results consist of quantitative strain, species, and genus level taxonomic classification of all microorganisms, and quantitative microbial gene and KO (KEGG Ortholog, KEGG = Kyoto Encyclopedia of Genes and Genomes [45]) expression levels. The matching blood samples were collected and analyzed as previously described [42]. Briefly, blood samples are collected by the study participants using the Viome commercial kits that included ambient temperature preservation solution and pre-paid return mailers. The kits include lancets and minivettes that enable easy and accurate collection of small volumes of blood from a finger prick. Transcriptomic analyses (RNA sequencing, RNAseq) were performed using an automated, clinically-validated laboratory and bioinformatics methods that require 50 microliters of capillary blood. Test results consist of quantitative human gene expression data.
All microorganisms that live in the intestines obtain their energy by converting chemical substrates into products, using metabolic pathways that consist of enzymes. Substrates are typically the molecular ingredients found in foods, and products are biochemicals usually referred to as secondary metabolites. Metatranscriptomic analysis of the gut microbiome enables the quantification of thousands of microbial pathways using the KEGG database. As part of the efficacy trials we describe here, we identified an average of 377 strains, 363 species, and 102 genera per stool sample, and a total of 2930 strains, 2,007 species, and 451 genera in all stool samples combined. In addition, we have identified an average of 1,916 KOs per stool sample, and 5,467 KOs in all stool samples combined. In blood samples, we quantified the expression of an average of 11,687 genes per sample and 15,434 genes in all samples combined.
Metabolic pathways and functional scores
We have designed functional scores to quantify certain biological phenomena; for example, leaky gut, inflammation, gas production, protein fermentation, cellular health, mitochondrial health, etc., that are relevant to human physiology and healthy homeostasis. Functional scores are weighted functions (Score = C1F1 + C2F2 + … + CnFn, where F is the feature and C is its weight) of components from the molecular data from the gut microbiome and/or blood transcriptome. The components that make up the functional scores can be taxa, microbial pathways, human pathways, or other functional scores. Functional scores range from 0 to 100, with 100 representing the highest possible activity. For example, a microbiome-derived functional score such as Butyrate Production Pathways is calculated as a weighted function consisting of the expression levels of many known butyrate-associated KOs (Fig. 2) [46]. The expression level of each KO from the Butyrate Production Pathways score is quantified using the metatranscriptomic stool test and the score is then computed using the weighted formula [47]. The weights attributed to each KO within a pathway are determined by a combination of domain knowledge and statistical analyses obtained from a collection of 200,000 stool and blood samples [48][49]. By quantifying each enzymatic member (KO) of the butyrate pathways using microbial gene expression data, accurate pathway activities can be calculated [50].
Table 1: Examples of specific foods and supplements, and reasons for recommendation to consume or avoid.
|
Food/supplement
|
Reasons to consume
|
Reasons to avoid
|
Artichoke
|
- Low Butyrate Production Pathways functional score, and
- Presence of specific microbial strains that convert inulin into butyrate
|
- High Gas Production functional score, or
- Small intestinal bacterial overgrowth (SIBO) symptoms
|
Broccoli
|
- High Inflammatory Activity functional score, or - Suspected liver and detox support needed
|
- High Sulfide Gas Production functional score, and - High Trimethylamine (TMA) Production functional score
|
Trout
|
High Inflammatory Activity functional score
|
High Uric Acid Production Pathways functional score
|
Nicotinamide Riboside (NR)
|
- Low Mitochondrial Health functiona score, or
- Low Mitochondrial Biogenesis Pathways functional score
|
- High Cellular Senescence functional score, and
-High Inflammatory Activity functional score
|
Curcumin
|
- High Inflammatory Activity functional score, or
- High Cellular Stress, or
- High Immune System Activation
|
High Bile Acid Production Pathway functional score
|
Making personalized nutritional recommendations
Precision nutritional recommendations are computed using the Viome AI Recommendation Engine and are designed to address an individual’s biological patterns at the functional level by boosting beneficial (health-associated) and suppressing harmful (disease-associated) activities with molecular ingredients from foods and supplements (Fig. 1). This approach is built on the concept that on a molecular level, a particular food or supplement may be beneficial for one person, but harmful to a different person. Table 1 shows examples of this concept. The Viome AI Recommendation Engine uses a complex set of algorithms to determine the final food and supplement recommendations. The algorithms are developed from the domain knowledge (publications on microbial and human physiology, food science, clinical trials, etc.), phenotypic information, and extensive clinical studies from which machine-learned models of nutritional modulation of the microbiome and human physiology were developed (e.g. [26]). These algorithms are applied to the functional scores and phenotype for each person whose stool, or stool and blood samples are analyzed. Phenotype is determined from information provided by a participant in the wellness questionnaire, such as symptom assessment, known health conditions, allergies, and medications.
The Viome AI Recommendation Engine considers compounds (molecular ingredients) in foods and supplements that can support the healthy functions of both the gut microbiome and the human. These compounds include specific polysaccharides, polyphenols, vitamins, minerals, amino acids, fatty acids, and many phytochemicals. This approach highlights the concept that a single food is more than simply its macronutrient content and that foods from the same family can have very different molecular compositions. For example, an almond is a source of many phytonutrients and compounds such as kaempferol (flavonoid), naringenin (flavonoid), ferulic acid (phenolic acid), oxalic acid, phytic acid, quercetin, procyanidin B2 and B3, magnesium, phytosterols such as retinol, a-tocopherol, vitamin K, vitamin D, and beta-sitosterol, fatty acids such as oleic acid, linoleic acid, and palmitic acid, and specific amino acids [51]. The decision to recommend a specific compound and its amount depends on the values of multiple functional scores. After considering all inputs, the recommendation engine classifies foods into one of four categories based on the molecular composition of each food. The food categories are superfoods, enjoy foods, minimize foods, and avoid foods, which are consumerized names that correspond to the recommended servings per day for each food.
Personalized supplement recommendations follow the same logic, considering all inputs to identify compounds that are beneficial or harmful to an individual’s functional scores and phenotype. Supplements include minerals, vitamins, botanicals or herbs, food extracts, enzymes, phospholipids, amino acids, prebiotics, and probiotics. When considering individual functional scores, supplement ingredients commonly believed to be beneficial may not be recommended. For example, turmeric is a commonly consumed supplement for its anti-inflammatory properties, but has also been shown to increase bile flow [52]. For individuals with a high Bile Acid Metabolism Pathway functional score, turmeric supplementation may be more harmful than beneficial. A high Bile Acid Metabolism Pathway score suggests that the microbial activity of transforming bile salts into bile acids is high. While such biotransformation is part of a balanced gut microbiota and bile acid homeostasis, excessive intestinal bile acids may promote a pro-inflammatory environment and play a role in the development of gastrointestinal diseases [53][54].
The process of categorizing foods and supplements (determining the servings or dose) includes prioritizing scores that need improvement and considering conflicts within the recommendations. An example is shown in Table 2: a low Energy Production Pathway functional score will yield recommendations based on compounds that contribute to the score activity, one of which is alpha-lipoic acid (ALA). Spinach and broccoli are recommended due to the ALA content that is a critical cofactor for mitochondrial energy production enzymes such as pyruvate dehydrogenase (PDH), alpha-ketoglutarate dehydrogenase (alpha-KGDH), and branched-chain ketoacid dehydrogenase (BCKDC) [55]. However, when considering additional score results, broccoli and spinach will be placed on the avoid food list due to broccoli’s glucosinolate content and spinach’s oxalate content. Instead, tomatoes and peas are recommended as sources of ALA to support the Energy Production Pathway functional score.
Table 2
Food Recommendations Case Example
Example Score Results:
High Sulfide Gas Production Pathways functional score
Low Butyrate Production Pathways functional score
Low Oxalate Metabolism Pathways functional score
Low Energy Production Pathways functional score
Self-reported information on wellness questionnaire:
Histamine intolerance
Pistachio allergy
|
Food
|
Compounds
|
Food categories (initial)
|
Final recommendation
|
Replacement food/supplement
|
Spinach
|
- Alpha-lipoic acid
- Oxalates
|
- Superfood, based on low Energy Production Pathways functional score
- Avoid, due to low Oxalate Metabolism Pathways functional score
|
Avoid
|
- Tomato and
- Alpha-lipoic acid supplement
|
Broccoli
|
- Alpha-lipoic acid
- Glucosinolates
|
- Superfood, based on low Energy Production Pathways functional score
- Avoid, due to high Sulfide Gas Production Pathways functional score
|
Avoid
|
- Peas and
- Alpha-lipoic acid supplement
|
Sauerkraut
|
Probiotics
|
- Superfood, based on low Butyrate Production Pathways functional score
- Minimize, due to self-reported histamine intolerance
|
Minimize
|
- Probiotic supplement that includes Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus rhamnosus
|
Pistachios
|
CoQ10
|
Superfood for Energy Production Pathways Low
Avoid due to self-reported pistachio allergy
|
Avoid
|
CoQ10 supplement
|
Grapes
|
Resveratrol
|
Superfood for Energy Production Pathways Low
|
Superfood
|
|
There are circumstances where a beneficial compound cannot be obtained from food due to an allergy or other health-related issue, or due to the lack of sufficient amounts in food. Personalized supplements help support those gaps in nutrition. In Table 2, pistachios are recommended due to their CoQ10 content. However, if an individual has an allergy to pistachios or exhibits small intestinal bacterial overgrowth (SIBO) symptoms, pistachios will be placed on the avoid or minimize food list. In this situation, CoQ10 can be provided through a supplement as the recommendation engine associates it as beneficial for the same score and/or phenotypic conditions.