Precision microbiome insights for families

Metagenomics is the study of genetic material from all organisms in a sample by a method called sequencing. Through metagenomic sequencing, Tiny Health uses the nucleic acids in microbial DNA to identify all microbes present in the sample, and in what proportions. This is often referred to as shotgun metagenomics. 

The greatest benefit to shotgun sequencing is that it is an unbiased hypothesis-free method [1]. Tests in traditional healthcare settings or from other microbiome companies typically limit their reports to specific species or strains. With Tiny Health, you get the complete picture of your gut or vaginal microbiome and a holistic plan to improve your microbiome health.

Once you receive your Tiny Health test, you may take your sample, activate your Kit ID on your Tiny Health account, and complete your intake surveys.  You will then mail your sample to our CLIA certified lab, where it will be processed using shotgun metagenomic sequencing technology. 

At the lab the microbial DNA is first extracted from your sample, and then it is run through the sequencer which spits out raw data files called fastQ files. This stage typically takes between 2-3 weeks, because the samples are processed in large batches. 

After sequencing, the lab delivers the raw data from your sample to Tiny Health and we process it on our proprietary bioinformatics pipeline. Our proprietary bioinformatics platform (eg thMP, thDB, thDD) allows us to create custom metrics and reports that pull in your survey data (such as age, pregnancy or not, etc.). Next it is run through thMAP to plot the right reference ranges. We then use machine learning to personalize the recommendations we offer based on your individual results and survey data. Your sample goes through a series of quality control checks. Finally, it runs through our expert highlight process and you are notified of your results via email and SMS. This stage typically takes between 2-3 business days. 

Tiny Health collection swabs do not need to be frozen before being returned to the lab for processing. Tiny Health microbiome tests report on microbial markers, by sequencing their DNA. DNA is very stable at ambient temperatures and therefore does not need to be frozen. 

We also use a collection swab with the latest fast-drying technology. This fast-drying technology allows the swab to preserve the microbes from your sample and inactivates them which stops them from continuing to grow in transit. This advanced technology allows us to accurately process samples stored at room temperature for up to four weeks.

If your physician would like you to add on Tiny Health PRO, which tests for non-microbial markers, this part of your sample would need to be frozen. Non-microbial markers such as proteins like sIgA, elastase, and calprotectin, will degrade at room temperature. These non-microbial markers therefore have to be flash-frozen before sending in your sample.

There are two types of parasites. The first are Protists parasites, or single-cell organisms that are not visible to the naked eye. The second type of parasite are Helminths. Helminths are parasitic worms, meaning they are multicellular and may be visible to the naked eye.  

Tiny Health tests for Protists parasites, but does not test for multicellular worms. Testing for worms would require obtaining part of the worm for sequencing during the collection process, which is unlikely with our current collection process.  If you suspect you have a parasitic worm please reach out to your provider for the appropriate testing.

For more information, visit our blog post, Gut Parasites - Should I be worried?

The short answer is yes, we test for both Candida and H. Pylori.  However, these microbes can be harder to detect for the following reasons, and PCR based stool tests may be more sensitive when it comes to detecting them. 

Candida is a fungus and with their rigid cell wall it can be harder to extract DNA from them than bacteria. However, we use a DNA extraction method that has been well documented to work for a wide variety of fungal species, including Candida and molds (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224647/). There are a number of papers that use a very similar extraction method for a wide variety of fungal species, and they do not have problems extracting DNA. One relevant paper: https://www.atsjournals.org/doi/abs/10.1164/ajrccm-conference.2018.197.1_MeetingAbstracts.A5491. We have confirmation of this in our taxa results, that we do find Candida in a number of kits. The tricky thing here is that relative abundance is potentially less informative when compared to bacterial genomes. So while we detect presence/absence, it could be in a higher abundance than our methodology suggests.

Helicobacter pylori is a gram-negative bacterium. It can cause stomach inflammation and infections in the small intestine. As these are in the upper GI tract, and our samples (stool) primarily carry bacteria from the lower GI tract. While H. pylori infection is associated with gut microbiome composition changes (https://www.nature.com/articles/s41598-019-56631-4), sequencing it from stool without PCR amplification is difficult. Concerns with extracting DNA from H. pylori, typically stem from medical samples that are trying to isolate H. pylori from intestinal epithelial cells, which results in an overabundance of human cells. Similar methods to our DNA extraction method has been shown to extract DNA from H. pylori (https://journals.asm.org/doi/pdf/10.1128/mbio.00955-19). However, if the cells are not making it through the large intestine, the likelihood of capturing these cells is low. We have not detected H. pylori in any of our samples yet.

Testing:
In both of these cases, PCR based tests of stool may be more sensitive.

PubMed Central (PMC)
Evaluation of three DNA extraction methods from fungal cultures

Polymerase chain reaction (PCR) based assays have been developed to amplify DNA of fungal pathogens as culture-based detection methods show low sensitivity. In order to perform a sensitive, specific, and reliable PCR based assay, the availability of pure ... (174 kB)
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6224647/

Nature
Helicobacter pylori infection associates with fecal microbiota composition and diversity

Scientific Reports - Helicobacter pylori infection associates with fecal microbiota composition and diversity (184 kB)

Shotgun sequencing can be used to identify strain-level differences between microbes. This is important because sometimes the same species are doing very different things depending on which strain they are, for example, many strains of E. coli are commensal and don't cause harm, but some strains are pathogenic.

For the argument of sequencing DNA vs. RNA, both can be very useful, but for different things.

Shotgun sequencing with DNA can tell you both who’s in your microbiome and what they're capable of doing since we're sequencing all microbial genes, not just taxa markers. This lets us tell you if your gut has the right capacity for important gut health functions like short-chain fatty acid production or protein breakdown. But this doesn't tell you how much of that gene is actively been expressed in that moment.

The issue we find with RNA sequencing for understanding gut health is that it is highly variable and really dependent on what's happening in that sample. For example, your microbial RNA could look dramatically different depending on time of day, what you ate last night, how much sleep you got. It could be very different the next day. This makes it hard to give users an understanding of their microbiome health and what they should do to make long-term changes.

However, RNA sequencing is great for controlled trials that are looking at how specific microbes are responding to medication or drugs, for example. You just have to sample frequently and design very specific controls.

Tiny Health tests look at all bacteria, viruses, fungi, and parasites in your microbiome using deep metagenomic sequencing. This is the latest and best technology for microbiome analysis used by top research institutions.

In comparison:

Quantitative polymerase chain reaction (qPCR / PCR) tests 

PCR tests like GI Map, GI Effects, Gut Zoomer, or Doctors Data 360 examine a tiny fraction of the microbes in your gut. This technology is useful to healthcare providers for quick diagnosis of a specific infection or condition. But it only identifies specific microbes that the test is looking for, at nearly double the cost. And PCR tests often emphasize pathogenic microbes, and focus less on beneficial or new microbe discoveries. 

These tests sometimes include a culture component, which is very good at detecting certain pathogens. But they can miss things that are difficult to culture, and they can’t give you a picture of the whole microbiome community (Silverman 2021).

Some tests also report on non-microbial stool chemistry like secretory IgA or calprotectin. We do not think this is a necessary part of understanding your overall gut health if you are not actively experiencing symptoms. But it may be helpful when trying to get to the root cause of more severe chronic conditions. Tiny Health PRO tests include these non-microbial stool chemistry measures and can be ordered by your practitioner. 

16s amplicon sequencing

Lower-cost and quicker turnaround time tests on the market, like Snapi and Ombre, use 16s amplicon sequencing which targets 16S rRNA genes to identify bacteria only. While this sequencing method was groundbreaking in the early 2000s, it’s now considered a limited technology, especially when characterizing the complex communities of the gut microbiome because of its low taxonomic resolution lack of functional gene insights, and higher false positive rates (Peterson, D., 2021).

mRNA / RNA tests (metatranscriptomics)

Some microbiome companies like Viome use mRNA (or RNA) tests. This technology, called metatranscriptomics, looks for the gene sequences that are active at the time of sampling. It’s great to see which genes are actively expressed for research purposes. The downside of RNA testing, however, is that mRNA degrades quickly and is highly variable (Bashiardes, 2016; Reck, 2015). Time of day or even the contents of your last meal can affect your test results. And an mRNA test will not capture microbes that may be temporarily dormant  (Bashiardes, 2016). 

There is limited evidence for long-term dietary, supplement, or lifestyle interventions based on mRNA technology, because of its high variability.

Other metagenomics companies

Other companies using metagenomics like Floré use a smaller database of microbes and/or reference ranges that aren’t appropriate for a baby or child. In addition, many companies try to upsell you their own supplements. We only recommend products that we have researched and can stand behind, based on evidence of their effectiveness among our users.

Tiny Health uses CLIA and CAP certified labs to sequence microbiome samples. In short, your samples are processed at a clinical-grade lab, held to the highest possible standards.

CLIA stands for Clinical Laboratory Improvement Amendments. It is a program regulating the quality and safety of U.S. clinical laboratories to ensure the accuracy, reliability, and timeliness of patient test results. CLIA has regulatory requirements for quality that all laboratories must meet. You can find more information on The Centers for Medicare & Medicaid Services (CMS) website. 

CAP stands for College of American Pathologists. CAP’s accreditation program helps laboratories maintain accuracy of test results and ensure accurate patient diagnosis; meet required standards from CLIA, FDA and OSHA; manage rapidly evolving changes in laboratory medicine and technology; exchange ideas and best practices among pathology and laboratory medicine peers; and offer professional development and learning opportunities for laboratory staff. You can find more information on the CAP website.