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Bushra's Science Blogs: How we decode your dog's gut, sequencing explained

Sequencing involves reading the nucleotide sequence of DNA. Using polymerase chain reaction (PCR), we amplify the gene of interest—in this case, the 16S rRNA gene specific to bacteria—so that we have numerous copies, which serve as technical replicates.

July 11, 2024 • 2 min read

Bushra Schuitemaker

Lead Microbiologist

What is Sequencing?

Figure 1: Schematic representation of the 16S rRNA gene, labelled with all hypervariable regions.

We can confidently determine the DNA sequence with hundreds of thousands of identical gene copies in one sample. We then match these sequences to bacterial genomes to identify which bacteria are present.

What is Being Sequenced?

We extract DNA from your dog's faeces and prepare it for sequencing in our labs. This process involves amplifying the 16S rRNA gene and creating a library, which is then sequenced.

The 16S gene is ubiquitous in bacteria and highly conserved, meaning it is present across different bacterial species but with slight variations unique to each species.

During sequencing, we read the individual base pairs that constitute these variations within the 16S gene for each species. These sequences are then mapped using a reference database to confirm their identity.

Figure 2: Workflow of standard Next Generation Sequencing protocol.

What Sequencing Platform Do We Use?

We use the Oxford Nanopore sequencing platform, which employs long-read sequencing technology. This allows us to sequence the entire 16S rRNA gene, encompassing variable regions 1-9.

In contrast, the Illumina platform, which uses short-read sequencing, can only capture variable regions 3-4 at most. Direct comparisons have shown that the Oxford Nanopore platform is more accurate and replicable for sequencing the 16S rRNA gene.

Figure 3: Diagram of the Oxford Nanopore sequencing technology.

Why We Do It Differently

Many researchers focus on the V3-4 regions of the 16S rRNA gene, as these middle sections are highly variable and make it easier to detect differences. However, some bacterial species have very similar genes, making it difficult to differentiate them based solely on the V3-4 regions.

By examining the entire 16S gene, we achieve accurate and replicable species-level taxonomic resolution, ensuring precise identification of bacterial species.

Taxonomic resolution refers to the ability to classify and identify organisms at various taxonomy levels. The broadest level is the domain, in this case, prokaryotic. Next is the kingdom, which is bacteria. From here, the classification varies depending on the specific bacteria. By sequencing variable regions 1-9 of the 16S gene, we can confidently identify individual bacterial species, providing a high level of taxonomic resolution.

References

López-Aladid, R., Fernández-Barat, L., Alcaraz-Serrano, V., Bueno-Freire, L., Vázquez, N., Pastor-Ibáñez, R., Palomeque, A., Oscanoa, P. and Torres, A., 2023. Determining the most accurate 16S rRNA hypervariable region for taxonomic identification from respiratory samples. Scientific reports, 13(1), p.3974.

Nanopore Is Preferable over Illumina for 16S Amplicon Sequencing of the Gut Microbiota When Species-Level Taxonomic Classification, Accurate Estimation of Richness, or Focus on Rare Taxa Is Required - PMC

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Frequently asked questions

The microbiome is the name given to the collection of microbes, mostly bacteria, but also fungi and protozoa, that exist within your dog’s gut. It is a diverse and complex microbial community which can directly affect health and wellbeing. We know that 90% of a human’s body cells are microbes, with only 10% being human cells – it’s just that human cells are markedly larger than the microbes. It’s similar for our four-legged friends. Testing the microbiome gives us an idea of exactly which bacteria are present in your dog’s gut and this can help indicate existing or future health problems.
A healthy diversity within the microbiome has been found to be an accurate indicator of overall health and wellbeing. If your dog appears healthy, but has an imbalance in their microbiome, then this could be an indicator of a potential future health issue. If your dog has any existing health complaints, then improving the health of their microbiome can help to improve immune system health and overall wellbeing, as well as improving disease symptoms.
Testing and treatment have the potential to help with a whole range of different health complaints. The immune system is very closely associated with the gut, so any imbalance in the microbiome can influence immune system health, overall vitality and wellbeing. Our supplement recommendations are also tailored to your individual dog, with specific ranges designed to help with gastrointestinal inflammation, joint problems, allergies and skin complaints, to name a few.
All you need to do is order a kit online and fill in our questionnaire about your dog and their general health. We will then send the kit out to you by post. You then just need to collect a sample and return it to us, again by post. Once the test is performed, we will email the results directly to you.
You do not need to get your vet’s permission to test, or talk to them about performing the test beforehand. We do recommend that you pass on a copy of your test results to your regular vets, as it may help them in understanding your pet’s current health, and any future complaints they may have.