The Microbiome

What Is 16S Microbiome Analysis?

16S microbiome analysis uses sequencing of the bacterial 16S ribosomal RNA gene to compare which bacterial groups are present in a sample and their relative proportions. It helps describe bacterial communities, but does not directly show what those bacteria are doing or measure their absolute numbers.

Science explainer

Content type

Science explainer

Focus

Bacterial composition and relative abundance

What question does 16S microbiome analysis answer?

The canine gut contains an enormous number of microorganisms.

But most of them cannot simply be identified by looking at a faecal sample under a microscope.

Modern microbiome research therefore relies heavily on genetic techniques.

One of the most widely used is known as 16S ribosomal RNA gene sequencing.

The basic question it helps answer is:

Which bacterial groups are present in a sample, and how does the bacterial community differ between samples or over time?

How does 16S ribosomal RNA gene sequencing work?

Bacteria contain a gene known as the 16S ribosomal RNA gene.

Parts of this gene are highly conserved across bacteria, while other regions vary between bacterial groups.

That combination makes it useful for identifying and comparing bacteria.

DNA extraction

Researchers first extract genetic material from a biological sample, such as canine faeces.

Amplification

Selected regions of the 16S gene are copied many times using a laboratory process known as polymerase chain reaction.

Sequencing

The resulting genetic material is sequenced.

Bioinformatic analysis

Computer-based methods compare the sequences and group them according to their similarities.

Researchers can then estimate the relative abundance of different bacterial groups within each sample.

This makes it possible to compare microbiome composition before and after an intervention, between individual dogs, or between different populations.

What information can 16S analysis provide?

16S analysis can provide several different types of information.

Relative abundance

Researchers can estimate what proportion of the bacterial sequences in a sample belong to particular groups.

Diversity

The data can be used to examine how many different bacterial groups are detected and how evenly they are distributed.

Differences between samples

Researchers can compare microbial communities between dogs, between diets, or at different points in time.

Taxonomic patterns

Depending on the sequencing method and quality of the data, bacterial groups may be classified at levels such as phylum, family or genus.

In some cases, species-level identification may be possible, although this is not always reliable with 16S sequencing alone.

How did 16S sequencing change microbiome research?

16S sequencing transformed microbiome research because it allowed scientists to study bacterial communities without needing to grow each organism in a laboratory.

That revealed far greater microbial diversity than traditional culture methods had suggested.

It also made large-scale comparisons much easier.

Researchers can now examine whether the relative abundance of bacterial groups changes alongside diet, age, environment or nutritional interventions.

However, the technique mainly tells us about bacterial composition.

It does not directly tell us what those bacteria are doing.

That distinction is important when interpreting microbiome studies.

What can 16S analysis reveal about bacterial communities?

16S analysis gives researchers a powerful way to examine the structure of a bacterial community.

It can help answer:

Who is there?

and

How does that community change?

This is why it has been useful in CaniNectar and Enzyme Rich Malt Extract research examining the canine gut microbiome.

But microbiome composition is only one layer of the gut ecosystem.

To understand more about microbial activity, researchers may combine 16S analysis with other approaches such as metabolomics.

That creates a useful distinction:

16S analysis → which bacterial groups are present

Metabolomics → which compounds are present

Together, these approaches can provide a richer picture of the gut environment.

What are the limitations of 16S analysis?

16S microbiome analysis has important limitations.

Relative rather than absolute abundance

The method usually reports bacteria as proportions of the microbial community.

An apparent increase in one group can therefore partly reflect a decrease in another group rather than a true increase in bacterial numbers.

Limited functional information

Knowing that a bacterium is present does not automatically reveal what it is doing.

Different bacteria can perform similar metabolic functions, while the same bacterial group may behave differently under different nutritional conditions.

Resolution

16S sequencing does not always distinguish reliably between closely related bacterial species.

Sampling

Faecal samples provide useful information about the lower gastrointestinal tract but do not represent every microbial community present throughout the digestive system.

For these reasons, 16S results are most useful when interpreted alongside other measurements rather than treated as a complete description of the microbiome.

Sources and further reading

References

Further reading:

Hooda S, Minamoto Y, Suchodolski JS, Swanson KS. Current state of knowledge: the canine gastrointestinal microbiome. Animal Health Research Reviews. 2012;13(1):78–88.

Pilla R, Suchodolski JS. The Role of the Canine Gut Microbiome and Metabolome in Health and Gastrointestinal Disease. Frontiers in Veterinary Science. 2020;6:498.

Suchodolski JS. Diagnosis and interpretation of intestinal dysbiosis in dogs and cats. The Veterinary Journal. 2016;215:30–37.

Waring RH, Dagi TF, Hunter JO. Innovative Approaches to Managing the Mammalian Microbiome: Evidence for the Role of Anabiomics. Journal of Modern Agriculture and Biotechnology. 2024;3:10.

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