The Microbiome

How Metabolomics and 16S Analysis Work Together

16S microbiome analysis and metabolomics answer different questions about the gut. One helps show which bacterial groups are present, while the other measures compounds produced, transformed or left behind. Used together, they provide a much richer picture of the gut ecosystem than either method alone.

Science explainer

How Metabolomics and 16S Analysis Work Together

Content type

Science explainer

Focus

Microbial composition, relative abundance, metabolites and gut functional activity

The big question

There are two very different questions researchers can ask about the gut microbiome.

The first is:

Which bacterial groups are present?

The second is:

What is happening chemically within the gut?

These questions are related, but they are not the same.

16S microbiome analysis is mainly used to study bacterial composition.

Metabolomics is used to study small molecules present within a biological sample.

Used together, they can help connect microbial community structure with biological activity.

The big question is:

Why is it more informative to measure both composition and metabolites than to rely on either one alone?

How do scientists study it?

The two methods examine different layers of the same system.

16S microbiome analysis

16S analysis examines regions of the bacterial 16S ribosomal RNA gene.

Differences in these genetic sequences allow researchers to classify bacterial groups and estimate their relative abundance within a sample.

The result is a picture of microbial community composition.

Metabolomics

Metabolomics examines small molecules present within a sample.

These molecules may come from the dog, diet, microbial activity or interactions between all three.

Depending on the analytical method, researchers may measure compounds including short-chain fatty acids, alcohols, organic acids and other metabolic products.

The result is a chemical picture of the sample.

Combining the data

Researchers can then ask whether changes in particular bacterial groups occur alongside changes in particular metabolites.

This does not automatically prove that one caused the other, but it can reveal biologically interesting relationships.

What can we measure?

The two methods provide complementary information.

16S analysis can measure

Relative abundance of bacterial groups

Differences in microbial composition between samples

Changes in diversity

Community-level shifts over time or between interventions

Metabolomics can measure

Short-chain fatty acids

Volatile organic compounds

Products of carbohydrate fermentation

Products of protein metabolism

Other small molecules present within the sample

Together, the methods can help researchers distinguish between:

Who is there?

and

What chemical activity is associated with that environment?

What have we learned?

A major lesson from microbiome research is that composition and function do not always change in parallel.

Similar microbiomes can behave differently

Two dogs may have broadly similar bacterial communities but different metabolite profiles.

That could happen because the available diet or substrate differs, changing what those microorganisms are doing.

Different microbiomes can produce similar functions

Different bacterial species can sometimes carry out overlapping metabolic roles.

A change in bacterial composition therefore does not necessarily mean that every aspect of microbial function has changed.

Relative abundance does not show activity

16S sequencing can show that a bacterial group became more or less relatively abundant.

It cannot directly show how metabolically active that group was.

Metabolites provide functional clues

Changes in compounds such as acetate, propionate, butyrate, ammonia or other metabolites can provide information about the chemical environment associated with microbial activity.

Together they create context

If a shift in microbial composition occurs alongside a corresponding shift in metabolite profile, the combined result can be more informative than either observation alone.

Why does this matter?

Using 16S analysis and metabolomics together helps researchers move from a simple list of microorganisms towards a more functional understanding of the gut ecosystem.

A simplified way to think about it is:

16S analysis → community structure

Metabolomics → chemical output

Together:

Community structure + Chemical output → richer picture of gut function

This is especially useful when investigating nutrition.

Diet and digestion alter the substrates entering the microbial environment.

Those substrates influence microbial activity.

Microbial activity can alter the metabolites detected downstream.

That creates a connected research framework:

Diet → Digestion → Substrate → Microbiome composition → Microbial activity → Metabolites

No single analytical method measures every step.

Combining methods is therefore one of the most useful ways to study the system as a whole.

What should we keep in mind?

Combining methods improves interpretation, but it does not remove uncertainty.

Correlation does not prove causation

If a bacterial group changes at the same time as a metabolite, that does not prove the bacterium produced the metabolite.

Many organisms can produce the same compound

Microbial metabolic pathways often overlap.

The same metabolite may be produced by several different organisms.

Metabolites can have multiple sources

A molecule found in faeces may come from diet, host metabolism, microbial activity or combinations of all three.

Faecal samples represent an end point

Both 16S and faecal metabolomics usually analyse what is present in stool.

They do not directly measure every process occurring earlier in the gastrointestinal tract.

Relative abundance has limits

16S sequencing commonly reports proportions rather than absolute bacterial numbers.

Timing matters

Microbial composition and metabolite concentrations can vary over time, so the sampling point can influence results.

The strongest interpretations therefore come from combining methods with careful study design, repeated sampling and biological context.

Sources and further reading

References

Further reading:

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.

Pilla R, Suchodolski JS. The Gut Microbiome of Dogs and Cats, and the Influence of Diet. Veterinary Clinics of North America: Small Animal Practice. 2021;51(3):605–621.

Wernimont SM, Radosevich J, Jackson MI, et al. The Effects of Nutrition on the Gastrointestinal Microbiome of Cats and Dogs: Impact on Health and Disease. Frontiers in Microbiology. 2020;11:1266.

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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