Introduction
If you have persistent digestive symptoms, you may wonder whether the microorganisms living in your gut could explain them. Microbiome analysis can describe aspects of those microbial communities, but a list of organisms is not, on its own, an explanation for symptoms.
Microbiome analysis is the study of microorganisms in a particular environment, including which microbes are present and, depending on the method, what they may be capable of doing. Researchers use it to study communities in the gut, on the skin and elsewhere in the human body.
This guide is for readers who want to understand the science or make sense of a microbiome report. We explain the main methods, what terms such as commensal and metagenomic mean, and why results need careful interpretation.
Our approach is to start with a GP conversation about persistent or concerning symptoms, keep a practical record of what you notice, and consider relevant testing later if it could support a discussion with a health professional.
What Is the Human Microbiome?
The human body is home to communities of microorganisms, including bacteria, archaea, fungi and viruses. Different parts of the body provide different environments, so the community in the mouth is not the same as the community on the skin or in the gut.
The word microbiota refers to the microorganisms themselves. Microbiome is often used more broadly for those communities and their genetic material. In everyday use, the terms are sometimes used interchangeably.
Microbes interact with their surroundings and with one another. Some gut microbes, for example, take part in breaking down substances that pass through the digestive system. Researchers also investigate how microbial communities relate to other aspects of health. A relationship found in a study does not automatically show that a particular microbe caused a symptom.
Microbiome analysis is used beyond human health too. Researchers study microbial communities in soil and water, for example. The same broad idea applies: collect material from an environment, examine the microbes or their products, and interpret the findings in context.
What Does Commensal Mean?
A commensal microorganism lives in association with a host without normally causing harm. The term is useful, but it should not be read as a permanent label of "good" or "bad".
A microbe's effects can depend on where it is found and the circumstances of its host. Commensal microbiome analysis examines organisms living as part of these communities, often looking at their distribution or possible roles. Finding a particular commensal organism does not, by itself, establish that someone is healthy or unwell.
What Does Microbiome Analysis Measure?
The answer depends on the sample and the method. A report may focus on one or more of the following:
- Composition: which types of microorganisms were detected.
- Relative abundance: the proportion of detected microbial material attributed to a particular group.
- Diversity: a summary of the variety of organisms detected within or between samples.
- Functional potential: genes that suggest what a community may be capable of doing.
- Activity or products: signals such as RNA, proteins or small molecules that can provide a different view of microbial activity.
These are related questions, but they are not interchangeable. Knowing which organisms were detected is different from knowing which genes are present. Knowing that a gene is present is different again from knowing whether it was active or what effect it had.
The Galaxy Training Network's introduction to microbiome analysis describes these approaches as different ways to examine microbial communities. Each offers a partial view rather than a complete account of everything happening in the body.
How Are Samples Collected and Analysed?
The sample must match the question. A stool sample is commonly used to study aspects of the gut microbiome, while a swab may be used to sample a surface such as the skin or mouth.
A stool sample can provide information about material passing through the gut. It should not be assumed to represent every location in the digestive system equally. Likewise, a skin swab describes the area sampled, not necessarily the whole body.
A typical research workflow has several stages:
- Define the question. Researchers decide which community, comparison or function they want to investigate.
- Collect and handle samples. They use a consistent collection and storage approach suited to the study.
- Extract material. The laboratory prepares DNA, RNA or another substance for examination.
- Measure or sequence it. The method may target a particular marker or examine a broader set of genetic material.
- Process the data. Computer methods filter and organise the measurements.
- Interpret the findings. Researchers consider the results alongside the study design, sample details and limitations.
Each stage can affect the final picture. Collection, storage and extraction methods can influence what is detected. Material introduced during handling can also complicate results, particularly when a sample contains relatively little microbial material.
For a research project, a useful protocol therefore records not only the sequencing method, but also how samples were collected, stored and processed. Controls can help researchers recognise contamination or technical problems. Following the same approach across comparable samples makes differences easier to interpret.
For an individual using a sample kit, the practical step is simpler: follow the instructions supplied for that specific kit. Ask the provider or your health professional if the collection instructions are unclear.
Microbiome Analysis Methods Explained
Several techniques can be used to study a microbial community. The choice depends on whether the question is primarily about identity, genetic potential or activity.
Targeted Marker Sequencing
Targeted sequencing examines a selected region of genetic material used as a marker for a group of organisms. The laboratory first makes copies of that region, then sequences it.
One commonly used approach looks at the 16S ribosomal RNA gene to profile bacteria and archaea. Other targets include the ITS region for fungi and parts of the 18S ribosomal RNA gene for certain eukaryotes, a broad group of organisms whose cells have a nucleus.
A targeted method can be useful when the question concerns a particular group. Its limitation is that it only examines what the chosen marker can capture. The region selected and the way it is copied can affect the results. It may also be difficult to distinguish closely related species or strains.
A 16S profile is therefore not a complete inventory of all bacteria, fungi and viruses in a sample. Nor does the marker itself directly measure what those organisms are doing.
Shotgun Metagenomic Sequencing
Shotgun metagenomics takes a broader approach. Instead of selecting one marker, it sequences fragments of DNA from the sample. Computer analysis then compares those fragments with reference information to help identify organisms and genes.
This can provide a more detailed picture of microbial composition and genetic potential than a single marker approach. It also creates more complex data. The sample may contain human DNA as well as microbial DNA, and the analysis depends partly on the reference databases available.
Not every organism or gene will necessarily be identified. A result also depends on what was present in the sample and what the method could detect. DNA sequencing alone should not be assumed to capture every virus, including those whose genetic material is RNA.
As the Galaxy training material explains, targeted and shotgun sequencing answer overlapping but different questions. The broader method is not automatically the better choice for every purpose.
Culture and Other Techniques
Some microorganisms can be grown in a laboratory using culture methods. This can help researchers examine living organisms under specified conditions. Not all microbes grow readily under the same conditions, however, so culture gives a different view from sequencing.
Laboratories can also use targeted molecular methods to look for particular genetic material. A targeted test and a broad community profile have different purposes: one asks whether selected targets are present, while the other aims to describe a wider community.
The method should fit the question. A report with more categories or technical detail is not necessarily more useful for an individual decision.
What Is Functional Microbiome Analysis?
Composition analysis asks who is there. Functional analysis asks a different question: what might the community be able to do, or what signs of activity can be measured?
For example, shotgun metagenomic data can be examined for genes associated with particular biological processes. This gives an estimate of functional potential. It does not prove that those genes were active when the sample was collected.
Other methods examine different layers:
- Metatranscriptomics looks at RNA transcripts, which can provide information about gene expression at the time of sampling.
- Metaproteomics examines proteins associated with the sample.
- Metabolomics examines small molecules. Their presence does not automatically show which organism produced them, because they can also come from the person or other sources.
These methods can be combined, but combining measurements still requires careful interpretation. A gene, an RNA signal and a measured molecule are different kinds of evidence. The Galaxy overview of microbiome methods sets out these distinctions.
A useful question to ask about any functional report is: Was this function directly measured, or predicted from the organisms or genes detected? A prediction can help generate a research question, but it is not the same as observing the process in the body.
How Sequencing Data Becomes a Report
Sequencing produces many short pieces of information, called reads. These are not a ready-made list of diagnoses. They need processing before researchers can compare samples or describe communities.
Checking and Classifying the Data
Researchers first check sequence quality and remove material that is unsuitable for the intended analysis. In a targeted sequencing workflow, they may also identify artificial sequences created during the copying step.
The remaining sequences can be organised in different ways. Two terms you may see are:
- Operational taxonomic units, or OTUs: groups of similar sequences used as a practical way to summarise the data.
- Amplicon sequence variants, or ASVs: distinct marker sequences identified after processing the data to account for errors.
These are analytical groupings, not always equivalent to named species. Researchers compare sequences with reference databases to assign names where possible. If a database is incomplete or sequences are very similar, the result may be identified only to a broader group.
For shotgun data, researchers may also compare sequences with databases of known genes and their possible functions. The result depends on the information available in those databases and the analytical choices made.
Comparing Samples
A research team might compare samples from different people, body sites or time points. To do this meaningfully, it needs to account for differences in sampling and sequencing.
The number of reads obtained from a sample is not simply the number of microbes originally present. A sample with more reads has been examined more deeply, but that alone does not establish that it contained more microorganisms.
Researchers also consider factors that could affect a comparison, such as medication use, age or differences in sample handling. If two groups differ in several ways, it can be difficult to know which factor is associated with an observed microbial difference.
An association is a starting point for investigation. It does not, on its own, establish cause and effect.
How to Read Composition and Diversity Results
Microbiome reports often present colourful charts and percentages. They can be useful descriptions, but their meaning is easy to overstate.
Relative Abundance Is a Proportion
Suppose a report attributes 40% of the microbial sequences it classified to one group. That figure describes a share of the classified material in that analysis. It does not necessarily tell you the number of living organisms in the original sample.
It also matters what happens to the rest of the community. A group's percentage can rise because another group's share falls, even if the first group's actual amount has not risen. An educational microbiome analysis workshop explains why sequencing proportions and absolute amounts should not be treated as the same measurement.
If a report compares your percentage with another sample, ask what was measured, which method was used and whether the comparison uses a compatible sample and process.
Alpha and Beta Diversity
Alpha diversity describes diversity within a sample. It can reflect how many different groups were detected and, depending on the measure, how evenly they were represented.
Beta diversity compares communities between samples. It helps researchers describe how similar or different those samples are.
Neither term is a diagnosis. A diversity value depends on the body site, method and measure used. It is not a simple score that can tell you on its own whether you are healthy. A difference between two communities also does not explain what caused the difference or whether it matters clinically.
Why Reference Comparisons Need Context
A report may compare a result with a reference group. Before drawing conclusions, consider who was included in that group, which body site was sampled and whether the same methods were used.
A value outside a displayed comparison range is a prompt to ask what the range means. It is not automatically evidence of a condition. Equally, a result that looks similar to a reference group does not explain away persistent symptoms.
Can Microbiome Analysis Explain Symptoms?
Microbiome research can help scientists investigate patterns, but an individual profile has limits. A sample reflects a particular site and point in time. The result is shaped by the collection method, laboratory process and analysis as well as the biology being studied.
Symptoms such as ongoing diarrhoea, abdominal discomfort or fatigue can have many possible explanations. A microbiome report cannot establish the cause on its own. It also should not replace an assessment of your symptoms, medical history and any investigations your GP considers appropriate.
Microbiome profiling is different from a clinician arranging a specific test to investigate a suspected infection or another medical concern. If you are unsure what a test is intended to answer, ask before relying on its result.
If symptoms are persistent, worsening or concerning, contact your GP. For sudden severe symptoms, breathing difficulty or collapse, seek urgent help through 999 or A&E.
Preparing for a Useful GP Conversation
You do not need to interpret a complicated report alone. Whether or not you have had microbiome analysis, a short record can help make an appointment more productive.
Note when symptoms began, how often they occur and whether they are changing. Depending on your concern, it may also help to record sleep, stress, energy, weight changes and relevant changes to medication or supplements. A brief record is enough. You do not need to monitor every detail.
If you have a report, take the complete document rather than only a highlighted score. Useful questions include:
- What did this test actually measure, and from which sample?
- Does the report distinguish measured findings from predictions?
- What does its comparison range represent?
- Could the result change the next step in my care?
- Are there other explanations for my symptoms that need assessment?
Your GP can consider the report alongside your history and symptoms. If a separate blood test is relevant, it can provide a snapshot of the markers it measures, but it does not analyse the microbiome. A broader set of tests is not automatically more useful than a focused investigation.
The Practical Next Step
Microbiome analysis can describe microbial communities and, with some methods, explore their genetic potential or activity. Its value depends on the question, sample and method. A detailed profile cannot by itself explain an individual's symptoms.
We recommend starting with the concern rather than the chart. Keep a simple record, bring any existing report with you and book a GP appointment to discuss persistent symptoms and the most relevant next step.
FAQ
Is microbiome analysis the same as a stool test?
Not always. A stool sample can be used for microbiome analysis, but microbiomes can also be studied using samples from other sites, such as the skin or mouth. Stool tests can have different purposes, so check what a particular test measures.
Can microbiome analysis identify every microorganism?
No method should be assumed to detect every microorganism. The sample, sequencing target, laboratory process and reference database all influence what can be identified. A report describes what its method detected and classified.
Does a higher diversity score mean a healthier microbiome?
Not necessarily. Diversity is a way to summarise a community, and its meaning depends on the body site, method and context. It cannot diagnose a condition or explain symptoms on its own.
Should I take a microbiome report to my GP?
Yes, if you have concerns or want help understanding it. Take the full report and a brief record of your symptoms. Your GP can discuss whether the findings are relevant and whether any further assessment is appropriate.
