16S vs. Shotgun Sequencing: What's the Difference?
If you want to analyse your microbiome, it is not only the sample that matters, but also the method used to examine it. Two of the best-known approaches in microbiome research are 16S sequencing and shotgun sequencing. But which approach is the bestt?
Both methods can provide important information about the gut microbiome. The difference lies in the depth of the analysis: while 16S mainly offers an overview of bacterial groups, shotgun sequencing analyses the microbial DNA present far more broadly, including bacteria, fungi, viruses, resistance genes and functional potential¹.
That is why the choice of method plays a major role if you want to understand your microbiome not just superficially, but on a deeper scientific level.
What is 16S sequencing?
16S sequencing is an established method for analysing bacterial communities. It does not examine the entire DNA of a sample, but instead targets a specific section of the bacterial 16S rRNA gene².
This gene occurs in bacteria and contains regions that differ between various bacterial groups. This makes it possible to estimate which bacterial groups are present in a sample.
16S sequencing has been used in microbiome research for many years and has made important contributions to our understanding of bacterial ecosystems. It can still be useful, especially for large studies, initial overviews or cost-conscious projects.
Its great advantage lies in its simplicity: the method is comparatively inexpensive, well established and provides a broad overview of bacterial compositions. At the same time, it has clear limitations, particularly when it comes to high taxonomic resolution, fungi, viruses or functional information.
What is shotgun sequencing?
Shotgun sequencing is a modern form of metagenomic sequencing. Unlike 16S, it does not examine just a single bacterial marker region, but the entire DNA present in a sample³.
This creates a much more comprehensive picture of the microbiome. In addition to bacteria, other microbial groups such as fungi, viruses, bacteriophages and archaea can also be analysed. Functional genetic potential can also be examined, for example metabolic pathways or resistance genes.
This method is more data-intensive and technically more demanding, but provides much deeper insights into microbial composition and function.
The key differences at a glance
| Aspect | 16S sequencing | Shotgun sequencing |
|---|---|---|
| Analysis principle | targeted bacterial marker region | entire microbial DNA |
| Focus | Bacteria only | Bacteria, fungi, viruses, phages, archaea, parasites, resistance genes & functional potential |
| Resolution | often genus level | often down to species level |
| Functional analysis | strongly limited | possible |
| Resistance genes | not analysable | analysable |
| Data volume | lower | significantly higher |
| Cost | very low | usually higher |
| Scientific depth | good for an overview | considerably more comprehensive |
The central difference between 16S and shotgun lies in how much genetic information is analysed.
16S sequencing looks specifically at one bacterial marker region. This provides a useful but limited overview of bacterial groups. Shotgun sequencing, on the other hand, analyses the entire microbial DNA of a sample and can therefore create a far more detailed picture of the microbiome⁴.
This is especially important when the goal is not only to answer the question “Which bacteria are present?”, but also:
- Which species are precisely present?
- What functional potential does the microbiome carry?
- Are there indications of resistance genes?
- What role do fungi, viruses or other microorganisms play?
For a modern microbiome analysis, this difference is decisive.
Why does resolution matter?
Many bacteria belong to the same genus but can have very different properties. This is exactly where the resolution of the method becomes important.
A simple example: if an analysis only identifies a bacterial genus, you do not necessarily know which specific species is behind it. Different species within the same genus can have different metabolic pathways, take on different ecological roles or interact differently with other microorganisms.
Shotgun sequencing can often enable a more precise taxonomic classification than classic 16S methods⁵. As a result, the microbiome becomes visible not just as a rough overview, but as a more complex ecosystem with many individual players.
This is particularly relevant when you want to understand microbial diversity, functional relationships or finer changes in the gut microbiome.
More than just bacteria
An important difference between 16S and shotgun is the view beyond bacteria.
16S sequencing focuses primarily on bacteria. That is scientifically sensible when this is exactly the question at the centre. However, the gut microbiome does not consist of bacteria alone. Fungi, viruses, bacteriophages and archaea are also part of the gut's microbial ecosystem⁶.
Shotgun sequencing can capture this broader microbial world more effectively. This creates a more comprehensive picture of the gut microbiome – not just as a list of bacteria, but as a complex network of different microorganisms.
In modern microbiome research in particular, these interactions are becoming increasingly important. Because the stability, diversity and function of the microbiome do not arise from individual bacteria alone, but from the interplay of many microbial groups.
Functional microbiome analyses
A modern microbiome analysis should not only show which microorganisms are present. It should also help you understand what functional potential this microbial ecosystem has.
This is where one of the biggest advantages of shotgun sequencing lies. Because many DNA segments from the entire microbiome are analysed, functional genes and metabolic pathways can also be examined bioinformatically⁷.
These include, for example, microbial potential for:
- Butyrate production
- Gas formation
- Mucin degradation
- Antibiotic resistance genes
- Inflammation-associated microbial patterns
This information is scientifically valuable because it does not just describe the microbiome, but classifies it functionally. It remains important to note: such analyses serve the educational and scientific interpretation of microbial patterns and do not replace a medical diagnosis.
Which method does Bactera use – and why?
At Bactera, we rely on modern shotgun sequencing for Microbiome 360°, because we want to analyse the gut microbiome as comprehensively and scientifically deeply as possible.
Our goal is not just a simple overview of bacterial groups. We want to give you a differentiated picture of your microbiome, with information on microbial diversity, bacterial composition, fungi, viruses, resistance genes and functional potential.
Shotgun metagenomics therefore fits better with our scientific standards: high-resolution, data-driven and future-oriented.
If you want to analyse your gut flora at home, Microbiome 360° enables a modern microbiome analysis with deeper biological classification than classic methods.
When is 16S still used?
Even though shotgun sequencing can provide significantly more information, that does not mean 16S sequencing is “bad”.
16S remains an important method in microbiome research. It can still be useful especially for large cohort studies, cost constraints or questions where a rough bacterial overview is sufficient.
The method is established, comparatively inexpensive and absolutely suitable for certain scientific questions. So the decisive factor is not whether 16S or shotgun is fundamentally “better”, but which question is to be answered.
For simple bacterial overviews, 16S can be sufficient. For a deeper, broader and more functional microbiome analysis, however, shotgun sequencing offers clear advantages.
Frequently asked questions about 16S and shotgun sequencing
What is the difference between 16S and shotgun sequencing?
16S targets a specific bacterial marker region. Shotgun sequencing examines all of the microbial DNA present in a sample.
Why does shotgun provide more information?
Because it does not analyse just a single DNA segment, but many DNA fragments from the entire microbiome.
Can 16S also detect fungi and viruses?
No, 16S is primarily aimed at bacteria. Fungi, viruses and bacteriophages are not comprehensively captured by it.
Which method is more accurate?
In many cases, shotgun sequencing offers higher taxonomic resolution and can more often analyse down to species level.
Why is shotgun sequencing more expensive?
Because significantly more DNA data has to be generated and processed bioinformatically. The analysis is technically and computationally more demanding.
What does species-level resolution mean?
It means that microorganisms can be classified not just roughly at group or genus level, but down to the specific species.
Can resistance genes be analysed?
With shotgun sequencing, certain resistance genes can be examined at the DNA level. 16S is not suitable for this.
What does a modern microbiome analysis show?
Depending on the method, bacterial diversity, microbial composition, fungi, viruses, resistance genes and functional potential can be made visible.
Which method does Bactera use?
Bactera uses modern shotgun sequencing for Microbiome 360°.
Is shotgun sequencing diagnostic?
No. The analysis serves the scientific and educational interpretation of microbial patterns and does not replace a medical diagnosis.
Scientific references
- Quince C et al. Shotgun metagenomics, from sampling to analysis. Nat Biotechnol. 2017.
- Johnson JS et al. Evaluation of 16S rRNA gene sequencing for species and strain-level microbiome analysis. Nat Commun. 2019.
- Sharpton TJ. An introduction to the analysis of shotgun metagenomic data. Front Plant Sci. 2014.
- Knight R et al. The microbiome and human biology. Annu Rev Genomics Hum Genet. 2017.
- Almeida A et al. A unified catalog of 204,938 reference genomes from the human gut microbiome. Nat Biotechnol. 2021.
- Huttenhower C et al. Structure, function and diversity of the healthy human microbiome. Nature. 2012.
- Lloyd-Price J et al. Multi-omics of the gut microbial ecosystem in inflammatory bowel diseases. Nature. 2019.
- Qin J et al. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010.