Amplicon Sequencing and shotgun metagenomics are two main approaches to studying the microbiome. Both allow researchers to characterise microbial communities, but they differ in the information they provide, taxonomic resolution, sample requirements and costs.
The choice should depend primarily on your research objectives. If you want to compare the composition of a particular group of microorganisms across many samples, amplicon sequencing of an appropriate marker is usually sufficient. If your project also involves assessing the functional potential of the microbial community as a whole, shotgun metagenomics is a better choice.
What is amplicon sequencing?
Amplicon sequencing is a targeted approach that involves amplifying a selected taxonomic marker using PCR, then sequencing the resulting products, known as amplicons.
The marker is selected according to the group of microorganisms being studied:
Amplicon sequencing primarily provides information about the taxonomic composition and relative abundance of microorganisms within the targeted group. It does not directly assess gene activity in the microorganisms being studied.
When should you choose amplicon sequencing?
Amplicon sequencing is suitable when:
- your aim is to characterise bacteria, archaea, fungi or another selected group of microorganisms;
- you want to compare microbial diversity across samples;
- you are analysing a large number of samples;
- only a small amount of DNA is available;
- you are conducting a screening study;
- keeping costs down is a priority;
- direct analysis of genes and metabolic pathways is not required.
Applications include microbiome studies involving soil, water, the gastrointestinal tract, plant material, food and clinical samples.
What is shotgun metagenomics?
Shotgun metagenomics sequences DNA extracted from a sample without first targeting a single taxonomic marker for amplification. It can therefore analyse genetic material from bacteria, archaea, fungi, DNA viruses and other organisms simultaneously, provided their DNA is present and sufficient sequencing depth is achieved.
Shotgun metagenomics can characterise taxonomic composition and identify genes and metabolic pathways, providing insight into the functional potential of the microbiome. It enables the analysis of antibiotic resistance genes and virulence-associated genes, and the inference of potential metabolic functions. It does not establish whether these genes are actively expressed.
The presence of a gene indicates functional potential, but does not demonstrate that the gene is actively expressed. Gene expression within microbial communities is investigated using metatranscriptomics.
When should you choose shotgun metagenomics?
Shotgun metagenomics is suitable when:
- you want to analyse multiple groups of microorganisms without restricting the study to a single marker;
- you need high taxonomic resolution;
- your aim is to assess the functional potential of the microbiome;
- you want to identify antibiotic resistance genes, virulence-associated genes or genes involved in selected metabolic pathways;
- you plan to reconstruct microbial genomes from metagenomic data to obtain metagenome-assembled genomes (MAGs);
- you want to avoid limitations associated with marker-specific primer selection and PCR amplification.
However, this approach generally requires a larger amount of data, more extensive bioinformatics analysis and a larger budget.
Key differences
| Criterion | Amplicon Sequencing | Shotgun metagenomics |
|---|---|---|
| Scope of analysis | A selected group of microorganisms | DNA from different groups of organisms present in the sample |
| Material analysed | A selected marker, such as the 16S rRNA gene, ITS region or 18S rRNA gene | Total DNA extracted from the sample |
| Bacteria and archaea | 16S rRNA gene | Analysed alongside DNA from other organisms |
| Fungi | ITS region | Analysed alongside DNA from other organisms |
| Taxonomic resolution | Depends on the marker and the region analysed | Usually higher resolution |
| Functional analysis | Limited; functions may be inferred | Direct identification of genes and assessment of potential functions |
| Targeted PCR amplification of a taxonomic marker | Yes | No |
| Data requirements | Lower | Higher |
| Bioinformatics analysis | Less complex | More complex |
| Cost | Generally lower | Generally higher |
| Analysis of large cohorts | Particularly cost-effective | Feasible, but generally more expensive |
What should you consider before choosing a method?
In amplicon sequencing, results depend on the selected marker, the region amplified, the primers used and the quality of reference databases. Differences in amplification efficiency can favour some taxa over others, affecting the observed community profile.
Standard workflows for analysing both bacteria and fungi require separate libraries targeting the 16S rRNA gene and the ITS region, prepared in separate amplification reactions.
For shotgun metagenomics, the proportion of host DNA is particularly important. In samples from humans, animals or plants, host-derived reads may account for a substantial proportion of the sequencing data, leaving fewer reads available for microbial analysis. This may require increased sequencing depth or the use of host DNA depletion methods.
Both approaches typically provide relative abundance estimates. If your aim is to determine the absolute abundance of microorganisms, consider incorporating quantitative standards or complementary methods such as quantitative PCR (qPCR).
Which method should you choose?
Choose Amplicon Sequencingif you want to compare the composition of a selected group of microorganisms across many samples cost-effectively. Use the 16S rRNA gene to analyse bacteria and archaea, or the ITS region to analyse fungi.
Choose shotgun metagenomicsif you need a broader characterisation of the microbiome without restricting the analysis to a single marker, together with information about the genes and functional potential of the community as a whole.
The most suitable approach also depends on the sample type, number of samples, expected proportion of host DNA, required taxonomic resolution and available budget. Tell us about your samples and research objectives, and we will help you select an appropriate marker, sequencing technology, sequencing depth and scope of bioinformatics analysis.
Selected projects and client publications
See which institutions we perform next-generation sequencing (NGS) for and explore scientific publications that draw on the data we have supplied.