Glossary · Laboratory techniques
Shotgun metagenomics
In one sentence
Shotgun metagenomics sequences all the DNA in a sample in an untargeted way, without amplifying a single marker gene. It can identify bacteria, DNA viruses, fungi and parasites, often to species level, and reveal their functional and resistance genes.
Also known as: shotgun sequencing · shotgun metagenomic sequencing · whole-metagenome sequencing · metagenómica shotgun
What is shotgun metagenomics?
The name comes from the scatter of a shotgun blast: the sample’s DNA is broken at random and every fragment is sequenced, whether it comes from bacteria, fungi, viruses, parasites or the patient. Bioinformatics then works out which organism and which gene each read belongs to. Unlike 16S rRNA gene sequencing, there is no pre-selected marker, so the approach is untargeted.
In their 2017 review in Nature Biotechnology, Quince, Walker, Simpson, Loman and Segata describe it as having transformed microbiology, while stressing that analysis remains challenging for complex samples and for organisms poorly represented in reference databases.
What it is used for
- Taxonomic resolution: usually to species and, with enough depth, strain level.
- Beyond bacteria: it can detect DNA viruses, fungi, protozoa and helminths.
- Functional potential: it shows which genes are present (for example, butyrate-production pathways), not just who is there.
- The resistome: it identifies antimicrobial resistance genes and, with long reads, the genetic element carrying them.
- Metagenome-assembled genomes, useful for organisms that cannot be cultured.
Limitations
- Host DNA: in blood, urine or tissue most reads are human and dilute the microbial signal.
- Cost and depth: finding minority organisms means sequencing a lot.
- Databases: poorly represented organisms are misclassified or missed.
- DNA is not activity: a gene shows capacity, not that it is being expressed or that the organism is alive.
- Contamination: low-biomass samples need negative controls.
Example
A gastroenterology team is assessing a patient with persistent diarrhoea after several courses of antibiotics. Beyond changes in bacterial composition, they want to know whether resistance genes or intestinal parasites are present. A 16S study would describe bacteria only; a shotgun analysis can report species, resistance genes and other organisms in one assay. The findings are weighed alongside stool culture, conventional parasitology and the clinical picture, and interpretation rests with the clinician.
Shotgun metagenomics at Xenogene
The product sheet for the gut microbiota test presents it as a metagenomic analysis using nanopore NGS that studies the microbial composition of stool and detects pathogenic bacteria, E. coli strains, parasites and helminths, as well as functional aspects of the microbiota. For questions about the technical scope of a specific analysis, clinicians can contact the laboratory before requesting it.
Frequently asked questions
Why is shotgun metagenomics more expensive than 16S?
Because far more sequencing is needed. All the DNA in the sample is read, including human DNA, and detecting low-abundance organisms requires depth. The bioinformatics and whole-genome reference databases are also more demanding than for 16S.
What is shallow shotgun sequencing?
It is shotgun sequencing at a lower depth per sample, designed to bring costs closer to 16S while keeping species-level profiling. It works reasonably for high-biomass samples such as stool, but it is less suited to detecting rare organisms or reconstructing genomes.
Does shotgun metagenomics detect RNA viruses?
Not with a DNA-only protocol. RNA viruses require a reverse-transcription step (metatranscriptomics or combined DNA and RNA workflows). It is worth checking which nucleic acids a given assay actually processes.
Related terms
- 16S rRNA gene sequencing
16S rRNA gene sequencing amplifies and reads a marker gene found in all bacteria and archaea to show which groups are present in a sample and in what proportions. It is inexpensive but usually resolves to genus level and does not measure function directly.
- NGS (next-generation sequencing)
NGS (next-generation sequencing) is a family of technologies that read millions of DNA or RNA fragments in parallel in a single run, making it possible to study whole genomes, targeted genes or entire microbial communities at scale.
- Clinical metagenomics
Clinical metagenomics (mNGS) is the untargeted sequencing of the genetic material in a patient sample to detect bacteria, viruses, fungi or parasites without choosing the pathogen in advance. It complements culture and susceptibility testing rather than replacing them.
- Nanopore sequencing
Nanopore sequencing is a third-generation technique that reads single DNA or RNA molecules as they pass through a nanometre-scale pore, by measuring changes in electrical current. It produces long reads and data in real time.
- Antimicrobial resistance
Antimicrobial resistance (AMR) is the ability of bacteria, viruses, fungi or parasites to stop responding to medicines that used to control them, making infections harder to treat.
Educational content for healthcare professionals and the general public. It does not replace assessment by a healthcare professional: ordering and interpreting any test is the responsibility of the requesting clinician.
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