Xenogene
Patient Test

Glossary · Laboratory techniques

16S rRNA gene sequencing

In one sentence

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.

Also known as: 16S sequencing · 16S rRNA sequencing · 16S rRNA gene · 16S rDNA · 16S amplicon sequencing · secuenciación 16S

What is 16S rRNA gene sequencing?

The 16S gene encodes part of the bacterial ribosome. It is around 1,500 bases long and mixes conserved regions, nearly identical across bacteria, with nine variable regions (V1 to V9) that differ between groups. The conserved stretches allow universal primers; the variable ones tell organisms apart. That combination made it the standard way to describe the bacterial microbiota, and it underpinned much of the Human Microbiome Project.

How it works

  1. DNA is extracted from the sample (faeces, saliva, vaginal swab and so on).
  2. One or more variable regions (for example V3–V4), or the full gene, are amplified by PCR.
  3. The amplicons are sequenced by NGS.
  4. Sequences are grouped into OTUs or ASVs, matched against reference databases and used to estimate relative abundance and diversity.

Taxonomic resolution

This is the main constraint. Johnson and colleagues (Nature Communications, 2019) showed that sequencing short variable regions cannot match the resolution of the full-length gene, and that full-length 16S, read with long-read platforms such as nanopore sequencing, can approach species and even strain level when intragenomic copy variants are handled properly. With short reads, genus-level interpretation is the prudent default.

Other limitations

  • It detects bacteria and archaea only.
  • Gene copy number varies between species (from one to more than ten), which skews proportions.
  • Primers do not amplify every group equally.
  • It does not measure functional genes; function is predicted by software rather than observed.

Example

A research team wants to compare the faecal bacterial composition of several hundred participants on a tight budget. 16S is a sensible choice: it shows differences in diversity and genus-level composition between groups. If the team later needs to know the exact species, or which resistance genes are present, shotgun metagenomics or other methods are needed.

16S sequencing at Xenogene

The product sheet for Xenogene’s gut microbiota test describes it as a metagenomic analysis using nanopore NGS that looks for bacteria, parasites and other organisms in stool, which is a broader scope than a conventional bacteria-only 16S study. Clinicians who want to know the exact technical approach of an analysis, the resolution it offers, or how it compares with an earlier 16S report can ask the laboratory before requesting it.

Frequently asked questions

Is 16S or shotgun sequencing better?

It depends on the question. 16S amplifies a single bacterial gene, so it is cheaper and copes well with low-biomass samples, but it usually stops at genus and sees only bacteria and archaea. Shotgun sequencing reads all the DNA, can reach species or strain level, picks up DNA viruses, fungi and parasites, and looks at functional genes, at a higher cost.

Does 16S sequencing detect fungi or viruses?

No. The 16S rRNA gene exists only in bacteria and archaea. Fungi are profiled with other markers such as the ITS region, and viruses have no universal marker gene, so they need metagenomic approaches or other tests.

Can 16S identify bacteria to species level?

Sometimes. Short reads covering one or two variable regions usually resolve only to genus. Sequencing the full-length gene with long reads improves species-level identification for many groups, although some closely related species remain indistinguishable.

Related terms

  • Shotgun metagenomics

    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.

  • 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.

  • OTU and ASV

    OTUs (operational taxonomic units) group similar DNA sequences, usually at 97% identity; ASVs (amplicon sequence variants) are exact sequences that can be told apart by a single nucleotide.

  • Relative abundance

    Relative abundance is the share of a microorganism in the total detected in a sample, usually given as a percentage. It reflects its weight within the community, not its absolute quantity.

  • 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.

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.

Updated on

← Back to the glossary