Glossary · Laboratory techniques
Nanopore sequencing
In one sentence
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.
Also known as: nanopore · Oxford Nanopore · ONT sequencing · secuenciación Nanopore
What is nanopore sequencing?
It is a form of NGS that does not need to synthesise copies of DNA in order to read it. A protein with a tiny pore is embedded in a synthetic membrane carrying an ionic current. As a DNA or RNA strand moves through the pore, each stretch of bases disrupts the current in a characteristic way, and a basecalling algorithm, based on neural networks, converts that signal into sequence.
Because it reads single, intact molecules, read length is limited not by the chemistry but by how intact the extracted DNA is. Reads of thousands to tens of thousands of bases are routine, and ultra-long protocols go well beyond a hundred thousand. Wang and colleagues reviewed its uses in Nature Biotechnology in 2021: genome assembly, full-length transcripts, base-modification detection, rapid clinical diagnosis and outbreak surveillance.
How it works in microbiology
- Long reads help identification at species level and show a resistance gene together with its genetic neighbourhood (chromosome or plasmid).
- Real time: data can be analysed during the run, which helps when turnaround matters.
- Flexibility: it is used for shotgun metagenomics and for sequencing the full-length 16S gene (around 1,500 bases), which resolves more than short variable regions.
- Accuracy was historically its weak point. With R10.4.1 chemistry the manufacturer reports single-read accuracy above 99% (Q20+), and consensus accuracy rises further with coverage.
Example
A study in Nature Biotechnology (Charalampous et al., 2019) combined human DNA depletion with nanopore metagenomics on respiratory samples, delivering results in about six hours. Compared with culture, sensitivity was 96.6% but specificity only 41.7%, improving after confirmatory testing. It is a good illustration of the technology: fast and broad, but findings still need confirmation and clinical interpretation.
Limitations
- In samples such as blood, the overwhelming amount of human DNA reduces the share of microbial reads.
- Reagent contamination matters more in low-biomass samples.
- Finding a resistance gene does not tell you the organism’s actual susceptibility; phenotypic susceptibility testing remains the reference.
Nanopore sequencing at Xenogene
Xenogene’s AMC in Blood uses metagenomic nanopore sequencing on venous blood to look for bacteria, viruses, fungi, parasites and mycobacteria, as well as resistance and virulence genes and plasmids. According to the product sheet, the protocol does not require PCR amplification. The sheet itself notes that a negative result does not rule out infection and that findings must be read in their clinical context by the requesting clinician.
Frequently asked questions
How accurate is nanopore sequencing?
Early versions had a high per-read error rate, but newer chemistries and basecalling models have improved it substantially; the manufacturer reports single-read accuracy above 99% with its R10.4.1 chemistry. The reliability of a clinical result, however, depends on the whole protocol, its controls and the laboratory's validation, not just the sequencer.
How does nanopore compare with Illumina?
Illumina produces short reads, usually 150 to 300 bases, with very high per-base accuracy. Nanopore produces much longer reads, from thousands to hundreds of thousands of bases, and data can be analysed while the run is still going. They are complementary, and the right choice depends on the question being asked.
Why do long reads matter in microbiology?
A long read spans more of the same genome, which makes it easier to assign to a species, to assemble genomes and to see the context of a resistance gene, for example whether it sits on a plasmid. With short reads that context is lost more easily.
Related terms
- 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.
- 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.
- 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.
- 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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