Glossary · Laboratory techniques
NGS (next-generation sequencing)
In one sentence
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.
Also known as: NGS · next-generation sequencing · high-throughput sequencing · massively parallel sequencing · secuenciación de nueva generación
What is NGS?
Next-generation sequencing emerged in the mid-2000s as an alternative to Sanger sequencing, which reads one fragment per reaction. Its defining feature is massive parallelism: millions of fragments are read in the same run. That dramatically lowered the cost per base and made it practical to study whole genomes, exomes, transcriptomes and complete microbial communities such as the gut microbiota.
As Goodwin, McPherson and McCombie described in their 2016 review in Nature Reviews Genetics, the field has split into two broad approaches: platforms that maximise throughput with short reads (tens to a few hundred bases) and platforms that prioritise long, contiguous reads, such as nanopore sequencing.
How it works
Each platform has its own chemistry, but the overall workflow is similar:
- Extraction of DNA (or RNA) from the sample: blood, faeces, urine, swabs and so on.
- Library preparation: the material is fragmented or amplified, and adapters recognised by the instrument are attached.
- Sequencing: the instrument reads the fragments and produces files containing millions of reads.
- Bioinformatics: reads are quality-filtered and compared against reference databases to work out which organism or gene they come from.
In microbiology there are two main strategies. 16S rRNA gene sequencing amplifies a single bacterial marker; it is inexpensive but has limited taxonomic resolution. Shotgun metagenomics sequences all the DNA in the sample, can reach species level and can look at functional and resistance genes, at a higher cost and with heavier analysis.
Example
A gastroenterologist requests a stool microbiota study for a patient with persistent bowel symptoms. The laboratory extracts DNA, sequences it by NGS and reports which bacterial groups dominate and in what proportion (relative abundance), together with community diversity. The clinician weighs those findings against the examination, blood tests and history; the report is not a diagnosis in itself.
Limitations
- Reliance on reference databases: organisms that are poorly represented are classified less reliably.
- Technical bias: extraction, PCR amplification and sequencing depth all shape the result.
- Detecting DNA is not proof of a living organism, nor that it is causing symptoms.
- Results from different laboratories may not be directly comparable if the methods differ.
NGS at Xenogene
Xenogene uses NGS based on nanopore technology for its microbiota analyses and for the AMC panels that detect microorganisms in clinical samples. For instance, the gut microbiota test analyses a stool sample with nanopore NGS to describe the microbial composition and to detect pathogenic bacteria, parasites and other organisms. Tests are carried out only on the request of a healthcare professional, who is responsible for interpreting the report.
Frequently asked questions
What is the difference between NGS and PCR?
PCR amplifies and detects specific sequences that you are deliberately looking for, so it answers a closed question: is this organism or gene present? NGS reads the sequence of many fragments at once and can reveal what is in a sample without knowing in advance. Many NGS workflows still use a PCR step to prepare the sample.
What is third-generation sequencing?
The term is used for technologies that read single, long DNA molecules, such as nanopore and PacBio sequencing, as opposed to short-read platforms. In everyday use they are often all grouped under NGS. The practical differences are read length and error profile.
Can an NGS test give a diagnosis on its own?
No. NGS produces data that must be processed with bioinformatics tools and reference databases. The result has to be interpreted by the requesting clinician alongside symptoms, examination and other tests.
Related terms
- 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.
- 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.
- 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.
- PCR and qPCR
PCR (polymerase chain reaction) is a technique that copies a chosen stretch of DNA millions of times so it can be detected. qPCR, or real-time PCR, tracks amplification cycle by cycle, allowing the target to be quantified.
- 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.
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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