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Short answer: pyrosequencing is a sequencing-by-synthesis method that reads DNA by detecting the light released each time a polymerase adds a nucleotide. Because the light signal is proportional to the nucleotide incorporated, it is used for short, targeted regions, especially to quantify single-nucleotide polymorphisms (SNPs) and the percentage of DNA methylation at individual CpG sites.
Definition and scope
Pyrosequencing is a real-time sequencing-by-synthesis system. A 2015 Methods in Molecular Biology chapter describes it as designed to quantify SNPs, and a 2023 forensic review in Electrophoresis describes it as sequencing by synthesis based on chemiluminescent detection of inorganic pyrophosphate (PPi). In practice it is applied to one short PCR amplicon at a time, not to whole genomes. Whole-gene or genome-scale questions are better served by Sanger or next-generation sequencing (see the comparison below and the Illumina vs Nanopore comparison for the high-throughput side).
How pyrosequencing works
A single-stranded, PCR-generated template is primed with a sequencing primer. The four nucleotides are then added one at a time in a known order, and each addition triggers a chain of reactions:
- DNA polymerase incorporates the nucleotide if it matches the template and releases PPi.
- ATP sulfurylase converts the PPi to ATP, using adenosine phosphosulfate (APS) as a substrate.
- Luciferase uses the ATP to convert luciferin to oxyluciferin, producing light. A CCD camera records it, and the signal is proportional to the amount of nucleotide incorporated.
- Apyrase degrades the unincorporated nucleotide and leftover ATP so the next nucleotide can be dispensed.
The peaks plotted against the dispensation order form a pyrogram, which represents the sequence. The table summarises each component as described in the Aspergillus resistance-assay review (J Fungi 2020).
| Component | Type | Role in the reaction |
|---|---|---|
| DNA polymerase | Enzyme | Adds the dispensed nucleotide to the primer and releases PPi |
| ATP sulfurylase | Enzyme | Converts PPi to ATP |
| Luciferase | Enzyme | Uses ATP to produce light from luciferin |
| Apyrase | Enzyme | Degrades excess nucleotide between dispensations |
| APS | Substrate | Used by sulfurylase with PPi to make ATP |
| Luciferin | Substrate | Converted by luciferase to generate light |
Worked example: reading methylation as a C/T ratio
Methylation analysis shows why pyrosequencing is called quantitative. Bisulfite treatment converts unmethylated cytosine to uracil, while 5-methylcytosine is protected. After PCR, uracil is read as thymine, so every CpG becomes an artificial C/T polymorphism. The pyrosequencer then compares the light from the C and T incorporations at that position. The relative height of the two peaks gives the percentage of methylation at that CpG. The 2015 chapter lists the workflow as: bisulfite-convert the genomic DNA, PCR the region of interest with one biotinylated primer, separate the strands to obtain single-stranded template, anneal the sequencing primer, then run the nucleotide dispensation. It reports that results can be obtained within a single day when starting from PCR.
Where it is used
- DNA methylation: local and global methylation measured in real time, as an alternative to HPLC, methylation-specific PCR, bisulfite sequencing and NGS.
- SNP and mutation detection: the J Fungi review applies it to azole-resistance mutations in Aspergillus fumigatus, where it can detect rare and mixed sequences.
- Forensics: the 2023 review names species identification, body fluid identification and smoking status among forensic applications.
Pyrosequencing vs Sanger vs NGS: fit for purpose
| Question | Better fit, per the cited sources |
|---|---|
| Quantify a known SNP or CpG methylation level in a short amplicon | Pyrosequencing (designed to quantify SNPs; methylation measured as percent at each CpG) |
| Detect rare or mixed mutations, with fast turnaround and lower cost | Pyrosequencing, which the J Fungi authors say is favoured over Sanger on sensitivity, limit of detection, rare and mixed mutations, turnaround time and cost |
| Sequence an entire target gene | Sanger or NGS; the same review notes the assay it describes reads up to 150 bp per reaction |
The 150 bp figure belongs to that specific assay, not to every pyrosequencing instrument.
Where it matters for lab planning
For a lab deciding whether to run an assay in-house, the sources imply a short checklist of inputs: a bisulfite conversion kit (for methylation work), PCR primers with one biotinylated primer for strand separation, a sequencing primer, and the enzyme and substrate reagents listed above. Amplicons from endpoint PCR need not be purified before the reaction, only checked for successful amplification by agarose gel electrophoresis, according to the J Fungi review. For primer design see the OligoAnalyzer guide; for sequence-analysis tooling see Geneious Prime; for the array-based cousin of genome-wide assays see ChIP-chip; and for published protocol formats see Bio-protocol. The epigenetic background is in genetics vs epigenetics.
Common misunderstandings
- “It sequences whole genomes.” It reads short amplicons; long or whole-gene sequencing is done with Sanger or NGS.
- “Bisulfite pyrosequencing shows all cytosine modifications.” Bisulfite conversion cannot discriminate 5-methylcytosine from 5-hydroxymethylcytosine, a stated pitfall of the method.
FAQ
What is pyrosequencing used for?
Quantifying SNPs and CpG methylation, detecting rare or mixed mutations in a short amplicon, and some forensic identification tasks.
Is pyrosequencing the same as Sanger sequencing?
No. Pyrosequencing detects light released as nucleotides are incorporated in a known dispensation order; the J Fungi review compares it with Sanger on sensitivity, limit of detection, turnaround and cost.
How long are pyrosequencing reads?
The assay in the cited J Fungi review generates reads of up to 150 bp per reaction; read length depends on the assay and system.
Does it need bisulfite treatment?
Bisulfite conversion is the step used for methylation analysis, where it creates the C/T difference that is measured. It is not part of the light-detection chemistry itself.
Sources
- Analysis of DNA Methylation by Pyrosequencing. Methods Mol Biol 2015. PMC4772880 (read 2026-11-07).
- Detecting Azole-Antifungal Resistance in Aspergillus fumigatus by Pyrosequencing. J Fungi 2020. PMC7151159 (read 2026-11-08).
- Pyrosequencing: Current forensic methodology and future applications, a review. Electrophoresis 2023. PubMed 36168852 (abstract read 2026-11-07).
