Quantifying sequence variations with pyrosequencing

The PyroMark platforms are used for rapid and accurate quantification of genetic and epigenetic DNA modifications using pyrosequencing assays. They can be used for advanced methylation, mutation and SNP quantification but also for verification and validation of samples from NGS and array experiments. The PyroMark platform consists of PyroMark Q24, PyroMark Q24 Advanced, PyroMark Q48 Autoprep, and PyroMark Q96 ID. PyroMark Q24 can analyze up to 24 samples in 15 minutes, can read up to 80 bp and performs mutation analysis and resistance typing. PyroMark Q24 Advanced shares those features but can read up to 140 bp and perform more complex mutation analyses, as well as epigenetic (CpG and CpN) analyses and microbial typing. PyroMark Q48 Autoprep with automated sample preparation is used for similar applications and can read equally long sequences, but can analyze up to 48 samples at a time. Finally, PyroMark Q96 ID has the capacity of analyzing up to 96 samples in each run.

From pyrosequencing to analysis reports

The pyrosequencing reaction relies on the release of light signals, where the amount of light is directly proportional to the number of incorporated nucleotides. The four nucleotides A, C, G and T are dispensed in a predefined order and as the DNA is elongated, pyrophosphate is released and converted to ATP, which is used by luciferase to generate light. The result of each dispensation of nucleotides is shown in the pyrogram, so if for example 3 nucleotides are incorporated (e.g. AAA), three ATPs are generated giving rise to triple peak in the pyrogram. If no nucleotides are incorporated, there is no light signal and thus no peak. The PyroMark software can be set up in different analysis modes in order to target analysis of for example SNPs, CpGs or perform base-calling of unknown sequences. When the analysis has been finalized, the software will generate comprehensive reports displaying the results as pyrograms as well as presenting statistics such as methylation percentage, allele frequencies and other analysis results.