Independent Clinical Epigenetics Laboratory, Pomeranian Medical University in Szczecin, Unii Lubelskiej 1, 71-252 Szczecin, Poland.
Department of Biomedicine, Aarhus University, Høegh-Guldbergs Gade 10 Build. 1116, 8000 Aarhus C, Denmark.
Genes (Basel). 2022 Aug 9;13(8):1418. doi: 10.3390/genes13081418.
Pyrosequencing is one of the technologies widely used for quantitative methylation assessment. The protocol of pyrosequencing experiment consists of PCR amplification of a locus of interest and subsequent sequencing via synthesis of the amplified PCR product. As the PCR in this protocol utilizes one primer set for the amplification of a template originating from both methylated and non-methylated versions of the analysed locus, the unequal amplification of one of the templates may affect the methylation level assessment by pyrosequencing. We have investigated whether the unequal amplification of one of the templates challenges the quantitative properties of the pyrosequencing technology. Our results show that the sensitivity and dynamic range of pyrosequencing can be significantly affected by unequal amplification of the methylated and non-methylated version of the locus of interest in an assay specific manner. Thus, the assessment of the effect of unequal template amplification on the performances of the specific pyrosequencing assay is necessary before using the assay either in research or especially in diagnostic settings.
焦磷酸测序是一种广泛用于定量甲基化评估的技术。焦磷酸测序实验的方案包括感兴趣的基因座的 PCR 扩增，以及通过扩增的 PCR 产物的合成进行后续测序。由于该方案中的 PCR 利用一组引物来扩增来自分析基因座的甲基化和非甲基化版本的模板，因此模板之一的不等扩增可能会影响焦磷酸测序的甲基化水平评估。我们研究了模板之一的不等扩增是否会挑战焦磷酸测序技术的定量特性。我们的结果表明，焦磷酸测序的灵敏度和动态范围可能会受到在特定测定中对感兴趣的基因座的甲基化和非甲基化版本的模板的不等扩增的显著影响。因此，在研究或特别是在诊断环境中使用该测定法之前，有必要评估不等模板扩增对特定焦磷酸测序测定法性能的影响。
