Institute of Legal Medicine, Faculty of Medicine and University Hospital Cologne, University of Cologne, Melatengürtel 60-62, D-50823, Cologne, Germany.
Forensic Sci Int Genet. 2020 Jan;44:102189. doi: 10.1016/j.fsigen.2019.102189. Epub 2019 Oct 14.
DNA methylation-based age estimation is a promising new tool for forensic molecular biology. There is growing understanding of the best predictive CpG loci and their performance in various sample types. Since forensic samples usually provide only small amounts of DNA, the sensitivity of the method is crucial. Pyrosequencing is one of the most sensitive methods but only capable to analyze different target regions separately. Thus, multiple input DNA samples are required for investigations of different target regions, which is required for all current age estimation models. To overcome this limitation, we developed a novel multiplex strategy for Pyrosequencing, which allows the investigation of different target regions from a single small amount of input DNA. A pre-amplification step was introduced to increase the amount of target-specific template for the subsequent sequencing PCR step. We tested this multiplex strategy for eight target regions including 15 age CpGs associated with the genes of ELOVL2, FHL2, CCDC102B, C1orf132, KLF14, EDARADD, PDE4C and SST. Except for FHL2, all target regions were successfully sequenced with the multiplex strategy and the precision in terms of reproducibility of the measurements was equal to the singleplex strategy. The measured methylation values at the age CpGs displayed borderline significant differences between both analytical strategies for six out of 14 CpG sites whereas both strategies delivered equal methylation values for the remaining eight age CpGs. In total, our results indicate that the multiplex strategy can act as a promising alternative for age estimation studies in cases when only limited amounts of DNA samples are available.
基于 DNA 甲基化的年龄估算是法医学中一种很有前途的新工具。人们越来越了解最佳预测性 CpG 位点及其在各种样本类型中的表现。由于法医样本通常只提供少量的 DNA，因此该方法的灵敏度至关重要。焦磷酸测序是最敏感的方法之一，但只能分别分析不同的目标区域。因此，对于不同的目标区域的研究需要多个输入 DNA 样本，这是所有当前年龄估计模型都需要的。为了克服这一限制，我们开发了一种新的焦磷酸测序多重策略，该策略允许从单个少量输入 DNA 中研究不同的目标区域。引入了预扩增步骤，以增加后续测序 PCR 步骤中靶特异性模板的数量。我们测试了这种多重策略用于八个目标区域，包括与 ELOVL2、FHL2、CCDC102B、C1orf132、KLF14、EDARADD、PDE4C 和 SST 基因相关的 15 个年龄 CpG。除了 FHL2 之外，所有目标区域都可以通过多重策略成功测序，并且在测量的重现性方面的精度与单重策略相同。在 14 个 CpG 位点中，有 6 个位点的年龄 CpG 测量的甲基化值在两种分析策略之间存在边界显著差异，而对于其余 8 个年龄 CpG 位点，两种策略都提供了相同的甲基化值。总的来说，我们的结果表明，在只有有限数量的 DNA 样本可用的情况下，多重策略可以作为年龄估计研究的一种很有前途的替代方法。
