Novel multiplex strategy for DNA methylation-based age prediction from small amounts of DNA via Pyrosequencing

[Display omitted] •Multiplex amplification strategy was designed for combined quantitative methylation analysis of several CpG regions via Pyrosequencing.•Targets (including 15 age CpG sites): ELOVL2, FHL2, CCDC102B, C1orf132, KLF14, EDARADD, PDE4C and SST.•Both strategies performed equally precise for all investigated age CpGs.•No measurement differences were observed for methylation values from eight out of fourteen age CpGs.•Multiplex strategy measurements for six out of fourteen age CpGs require adjustments before applying to age estimation models. 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.