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Individual age estimation can be applied to criminal, legal, and anthropological investigations. DNA methylation has been established as the biomarker of choice for age prediction, since it was observed that specific CpG positions in the genome show systematic changes during an individual’s lifetime, with progressive increases or decreases in methylation levels. Subsequently, several forensic age prediction models have been reported, providing average age prediction error ranges of ±3–4 years, using a broad spectrum of technologies and underlying statistical analyses. DNA methylation assessment is not categorical but quantitative. Therefore, the detection platform used plays a pivotal role, since quantitative and semi-quantitative technologies could potentially result in differences in detected DNA methylation levels. In the present study, we analyzed as a shared sample pool, 84 blood-based DNA controls ranging from 18 to 99 years old using four different technologies: EpiTYPER ® , pyrosequencing, MiSeq, and SNaPshot TM . The DNA methylation levels detected for CpG sites from ELOVL2 , FHL2 , and MIR29B2 with each system were compared. A restricted three CpG-site age prediction model was rebuilt for each system, as well as for a combination of technologies, based on previous training datasets, and age predictions were calculated accordingly for all the samples detected with the previous technologies. While the DNA methylation patterns and subsequent age predictions from EpiTYPER ® , pyrosequencing, and MiSeq systems are largely comparable for the CpG sites studied, SNaPshot TM gives bigger differences reflected in higher predictive errors. However, these differences can be reduced by applying a z-score data transformation.

We detail the development of the ancestry informative single nucleotide polymorphisms (SNPs) panel forming part of the VISAGE Basic Tool (BT), which combines 41 appearance predictive SNPs and 112 ancestry predictive SNPs (three SNPs shared between sets) in one massively parallel sequencing (MPS) multiplex, whereas blood-based age analysis using methylation markers is run in a parallel MPS analysis pipeline. The selection of SNPs for the BT ancestry panel focused on established forensic markers that already have a proven track record of good sequencing performance in MPS, and the overall SNP multiplex scale closely matched that of existing forensic MPS assays. SNPs were chosen to differentiate individuals from the five main continental population groups of Africa, Europe, East Asia, America, and Oceania, extended to include differentiation of individuals from South Asia. From analysis of 1000 Genomes and HGDP-CEPH samples from these six population groups, the BT ancestry panel was shown to have no classification error using the Bayes likelihood calculators of the Snipper online analysis portal. The differentiation power of the component ancestry SNPs of BT was balanced as far as possible to avoid bias in the estimation of co-ancestry proportions in individuals with admixed backgrounds. The balancing process led to very similar cumulative population-specific divergence values for Africa, Europe, America, and Oceania, with East Asia being slightly below average, and South Asia an outlier from the other groups. Comparisons were made of the African, European, and Native American estimated co-ancestry proportions in the six admixed 1000 Genomes populations, using the BT ancestry panel SNPs and 572,000 Affymetrix Human Origins array SNPs. Very similar co-ancestry proportions were observed down to a minimum value of 10%, below which, low-level co-ancestry was not always reliably detected by BT SNPs. The Snipper analysis portal provides a comprehensive population dataset for the BT ancestry panel SNPs, comprising a 520-sample standardised reference dataset; 3445 additional samples from 1000 Genomes, HGDP-CEPH, Simons Foundation and Estonian Biocentre genome diversity projects; and 167 samples of six populations from in-house genotyping of individuals from Middle East, North and East African regions complementing those of the sampling regimes of the other diversity projects.

The development of microhaplotype (MH) panels for massively parallel sequencing (MPS) platforms is gaining increasing relevance for forensic analysis. Here, we expand the applicability of a 102 autosomal and 11 X-chromosome panel of MHs, previously validated with both MiSeq and Ion S5 MPS platforms and designed for identification purposes. We have broadened reference population data for identification purposes, including data from 240 HGDP-CEPH individuals of native populations from North Africa, the Middle East, Oceania and America. Using the enhanced population data, the panel was evaluated as a marker set for bio-geographical ancestry (BGA) inference, providing a clear differentiation of the five main continental groups of Africa, Europe, East Asia, Native America, and Oceania. An informative degree of differentiation was also achieved for the population variation encompassing North Africa, Middle East, Europe, South Asia, and East Asia. In addition, we explored the potential for individual BGA inference from simple mixed DNA, by simulation of mixed profiles followed by deconvolution of mixture components.

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