CSYseq: The first Y-chromosome sequencing tool typing a large number of Y-SNPs and Y-STRs to unravel worldwide human population genetics.

Male-specific Y-chromosome (chrY) polymorphisms are interesting components of the DNA for population genetics. While single nucleotide polymorphisms (Y-SNPs) indicate distant evolutionary ancestry, short tandem repeats (Y-STRs) are able to identify close familial kinships. Detailed chrY analysis provides thus both biogeographical background information as paternal lineage identification. The rapid advancement of high-throughput massive parallel sequencing (MPS) technology in the past decade has revolutionized genetic research. Using MPS, single-base information of both Y-SNPs as Y-STRs can be analyzed in a single assay typing multiple samples at once. In this study, we present the first extensive chrY-specific targeted resequencing panel, the 'CSYseq', which simultaneously identifies slow mutating Y-SNPs as evolution markers and rapid mutating Y-STRs as patrilineage markers. The panel was validated by paired-end sequencing of 130 males, distributed over 65 deep-rooted pedigrees covering 1,279 generations. The CSYseq successfully targets 15,611 Y-SNPs including 9,014 phylogenetic informative Y-SNPs to identify 1,443 human evolutionary Y-subhaplogroup lineages worldwide. In addition, the CSYseq properly targets 202 Y-STRs, including 81 slow, 68 moderate, 27 fast and 26 rapid mutating Y-STRs to individualize close paternal relatives. The targeted chrY markers cover a high average number of reads (Y-SNP = 717, Y-STR = 150), easy interpretation, powerful discrimination capacity and chrY specificity. The CSYseq is interesting for research on different time scales: to identify evolutionary ancestry, to find distant family and to discriminate closely related males. Therefore, this panel serves as a unique tool valuable for a wide range of genetic-genealogical applications in interdisciplinary research within evolutionary, population, molecular, medical and forensic genetics.

At the Interface of Life and Death: Post-mortem and Other Applications of Vaginal, Skin, and Salivary Microbiome Analysis in Forensics.

Microbial forensics represents a promising tool to strengthen traditional forensic investigative methods and fill related knowledge gaps. Large-scale microbiome studies indicate that microbial fingerprinting can assist forensics in areas such as trace evidence, source tracking, geolocation, and circumstances of death. Nevertheless, the majority of forensic microbiome studies focus on soil and internal organ samples, whereas the microbiome of skin, mouth, and especially vaginal samples that are routinely collected in sexual assault and femicide cases remain underexplored. This review discusses the current and emerging insights into vaginal, skin, and salivary microbiome-modulating factors during life (e.g., lifestyle and health status) and after death (e.g., environmental influences and post-mortem interval) based on next-generation sequencing. We specifically highlight the key aspects of female reproductive tract, skin, and mouth microbiome samples relevant in forensics. To fill the current knowledge gaps, future research should focus on the degree to which the post-mortem succession rate and profiles of vaginal, skin, and saliva microbiota are sensitive to abiotic and biotic factors, presence or absence of oxygen and other gases, and the nutrient richness of the environment. Application of this microbiome-related knowledge could provide valuable complementary data to strengthen forensic cases, for example, to shed light on the circumstances surrounding death with (post-mortem) microbial fingerprinting. Overall, this review synthesizes the present knowledge and aims to provide a framework to adequately comprehend the hurdles and potential application of vaginal, skin, and salivary post-mortem microbiomes in forensic investigations.

Cell survival and DNA damage repair are promoted in the human blood thanatotranscriptome shortly after death.

RNA analysis of post-mortem tissues, or thanatotranscriptomics, has become a topic of interest in forensic science due to the essential information it can provide in forensic investigations. Several studies have previously investigated the effect of death on gene transcription, but it has never been conducted with samples of the same individual. For the first time, a longitudinal mRNA expression analysis study was performed with post-mortem human blood samples from individuals with a known time of death. The results reveal that, after death, two clearly differentiated groups of up- and down-regulated genes can be detected. Pathway analysis suggests active processes that promote cell survival and DNA damage repair, rather than passive degradation, are the source of early post-mortem changes of gene expression in blood. In addition, a generalized linear model with an elastic net restriction predicted post-mortem interval with a root mean square error of 4.75 h. In conclusion, we demonstrate that post-mortem gene expression data can be used as biomarkers to estimate the post-mortem interval though further validation using independent sample sets is required before use in forensic casework.

YMrCA: Improving Y-chromosomal ancestor time estimation for DNA kinship research.

The Y-chromosome is a valuable kinship indicator in family history and forensic research. To reconstruct genealogies, the time to the most recent common ancestor (tMRCA) between paternal relatives can be estimated through Y-STR analysis. Existing models are the stepwise mutation model (SMM, only one-step Y-STR changes) and the infinite allele model (IAM, new allele per Y-STR change). In this study, these mutation models and all existing tMRCA calculators were validated through a genetic-genealogy database containing 1,120 biologically related genealogical pairs confirmed by 46 Y-STRs with known tMRCA (18,109 generations). Consistent under- and overestimation and broad confidence intervals were observed, leading to dubious tMRCA estimates. This is because they do not include individual mutation rates or multi-step changes and ignore hidden multiple, back, or parallel modifications. To improve tMRCA estimation, we developed a user-friendly calculator, the "YMrCA", including all previously mentioned mutation characteristics. After extensive validation, we observed that the YMrCA calculator demonstrated a promising performance. The YMrCA yields a significantly higher tMRCA success rate (96%; +20%) and a lower tMRCA error (7; -3) compared to the mutation models and all online tMRCA calculators. Therefore, YMrCA offers the next step towards more objective tMRCA estimation for DNA kinship research.

Ysurnames? The patrilineal Y-chromosome and surname correlation for DNA kinship research.

The Y-chromosome is a widely studied and useful small part of the genome providing different applications for interdisciplinary research. In many (Western) societies, the Y-chromosome and surnames are paternally co-inherited, suggesting a corresponding Y-haplotype for every namesake. While it has already been observed that this correlation may be disrupted by a false-paternity event, adoption, anonymous sperm donor or the co-founding of surnames, extensive information on the strength of the surname match frequency (SMF) with the Y-chromosome remains rather unknown. For the first time in Belgium and the Netherlands, we were able to study this correlation using 2,401 males genotyped for 46 Y-STRs and 183 Y-SNPs. The SMF was observed to be dependent on the number of Y-STRs analyzed, their mutation rates and the number of Y-STR differences allowed for a kinship. For a perfect match, the Yfiler® Plus and our in-house YForGen kit gave a similar high SMF of 98%, but for non-perfect matches, the latter could overall be identified as the best kit. The SMF generally increased due to less mismatches when encountering [1] deep Y-subhaplogroups, [2] less frequently occurring surnames, and [3] small geographical distances between relatives. This novel information enabled the design of a surname prediction model based on genetic and geographical distances of a kinship. The prediction model has an area under the curve (AUC) of 0.9 and is therefore useable for DNA kinship priority listing in estimation applications like forensic familial searching.

The Dutch Y-chromosomal landscape.

Previous studies indicated existing, albeit limited, genetic-geographic population substructure in the Dutch population based on genome-wide data and a lack of this for mitochondrial SNP based data. Despite the aforementioned studies, Y-chromosomal SNP data from the Netherlands remain scarce and do not cover the territory of the Netherlands well enough to allow a reliable investigation of genetic-geographic population substructure. Here we provide the first substantial dataset of detailed spatial Y-chromosomal haplogroup information in 2085 males collected across the Netherlands and supplemented with previously published data from northern Belgium. We found Y-chromosomal evidence for genetic-geographic population substructure, and several Y-haplogroups demonstrating significant clinal frequency distributions in different directions. By means of prediction surface maps we could visualize (complex) distribution patterns of individual Y-haplogroups in detail. These results highlight the value of a micro-geographic approach and are of great use for forensic and epidemiological investigations and our understanding of the Dutch population history. Moreover, the previously noted absence of genetic-geographic population substructure in the Netherlands based on mitochondrial DNA in contrast to our Y-chromosome results, hints at different population histories for women and men in the Netherlands.

Correction to: The Dutch Y-chromosomal landscape.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A Historical-Genetic Reconstruction of Human Extra-Pair Paternity.

Paternity testing using genetic markers has shown that extra-pair paternity (EPP) is common in many pair-bonded species [1, 2]. Evolutionary theory and empirical data show that extra-pair copulations can increase the fitness of males as well as females [3, 4]. This can carry a significant fitness cost for the social father, who then invests in rearing offspring that biologically are not his own [5]. In human populations, the incidence and correlates of extra-pair paternity remain highly contentious [2, 6, 7]. Here, we use a population-level genetic genealogy approach [6, 8] to reconstruct spatiotemporal patterns in human EPP rates. Using patrilineal genealogies from the Low Countries spanning a period of over 500 years and Y chromosome genotyping of living descendants, our analysis reveals that historical EPP rates, while low overall, were strongly impacted by socioeconomic and demographic factors. Specifically, we observe that estimated EPP rates among married couples varied by more than an order of magnitude, from 0.4% to 5.9%, and peaked among families with a low socioeconomic background living in densely populated cities of the late 19th century. Our results support theoretical predictions that social context can strongly affect the outcomes of sexual conflict in human populations by modulating the incentives and opportunities for engaging in extra-pair relationships [9-11]. These findings show how contemporary genetic data combined with in-depth genealogies open up a new window on the sexual behavior of our ancestors.

An interdisciplinary study around the reliquary of the late cardinal Jacques de Vitry.

The reliquary of Jacques de Vitry, a prominent clergyman and theologian in the early 13th century, has experienced several transfers over the last centuries, which seriously question the attribution of the remains to the late Cardinal. Uncertainty about the year of his birth poses an additional question regarding his age at death in 1240. The reliquary, located in the Saint Marie d'Oigines church, Belgium, was reopened in 2015 for an interdisciplinary study around his relics as well as the Treasure of Oignies, a remarkable cultural heritage notably built from Jacques de Vitry's donation. Anthropological, isotopic and genetic analyses were performed independently on the remains found in the reliquary. Results of the analyses provided evidence that the likelihood that these remains are those of Jacques de Vitry is very high: the remains belong to the same human male individual and the historical tradition about his age is confirmed. In addition, a separate relic (left tibia) was analysed and found to match with the remains of the reliquary (right tibia). The unique Jacques de Vitry's mitre, made of parchment, was sampled non-destructively and the extracted parchment collagen was analysed by a proteomic method in order to determine the animal species. The results showed that, surprisingly, not all parts of the mitre were made from the same species. All together, these findings are expected to fertilize knowledge carried by historical tradition around the relics of Jacques de Vitry and his related cultural heritage.

A game of hide and seq: Identification of parallel Y-STR evolution in deep-rooting pedigrees.

Short tandem repeats on the Y-chromosome (Y-STRs) are common DNA polymorphisms useful for genetic genealogy, population and evolutionary genetics, human genetics, pathology and forensic sciences. It is important to identify all Y-STR variants and to have knowledge of Y-STR mutation rates in order to correctly estimate the time to the most recent common ancestor (tMRCA) between paternally related individuals. When capillary electrophoresis (CE) is performed to analyze genealogical pairs, Y-STR sequence variations remain hidden when the number of repeats is identical. These hidden variations could be due to parallel Y-STR changes or modifications (PM) that occur independently in different lineages leading to alleles with identical number of repeats. In this study, we detect for the first time twelve PM by analyzing 133 males (960 meiosis) in extended deep-rooting family pedigrees on 42 Y-STRs. These PM were observed in nine Y-STR loci with mutation rates of at least 5.94 × 10-3 per generation. Sequencing analysis made it possible to distinguish insertions/deletions in different repeat regions revealing the presence of two unique changes in three PM on rapidly mutating and complex Y-STRs DYS724-ab and DYS518. Sequencing unraveled more information concerning the identity of alleles, and increased allelic discrimination possibilities which is of great importance in population genetics and forensic analysis. Limiting the analysis to CE could lead to wrong ancestral allele assumptions, to false negative interpretations and to tMRCA underestimations. These observations highlight the importance and added value of sequencing analysis and suggest a shift in genotyping methods from CE to next generation sequencing.

Evaluation of three statistical prediction models for forensic age prediction based on DNA methylation.

DNA methylation is a promising biomarker for forensic age prediction. A challenge that has emerged in recent studies is the fact that prediction errors become larger with increasing age due to interindividual differences in epigenetic ageing rates. This phenomenon of non-constant variance or heteroscedasticity violates an assumption of the often used method of ordinary least squares (OLS) regression. The aim of this study was to evaluate alternative statistical methods that do take heteroscedasticity into account in order to provide more accurate, age-dependent prediction intervals. A weighted least squares (WLS) regression is proposed as well as a quantile regression model. Their performances were compared against an OLS regression model based on the same dataset. Both models provided age-dependent prediction intervals which account for the increasing variance with age, but WLS regression performed better in terms of success rate in the current dataset. However, quantile regression might be a preferred method when dealing with a variance that is not only non-constant, but also not normally distributed. Ultimately the choice of which model to use should depend on the observed characteristics of the data.

Determining Y-STR mutation rates in deep-routing genealogies: Identification of haplogroup differences.

Knowledge of Y-chromosomal short tandem repeat (Y-STR) mutation rates is essential to determine the most recent common ancestor (MRCA) in familial searching or genealogy research. Up to now, locus-specific mutation rates have been extensively examined especially for commercially available forensic Y-STRs, while haplogroup specific mutation rates have not yet been investigated in detail. Through 450 patrilineally related namesakes distributed over 212 deep-rooting genealogies, the individual mutation rates of 42 Y-STR loci were determined, including 27 forensic Y-STR loci from the Yfiler® Plus kit and 15 additional Y-STR loci (DYS388, DYS426, DYS442, DYS447, DYS454, DYS455, DYS459a/b, DYS549, DYS607, DYS643, DYS724a/b and YCAIIa/b). At least 726 mutations were observed over 148,596 meiosis and individual Y-STR mutation rates varied from 2.83 × 10-4 to 1.86 × 10-2. The mutation rate was significantly correlated with the average allele size, the complexity of the repeat motif sequence and the age of the father. Significant differences in average Y-STR mutations rates were observed when haplogroup 'I & J' (4.03 × 10-3 mutations/generation) was compared to 'R1b' (5.35 × 10-3 mutations/generation) and to the overall mutation rate (5.03 × 10-3 mutations/generation). A difference in allele size distribution was identified as the only cause for these haplogroup specific mutation rates. The haplogroup specific mutation rates were also present within the commercially available Y-STR kits (Yfiler®, PowerPlex® Y23 System and Yfiler® Plus). This observation has consequences for applications where an average Y-STR mutation rate is used, e.g. tMRCA estimations in familial searching and genealogy research.

The black legend on the Spanish presence in the low countries: Verifying shared beliefs on genetic ancestry.

OBJECTIVES: War atrocities committed by the Spanish army in the Low Countries during the 16th century are so ingrained in the collective memory of Belgian and Dutch societies that they generally assume a signature of this history to be present in their genetic ancestry. Historians claim this assumption is a consequence of the so-called "Black Legend" and negative propaganda portraying and remembering Spanish soldiers as extreme sexual aggressors. The impact of the presence of Spaniards during the Dutch Revolt on the genetic variation in the Low Countries has been verified in this study. MATERIALS AND METHODS: A recent population genetic analysis of Iberian-associated Y-chromosomal variation among Europe is enlarged with representative samples of Dutch (N = 250) and Flemish (N = 1,087) males. Frequencies of these variants are also compared between donors whose oldest reported paternal ancestors lived in-nowadays Flemish-cities affected by so-called Spanish Furies (N = 116) versus other patrilineages in current Flemish territory (N = 971). RESULTS: The frequencies of Y-chromosomal markers Z195 and SRY2627 decline steeply going north from Spain and the data for the Flemish and Dutch populations fits within this pattern. No trend of higher frequencies of these variants has been found within the well-ascertained samples associated with Spanish Fury cities. DISCUSSION: Although sexual aggression did occur in the 16th century, these activities did not leave a traceable "Spanish" genetic signature in the autochthonous genome of the Low Countries. Our results support the view that the 'Black Legend' and historical propaganda on sexual aggression have nurtured today's incorrect assumptions regarding genetic ancestry.

Defining Y-SNP variation among the Flemish population (Western Europe) by full genome sequencing.

Y-chromosomal single nucleotide polymorphisms (Y-SNPs) represent a powerful tool in forensic research and casework, especially for inferring paternal ancestry of unknown perpetrators and unidentified bodies. However, the wealth of recently discovered Y-SNPs, the 'jungle' of different evolutionary lineage trees and nomenclatures, and the lack of population-wide data of many phylogenetically mapped Y-SNPs, limits the use of Y-SNPs in routine forensic approaches. Recently, a concise reference phylogeny of the human Y chromosome, the 'Minimal Reference Y-tree', was introduced aiming to provide a stable phylogeny with optimal global discrimination capacity by including the most resolving Y-SNPs. Here, we obtained a representative sample of 270 whole-genome sequences (WGS) to grasp the Y-SNP variation within the autochthonous Flemish population (Belgium, Western Europe) according to this reference Y-tree. The high quality of the Y-SNP calling was guaranteed for the WGS sample as well as its representativeness for the Flemish population based on the comparison of the main haplogroup frequencies with those from earlier studies on Flanders and the Netherlands. The 270 Flemish Y chromosomes were assigned to 98 different sub-haplogroups of the Minimal Reference Y-tree, showing its high potential of discrimination and confirming the spectrum of evolutionary lineages within Western Europe in general and within Flanders in particular. The full database with all Y-SNP calls of the Flemish sample is public available for future updates including forensic and population genetic studies. New initiatives to categorise Y-SNP variation in other populations according to the reference phylogeny of the Y chromosome are highly encouraged for forensic applications. Recommendations to realise such future population sample sets are discussed based on this study.

Genetic-genealogy approach reveals low rate of extrapair paternity in historical Dutch populations.

OBJECTIVE: Evolutionary theory has shown that seeking out extrapair paternity (EPP) can be a viable reproductive strategy for both sexes in pair-bonded species, also in humans. As yet, estimates of the contemporary or historical EPP rate in human population are still rare. In the present study, we estimated the historical EPP rate in the Dutch population over the last 400 years and compared the rate with those obtained for other human populations to determine the evolutionary, cultural, and socio-demographic factors that influence human cuckoldry behavior. METHODS: We estimated the historical EPP rate for the Dutch population via the "genealogical pair method", in which the EPP rate is derived from Y-chromosome mismatches between pairs of individuals that, based on genealogical evidence, share a common paternal ancestor. RESULTS: Based on the analysis of 68 representative genealogical pairs, separated by a total of 1013 fertilization events, we estimated that the historical EPP rate for the Dutch population over the last 400 years was 0.96% per generation (95% confidence interval 0.46%-1.76%). CONCLUSION: The Dutch EPP rate fits perfectly within the range reported for other contemporary and historical populations in Western Europe and was highly congruent with that estimated for neighboring Flanders, despite the socio-economic and religious differences between both populations. The estimated low EPP rate challenges the "dual mating strategy hypothesis" that states that women could obtain fitness benefits by securing investment from one man while cuckolding him to obtain good genes from an affair partner.

Bight of Benin: a Maternal Perspective of Four Beninese Populations and their Genetic Implications on the American Populations of African Ancestry.

The understanding of the first movements of the ancestral populations within the African continent is still unclear, particularly in West Africa, due to several factors that have shaped the African genetic pool across time. To improve the genetic representativeness of the Beninese population and to better understand the patterns of human settlement inside West Africa and the dynamics of peopling of the Democratic Republic of Benin, we analyzed the maternal genetic variation of 193 Beninese individuals belonging to Bariba, Berba, Dendi, and Fon populations. Results support the oral traditions indicating that the western neighbouring populations have been the ancestors of the first Beninese populations, and the extant genetic structure of the Beninese populations is most likely the result of admixture between populations from neighbouring countries and native people. The present findings highlight how the Beninese populations contributed to the gene pool of the extant populations of some American populations of African ancestry. This strengthens the hypothesis that the Bight of Benin was not only an assembly point for the slave trade during the Trans-Atlantic Slave Trade but also an important slave trapping area.

Biohistorical materials and contemporary privacy concerns-the forensic case of King Albert I.

The rapid advancement of technology in genomic analysis increasingly allows researchers to study human biohistorical materials. Nevertheless, little attention has been paid to the privacy of the donor's living relatives and the negative impact they might experience from the (public) availability of genetic results, even in cases of scientific, forensic or historical relevance. This issue has become clear during a cold case investigation of a relic attributed to Belgian King and World War I-hero Albert I who died, according to the official version, in a solo climbing accident in 1934. Authentication of the relic with blood stains assigned to the King and collected on the place where his body was discovered is recognised as one of the final opportunities to test the plausibility of various conspiracy theories on the King's demise. While the historical value and current technological developments allow the genomic analysis of this relic, publication of genetic data would immediately lead to privacy concerns for living descendants and relatives of the King, including the Belgian and British royal families, even after more than 80 years. Therefore, the authentication study of the relic of King Albert I has been a difficult exercise towards balancing public research interests and privacy interests. The identification of the relic was realised by using a strict genetic genealogical approach including Y-chromosome and mitochondrial genome comparison with living relatives, thereby limiting the analysis to genomic regions relevant for identification. The genetic results combined with all available historical elements concerning the relic, provide strong evidence that King Albert I was indeed the donor of the blood stains, which is in line with the official climbing accident hypothesis and contradicts widespread 'mise-en-scène' scenarios. Since publication of the haploid data of the blood stains has the potential to violate the privacy of living relatives, we opted for external and independent reviewing of (the quality of) our data and statistical interpretation by external forensic experts in haploid markers to guarantee the objectivity and scientific accuracy of the identification data analysis as well as the privacy of living descendants and relatives. Although the cold case investigation provided relevant insights into the circumstances surrounding the death of King Albert I, it also revealed the insufficient ethical guidance for current genomic studies of biohistorical material.

Comparing maternal genetic variation across two millennia reveals the demographic history of an ancient human population in southwest Turkey.

More than two decades of archaeological research at the site of Sagalassos, in southwest Turkey, resulted in the study of the former urban settlement in all its features. Originally settled in late Classical/early Hellenistic times, possibly from the later fifth century BCE onwards, the city of Sagalassos and its surrounding territory saw empires come and go. The Plague of Justinian in the sixth century CE, which is considered to have caused the death of up to a third of the population in Anatolia, and an earthquake in the seventh century CE, which is attested to have devastated many monuments in the city, may have severely affected the contemporary Sagalassos community. Human occupation continued, however, and Byzantine Sagalassos was eventually abandoned around 1200 CE. In order to investigate whether these historical events resulted in demographic changes across time, we compared the mitochondrial DNA variation of two population samples from Sagalassos (Roman and Middle Byzantine) and a modern sample from the nearby town of Aglasun. Our analyses revealed no genetic discontinuity across two millennia in the region and Bayesian coalescence-based simulations indicated that a major population decline in the area coincided with the final abandonment of Sagalassos, rather than with the Plague of Justinian or the mentioned earthquake.

The Paternal Landscape along the Bight of Benin - Testing Regional Representativeness of West-African Population Samples Using Y-Chromosomal Markers.

Patterns of genetic variation in human populations across the African continent are still not well studied in comparison with Eurasia and America, despite the high genetic and cultural diversity among African populations. In population and forensic genetic studies a single sample is often used to represent a complete African region. In such a scenario, inappropriate sampling strategies and/or the use of local, isolated populations may bias interpretations and pose questions of representativeness at a macrogeographic-scale. The non-recombining region of the Y-chromosome (NRY) has great potential to reveal the regional representation of a sample due to its powerful phylogeographic information content. An area poorly characterized for Y-chromosomal data is the West-African region along the Bight of Benin, despite its important history in the trans-Atlantic slave trade and its large number of ethnic groups, languages and lifestyles. In this study, Y-chromosomal haplotypes from four Beninese populations were determined and a global meta-analysis with available Y-SNP and Y-STR data from populations along the Bight of Benin and surrounding areas was performed. A thorough methodology was developed allowing comparison of population samples using Y-chromosomal lineage data based on different Y-SNP panels and phylogenies. Geographic proximity turned out to be the best predictor of genetic affinity between populations along the Bight of Benin. Nevertheless, based on Y-chromosomal data from the literature two population samples differed strongly from others from the same or neighbouring areas and are not regionally representative within large-scale studies. Furthermore, the analysis of the HapMap sample YRI of a Yoruban population from South-western Nigeria based on Y-SNPs and Y-STR data showed for the first time its regional representativeness, a result which is important for standard population and forensic genetic applications using the YRI sample. Therefore, the uniquely and powerful geographical information carried by the Y-chromosome makes it an important locus to test the representativeness of a certain sample even in the genomic era, especially in poorly investigated areas like Africa.

Improved age determination of blood and teeth samples using a selected set of DNA methylation markers.

Age estimation from DNA methylation markers has seen an exponential growth of interest, not in the least from forensic scientists. The current published assays, however, can still be improved by lowering the number of markers in the assay and by providing more accurate models to predict chronological age. From the published literature we selected 4 age-associated genes (ASPA, PDE4C, ELOVL2, and EDARADD) and determined CpG methylation levels from 206 blood samples of both deceased and living individuals (age range: 0-91 years). This data was subsequently used to compare prediction accuracy with both linear and non-linear regression models. A quadratic regression model in which the methylation levels of ELOVL2 were squared showed the highest accuracy with a Mean Absolute Deviation (MAD) between chronological age and predicted age of 3.75 years and an adjusted R(2) of 0.95. No difference in accuracy was observed for samples obtained either from living and deceased individuals or between the 2 genders. In addition, 29 teeth from different individuals (age range: 19-70 years) were analyzed using the same set of markers resulting in a MAD of 4.86 years and an adjusted R(2) of 0.74. Cross validation of the results obtained from blood samples demonstrated the robustness and reproducibility of the assay. In conclusion, the set of 4 CpG DNA methylation markers is capable of producing highly accurate age predictions for blood samples from deceased and living individuals.