Collaborative exercise: analysis of age estimation using a QIAGEN protocol and the PyroMark Q48 platform.

Human age estimation from trace samples may give important leads early in a police investigation by contributing to the description of the perpetrator. Several molecular biomarkers are available for the estimation of chronological age, and currently, DNA methylation patterns are the most promising. In this study, a QIAGEN age protocol for age estimation was tested by five forensic genetic laboratories. The assay comprised bisulfite treatment of the extracted DNA, amplification of five CpG loci (in the genes of ELOVL2, C1orf132, TRIM59, KLF14, and FHL2), and sequencing of the amplicons using the PyroMark Q48 platform. Blood samples from 49 individuals with ages ranging from 18 to 64 years as well as negative and methylation controls were analyzed. An existing age estimation model was applied to display a mean absolute deviation of 3.62 years within the reference data set. Key points: Age determination as an intelligence tool during investigations can be a powerful tool in forensic genetics.In this study, five laboratories ran 49 samples and obtained a mean absolute deviation of 3.62 years.Five markers were analyzed on a PyroMark Q48 platform.

Cleaning protocols in forensic genetic laboratories.

It is pivotal to avoid cross-sample contamination in forensic genetic laboratories and optimal cleaning protocols for the removal of DNA are essential. A survey was performed, and ten forensic genetic laboratories shared their cleaning protocols in pre-PCR and post-PCR laboratories. The cleaning frequencies on different surface areas were somewhat similar, whereas none of the laboratories used the same cleaning reagents. Therefore, the efficiencies of the cleaning protocol utilised were tested and compared. The results showed that freshly made household bleach and Virkon® removed all amplifiable DNA from the surfaces, whereas DNA AWAY™ and the disinfection reagents ethanol, isopropanol, and ChemGene HLD4L did not.

Performance of a 74-Microhaplotype Assay in Kinship Analyses.

Microhaplotypes (MHs) consisting of multiple SNPs and indels on short stretches of DNA are new and interesting loci for forensic genetic investigations. In this study, we analysed 74 previously defined MHs in two of the populations that our laboratory provides with forensic genetic services, Danes and Greenlanders. In addition to the 229 SNPs that originally made up the 74 MHs, 66 SNPs and 3 indels were identified in the two populations, and 45 of these variants were included in new definitions of the MHs, whereas 24 SNPs were considered rare and of little value for case work. The average effective number of alleles (Ae) was 3.2, 3.0, and 2.6 in Danes, West Greenlanders, and East Greenlanders, respectively. High levels of linkage disequilibrium were observed in East Greenlanders, which reflects the characteristics of this population that has a small size, and signs of admixture and substructure. Pairwise kinship simulations of full siblings, half-siblings, first cousins, and unrelated individuals were performed using allele frequencies from MHs, STRs and SNPs from Danish and Greenlandic populations. The MH panel outperformed the currently used STR and SNP marker sets and was able to differentiate siblings from unrelated individuals with a 0% false positive rate and a 1.1% false negative rate using an LR threshold of 10,000 in the Danish population. However, the panel was not able to differentiate half-siblings or first cousins from unrelated individuals. The results generated in this study will be used to implement MHs as investigative markers for relationship testing in our laboratory.

Longitudinal changes and variation in human DNA methylation analysed with the Illumina MethylationEPIC BeadChip assay and their implications on forensic age prediction.

DNA methylation, a pivotal epigenetic modification, plays a crucial role in regulating gene expression and is known to undergo dynamic changes with age. The present study investigated epigenome-wide methylation profiles in 64 individuals over two time points, 15 years apart, using the Illumina EPIC850k arrays. A mixed-effects model identified 2821 age-associated differentially methylated CpG positions (aDMPs) with a median rate of change of 0.18% per year, consistent with a 10-15% change during a human lifespan. Significant variation in the baseline DNA methylation levels between individuals of similar ages as well as inconsistent direction of change with time across individuals were observed for all the aDMPs. Twenty-three of the 2821 aDMPs were previously incorporated into forensic age prediction models. These markers displayed larger changes in DNA methylation with age compared to all the aDMPs and less variation among individuals. Nevertheless, the forensic aDMPs also showed inter-individual variations in the direction of DNA methylation changes. Only cg16867657 in ELOVL2 exhibited a uniform direction of the age-related change among the investigated individuals, which supports the current knowledge that CpG sites in ELOVL2 are the best markers for age prediction.

Development of an Okinawa panel for biogeographic inference of Okinawans.

BACKGROUND: The Precision ID Ancestry Panel with 165 SNP markers was unable to differentiate between mainland Japanese and Okinawa Japanese or to distinguish either of them from other East Asian populations. AIM: An Okinawa panel was developed with the aim of further separating Okinawa Japanese individuals from mainland Japanese and other Asian groups. Seventy-five SNPs were selected using the most informative markers from the literature. Further, 22 SNPs were selected to separate Okinawa Japanese at minimum SNPs. SUBJECTS AND METHODS: Samples were collected from 48 unrelated individuals from mainland Japan and 46 unrelated residents of the Okinawa prefecture. Data were evaluated by STRUCTURE, principal component, and GenoGeographer analyses. RESULTS: The 22 SNP set had similar levels of differentiation in STRUCTURE and PCA analyses as the 75 SNP set. GenoGeographer analysis showed that, out of the 46 Okinawa Japanese individuals, the 75 SNP and 22 SNP sets correctly assigned the Okinawan population as the most likely population of origin for 32 and 31 individuals, respectively. CONCLUSION: Neither SNP set could completely differentiate between Okinawa Japanese and other Asian groups, however, these sets should be useful for crime investigation, when the sample, cost and time are limited.

SNPtotree-Resolving the Phylogeny of SNPs on Non-Recombining DNA.

Genetic variants on non-recombining DNA and the hierarchical order in which they accumulate are commonly of interest. This variant hierarchy can be established and combined with information on the population and geographic origin of the individuals carrying the variants to find population structures and infer migration patterns. Further, individuals can be assigned to the characterized populations, which is relevant in forensic genetics, genetic genealogy, and epidemiologic studies. However, there is currently no straightforward method to obtain such a variant hierarchy. Here, we introduce the software SNPtotree v1.0, which uniquely determines the hierarchical order of variants on non-recombining DNA without error-prone manual sorting. The algorithm uses pairwise variant comparisons to infer their relationships and integrates the combined information into a phylogenetic tree. Variants that have contradictory pairwise relationships or ambiguous positions in the tree are removed by the software. When benchmarked using two human Y-chromosomal massively parallel sequencing datasets, SNPtotree outperforms traditional methods in the accuracy of phylogenetic trees for sequencing data with high amounts of missing information. The phylogenetic trees of variants created using SNPtotree can be used to establish and maintain publicly available phylogeny databases to further explore genetic epidemiology and genealogy, as well as population and forensic genetics.

Pitfalls and challenges with population assignments of individuals from admixed populations: Applying Genogeographer on Brazilian individuals.

The assignment of individuals to a population can be of importance for the identification of mass disaster victims or criminal offenders in the field of forensic genetics. This assignment is based on biostatistical methods that process data of ancestry informative markers (AIMs), which are selected based on large allele frequency differences between the populations of interest. However, population assignments of individuals with an admixed genetic background are challenging. Admixed individuals are genetic mosaics of chromosomal segments from the parental populations, which may lead to ambiguous or no population assignment. This is problematic since admixture events are a substantial part of human history. In this study, we present challenges of interpreting the evidential weight of population assignments. We used Genogeographer for likelihood ratio (LR) calculations and Brazilians as examples of admixed individuals. Brazilians are a very heterogenous population representing a three-way admixture between Native Americans, Europeans, and Africans. Ancestry informative markers were typed in a total of 589 individuals from Brazil using the Precision ID Ancestry Panel. The Brazilians were assigned to six metapopulations (East Asia, Europe, Middle East, North Africa, South-Central Asia, Sub-Saharan Africa) defined in the Genogeographer software and LRs were calculated if the AIM profile was not an outlier in all metapopulations and simulated two-way (1:1) admixtures of the six metapopulations. Population assignments failed for 55% of the samples. These samples had significantly higher genetic contributions from East Asia, South-Central Asia and Sub-Saharan Africa, and significantly lower genetic contributions from Europe. Most of the individuals with population assignments were assigned to the metapopulations of Middle East (58%) or North Africa (36%), followed by Europe (4%), South-Central Asia (1%), and Sub-Saharan Africa (1%). For 8% of the samples, population assignments were only possible when assignments to simulated two-way (1:1) admixtures of the six metapopulations were considered. Most of these individuals were assigned to two-way admixtures of North Africa, South-Central Asia, or Sub-Saharan Africa. Relatively low median likelihood ratios (LRs<1000) were observed when comparing population likelihoods for Europe, Middle East, North Africa, South-Central Asia, or simulated 1:1 admixtures of these metapopulations. Comparisons including East Asian or Sub-Saharan African populations resulted in larger median LRs (LR>1010). The results suggested that the Precision ID Ancestry Panel provided too little information and that additional markers specifically selected for sub-continental differentiation may be required for accurate population assignment of admixed individuals. Furthermore, a Genogeographer database with additional populations including admixed populations would be advantageous for interpretation of admixed AIM profiles. It would likely increase the number of population assignments and illustrate alternatives to the most likely population, which would be valuable information for the case officer when writing the case report.

Prediction of chronological age and its applications in forensic casework: methods, current practices, and future perspectives.

Estimating an individual's age can be relevant in several areas primarily related to the clinical and forensic fields. In the latter, estimation of an individual's chronological age from biological material left by the perpetrator at a crime scene may provide helpful information for police investigation. Estimation of age is also beneficial in immigration cases, where age can affect the person's protection status under the law, or in disaster victim identification to narrow the list of potential missing persons. In the last decade, research has focused on establishing new approaches for age prediction in the forensic field. From the first forensic age estimations based on morphological inspections of macroscopic changes in bone and teeth, the focus has shifted to molecular methods for age estimation. These methods allow the use of samples from human biological material that does not contain morphological age features and can, in theory, be investigated in traces containing only small amounts of biological material. Molecular methods involving DNA analyses are the primary choice and estimation of DNA methylation levels at specific sites in the genome is the most promising tool. This review aims to provide an overview of the status of forensic age prediction using molecular methods, with particular focus in DNA methylation. The frequent challenges that impact forensic age prediction model development will be addressed, together with the importance of validation efforts within the forensic community.

Association between Variants in the OCA2-HERC2 Region and Blue Eye Colour in HERC2 rs12913832 AA and AG Individuals.

The OCA2-HERC2 region is strongly associated with human pigmentation, especially eye colour. The HERC2 SNP rs12913832 is currently the best-known predictor for blue and brown eye colour. However, in a previous study we found that 43 of 166 Norwegians with the brown eye colour genotype rs12913832:AA or AG, did not have the expected brown eye colour. In this study, we carried out massively parallel sequencing of a ~500 kbp HERC2-OCA2 region in 94 rs12913832:AA and AG Norwegians (43 blue-eyed and 51 brown-eyed) to search for novel blue eye colour variants. The new candidate variants were subsequently typed in a Norwegian biobank population (total n = 519) for population specific association analysis. We identified five new variants, rs74409036:A, rs78544415:T, rs72714116:T, rs191109490:C and rs551217952:C, to be the most promising candidates for explaining blue eye colour in individuals with the rs12913832:AA and AG genotype. Additionally, we confirmed the association of the missense variants rs74653330:T and rs121918166:T with blue eye colour, and observed lighter skin colour in rs74653330:T individuals. In total, 37 (86%) of the 43 blue-eyed rs12913832:AA and AG Norwegians could potentially be explained by these seven variants, and we suggest including them in future prediction models.

Comparison of global DNA methylation analysis by whole genome bisulfite sequencing and the Infinium Mouse Methylation BeadChip using fresh and fresh-frozen mouse epidermis.

Until recently, studying the murine methylome was restricted to sequencing-based methods. In this study we compared the global DNA methylation levels of hairless mouse epidermis using the recently released Infinium Mouse Methylation BeadChip from Illumina and whole genome bisulphite sequencing (WGBS). We also studied the effect of sample storage conditions by using fresh and fresh-frozen epidermis. The DNA methylation levels of 123,851 CpG sites covered by both the BeadChip and WGBS were compared. DNA methylation levels obtained with WGBS and the BeadChip were strongly correlated (Pearson correlation r = 0.984). We applied a threshold of 15 reads for the WGBS methylation analysis. Even at a threshold of 10 reads, we observed no substantial difference in DNA methylation levels compared with that obtained with the BeadChip. The DNA methylation levels from the fresh and the fresh-frozen samples were strongly correlated when analysed with both the BeadChip (r = 0.999) and WGBS (r = 0.994). We conclude that the two methods of analysis generally work equally well for studies of DNA methylation of mouse epidermis and find that fresh and fresh-frozen epidermis can generally be used equally well. The choice of method will depend on the specific study's aims and the available resources in the laboratory.

Anthropological analyses of 30 insertion/deletion autosomal markers in five major ethnic groups of Pakistan.

We investigated the forensic efficacy of the 30 insertion/deletion (Indel) markers included in the Qiagen Investigator® DIPplex kit in 529 Pakistani individuals from five major subpopulations in Pakistan (Punjabi, Pashtun, Sindhi, Saraiki, and Baloch). In the Sindhi population, the distribution of HLD81 and HLD97 alleles deviated from Hardy-Weinberg equilibrium after Bonferroni correction. The combined match probability ranged from 2.0E-12 (Pashtun and Baloch) to 1.0E-12 (Sindhi), and the mean paternity exclusion power varied from 0.995 (Punjabi, Sindhi, and Saraiki) to 0.996 (Pashtun and Baloch). The high combined power of discrimination (0.999 999 999 999 97) and low combined match probability (1.7E-12) for all subpopulations studied support the utility of the 30 Indel markers for forensic identification in the studied subpopulations. The allele frequencies of the Indel markers in the Pakistani subpopulations were compared with those from 18 other populations. The results show that the populations clustered according to geography. The subpopulations investigated in this work showed a close genetic relationship with others from Pakistan, as well as with South Central Asian and Middle Eastern populations. The results suggest that the Investigator® DIPplex kit can be useful as a supplementary tool for human identification in the five Pakistani subpopulations investigated in this study. Supplemental data for this article is available online at https://doi.org/10.1080/20961790.2021.1933366 .

Sequencing of human identification markers in an Uyghur population using the MiSeq FGxTM Forensic Genomics System.

Massively parallel sequencing (MPS) offers a useful alternative to capillary electrophoresis (CE) based analysis of human identification markers in forensic genetics. By sequencing short tandem repeats (STRs) instead of determining the fragment lengths by CE, the sequence variation within the repeat region and the flanking regions may be identified. In this study, we typed 264 Uyghur individuals using the MiSeq FGx™ Forensic Genomics System and Primer Mix A of the ForenSeq™ DNA Signature Prep Kit that amplifies 27 autosomal STRs, 25 Y-STRs, seven X-STRs, and 94 HID-SNPs. STRinNGS v.1.0 and GATK 3.6 were used to analyse the STR regions and HID-SNPs, respectively. Increased allelic diversity was observed for 33 STRs with the PCR-MPS assay. The largest increases were found in DYS389II and D12S391, where the numbers of sequenced alleles were 3-4 times larger than those of alleles determined by repeat length alone. A relatively large number of flanking region variants (28 SNPs and three InDels) were observed in the Uyghur population. Seventeen of the flanking region SNPs were rare, and 12 of these SNPs had no accession number in dbSNP. The combined mean match probability and typical paternity index based on 26 sequenced autosomal STRs were 3.85E-36 and 1.49E + 16, respectively. This was 10 000 times lower and 1 000 times higher, respectively, than the same parameters calculated from STR repeat lengths.Key PointsSequencing data on STRs and SNPs used for human identification are presented for the Uyghur population.STRinNGS v.1.0 was used to analyse the flanking regions of STRs.The concordance between PCR-CE and PCR-MPS results was 99.86%.Detection of sequence variation in STRs and their flanking regions increased the allelic diversity.

Testing the Ion AmpliSeq™ HID Y-SNP Research Panel v1 for performance and resolution in admixed South Americans of haplogroup Q.

Y haplogroups, defined by Y-SNPs, allow the reconstruction of the human Y chromosome genealogy, which is important for population, evolutionary and forensic genetics. In this study, Y-SNPs were typed and haplogroups inferred with the MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1, as a high-throughput approach. Firstly, the performance of the panel was evaluated with different DNA input amounts, reagent volumes and cycle numbers. DNA-inputs from 0.5 to 1 ng generated the most balanced read depth. Combined with full reagent and 19 cycles, this offered the highest number of amplicons with a sequencing read depth of at least 20 reads. Secondly, the sub-haplogroups of 182 admixed South Americans and Greenlanders belonging to haplogroup Q were inferred and tested for potential improvement in resolution. Most samples were assigned to lineage Q-M3 with some samples assigned to lineages upstream (Q-M346, L56, L57; Q-L331, L53; Q-L54; Q-CTS11969, CTS11970) or parallel (Q-L330, L334; Q-Z780/M971) to Q-M3. Only one sample was assigned to a downstream lineage (Q-Z35615, Z35616). Most individuals of haplogroup Q with NAM ancestry could neither be distinguished from each other, nor from half of the Greenlandic samples. Typing additional, known SNPs within lineage Q-M3, Z19483 and SA05, increased the resolution of predicted haplogroups. The search for novel variants in the sequenced regions allowed the detection of 42 variants and the subdivision of lineage Q-M3 into new subclades. The variants found in six of these subclades were exclusive to certain South American countries. In light of the limited differentiation of haplogroup Q samples, the additional information on known or novel SNPs disclosed in this study when using MPS Ion AmpliSeq™ HID Y-SNP Research Panel v1 should be included in the Yleaf software, to increase the differentiation of lineage Q-M3.

Prediction of Eye Colour in Scandinavians Using the EyeColour 11 (EC11) SNP Set.

Description of a perpetrator's eye colour can be an important investigative lead in a forensic case with no apparent suspects. Herein, we present 11 SNPs (Eye Colour 11-EC11) that are important for eye colour prediction and eye colour prediction models for a two-category reporting system (blue and brown) and a three-category system (blue, intermediate, and brown). The EC11 SNPs were carefully selected from 44 pigmentary variants in seven genes previously found to be associated with eye colours in 757 Europeans (Danes, Swedes, and Italians). Mathematical models using three different reporting systems: a quantitative system (PIE-score), a two-category system (blue and brown), and a three-category system (blue, intermediate, brown) were used to rank the variants. SNPs with a sufficient mean variable importance (above 0.3%) were selected for EC11. Eye colour prediction models using the EC11 SNPs were developed using leave-one-out cross-validation (LOOCV) in an independent data set of 523 Norwegian individuals. Performance of the EC11 models for the two- and three-category system was compared with models based on the IrisPlex SNPs and the most important eye colour locus, rs12913832. We also compared model performances with the IrisPlex online tool (IrisPlex Web). The EC11 eye colour prediction models performed slightly better than the IrisPlex and rs12913832 models in all reporting systems and better than the IrisPlex Web in the three-category system. Three important points to consider prior to the implementation of eye colour prediction in a forensic genetic setting are discussed: (1) the reference population, (2) the SNP set, and (3) the reporting strategy.

Analysis of Skin Pigmentation and Genetic Ancestry in Three Subpopulations from Pakistan: Punjabi, Pashtun, and Baloch.

Skin pigmentation is one of the most prominent and variable phenotypes in humans. We compared the alleles of 163 SNPs and indels from the Human Pigmentation (HuPi) AmpliSeq™ Custom panel, and biogeographic ancestry with the quantitative skin pigmentation levels on the upper arm, lower arm, and forehead of 299 Pakistani individuals from three subpopulations: Baloch, Pashtun, and Punjabi. The biogeographic ancestry of each individual was estimated using the Precision ID Ancestry Panel. All individuals were mainly of mixed South-Central Asian and European ancestry. However, the Baloch individuals also had an average proportion of Sub-Saharan African ancestry of approximately 10%, whereas it was <1% in the Punjabi and Pashtun individuals. The pairwise genetic distances between the Pashtun, Punjabi, and Baloch subpopulations based on the ancestry markers were statistically significantly different. Individuals from the Pashtun subpopulation had statistically significantly lower skin pigmentation than individuals from the Punjabi and Baloch subpopulations (p < 0.05). The proportions of European and Sub-Saharan African ancestry and five SNPs (rs1042602, rs10831496, rs1426654, rs16891982, and rs12913832) were statistically significantly associated with skin pigmentation at either the upper arm, lower arm or forehead in the Pakistani population after correction for multiple testing (p < 10-3). A model based on four of these SNPs (rs1426654, rs1042602, rs16891982, and rs12913832) explained 33% of the upper arm skin pigmentation. The four SNPs and the proportions of European and Sub-Saharan African ancestry explained 37% of the upper arm skin pigmentation. Our results indicate that the four likely causative SNPs, rs1426654, rs1042602, rs16891982, and rs12913832 located in SLC24A5, TYR, SLC45A2, and HERC2, respectively, are essential for skin color variation in the admixed Pakistani subpopulations.

Gene expressions in cerebral palsy subjects reveal structural and functional changes in the gastrocnemius muscle that are closely associated with passive muscle stiffness.

Cerebral palsy (CP) is a non-progressive motor disorder that affects posture and gait due to contracture development. The purpose of this study is to analyze a possible relation between muscle stiffness and gene expression levels in muscle tissue of children with CP. Next-generation sequencing (NGS) of gene transcripts was carried out in muscle biopsies from gastrocnemius muscle (n = 13 children with CP and n = 13 typical developed (TD) children). Passive stiffness of the ankle plantarflexors was measured. Structural changes of the basement membranes and the sarcomere length were measured. Twelve pre-defined gene target sub-categories of muscle function, structure and metabolism showed significant differences between muscle tissue of CP and TD children. Passive stiffness was significantly correlated to gene expression levels of HSPG2 (p = 0.02; R2 = 0.67), PRELP (p = 0.002; R2 = 0.84), RYR3 (p = 0.04; R2 = 0.66), C COL5A3 (p = 0.0007; R2 = 0.88), ASPH (p = 0.002; R2 = 0.82) and COL4A6 (p = 0.03; R2 = 0.97). Morphological differences in the basement membrane were observed between children with CP and TD children. The sarcomere length was significantly increased in children with CP when compared with TD (p = 0.04). These findings show that gene targets in the categories: calcium handling, basement membrane and collagens, were significantly correlated to passive muscle stiffness. A Reactome pathway analysis showed that pathways involved in DNA repair, ECM proteoglycans and ion homeostasis were amongst the most upregulated pathways in CP, while pathways involved in collagen fibril crosslinking, collagen fibril assembly and collagen turnover were amongst the most downregulated pathways when compared with TD children. These results underline that contracture formation and motor impairment in CP is an interplay between multiple factors.

Genetic analysis of sixteen autosomal STR loci in three Tunisian populations from Makthar, Nabeul and Sousse.

BACKGROUND: Due to its strategic location, Tunisia witnessed the succession and influence of many civilisations throughout history. However, the majority of studies carried out on Tunisia are focussed on Barbarian ethnicity. AIM: To estimate genetic diversity and genetic structure of three Tunisian populations using autosomal STRs. SUBJECTS AND METHODS: 278 individuals were analysed for sixteen STRs. Allele frequencies and statistical parameters were determined. RESULTS: The studied populations showed genetic affinity with geographically close populations. AMOVA showed no genetic difference between the Tunisian populations. Nevertheless, the variance between the populations of the same group was significant, reflecting their heterogeneity even though they came from the same geographical area, and had the same ethnicity and complex demographic history. CONCLUSION: Our results strongly supported the application of autosomal genetic markers in anthropological and forensic studies. The analyses conducted at the 15-loci level provide the resolution to assess the phylogenetic relationships among the populations examined and other geographically targeted worldwide populations, while the results resulting from the 10-loci studies provide an understanding of the relationships and origins of the North African populations. Furthermore, the current report demonstrates that the battery of autosomal STRs reported are useful, providing the power of discrimination for forensic and paternity analyses.

Forensic application and genetic diversity of 21 autosomal STR loci in five major population groups of Pakistan.

OBJECTIVES: Investigation of genetic diversity of the 21 autosomal STR loci included in the GlobalFilerTM PCR Amplification Kit in 529 Pakistani individuals belonging to the Punjabi, Pashtun, Sindhi, Saraiki, and Baloch ethnic groups. Population genetic parameters and forensic informative metrics for each group were evaluated. RESULTS: SE33 showed the greatest power of discrimination in all populations studied. The combined match probability ranged from 8.06E-27 (Saraiki) to 1.05E-26 (Baloch), and the combined power of exclusion ranged from 0.99999999902 (Punjabi) to 0.99999999964 (Pashtun). D12S391 in the Baloch population and D2S441 in the Saraiki population showed deviation from Hardy-Weinberg equilibrium. CONCLUSION: Significant genetic distances were observed between the Punjabi, Pashtun, and Baloch populations. This study supports the utilization of the GlobalFilerTM STR kit for forensic applications in Pakistan.

Evaluation of a custom GeneRead™ massively parallel sequencing assay with 210 ancestry informative SNPs using the Ion S5™ and MiSeq platforms.

A custom GeneRead DNAseq SNP panel with 210 markers was evaluated using the Ion S5 and MiSeq sequencing platforms. Sensitivity, PCR cycle number, and the use of half volume of reagents for target enrichment and library preparation were tested. Furthermore, genotype concordance between results obtained with the different sequencing platforms and with known profiles generated using other sequencing assays was analysed. The GeneRead DNASeq SNP assay gave reproducible results with an input of 200 pg DNA on both platforms. A total of 204 loci were successfully sequenced. Three loci failed completely in the PCR amplification, and three additional loci displayed frequent locus drop-outs due to low read depth or high heterozygote imbalance. Overall, the read depth across the loci was more well-balanced with the MiSeq, while the heterozygote balance was less variable with the Ion S5. Noise levels were low on both platforms (median< 0.2 %). Two simple criteria for genotyping were applied: A minimum threshold of 45 reads and an acceptable heterozygote balance range of 0.3-3.0. Complete concordance between platforms was observed except for three genotypes in one of the poorly performing loci, rs1470637. This locus had relatively low read depths on both platforms, skewed heterozygote balance, and frequent locus drop-outs. There was also full genotype concordance between the results from the GeneRead assay and known profiles generated with the QIAseq and Ion AmpliSeq assays. The few discordant results were either due to locus drop-outs in the poorly performing loci or allele drop-outs in the QIAseq assay. Profiles with a minimum of 179 SNPs were obtained from four challenging case work samples (blood swabs, bone, or blood from a corpse). Overall, the GeneRead DNASeq assay showed considerable potential and could provide a reliable method for SNP genotyping in cases involving identification of individuals, prediction of phenotypic traits, and ancestry inference.

Association between brown eye colour in rs12913832:GG individuals and SNPs in TYR, TYRP1, and SLC24A4.

The genotype of a single SNP, rs12913832, is the primary predictor of blue and brown eye colours. The genotypes rs12913832:AA and rs12913832:GA are most often observed in individuals with brown eye colours, whereas rs12913832:GG is most often observed in individuals with blue eye colours. However, approximately 3% of Europeans with the rs12913832:GG genotype have brown eye colours. The purpose of the study presented here was to identify variants that explain brown eye colour formation in individuals with the rs12913832:GG genotype. Genes and regulatory regions surrounding SLC24A4, TYRP1, SLC24A5, IRF4, TYR, and SLC45A2, as well as the upstream region of OCA2 within the HERC2 gene were sequenced in a study comprising 40 individuals with the rs12913832:GG genotype. Of these, 24 individuals were considered to have blue eye colours and 16 individuals were considered to have brown eye colours. We identified 211 variants within the SLC24A4, TYRP1, IRF4, and TYR target regions associated with eye colour. Based on in silico analyses of predicted variant effects we recognized four variants, TYRP1 rs35866166:C, TYRP1 rs62538956:C, SLC24A4 rs1289469:C, and TYR rs1126809:G, to be the most promising candidates for explanation of brown eye colour in individuals with the rs12913832:GG genotype. Of the 16 individuals with brown eye colours, 14 individuals had four alleles, whereas the alleles were rare in the blue eyed individuals. rs35866166, rs62538956, and rs1289469 were for the first time found to be associated with pigmentary traits, whilst rs1126809 was previously found to be associated with pigmentary variation. To improve prediction of eye colours we suggest that future eye colour prediction models should include rs35866166, rs62538956, rs1289469, and rs1126809.