Review of the Forensic Applicability of Biostatistical Methods for Inferring Ancestry from Autosomal Genetic Markers

The inference of ancestry has become a part of the services many forensic genetic laboratories provide. Interest in ancestry may be to provide investigative leads or identify the region of origin in cases of unidentified missing persons. There exist many biostatistical methods developed for the study of population structure in the area of population genetics. However, the challenges and questions are slightly different in the context of forensic genetics, where the origin of a specific sample is of interest compared to the understanding of population histories and genealogies. In this paper, the methodologies for modelling population admixture and inferring ancestral populations are reviewed with a focus on their strengths and weaknesses in relation to ancestry inference in the forensic context.

Evaluation of Forensic Genetic Parameters of 24 STR Loci and Y indel in a Southern Region Saudi Population Sample Using GlobalFiler™ PCR Amplification Kit

The last three decades have seen rapid advances in the field of short tandem repeats (STRs) genotyping technology. Autosomal STRs have emerged as a powerful tool in forensic identification and paternity investigations. The indigenous population of Saudi Arabia is irregularly distributed and has historically been organized into geographically distinct groups or tribes of patrilineal descent. So far, there has been no detailed investigation of the southern region Saudi population to assist in the interpretation of DNA-based forensic evidence and in the construction of DNA database. The objective of this study is to investigate the genetic structure in 154 unrelated healthy Saudi subjects within three generations from the southern Saudi regions using a GlobalFiler™ PCR Amplification kit. Intra- and Inter-population genetic diversity as well as the forensic genetics parameters were analyzed. Our results showed that SE33 and TPOX loci were the most and the least polymorphic loci, respectively. The PIC, PE, TPI, Ho and He varied from 0.56116 (TPOX) to 0.94393 (SE33), 0.26638 (TPOX) to 0.83859 (SE33), 1.1875 (TPOX) to 6.33333 (SE33), 0.57894 (TPOX) to 0.92105 (SE33) and 0.6169 (TPOX) to 0.952 (SE33), respectively. The highest PM was observed for D22S1045 (0.223944) and the highest PD for SE33 (0.98935). The combined PD was 99.99999999% and the combined PM was equal to 3.19021E-25. Phylogenetic parameters showed that the southern region Saudi population had the closest genetic relationship with the Saudi, Emirati, Kuwaiti, and Bahraini populations. The study offers some important insights into the southern region Saudi population structure using GlobalFiler™ PCR Amplification kit.

Ethics as Lived Practice. Anticipatory Capacity and Ethical Decision-Making in Forensic Genetics

Greater scrutiny and demands for innovation and increased productivity place pressures on scientists. Forensic genetics is advancing at a rapid pace but can only do so responsibly, usefully, and acceptably within ethical and legal boundaries. We argue that such boundaries require that forensic scientists embrace ‘ethics as lived practice’. As a starting point, we critically discuss ‘thin’ ethics in forensic genetics, which lead to a myopic focus on procedures, and to seeing ‘privacy’ as the sole ethical concern and technology as a mere tool. To overcome ‘thin’ ethics in forensic genetics, we instead propose understanding ethics as an intrinsic part of the lived practice of a scientist. Therefore, we explore, within the context of three case studies of emerging forensic genetics technologies, ethical aspects of decision-making in forensic genetics research and in technology use. We discuss the creation, curation, and use of databases, and the need to engage with societal and policing contexts of forensic practice. We argue that open communication is a vital ethical aspect. Adoption of ‘ethics as lived practice’ supports the development of anticipatory capacity—empowering scientists to understand, and act within ethical and legal boundaries, incorporating the operational and societal impacts of their daily decisions, and making visible ethical decision making in scientific practice.

Molecular Markers for Human Sex Determination in Forensic Genetics Analysis

Sex determination is indispensable in forensic anthropology, sexual disorder, and also as part of large-scale genetic population studies. The purpose of this investigation is to determine the human sex from whole blood using multiplex PCR analysis. Blood samples from 75 male and 70 female healthy volunteers were taken from Tikrit city, Iraq. Our study identified a reliable set of three primer locus, namely SRY, ALT1 (internal control) and amelogenin locus. The SRY primer on the Y chromosome showed a 254 bp of PCR product, with 100% accuracy for human male identification. Thus, the pair of SRY primers was considered a strong genetic marker for human sex identification. Amelogenin regions in the Y chromosome showed a true positive band (236 bp) with 100% accuracy on sex identification. Amelogenin regions in X chromosome also showed positive bands (330 bp) in female samples and positive band in male samples except for two samples showed a negative band (null bands). The most obvious finding from this study is that multiplex PCR of ALT1 and SRY is consider as a reliable genetic marker for human sex identification. The research has also shown that amelogenin is good genetic marker for human sex identification.

First experiences with the Spectrum Compact CE System

The newly released Spectrum Compact CE System by Promega is a capillary electrophoresis instrument developed for DNA-fragment separation and sequencing. In this study, its compatibility to 8 commercial short tandem repeat (STR) kits from 4 different manufacturers, reproducibility (sizing precision, accuracy and concordance) and robustness (sensitivity and mixture resolution) were tested and compared to the ABI PRISM® 310 Genetic Analyzer. The instrument was able to successfully analyse amplicons of all tested kits, proved to be as precise as claimed by manufacturer specifications and was shown to be more robust than the ABI PRISM® 310 Genetic Analyzer in some aspects. Analyses on the Spectrum Compact CE System were able to resolve peaks with length differences of 1-basepair in the short and long fragment range and mixtures of mixture ratios down to 1:30. We describe the advantages and limitations we have observed so far working with this instrument in our forensic genetics laboratory.

Vibration as a pitfall in pyrosequencing analyses

Since methylation analysis has become an important tool in forensic genetics, the reliability and credibility of the method must be ensured. After a successful validation and establishment of several pyrosequencing assays using a PyroMark® Q48 Autoprep instrument (Qiagen, Hilden, Germany), we decided to expand the method further purchasing a second instrument. But after initializing this second instrument side by side with the first, the majority of analyses failed (97 samples of 133 samples (73%)). The number of error messages increased rapidly and the average RFU values decreased. After purchasing two anti-vibration weighing tables for the PyroMark® instruments and repeating the analyses under the same conditions and with identical samples the results improved considerably, 115 samples of 130 samples (88%) showed successful and reproducible results. These findings demonstrate the impact of vibrations and percussions on PyroMark® Q48 Autoprep performance and the reliability of methylation analyses.

Open-Access STRS Database Of Populations From The 1000 Genomes Project Using High Coverage Phase 3 Data

Accurate STR genotyping from next-generation sequencing (NGS) data has been challenging. Haplotype inference and phasing for STRs (HipSTR) was specifically developed to deal with genotyping errors and obtain reliable STR genotypes from whole-genome sequencing datasets. The objective of this investigation was to perform a comprehensive genotyping analysis of a set of STRs of broad forensic interest from the 1000 Genomes populations and release a reliable open-access STR database to the forensic genetics community. A set of 22 STR markers were analyzed using the CRAM files of the 1000 Genomes Project Phase 3 high-coverage (30x) dataset generated by the New York Genome Center (NYGC). HipSTR was used to call genotypes from 2,504 samples from 26 populations organized into five groups: African, East Asian, European, South Asian, and admixed American. The D21S11 marker could not be detected in the present study. Moreover, the Hardy-Weinberg equilibrium analysis, coupled with a comprehensive analysis of allele frequencies, revealed that HipSTR could not identify longer Penta E (and Penta D at a lesser extent) alleles. This issue is probably due to the limited length of sequencing reads available for genotype calling, resulting in heterozygote deficiency. Notwithstanding that, AMOVA, a clustering analysis using STRUCTURE, and a Principal Coordinates Analysis revealed a clear-cut separation between the four major ancestries sampled by the 1000 Genomes Consortium (AFR, EUR, EAS, SAS). Meanwhile, the AMOVA results corroborated previous reports that most of the variance is (97.12%) observed within populations. This set of analyses revealed that except for larger Penta D and Penta E alleles, allele frequencies and genotypes defined by HipSTR from the 1000 Genomes Project phase 3 data and offered as an open-access database are consistent and highly reliable.

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.

Comparative analysis of allele variation using allele frequencies according to sample size in Korean population

Background Allele frequency using short tandem repeats (STRs) is used to calculate likelihood ratio for database match, to interpret DNA mixture and to estimate ethnic groups in forensic genetics. In Korea, three population studies for 23 STR loci have been conducted with different sample size for forensic purposes. Objective We performed comparative analysis to determine how the difference of sample size affects the allele frequency and allele variation within same ethnic population (i.e. Korean). Furthermore, this study was conducted to check how the sampling group and multiplex kit also affect allele variation such as rare alleles and population specific alleles. Methods To compare allele variation, we used allele frequencies of three population data published from three Korean forensic research groups. Allele frequencies were calculated using different sample sizes and multiplex kits: 526, 1000, and 2000 individuals, respectively. Results The results showed the different distribution of allele frequencies in some loci. There was also a difference in the number of rare alleles observed by the sample size and sampling bias. In particular, an allele of 9.1 in the D2S441 locus was not observed in population study with 526 individuals due to multiplex kits. Conclusion Because the allele frequencies play an important role in forensic genetics, even if the samples are derived from the same population, it is important to consider the effects of sample size, sampling bias, and selection of multiplex kits in population studies.