Forensic Genetic Analysis Research Articles

Evaluation of Four Forensic Investigative Genetic Genealogy Analysis Approaches with Decreased Numbers of SNPs and Increased Genotyping Errors

Background: Forensic investigative genetic genealogy (FIGG) has developed rapidly in recent years and is considered a novel tool for crime investigation. However, crime scene samples are often of low quality and quantity and are challenging to analyze. Deciding which approach should be used for kinship inference in forensic practice remains a troubling problem for investigators. Methods: In this study, we selected four popular approaches—KING, IBS, TRUFFLE, and GERMLINE—comprising one method of moment (MoM) estimator and three identical by descent (IBD) segment-based tools and compared their performance at varying numbers of SNPs and levels of genotyping errors using both simulated and real family data. We also explored the possibility of making robust kinship inferences for samples with ultra-high genotyping errors by integrating MoM and the IBD segment-based methods. Results: The results showed that decreasing the number of SNPs had little effect on kinship inference when no fewer than 164 K SNPs were used for all four approaches. However, as the number decreased further, decreased efficiency was observed for the three IBD segment-based methods. Genotyping errors also had a significant effect on kinship inference, especially when they exceeded 1%. In contrast, MoM was much more robust to genotyping errors. Furthermore, the combination of the MoM and the IBD segment-based methods showed a higher overall accuracy, indicating its potential to improve the tolerance to genotyping errors. Conclusions: In conclusion, this study shows that different approaches have unique characteristics and should be selected for different scenarios. More importantly, the integration of the MoM and the IBD segment-based methods can improve the robustness of kinship inference and has great potential for applications in forensic practice.

Exploring nanopore direct sequencing performance of forensic STRs, SNPs, InDels, and DNA methylation markers in a single assay

IntroductionThe field of forensic DNA analysis has undergone rapid advancements in recent decades. The integration of massively parallel sequencing (MPS) has notably expanded the forensic toolkit, moving beyond identity matching to predicting phenotypic traits and biogeographical ancestry. This shift is of particular significance in cases where conventional DNA profiling fails to identify a single suspect. Supplementing forensic analyses with estimated biological age may be valuable but involves a complex and time-consuming DNA methylation analysis. This study explores and validates the performance of a comprehensive forensic third-generation sequencing assay utilizing Oxford Nanopore Technologies (ONT) in an adaptive and direct sequencing approach. We incorporated the most widely used forensic markers, i.e., STRs, SNPs, InDels, mitochondrial DNA (mtDNA), and two methylation-based clock classifiers, thereby combining forensic genetic and epigenetic analysis in one single workflow. Methods and resultsIn our investigation, DNA from six anonymous individuals was sequenced using the ONT standard adaptive direct sequencing approach, reaching a mean percentage of on-target reads ranging from 6.6 % to 7.7 % per sample. ONT data was compared to standard MPS data and Illumina EPIC DNA methylation profiles. Basecalling employed recommended ONT software packages. TREAT was used for ONT-based analysis of autosomal and Y-chromosome STRs, achieving 90–92 % correct calls depending on allelic read depth thresholds. InDel analyses for two lower-quality samples proved challenging due to inadequate read depth, while the remaining four samples significantly contributed to the observed percentage markers (60.9 %) and correct calls (97.8 %). SNP analysis achieved a 98 % call rate, with only two mismatches and two missed alleles. ONT-generated DNA methylation data demonstrated Pearson’s correlation coefficients with EPIC data ranging from 0.67 to 0.97 for Horvath’s clock. Additional age-associated markers exhibited Pearson’s correlation coefficients with chronological age between 0.14 (ELOVL2) and 0.96 (FHL2) at read depths of <30 and <20, respectively. Despite excluding mtDNA from our targeted sequencing approach, adaptive proof-reading fragments covered the complete mtDNA with an average read depth of 21–72, showing 100 % concordance with reference data. DiscussionOur exploratory study using ONT adaptive sequencing for conventional forensic and age associated DNA methylation markers showed high sequencing accuracy for a significant number of markers, showcasing ONT as a promising (epi)genetic forensic method. Future studies must address three critical aspects: determining clear quantity and quality measures and detection thresholds for accuracy, optimizing input DNA quantity for forensic casework expectations, and addressing ethical considerations associated with phenotype and ancestry analysis to prevent ethnic biases.

Comprehensive body fluid identification and contributor assignment by combining targeted sequencing of mRNA and coding region SNPs

Forensic genetic analyses aim to retrieve as much information as possible from biological trace material recovered from crime scenes. While standard short tandem repeat (STR) profiling is essential to individualize biological traces, its significance is diminished in crime scenarios where the presence of a suspect's DNA is acknowledged by all parties. In such cases, forensic (m)RNA analysis can provide crucial contextualizing information on the source level about a trace’s composition, i.e., body fluids/tissues, and has therefore emerged as a powerful tool for modern forensic investigations. However, the question which of several suspects contributed a specific component (body fluid) to a mixed trace cannot be answered by RNA analysis using conventional methods. This individualizing information is stored within the sequence of the mRNA transcripts. Massively parallel sequencing (MPS) represents a promising alternative, offering not only higher multiplex capacity, but also the typing of individual coding region SNPs (cSNPs) to enable the assignment of contributors to mixture components, thereby reducing the risk of association fallacies. Herein, we describe the development of an extensive mRNA/cSNP panel for targeted sequencing on the IonTorrent S5 platform. Our panel comprises 30 markers for the detection of six body fluids/tissues (blood, saliva, semen, skin, vaginal and menstrual secretion), along with 70 linkage-controlled cSNPs for contributor assignment. It exhibited high reliable detection sensitivity with RNA inputs down to 0.75 ng and a conservatively calculated probability of identity of 0.03 – 6 % for individual body fluid-specific cSNP profiles. Limitations and areas for future work include RNA-related allele imbalances, inclusion of markers to correctly identify rectal mucosa and the optimization of specific markers. In summary, our new panel is intended to be a major step forward to interpret biological evidence at sub-source and source level based on cSNP attribution of a body fluid component to a suspect and victim, respectively.

Identification of individuals from low template blood samples using whole transcriptome shotgun sequencing

Biological trace samples consisting of very few cells pose a challenge to conventional forensic genetic DNA analysis. RNA may be an alternative to DNA when handling low template samples. Whereas each cell only contains two copies of an autosomal DNA segment, the transcriptome retains much of the genomic variation replicated in abundant RNA fragments. In this study, we describe the development of a prototype RNA-based SNP selection set for forensic human identification from low template samples (50 pg gDNA). Whole blood from a subset of the Danish population (41 individuals) and blood stains subjected to degradation at room temperature for up to two weeks were analysed by whole transcriptome shotgun sequencing. Concordance was determined by DNA genotyping with the Infinium Omni5–4 SNP chip. In the 100 protein-coding genes with the most reads, 5214 bi-allelic SNPs with gnomAD minor allele frequencies > 0.1 in the African/African American, East Asian, and (non-Finnish) European populations were identified. Of these, 24 SNPs in 21 genes passed screening in whole blood and degraded blood stains, with a resulting mean match probability of 4.5 ∙ 10−9. Additionally, ancestry informative SNPs and SNPs in genes useful for body fluid identification were identified in the transcriptome. Consequently, shotgun sequencing of RNA from low template samples may be used for a vast host of forensic genetics purposes, including simultaneous human and body fluid identification, leading to direct donor identification in the identified body fluid.

MHBase: A comprehensive database of short microhaplotypes for advancing forensic genetic analysis

Microhaplotypes (MHs) were first recommended by Prof. Kidd for use in forensics because they can improve human identification, kinship analysis, mixture deconvolution, and ancestry prediction. Since their introduction, extensive research has demonstrated the advantages of MHs in forensic applications and provided useful data for different populations. Currently, two databases, ALFRED (ALlele FREquency Database) and MicroHapDB (MicroHaplotype DataBase), house the published MH information and population data. We previously constructed a single nucleotide polymorphism SNP-SNP MH database (D-SNPsDB) of MHs within 50 bp on the whole human genome for 26 populations integrating basic data such as physical genome positions, mapping of variant identifiers (rsIDs), allele frequencies, and basic variant information. Building upon the previous research, we further selected MHs containing at least two variants (SNPs and/or insertions/deletions [InDels]) within a short DNA fragment (≤ 50 bp) in 26 populations based on the 1000 Genomes Project dataset (Phase 3) to construct a more comprehensive database. Subsequently, we established a user-friendly website that allows users to search the MH database (MHBase) based on their research objectives and study population to find suitable loci and provides other functions such as querying reported loci, performing online calculations using the PHASE software, and calculating ancestral-related parameters. The loci in the database are classified as SNP-based MHs, which include only SNPs, and InDel-including MHs, which contain at least one InDel. Here, we provide a detailed overview of the MHBase and an analysis of shared loci at the global and continental levels, ancestral markers, the genetic distance within loci, and mapping with the genome annotation file. The website is an accessible and useful tool for researchers engaged in marker discovery, population studies, assay development, and panel design.

Forensic genetics associated with hair analysis as a tool for jaguar (Panthera onca) identification

Animal smuggling and illegal wildlife trade comprise one of the most common crimes being committed on an international level. This action can affect biodiversity and most certainly increases the risk of extinction of several endangered species. Wildlife taxonomic identification is becoming routine to forensic experts. The aim of this study was to identify a possible jaguar sample (Panthera onca Linnaeus, 1958) from legal evidence using hair morphology and mitochondrial DNA (mtDNA) to help in an official animal identification. Scanning and optical microscope images of hair samples were used to compare diagnostic characteristics of felines to reference studies. A preserved region, (cytochrome oxidase subunit 1 (COI)), from mtDNA was sequenced and compared to a DNA database (GenBank) to perform genetic identification. Forensic genetic and hair analysis techniques combined positively identified the sample as jaguar. These methods can help practitioners and forensics experts to identify evidence collected in wildlife crimes suspected to involve jaguars.

The effect of fingerprint enhancement methods applied on adhesive surfaces on DNA recovery: a preliminary study

The presence of body fluids such as blood, saliva, semen or urine during fingerprint research on the evidence taken from the crime scene makes it necessary to protect biological materials to examine the evidence in multiple ways. Therefore, it is crucial that fingerprint development (FD) techniques do not disrupt the structure of biological materials during FD procedures. In this sense, it is essential to determine whether biological material or fingerprints should be the priority during the collection of evidentiary materials, to determine the systematic order and to determine whether the FD methods to be applied cause damage to the genetic material used in the identification of individuals and to evaluate them in terms of their evidentiary quality. This study investigated the effects of the application of trace detection methods on DNA profiling processes in evidence where fingerprints and biological samples are found at the same time. In this study, blood, saliva, semen and urine samples were taken from a male individual who signed an informed consent form at the laboratory stage. The samples were applied 50 µL on the adhesive tape surface. After application, the samples were treated with crystal violet (CV) and sticky side (SS) fingerprint development chemicals suitable for the surface type. The prepared samples were dried under room conditions. After 1 day and 45 days under normal room conditions, silica-based DNA extraction was performed. After extraction, DNA quantification was performed using the fluorimetry method. In the study, biological samples with known DNA content were used to focus on DNA quantification. Among the fresh samples prepared in the study, DNA recovery was higher in the SS-treated urine, blood and saliva samples and in the CV-treated semen sample group compared to the other groups. This shows that chemical treatment of some biological samples on adhesive tape increases the efficiency of DNA recovery. When the 45-day waiting samples were compared with the control group samples, DNA recovery decreased in CV-treated urine and blood samples, while DNA recovery increased in SS-treated urine and blood samples. In semen samples, both CV and SS treatment negatively affected DNA recovery. In saliva samples, DNA recovery increased ~2-fold in the CV-treated sample group, while SS treatment caused a ~75% decrease in DNA recovery. The results show that the non-porous adhesive tape does not adversely affect the amount of DNA in terms of STR profiling of latent FD chemicals used on the surfaces and that adhesive tape treated with fingerprint enhancement chemicals can actually be used for advanced forensic genetic analyses for DNA extraction on surfaces.

Retraction: Forensic characteristics and genetic affinity analyses of Xinjiang Mongolian group using a novel six fluorescent dye-labeled typing system including 41 Y-STRs and 3 Y-InDels.

Liu Y., Yu T., Mei S., Jin X., Lan Q., Zhou Y., Fang Y., Xie T., Huang J., Zhu B. (2020). Forensic characteristics and genetic affinity analyses of Xinjiang Mongolian group using a novel six fluorescent dye-labeled typing system including 41 Y-STRs and 3 Y-InDels. Molecular Genetics & Genomic Medicine, 8(2), e1097. https://doi.org/10.1002/mgg3.1097 The above article, published online on 26 December 2019 in Wiley Online Library (wileyonlinelibrary.com), has been retracted by agreement between the journal Editor in Chief, Suzanne Hart, and Wiley Periodicals LLC. Following publication of this article, concerns were raised by a third party regarding consent and ethical approval for the research undertaken and reported in the article. The publisher and representatives of the journal's editorial board undertook a review of the consent documentation provided, against the research performed and reported in the article. The review uncovered inconsistencies between the consent documentation and the research reported; the documentation was not sufficiently detailed to resolve the concerns raised. As a result, the parties have made the decision to retract the article. In addition, consent documentation did not give approval for data associated with this article to be shared publicly. The associated data for this article has been withdrawn from the publication record following a concern that the data could be used to identify those who have participated in the research. Yanfang Liu, Xiaoye Jin, and Bofeng Zhu replied on behalf of all of the authors, who disagree with the retraction.

Optimizing Analytical Thresholds for Low-Template DNA Analysis: Insights from Multi-Laboratory Negative Controls.

When analyzing challenging samples, such as low-template DNA, analysts aim to maximize information while minimizing noise, often by adjusting the analytical threshold (AT) for optimal results. A potential approach involves calculating the AT based on the baseline signal distribution in electrophoresis results. This study investigates the impact of reagent kits, testing quarters, environmental conditions, and amplification cycles on baseline signals using historical records and experimental data on low-template DNA. Variations in these aspects contribute to differences in baseline signal patterns. Analysts should remain vigilant regarding routine instrument maintenance and reagent replacement, as these may affect baseline signals. Prompt analysis of baseline status and tailored adjustments to ATs under specific laboratory conditions are advised. A comparative analysis of published methods for calculating the optimal AT from a negative signal distribution highlighted the efficiency of utilizing baseline signals to enhance forensic genetic analysis, with the exception of extremely low-template samples and high-amplification cycles. Moreover, a user-friendly program for real-time analysis was developed, enabling prompt adjustments to ATs based on negative control profiles. In conclusion, this study provides insights into baseline signals, aiming to enhance genetic analysis accuracy across diverse laboratories. Practical recommendations are offered for optimizing ATs in forensic DNA analysis.

Revealing Y-STR Diversity of Koli Populations (Gujarat) by Studying 23 Y-STR Loci

Genetic analysis of Y-STR loci is pivotal for forensic libraries and genetic analysis. The Koli population in Gujarat, India, however, lacks such genetic characterization. This study aims to develop an allele frequency database for 23 Y-STR loci in the Koli population, examining forensic parameters and assessing genetic connections with neighboring tribes. A total of 153 unrelated Koli males were genotyped using the PowerPlex®Y23 multiplex commercial kit. We identified 117 distinct haplotypes. The Haplotype Diversity (HD) and Discrimination Capacity (DC) for the 23 Y-STR loci were 0.993 and 0.8034, respectively. DYS385b locus exhibited the highest allele variability (10 alleles), whereas DYS391, DYS389I, and DYS437 showed the least (4 alleles each). The highest Polymorphic Information Content (PIC) was observed in DYS385b (0.775), with the lowest in DYS391 (0.386). The dominant haplogroup R1a accounted for 45% of the population. Comparative analysis with other Indian populations from YHRD revealed two distinct clusters, placing the Koli population in cluster 2, indicating significant genetic similarity within this group. This inaugural study of Y-STRs in the Koli population demonstrates the utility of the Y23 kit in male identification, highlighted by substantial haplotype diversity and discrimination capacity.

Types of human trace evidences and the importance of their forensic genetic analysis in Hungarian criminal and administrative proceedings – Case reports

Aim: The aim of the study is to present widely and successfully analysed exhibits and crime scene samples of human origin in forensic genetic examinations through short case reports of various types of Hungarian cases from the last decades. By presenting actual cases the study also aims to comprehensively depict the great importance of forensic genetic analysis in Hungarian jurisdiction.Methodology: For this study, 51 cases representing 22 case types were chosen out of several thousand closed cases investigated by the Hungarian Institute for Forensic Science. Forensic genetic analysis of the presented exhibits and traces gave investigative leads and evidence for solving crime cases or for the identificationof unknown bodies, if the relevant biological material remains can be found and recorded at the scene.Findings: Since the launch of forensic genetic analysis in Hungary in 1992, it has gradually become possible to provide genetic evidence for criminal and public administration proceedings by DNA analysis of an increasing number and variety of human biological material residues in a growing number and variety of cases (from theft to serial murders). The presented 51 cases of 22 case types may provide proof of the applicability of forensic genetic methodology in almost any case in Hungary, provided the relevant exhibits and biological traces are found and collected at the (crime) scenes.Value: This is the first comprehensive case report review that presents the various types and the importance of human trace pieces of evidence analysed with forensic genetic methods in 22 case types of 51 Hungarian criminal and administrative proceedings.

Mitogenome sequences of domestic cats demonstrate lineage expansions and dynamic mutation processes in a mitochondrial minisatellite

BackgroundAs a population genetic tool, mitochondrial DNA is commonly divided into the ~ 1-kb control region (CR), in which single nucleotide variant (SNV) diversity is relatively high, and the coding region, in which selective constraint is greater and diversity lower, but which provides an informative phylogeny. In some species, the CR contains variable tandemly repeated sequences that are understudied due to heteroplasmy. Domestic cats (Felis catus) have a recent origin and therefore traditional CR-based analysis of populations yields only a small number of haplotypes.ResultsTo increase resolution we used Nanopore sequencing to analyse 119 cat mitogenomes via a long-amplicon approach. This greatly improves discrimination (from 15 to 87 distinct haplotypes in our dataset) and defines a phylogeny showing similar starlike topologies within all major clades (haplogroups), likely reflecting post-domestication expansion. We sequenced RS2, a CR tandem array of 80-bp repeat units, placing RS2 array structures within the phylogeny and increasing overall haplotype diversity. Repeat number varies between 3 and 12 (median: 4) with over 30 different repeat unit types differing largely by SNVs. Five SNVs show evidence of independent recurrence within the phylogeny, and seven are involved in at least 11 instances of rapid spread along repeat arrays within haplogroups.ConclusionsIn defining mitogenome variation our study provides key information for the forensic genetic analysis of cat hair evidence, and for the first time a phylogenetically informed picture of tandem repeat variation that reveals remarkably dynamic mutation processes at work in the mitochondrion.

Amplification Failure of the Amelogenin X Gene Caused by a Rare Mutation in the Primer-Binding Region.

The study of gender markers is essential in forensic genetic analysis. Mutations in the X or Y homologs of the amelogenin gene can be misleading, resulting in serious mistakes in forensic genetic analysis. We recently discovered two male cases of the X homolog of the amelogenin (AMELX) allelic dropout while analyzing short tandem repeat genotypes obtained from crime scene evidence. Subsequently, we evaluated the molecular characteristics of AMELX allelic dropout in this study. We used two previously reported amelogenin primers to verify a half level of amelogenin gene amplification intensity in the two male cases, which we confirmed was caused by AMELX allelic dropout. We then characterized the point mutation using Sanger sequencing and designed mutation-specific primers that could overcome AMELX allelic dropout. Short tandem repeat genotyping analysis confirmed that the AMELX allelic dropout was recovered by the mutation-specific primer designed specifically for this case. The sequencing of the AMELX allele revealed a single-point variant from A→G at base position 7 downstream from the 3' end in the amelogenin forward primer-binding region. This point mutation was identically found in two different male cases, resulting in AMELX allelic dropout. To our knowledge, these mutations and the X homolog amplification failure of amelogenin have not been reported in the Korean population. Our study provides a reliable approach to AMELX allelic dropout due to rare case mutations and could enable the better interpretation of gender markers for forensic samples.

Forensic genetic analysis of single-nucleotide polymorphisms and microhaplotypes in Koreans through next-generation sequencing using precision ID identity panel.

Forensic DNA analysis has seen remarkable advancements with the advent of Next Generation Sequencing (NGS). In particular, NGS analysis of single nucleotide polymorphisms (SNPs) offers significant advantages in the analysis of challenging samples compared to conventional STR analysis. This study aimed to investigate the SNPs of the Precision ID Identity Panel, a commercially available NGS panel for personal identification, by generating genetic profiles of 298 Koreans and comparing them with other global populations. A total of 124 SNPs, including 90 autosomal and 34 Y-SNPs, were analyzed using the Precision ID Identity Panel, and forensic parameters, microhaplotypes, and population differences were investigated. The NGS data were successfully obtained from 298 Koreans. The analysis of forensic parameters exhibited a low combined match probability of 1.532 × 10- 34, which is comparable to that obtained from commonly used STR analysis. Additionally, the microhaplotype analysis revealed that the use of 16 microhaplotypes provided higher discriminatory power compared to single target SNPs. Furthermore, the adoption of microhaplotype data resulted in an increase of over 20% in expected heterozygosity at five loci. Inter-population analysis showed a close genetic relationship between Koreans and individuals from China and Myanmar in East and Southeast Asia, which are geographically adjacent to Korea. The results of this study show that the Precision ID Identity panel can be a useful alternative where traditional STR typing is not feasible. Also, the data from our study will be useful as a reference for Koreans in forensic investigations and the prosecution of criminal justice.

Comparison of three massively parallel sequencing platforms for single nucleotide polymorphism (SNP) genotyping in forensic genetics.

Three MPS platforms are being used in forensic genetic analysis, i.e., MiSeq FGx, Ion S5 XL, and MGISEQ-2000. However, few studies compared their performance. In this study, we sequenced 83 common SNPs of 71 samples using the ForenSeq™ DNA Signature Prep Kit on MiSeq FGx, the Precision ID Identity Panel on Ion S5 XL, and the MGIEasy Signature Identification Library Prep Kit on MGISEQ-2000 and then the performance was compared. Results showed that the MiSeq FGx had the highest sequence quality but the lowest sequencing depth and allele balance. Discordant genotypes were observed at six SNPs, which may be caused by variants at primer binding regions, indel errors, or misalignments. Besides, two kinds of background noises, allele-specific miscalled reads (ASMR) and allele-nonspecific miscalled reads (ANMR), were characterized. MGISEQ-2000 showed the highest level of ASMR while Ion S5 XL had the highest level of ANMR. Site- and genotype-dependent miscalled patterns were observed at several SNPs on Ion S5 XL and MGISEQ-2000, but few on MiSeq FGx. In conclusion, the three MPS platforms perform differently with respect to sequencing quality, sequencing depth, allele balance, concordance, and background noise. These findings may be useful for data comparison, mixture deconvolution, and heteroplasmy analysis in forensic genetics.

Systematically exploring the performance of a self-developed Multi-InDel system in forensic identification, ancestry inference and genetic structure analysis of Chinese Manchu and Mongolian groups

The insertion/deletion (InDel) polymorphism has promising applications in forensic DNA analysis. However, the insufficient forensic efficiencies of the present InDel-based systems restrict their applications in parentage testing, due to the lower genetic polymorphism of the biallelic InDel locus and the limited number of InDel loci in a multiplex amplification system. Here, we introduced an in-house developed system which contained 41 polymorphic Multi-InDel markers (equivalent to 82 InDels in total), to serve as an efficient and reliable tool for different forensic applications in the Manchu and Mongolian groups. We demonstrated that the new system exhibited potential efficiencies for personal identification, parentage testing, two-person DNA mixture interpretation and ancestry inference of intercontinental populations. Meanwhile, we explored the genetic backgrounds of the Manchu and Mongolian groups by conducting a series of population genetic analyses. We showed that the Manchu and Mongolian groups shared closer genetic relationships with East Asian populations, especially Han Chinese populations in northern China. Moreover, more similar genetic compositions were detected between the Manchu group and the northern Han populations in this study, suggesting that the Manchu group had higher genetic affinities with northern Han populations than the Mongolian group. Overall. this study provided the necessary evidence that these Multi-InDel genetic markers could play an important role in forensic applications.

Assessing population substructure in the Lebanese population: A population study using data on 23 autosomal short tandem repeats.

Understanding the genetic substructure of a population is essential for proper forensic genetic analysis. The Lebanese population has a high rate of endogamous and consanguineous marriages and is segregated based on religious belongings. However, no genetic population studies have been performed up to date to estimate the degree of genetic substructure and inbreeding coefficients. The present study analyzed a compendium of 23 autosomal STRs typed in 1400 individuals belonging to the seven major Lebanese religious subcommunities. Inbreeding coefficients such as F statistics were estimated. Results showed a Fst (theta) value of 0.002, indicating a low genetic subdivision within the Lebanese population. This study aimed at assessing the genetic substructure of the Lebanese population by analyzing 1400 Lebanese citizens' samples using 23 STR markers. F statistics were computed to evaluate the degree of genetic substructure. Results showed a Fst value of 0.002, indicating a low genetic subdivision within the Lebanese population.

Calculation and implementation of sample-wide stochastic thresholds for forensic genetic analysis of STRs and SNPs for massively parallel sequencing platforms

Capillary electrophoresis (CE) analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) use a stochastic threshold to consider the possibility of missing alleles (dropouts) or detecting additional alleles (drop-ins). In CE, this threshold may be approximately 200 RFU, and peak heights are assessed relative to this threshold. In next generation sequencing (NGS), also known as massively parallel sequencing (MPS), STRs are identified by their sequence, and specific alleles are identified by their repeat number and intra-allelic variation. Abundance is approximated by the number of sequence reads for each allele. The total number of reads generated for each marker in a sample depends on factors such as the numbers of samples pooled for sequencing, the number of markers in the assay, the integrity and quantity of the input DNA sample, and the inter-locus balance of the assay. For multiplexes that contain both autosomal and sex-linked markers, the biological sex of the sample also influences total reads per locus. To normalize these variables and better establish a robust stochastic threshold, a sample-wide metric is proposed for estimating the possibility of dropouts or drop-ins based on the variance of the inter-locus balance of the markers across a sample. The intuition is that samples with variable allele balance globally are more likely to have noisier data and therefore require more stringent read count thresholds. This method is robust to sequencing multiplexity, biological sex and manufacturing lot variation.

Forensic DNA phenotyping in Europe: How far may it go?

The fast evolution of genetic sequencing techniques led to new applications in forensic genetics, one of these being the prediction of the physical appearance of a possible perpetrator from biological traces found at the crime scene. Some European countries recently changed their legislations, to permit this technique, also known as Forensic DNA Phenotyping (FDP). The phenotypical traits that may be analyzed under those revised domestic laws are usually restricted to include no information about the suspect’s health. This article elaborates whether the European legal framework, as set by the Council of Europe and the European Union (EU), defines any boundaries for the analytical scope of FDP. After a brief introduction to FDP and a description of the type of data collected through predictive forensic genetics, this article discusses the relevant European legislation and the case law of the European Court of Human Rights (ECtHR) and the Court of Justice of the European Union (CJEU) around privacy, data protection and the use of genetic data. The article attempts to define possible limits for forensic genetic analysis, by eventually trying to predict the jurisprudence of the two European courts.

Population structure and forensic genetic analyses in Guarani and Kaingang Amerindian populations from Brazil

When DNA profile comparisons between a crime scene trace and a reference sample generate correspondence, the match probability has to be estimated, so that evaluation of the strength of the forensic DNA evidence can be made. The random match probability estimations require information on allele frequencies and an adjustment factor, referred to as theta (θ) or Fst, a co-ancestry correction factor for subpopulation effects. The θ value has been standardized for urban and isolated populations, but inconsistencies have been reported when it is specifically calculated for smaller and isolated populations, including Amerindian populations. Notably, attempts to characterize forensic markers of these minor populations have been extensively limited and more conservative estimates of the correction factor may be generated for each of them. Therefore, we estimate allele frequencies of 21 autosomal STR markers used for forensic testing and calculated relevant forensic parameters for the set. In addition, we featured the possible structure of five Brazilian Amerindian populations that have been genetically isolated for centuries so we could obtain the appropriate θ value for them. The sample consisted of 319 individuals: (1) 121 Kaingang, from two communities: Ivaí (KIV=61) and Rio das Cobras (KRC=60); and (2) 198 Guaranis from three communities: Mbya from Rio das Cobras (GRC=51), Guarani Ñandeva (GND=71) and Guarani Kaiowá (GKW=76). Between Guarani populations low (Rst=0.0402, p < 10−4) to high (Rst=0.1557, p < 10−5) differentiation was found. Regarding Guarani and Kaingang populations, intermediate (Rst=0.0590, p < 10−5) to high (Rst=0.1604, p < 10−5) differentiation was found. The two Kaingang populations showed very low differentiation between them (Rst=0.0017, p = 0.27), which justifies the union of both genetic data for forensic databases and calculations. The combined power of discrimination (PD) and the combined power of exclusion (PE) were calculated for each population, demonstrating the usefulness of this set of markers in forensic and kinship analysis regarding these populations. Considering the demographic heterogeneity of Amerindian populations in general, the Fst mean value (0.03) was evaluated regarding 43 different indigenous populations from the Americas, including Guaranis and Kaingangs. This result confirms the adequacy of the standardized θ value for the forensic random match probability estimations involving Amerindian populations.