Social and scientific motivations to move beyond groups in allele frequencies: The TOPMed experience.

For the genomics community, allele frequencies within defined groups (or "strata") are useful across multiple research and clinical contexts. Benefits include allowing researchers to identify populations for replication or "look up" studies, enabling researchers to compare population-specific frequencies to validate findings, and facilitating assessment of variant pathogenicity in clinical contexts. However, there are potential concerns with stratified allele frequencies. These include potential re-identification (determining whether or not an individual participated in a given research study based on allele frequencies and individual-level genetic data), harm from associating stigmatizing variants with specific groups, potential reification of race as a biological rather than a socio-political category, and whether presenting stratified frequencies-and the downstream applications that this presentation enables-is consistent with participants' informed consents. The NHLBI Trans-Omics for Precision Medicine (TOPMed) program considered the scientific and social implications of different approaches for adding stratified frequencies to the TOPMed BRAVO (Browse All Variants Online) variant server. We recommend a novel approach of presenting ancestry-specific allele frequencies using a statistical method based upon local genetic ancestry inference. Notably, this approach does not require grouping individuals by either predominant global ancestry or race/ethnicity and, therefore, mitigates re-identification and other concerns as the mixture distribution of ancestral allele frequencies varies across the genome. Here we describe our considerations and approach, which can assist other genomics research programs facing similar issues of how to define and present stratified frequencies in publicly available variant databases.

Assisting the analysis of insertions and deletions using regional allele frequencies.

Accurate estimation of population allele frequency (AF) is crucial for gene discovery and genetic diagnostics. However, determining AF for frameshift-inducing small insertions and deletions (indels) faces challenges due to discrepancies in mapping and variant calling methods. Here, we propose an innovative approach to assess indel AF. We developed CRAFTS-indels (Calculating Regional Allele Frequency Targeting Small indels), an algorithm that combines AF of distinct indels within a given region and provides "regional AF" (rAF). We tested and validated CRAFTS-indels using three independent datasets: gnomAD v2 (n=125,748 samples), an internal dataset (IGM; n=39,367), and the UK BioBank (UKBB; n=469,835). By comparing rAF against standard AF, we identified rare indels with rAF exceeding standard AF (sAF≤10-4 and rAF>10-4) as "rAF-hi" indels. Notably, a high percentage of rare indels were "rAF-hi", with a higher proportion in gnomAD v2 (11-20%) and IGM (11-22%) compared to the UKBB (5-9% depending on the CRAFTS-indels' parameters). Analysis of the overlap of regions based on their rAF with low complexity regions and with ClinVar classification supported the pertinence of rAF. Using the internal dataset, we illustrated the utility of CRAFTS-indel in the analysis of de novo variants and the potential negative impact of rAF-hi indels in gene discovery. In summary, annotation of indels with cohort specific rAF can be used to handle some of the limitations of current annotation pipelines and facilitate detection of novel gene disease associations. CRAFTS-indels offers a user-friendly approach to providing rAF annotation. It can be integrated into public databases such as gnomAD, UKBB and used by ClinVar to revise indel classifications.

Genetic profile of RHCE, Kell, Duffy, Kidd, Diego and MNS hybrid glycophorins blood groups in ethnic northeastern Thais: Alleles, genotypes and risk of alloimmunisation.

BACKGROUND: Antibodies against blood group antigens play a key role in the pathophysiology of haemolytic transfusion reactions (HTRs) and haemolytic disease of the fetus and newborn (HDFN). This study aimed to determine the frequencies of alleles, genotypes, and risk of alloimmunisation of clinically significant blood group systems in ethnic northeastern Thais. METHODS: In total, 345 unrelated, healthy, ethnic northeastern Thais were tested using the in-house PCR-sequence specific primers (PCR-SSP) method for simultaneously genotyping of RHCE, Kell, Duffy, Kidd, Diego and MNS glycophorin hybrids and results confirmed by Sanger sequencing. RESULTS: In this cohort, the alleles RHCE*C (81.0%) and RHCE*e (84.8%) were more prevalent than RHCE*c (19.0%) and RHCE*E (15.2%). The most common predicted haplotype combinations of the RHCE alleles were C+c-E-e+(R1R1) (59.4%) followed by the C+c+E+e+ (R1R2) (20.6%) and C+c+E-e+ (R1r) (11.3%). The KEL*01 allele was not found in this study. The frequencies of FY*01 and FY*02 were 88.3% and 11.7%, respectively. The genotype FY*02/02 was found in four samples (1.2%). The frequencies of JK*01 and JK*02 were 52.5% and 47.5%, respectively. Homozygous JK*02/02 was found in 81 samples (23.5%). The frequencies of DI*01 and DI*02 were 0.6% and 99.4%, respectively. In total, 64 samples (18.6%) were found to carry the MNS glycophorin hybrids. CONCLUSIONS: Our results indicated a possible high risk of c, E, Fyb, Jka, Jkb and Mia alloimmunisation in these populations. Moreover, methods established for genotyping clinically significant blood groups in this study can now be utilised in routine clinical application.

High Resolution HLA-A, HLA-B, and HLA-C Allele Frequencies in Romanian Hematopoietic Stem Cell Donors.

The HLA genes are associated with various autoimmune pathologies, with the control of the immune response also being significant in organs and cells transplantation. The aim of the study is to identify the HLA-A, HLA-B, and HLA-C alleles frequencies in the analyzed Romanian cohort. We performed HLA typing using next-generation sequencing (NGS) in a Romanian cohort to estimate class I HLA allele frequencies up to a six-digit resolution. A total of 420 voluntary donors from the National Registry of Voluntary Hematopoietic Stem Cell Donors (RNDVCSH) were included in the study for HLA genotyping. Peripheral blood samples were taken and brought to the Fundeni Clinical Institute during 2020-2021. HLA genotyping was performed using the Immucor Mia Fora NGS MFlex kit. A total of 109 different alleles were detected in 420 analyzed samples, out of which 31 were for HLA-A, 49 for HLA-B, and 29 for HLA-C. The most frequent HLA-A alleles were HLA-A*02:01:01 (26.11%), HLA-A*01:01:01 (12.5%), HLA-A*24:02:01 (11.67%), HLA-A*03:01:01 (9.72%), HLA-A*11:01:01, and HLA-A*32:01:01 (each with 8.6%). For the HLA-B locus, the most frequent allele was HLA-B*18:01:01 (11.25%), followed by HLA-B*51:01:01 (10.83%) and HLA-B*08:01:01 (7.78%). The most common HLA-C alleles were HLA-C*07:01:01 (17.36%), HLA-C*04:01:01 (13.47%), and HLA-C*12:03:01 (10.69%). Follow-up studies are ongoing for confirming the detected results.

HLA Polymorphisms and Clinical Manifestations in IgA Vasculitis.

Studies concerning the genetic background of IgA vasculitis (IgAV), a small-vessel vasculitis occurring predominantly in childhood, have confirmed that the HLA-DRB1 gene showed a strong association with disease susceptibility. The objective was to investigate human leukocyte antigen (HLA) polymorphisms among Croatian patients with IgAV and their influence on disease susceptibility and clinical heterogeneity. Thus, 130 children with IgAV and 202 unrelated healthy individuals were enrolled in the study. Genomic DNA was extracted from whole peripheral blood, and HLA-A, -B, -DRB1 and -DQB1 gene polymorphism analysis was performed. HLA-A*03 (21.4% vs. 12.38%, p = 0.0092), HLA-B*37 (2.9% vs. 0.2%, p = 0.0054) and HLA-DRB1*12 (3.1% vs. 0.7%, p = 0.0216) alleles were significantly more frequent in IgAV patients than in controls. High-resolution typing revealed significantly higher frequency of HLA-DRB1*10:01 and -DRB1*11:03 among IgAV patients with gastrointestinal manifestations of the disease in comparison to controls (p = 0.0021 and p = 0.0301, respectively), while HLA-DRB1*14:01P occurred significantly more often in the group of patients who developed nephritis during the course of the disease (17.5% vs. 4.5%, p = 0.0006). Our results demonstrated that there is an association of HLA-A*03, HLA-B*37 and HLA-DRB1*12 alleles with susceptibility to IgAV in the examined Croatian pediatric population. Studies which aim to determine the HLA profile may contribute to the elucidation of the genetic background of autoimmune diseases, including IgAV.

High-Resolution HLA-DRB1 Allele Frequencies in a Romanian Cohort of Stem Cell Donors.

The goal of the current study was to determine the high-resolution frequencies of the HLA-DRB1 alleles among the analyzed Romanian cohort of healthy stem cell donors. Using Next Generation Sequencing (NGS), we estimated class II HLA-DRB1 allele frequencies to a 6-digit resolution through HLA typing in a Romanian cohort of healthy individuals. The study for HLA genotyping included 420 willing donors from the National Registry of Voluntary Hematopoietic Stem Cell Donors (RNDVCSH). In 2020 and 2021, peripheral blood samples were collected and transported to the Fundeni Clinical Institute. We used the Immucor Mia Fora NGS MFlex kit for HLA genotyping. Forty-one different alleles were detected in 420 analyzed samples, out of which the most frequent HLA-DRB1 alleles were DRB1*16:01:01 (12.6%), DRB1*11:04:01 (12.1%) and DRB1*03:01:01 (12%). The HLA-DRB1*11:01:02 and -DRB1*08:04:01, -DRB1*05:01:01, -DRB1*13:05:01, -DRB1*14:07:01, -DRB1*09:01:02, -DRB1*11:02:01, -DRB1*04:07:01, -DRB1*15:03:01, -DRB1*03:02:01, -DRB1*04:06:02, -DRB1*04:08:01, -DRB1*14:05:01 were identified only once. The results revealed similarities with countries belonging to the Eastern Europe, the Balkans and the Caucasus regions. Further studies on larger Romanian cohorts are needed for confirming the current results.

Population Pharmacogenomics in Croatia: Evaluating the PGx Allele Frequency and the Impact of Treatment Efficiency.

BACKGROUND: Adverse drug reactions (ADRs) are a significant cause of mortality, and pharmacogenomics (PGx) offers the potential to optimize therapeutic efficacy while minimizing ADRs. However, there is a lack of data on the Croatian population, highlighting the need for investigating the most common alleles, genotypes, and phenotypes to establish national guidelines for drug use. METHODS: A single-center retrospective cross-sectional study was performed to examine the allele, genotype, and phenotype frequencies of drug-metabolizing enzymes, receptors, and other proteins in a random sample of 522 patients from Croatia using a 28-gene PGx panel. RESULTS: Allele frequencies, genotypes, and phenotypes for the investigated genes were determined. No statistically significant differences were found between the Croatian and European populations for most analyzed genes. The most common genotypes observed in the patients resulted in normal metabolism rates. However, some genes showed higher frequencies of altered metabolism rates. CONCLUSIONS: This study provides insights into the allele, genotype, and phenotype frequencies of drug-metabolizing enzymes, receptors, and other associated proteins in the Croatian population. The findings contribute to optimizing drug use guidelines, potentially reducing ADRs, and improving therapeutic efficacy. Further research is needed to tailor population-specific interventions based on these findings and their long-term benefits.

Like Wings of a Bird: Functional Divergence and Complementarity between HLA-A and HLA-B Molecules.

Human leukocyte antigen (HLA) genes are among the most polymorphic of our genome, as a likely consequence of balancing selection related to their central role in adaptive immunity. HLA-A and HLA-B genes were recently suggested to evolve through a model of joint divergent asymmetric selection conferring all human populations, including those with severe loss of diversity, an equivalent immune potential. However, the mechanisms by which these two genes might undergo joint evolution while displaying very distinct allelic profiles in populations are still unknown. To address this issue, we carried out extensive data analyses (among which factorial correspondence analysis and linear modeling) on 2,909 common and rare HLA-A, HLA-B, and HLA-C alleles and 200,000 simulated pathogenic peptides by taking into account sequence variation, predicted peptide-binding affinity and HLA allele frequencies in 123 populations worldwide. Our results show that HLA-A and HLA-B (but not HLA-C) molecules maintain considerable functional divergence in almost all populations, which likely plays an instrumental role in their immune defense. We also provide robust evidence of functional complementarity between HLA-A and HLA-B molecules, which display asymmetric relationships in terms of amino acid diversity at both inter- and intraprotein levels and in terms of promiscuous or fastidious peptide-binding specificities. Like two wings of a flying bird, the functional complementarity of HLA-A and HLA-B is a perfect example, in our genome, of duplicated genes sharing their capacity of assuming common vital functions while being submitted to complex and sometimes distinct environmental pressures.

Allele frequencies and minor contributor match statistic convergence using simulated population replicates.

Probabilistic genotyping permits a comparison of forensic evidence given hypotheses regarding the origin of observed short tandem repeat alleles in a mixed DNA profile. Using the publicly available R package forensim, it has been proposed that mixtures with non-contributors from low genetic diversity populations are more likely to be mistakenly identified as contributors to a mixture than non-contributors from high genetic diversity populations. We hypothesized that these observations are attributed to the unique distribution of alleles in the reference population and may not generalize to other samplings of the same population. We used forensim to simulate 200 US populations (50 each of self-reported African-American, Asian-American, European-American, and Hispanic descent). We compared likelihood ratios for 2400 mixtures to those derived from published data and identified stark differences. A minimum of ten population replicates were required to reduce observed differences relative to published data. Deviations from Hardy-Weinberg equilibrium and allele frequency distributions suggest that simulated populations should be sufficiently evaluated for expectations of population genetic parameters prior to use in DNA mixture modeling experiments. Overall, our findings support the utility of forensim and further describe its suitability to model population genetic parameters but suggest that a single population replicate (directly ascertained or simulated) may be insufficient to make conclusions about a given DNA mixture.

Population data for 21 STRs for the Salvadoran population using GlobalFiler Express and GlobalFiler STR Amplification Kits.

With the advent of expanded STR (short tandem repeats) typing kits, it was necessary to determine allele frequencies and other appropriate population data parameters for El Salvador. Samples were collected from the central, east, and west regions of the country and typed for 21 forensically relevant STR loci. The data indicate that all loci are highly polymorphic, the three regions are genetically similar, and the population data are similar to those of US Hispanics. The results of this study support that the allele frequency data described herein can be used for statistical calculations for human identity testing in El Salvador.

Population data for 15 autosomal STR loci in Orang Asli subgroups of Peninsular Malaysia.

Short tandem repeats (STRs) data for the Orang Asli population in Peninsular Malaysia is still scanty, especially for specific Orang Asli subgroups. The Orang Asli is believed as the earliest population arrived in Peninsular Malaysia about 50,000 years ago and currently makes up only 0.6% of the total population of Malaysia. This study reports the allele frequencies and several forensic statistical parameters for 15 autosomal STR loci for six Orang Asli subgroups. A total of 164 Orang Asli individuals representing the Semai, Che Wong, Orang Kanaq, Lanoh, Bateq, and Kensui subgroups were recruited for this study. This STR data will enrich the existing Malaysian autosomal STR database and will be useful for kinship testing and forensic applications.

Genetic epidemiology of human neutrophil antigen variants suggests significant global variability.

Human neutrophil antigens possess significant clinical implications especially in the fields of transfusion and transplantation medicine. Efforts to estimate the prevalence of genetic variations underpinning the antigenic expression are emerging. However, there lacks a precise capture of the global frequency profiles. Our article emphasizes the potential utility of maintaining an organized online repository of evidence on neutrophil antigen-associated genetic variants from published literature and reports. This, in our opinion, is an emerging area and would significantly benefit from the awareness and understanding of population-level diversities.

Effects of DGAT1 on milk performance in Sudanese Butana × Holstein crossbred cattle.

The improvement of milk production of indigenous Sudanese cattle such as Bos indicus Butana and its cross with Holstein is a major goal of the Sudanese government to ensure sufficient healthy nutrition in the country. In this study, we investigated the K232A polymorphism of diacylglycerol acyltransferase (DGAT1), a well-known modulator of milk production in other breeds. We determined allele frequencies and the allele effects on milk production. Therefore, 93 purebred Butana and 203 Butana × Holstein crossbred cattle were genotyped using competitive allele-specific PCR assays. Association analysis was performed using a linear mixed model in R. In purebred Butana cattle, the lysine DGAT1 protein variant K232, which is found to be associated with higher fat and protein contents, as well as higher fat yield was highly frequent at 0.929, while its frequency in Butana × Holstein crossbred cattle was 0.394. Significant effects were found on milk yield (P = 7.6 × 10-20), fat yield (P = 2.2 × 10-17), protein yield (P = 2.0 × 10-19) and lactose yield (P = 4.0 × 10-18) in crossbred cattle. As expected, the protein variant K232 was disadvantageous since it was decreasing milk, protein, and lactose yields by 1.741 kg, 0.063 kg and 0.084 kg, respectively. No significant effects were found for milk fat, protein, and lactose contents. The high frequency of the lysine DGAT1 protein variant K232 in Butana cattle could contribute to their high milk fat content in combination with low milk yield. In Butana × Holstein crossbred cattle, the DGAT1 marker can be used for effective selection and thus genetic improvement of milk production.

Allelic frequencies with 23 autosomic STRS in the Aymara population of Peru.

Population data of the Aymara in the province of Puno were established for 23 autosomal STR markers. DNA was obtained from unrelated individuals (n = 190) who reside in three areas of the Floating Islands of Lake Titicaca, residents on the border with Bolivia and residents who are not from the border with Bolivia. The PENTA E marker presented the highest PD (0.9738), PIC (0.8793), and PM (0.7847) values. The combined PD was greater than 0.99999999 and the combined PE was 0.99999994. The largest distance, based on Fst values, was between the Aymara population and the Ashaninca population (0.04022), and the smallest distance was with the populations of Bolivia (0.00136) and Peru (0.00525).

Exploring STR sequencing for forensic DNA intelligence databasing using the Austrian National DNA Database as an example.

Here, we present the results from a population study that evaluated the performance of massively parallel sequencing (MPS) of short tandem repeats (STRs) with a particular focus on DNA intelligence databasing purposes. To meet this objective, 247 randomly selected reference samples, earlier being processed with conventional capillary electrophoretic (CE) STR sizing from the Austrian National DNA Database, were reanalyzed with the PowerSeq 46Y kit (Promega). This sample set provides MPS-based population data valid for the Austrian population to increase the body of sequence-based STR variation. The study addressed forensically relevant parameters, such as concordance and backward compatibility to extant amplicon-based genotypes, sequence-based stutter ratios, and relative marker performance. Of the 22 autosomal STR loci included in the PowerSeq 46GY panel, 99.98% of the allele calls were concordant between MPS and CE. Moreover, 25 new sequence variants from 15 markers were found in the Austrian dataset that are yet undescribed in the STRSeq online catalogue and were submitted for inclusion. Despite the high degree of concordance between MPS and CE derived genotypes, our results demonstrate the need for a harmonized allele nomenclature system that is equally applicable to both technologies, but at the same time can take advantage of the increased information content of MPS. This appears to be particularly important with regard to database applications in order to prevent false exclusions due to varying allele naming based on different analysis platforms and ensures backward compatibility.

Documentation of clinically relevant genomic biomarker allele frequencies in the next-generation FINDbase worldwide database.

FINDbase (http://www.findbase.org) is a comprehensive data resource recording the prevalence of clinically relevant genomic variants in various populations worldwide, such as pathogenic variants underlying genetic disorders as well as pharmacogenomic biomarkers that can guide drug treatment. Here, we report significant new developments and technological advancements in the database architecture, leading to a completely revamped database structure, querying interface, accompanied with substantial extensions of data content and curation. In particular, the FINDbase upgrade further improves the user experience by introducing responsive features that support a wide variety of mobile and stationary devices, while enhancing computational runtime due to the use of a modern Javascript framework such as ReactJS. Data collection is significantly enriched, with the data records being divided in a Public and Private version, the latter being accessed on the basis of data contribution, according to the microattribution approach, while the front end was redesigned to support the new functionalities and querying tools. The abovementioned updates further enhance the impact of FINDbase, improve the overall user experience, facilitate further data sharing by microattribution, and strengthen the role of FINDbase as a key resource for personalized medicine applications and personalized public health.

Comparison and development of machine learning tools for the prediction of chronic obstructive pulmonary disease in the Chinese population.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a major public health problem and cause of mortality worldwide. However, COPD in the early stage is usually not recognized and diagnosed. It is necessary to establish a risk model to predict COPD development. METHODS: A total of 441 COPD patients and 192 control subjects were recruited, and 101 single-nucleotide polymorphisms (SNPs) were determined using the MassArray assay. With 5 clinical features as well as SNPs, 6 predictive models were established and evaluated in the training set and test set by the confusion matrix AU-ROC, AU-PRC, sensitivity (recall), specificity, accuracy, F1 score, MCC, PPV (precision) and NPV. The selected features were ranked. RESULTS: Nine SNPs were significantly associated with COPD. Among them, 6 SNPs (rs1007052, OR = 1.671, P = 0.010; rs2910164, OR = 1.416, P < 0.037; rs473892, OR = 1.473, P < 0.044; rs161976, OR = 1.594, P < 0.044; rs159497, OR = 1.445, P < 0.045; and rs9296092, OR = 1.832, P < 0.045) were risk factors for COPD, while 3 SNPs (rs8192288, OR = 0.593, P < 0.015; rs20541, OR = 0.669, P < 0.018; and rs12922394, OR = 0.651, P < 0.022) were protective factors for COPD development. In the training set, KNN, LR, SVM, DT and XGboost obtained AU-ROC values above 0.82 and AU-PRC values above 0.92. Among these models, XGboost obtained the highest AU-ROC (0.94), AU-PRC (0.97), accuracy (0.91), precision (0.95), F1 score (0.94), MCC (0.77) and specificity (0.85), while MLP obtained the highest sensitivity (recall) (0.99) and NPV (0.87). In the validation set, KNN, LR and XGboost obtained AU-ROC and AU-PRC values above 0.80 and 0.85, respectively. KNN had the highest precision (0.82), both KNN and LR obtained the same highest accuracy (0.81), and KNN and LR had the same highest F1 score (0.86). Both DT and MLP obtained sensitivity (recall) and NPV values above 0.94 and 0.84, respectively. In the feature importance analyses, we identified that AQCI, age, and BMI had the greatest impact on the predictive abilities of the models, while SNPs, sex and smoking were less important. CONCLUSIONS: The KNN, LR and XGboost models showed excellent overall predictive power, and the use of machine learning tools combining both clinical and SNP features was suitable for predicting the risk of COPD development.

Population genetic study of a Peruvian population using human identification STRs.

In this study, allele frequencies were determined in a Peruvian population for application to human identification. A population of 601 unrelated individuals was analyzed (400 individuals with the GlobalFiler Express kit and 201 individuals with the VeriFiler Express kit). The locus with the highest power of discrimination (PD) was SE33 (0.9851, 31 alleles), while the least polymorphic locus was D22S1045 (0.75810, 11 alleles). The PE in a similar fashion ranged from 0.2421 (D22S1045) to 0.7818 (SE33). Under the assumption of independence, the combined PD was > 0.9999999999 while the combined PE = 0.9999999933. When comparing the population studied with different populations of Latin America, the greatest Fst genetic distance was obtained with a Venezuelan population (0.052), and the shortest distance was with a Bolivian and Peruvian population (0.004).

HLA-B*15:47:01 allele with undefined serological equivalent considered as B Blank.

We report a discordance between complement-dependent cytotoxicity and next-generation sequencing molecular typing revealing HLA-B*15:47:01 allele with undefined serological equivalent confirmed by high-level immunization against the B15 serotype. Due to the high-level immunization against HLA-B15 and B70 antigens, we considered the HLA-B*15:47:01 allele to be B Blank and not as B15 or B70 serological specificity.

Genetic characterization of 15 autosomal STRs in the interior Sindhi population of Pakistan and their phylogenetic relationship with other populations.

Genetic structure of a population can be influenced by evolutionary processes and cultural histories which can alter the frequencies of different variants at particular genetic markers. These characteristics make DNA evidence suitable for forensic applications. Little relevant data are available from the interior Sindhi population; thus, in the current study, we have investigated 15 autosomal STRs in 181 unrelated individuals belonging to the interior parts of Sindh Pakistan, to establish its lineage and parameters of forensic interest. These STRs revealed a high power of discrimination (CPD), power of exclusion (CPE) and matching probability (CMP) are 0.9999999999999999968997, 0.99998612 and 3.1003 × 10-18 respectively. The genetic distances, neighbour-joining (NJ) tree, interactivity test and principal component analysis (PCA) based on 15 autosomal STR loci showed that the interior Sindhi population had a closer genetic relationship with Pakistani populations and distant relationships with regional (India and Afghanistan) populations. The present findings exhibited that STRs included in AmpFLSTR Identifiler kit (Applied Biosystems) are genetically polymorphic in the interior Sindhi population of Pakistan. This study provides valuable population genetic data for the genetic information study, forensic human individual identification and paternity testing.