Extended evaluation of the impact of dropout probability on likelihood ratios calculated in parent-child and full sibling analysis.

Population genetic analysis is a fundamental component in forensic genetics for ensuring the reliability and validity of DNA evidence interpretation. Here, we present a comprehensive forensic genetic analysis of a contemporary Swedish population, based on 37 autosomal, 12 X-chromosomal and 23 Y-chromosomal short tandem repeat (STR) loci. Blood samples were systematically collected from individuals across all 21 counties of Sweden. Five commercial forensic STR kits (PowerPlex Fusion 6C System, PowerPlex Y23 System, Investigator HDplex kit, Investigator X12QS kit and Mainstay SE) were analysed. In total, autosomal STR allele frequencies were generated from 538 individuals, and X- and Y-chromosomal haplotypes were established from 513 males. High genotype concordance (> 99.95%) was observed across overlapping loci between kits, confirming the robustness and reliability of the investigated systems. A few discrepancies were identified, primarily due to STR nomenclature differences between capillary electrophoresis and sequencing generated alleles. Both fragment length-based and sequence-based allele frequencies were obtained. Statistical analysis revealed high discriminatory power, no signs of deviations from Hardy-Weinberg equilibrium, and no evidence of linkage disequilibrium between any pair of loci. Minimal genetic differentiation was observed within Sweden and between Sweden and geographically close populations, whereas significant genetic differences were found when comparing with more distant populations. These findings confirm that the analysed STR loci are well suited for forensic applications in the Swedish population. The resulting allele and haplotype frequency data establish an updated and expanded population-specific reference database, which is expected to serve as a valuable resource for forensic casework in Sweden.

Short tandem repeats (STRs) are hotspots of genomic instability that mutate at rates orders of magnitude greater than non-repetitive loci due to frequent replication slippage. Expansions at some STR loci cause Mendelian diseases, while variation at other noncoding loci may affect complex traits, possibly by altering transcription factor occupancy of nearby binding sites. Accordingly, some STRs are inferred to be under purifying selection, regardless of their instability. One or more "interruptions", or bases that disrupt the locus's canonical repeat, significantly decrease an STR's mutability. For example, the onset of Huntington's Disease, a neurodegenerative disorder associated with somatic expansions of a trinucleotide coding STR, is delayed in individuals whose inherited alleles contain interruptions. Thus, interruptions that decrease mutation rate at some coding loci may broadly protect against deleterious phenotypes associated with locus instability. However, interruptions may themselves be deleterious at constrained loci, particularly at noncoding loci in gene regulatory elements, possibly disrupting the formation of secondary structures key to their function. We therefore hypothesized that the frequency of interruptions could depend on a locus's functional importance-at constrained loci, the fitness effects of expansions but also interruptions could be more deleterious than at neutral loci. To test this hypothesis, we examined the distribution of interruptions at ∼650,000 autosomal STRs. In the ∼2,500 3- or 6bp-motif coding STRs, we find that synonymous interruption density increases with purifying selection on the gene, while the opposite is true for missense-causing interruptions. In contrast, noncoding STRs in gene regulatory elements harbor fewer interruptions than elements that are unassociated with gene regulation and thus more likely to be evolving neutrally. Our findings indicate that the abundance of interruptions may be partially explained at coding STRs by the benefit of lower instability, whereas maintaining a core stretch of uninterrupted repeat may be key to the function of regulatory noncoding STRs, outweighing the benefits of stability.

The analysis of autosomal short tandem repeat markers (STRs) using capillary electrophoresis (CE) technology remains the dominant method for forensic investigations globally. However, next-generation sequencing technologies are increasingly being adopted as they enable simultaneous amplification of both STR and SNP (single-nucleotide polymorphisms) loci in large panels, amongst other benefits. Unlike STRs, which are highly variable or polymorphic, the majority of SNPs are biallelic, meaning there are only two allelic variants. A single SNP marker on its own is unlikely to provide as much evidentiary value as a single STR marker. However, the interpretation of SNP markers from mixed DNA evidence is computationally simpler than that of STR markers because they do not produce stutter artefacts and, being biallelic, there are fewer possible genotype combinations. SNPs are increasingly recognised as valuable markers, complementary to STRs, and have been proven to be useful for applications including kinship analyses, informing ancestry, providing phenotypic characters for investigative leads, or forensic investigative genetic genealogy. The most common approach for the interpretation of autosomal STR (aSTR) profiles developed using CE technology within the US, UK, and Australasia is now based on probabilistic genotyping methods. Some of these published and tested models have been applied to aSTR profiles developed using NGS technologies and in this paper, we investigate the application of one such model (STRmix™ NGS) to the interpretation of mixed SNP profiles. Comparisons of likelihood ratios (LRs) assigned to donors and non-donors show expected trends in single-source profiles. However, in more complex mixtures, the system's ability to differentiate between true and false donors diminishes quickly. This is consistent with theoretical limitations for biallelic markers. While this study is limited to the 94 iiSNPs (identity informative SNPs) included in the ForenSeq kit, the findings suggest that simple SNP profiles can be interpreted within the existing STRmix™ NGS framework. This provides another potential tool for SNP profile analysis and lays the foundation for future joint interpretation of STR and SNP markers, or assays with more SNP markers.

Autosomal short tandem repeat (STR) markers remain the cornerstone of modern forensic genetics, providing exceptional power for individualization, kinship verification, and reconstruction of complex investigative cases. Over the last decade, the field has undergone a major technological transition from length-based capillary electrophoresis (CE) toward sequence-level characterization using massively parallel sequencing (MPS), enabling detection of internal sequence variants (isoalleles) and flanking-region polymorphisms that substantially increase discriminatory power in many forensic contexts. Although MPS is increasingly adopted in forensic laboratories, implementation remains dependent on infrastructure, cost considerations, validation requirements, and jurisdiction-specific legal frameworks. This review synthesizes the molecular mechanisms underlying STR variability, including replication slippage and mutation processes, and critically evaluates the transition to sequencing-based analysis. Particular attention is given to analytical challenges such as stochastic effects in ultra-low-template DNA and PCR inhibition in degraded samples. Special emphasis is placed on identification of skeletal remains from mass graves and historical contexts, where hierarchical analytical strategies-from mini-STR approaches to MPS-based workflows-enable recovery of highly fragmented DNA. The review also examines the evolution of probabilistic genotyping (PG), highlighting the importance of algorithmic transparency and reproducible analytical frameworks for judicial applications. By integrating technological advances with practical forensic challenges, this review outlines a comprehensive framework for implementing high-resolution STR analysis in contemporary genomic casework. As a narrative synthesis, the conclusions reflect currently available published evidence and acknowledge variability in validation status, implementation practices, and regional forensic infrastructures.

Background/Objectives: The Casablanca-Settat region of Morocco, located at the interface between Arab and Amazigh cultural zones, has only recently been investigated using autosomal short tandem repeat (STR) markers. The objective of this study was to characterize the genetic diversity and forensic efficiency of 15 autosomal STR loci in the Casablanca-Settat population and to evaluate its genetic relationships with other Moroccan populations. Methods: Fifteen autosomal STR loci were genotyped in 138 unrelated Arabic-speaking individuals from the Casablanca-Settat region. Allele frequencies, Hardy–Weinberg equilibrium, and standard forensic parameters were calculated. The genetic structure of the population was further examined through comparative analyses with 12 previously published Moroccan reference populations using multivariate and phylogenetic approaches. Results: A total of 146 distinct alleles were identified across the 15 loci. D18S51 was the most polymorphic marker (Ho = 0.9203), whereas D3S1358, TPOX, D5S818, and D16S539 exhibited lower allelic diversity. No statistically significant deviation from Hardy–Weinberg equilibrium was detected after correction for multiple testing. The combined power of discrimination exceeded 0.99, and the combined power of exclusion reached 0.99999965, demonstrating the high forensic efficiency of the STR panel. Population structure analyses positioned the Casablanca-Settat population within an intermediate genetic cluster, closely related to central Moroccan populations, consistent with historical gene flow and admixture. Conclusions: This study provides robust autosomal STR reference data for the Casablanca-Settat population, confirming the suitability of these markers for forensic identification in Morocco and offering valuable insights into regional population structure and genetic diversity.

This study reports novel 21 aSTR (autosomal Short Tandem Repeats) allele frequencies from 538 individuals, as well as 11 triallelic profiles, representing seven Bantu-speaking groups in Southern Africa (Ndebele, Pedi, Phuthi, Tsonga, Sotho, Swati, and Xhosa). These data contributed to a comprehensive representation of the Southern Bantu (SB). The defined SB reference database was evaluated for various forensic uses and applications: extant diversity, population structure, adequacy of alternative reference databases, and continental biogeographical ancestry prediction. Different analytical methods-including summary statistics, multivariate analyses (Multidimensional Scaling, MDS; Discriminant Analysis of Principal Components, DAPC), and Bayesian clustering-detected continental structure, identifying four major clusters: Southern, Eastern, Western, and Horn of Africa. This observation motivated the evaluation of two practical applications of this information: one methodological (alternative reference frequency database) and one predictive (biogeographic assignment). The adequacy of alternative reference databases for representing SB populations-STRidER South Africa, STRidER Africa, African American, and global datasets-was assessed by comparing reciprocal allelic coverage and shifts in random match probabilities (RMPs). Of the databases tested, the STRidER Africa database provided the closest representation of the SB. Population-level analyses evidenced the need for a stratification correction (θ = 0.005 or 0.01) for SB populations. Intracontinental biogeographic prediction was assessed using an XGBoost machine learning classification model across four major African regions. The model's predictive balanced accuracy ranged from 80 % to 94 % across African regions (94 % for the Horn of Africa, 87 % for Southern Africa, 84 % for Western Africa, and 80 % for Eastern Africa). The accuracy and limitations of this practice are discussed, along with its ethical implications. The assessment of reference databases can be extended to more general applications across Africa.

Efficiency of STR markers in complex kinship investigations.

Nanopore sequencing has emerged as a promising technology due to its real-time data acquisition, portability, and high throughput. However, the genotyping capabilities of the latest flow cell, R10.4.1, for short tandem repeats (STRs) remain insufficiently validated, and its accuracy in typing insertions or deletions (InDels) in earlier versions has been rarely investigated. To bridge these gaps, we developed the NanoID panel, a multiplex amplification system incorporating 29 autosomal STRs, 29 Y-chromosome STRs, 61 autosomal InDels, 2 Y-chromosome InDels, and amelogenin. We conducted genotyping on 112 unrelated individual samples and screened 114 loci with 100% accuracy. Subsequently, we evaluated these 114 loci for reproducibility, sensitivity, kinship inference, and species specificity. All loci were consistently and accurately genotyped across triplicate experiments. NanoID achieved an accuracy rate exceeding 99.12% (based on the 2-out-of-3 rule) when the DNA input was ≥ 50 pg. The combined power of discrimination and the cumulative probability of exclusion were 1 - 7.990 × 10-57 and 1 - 2.299 × 10-16, respectively. For full-sibling kinship testing, the sensitivity, specificity, and accuracy reached 100% at likelihood ratio (LR) cutoff values of 0.0001 and 10000. Nonhuman samples were clearly distinguishable from human samples. These findings strongly support the NanoID system's effectiveness for individual identification and full-sibling kinship analysis using nanopore sequencing.

Revisiting guidance on population sampling for highly polymorphic STR loci.

Short Tandem Repeats (STRs) are the gold standard for human identification. They are widely used in crime scene investigations, kinship testing, and disaster victim identification due to their high polymorphism, sensitivity, and discrimination power. Combining autosomal STRs and sex-specific STRs (Y-STRs and X-STRs) across multiple tissues yields highly informative genetic profiles. Y-STRs, which target Y-chromosome DNA inherited through the paternal line, are particularly useful when autosomal STR profiling is inconclusive. They enhance the reliability of the results in establishing patrilineal relationships of male offspring, paternal kinship testing, and missing persons and disaster victim identification, as well as in sexual assault cases when male DNA is masked by female DNA. In complex mixtures involving male victims or multiple male contributors, autosomal DNA profiling is challenging. Even with advanced probabilistic genotyping software, separating these mixtures can still be limited due to issues like template imbalance, low quantity DNA, or degraded DNA. In such cases, Y-STR analysis serves as a valuable complementary tool. However, Y-STRs cannot distinguish between closely related males within the same paternal lineage, limiting their individualising power. Their evidentiary value also depends on robust population-specific databases for accurate statistical interpretation. This review outlines the practical uses and limitations of Y-STR analysis, emphasising the importance of population databases such as the Y Chromosome Haplotype Reference Database in evaluating evidentiary weight. Based on these considerations, this study highlights the effectiveness of the PowerPlex® Y23 system, as a valuable complementary tool to autosomal STRs, particularly in challenging cases where autosomal results alone are insufficient.

Short tandem repeats (STRs) are central to human identification and kinship analysis owing to their high polymorphism. We report the developmental validation of the SF28CS typing system, a novel six-dye multiplex PCR assay that simultaneously amplifies 28 loci, including 23 autosomal STRs (18 expanded CODIS loci, Penta D, Penta E, and three additional markers), three Y-STRs (DYS391, DYS576, DYS518), one Y-chromosomal insertion/deletion marker, and the Amelogenin locus for sex determination. Validation was conducted in accordance with SWGDAM guidelines using the Applied Biosystems 3500 Genetic Analyzer. The system demonstrated high sensitivity, generating complete profiles with as little as 62.5 pg of DNA, and successfully genotyped challenging forensic samples such as bloodstains, semen stains, and decade-old materials. It showed strict species specificity and stability under common PCR inhibitors, including calcium and EDTA. Mixture studies indicated reliable resolution at contributor ratios up to 1:4. A population study of 500 unrelated individuals further confirmed the high discriminatory power and informativeness of the assay, supporting its application in forensic databases. Compared with existing multiplex kits, the SF28CS exhibited superior robustness for degraded samples and complex mixtures, while its inclusion of Y-chromosomal markers enhances its utility in kinship testing and male identification. These results establish the SF28CS system as a sensitive, reliable, and comprehensive tool for forensic casework and human identification.

Background: The Tawahka ethnic group, with approximately 2690 individuals in northeastern Honduras, represents one of the country’s smallest indigenous communities. No genetic studies have been published on this population, and population-specific databases are essential for forensic applications. Methods: Allele frequencies for 23 autosomal short tandem repeats (STRs) loci were analyzed in 100 unrelated Tawahka individuals (61 females, 39 males) from the municipality of Wampusirpi. Deoxyribonucleic acid (DNA) was extracted from blood on Fast Technology for Analysis of nucleic acids (FTA) cards and amplified using the PowerPlex Fusion 6C System. Statistical parameters were calculated using Genepop v4.2 and Arlequin v5.3.2.2. Results: All loci showed substantial polymorphism with no Hardy–Weinberg equilibrium deviations after Bonferroni correction (α = 0.0022). Expected heterozygosity ranged from 0.4968 to 0.8812. Combined power of discrimination was 99.9999% and combined chance of exclusion was 99.99%. Conclusions: This first genetic characterization of the Tawahka population provides essential reference data for forensic identification, paternity testing, and population genetics studies. The dataset contributes to understanding indigenous Central American genetic diversity and ensures accurate forensic analyses for individuals of Tawahka ancestry following Combined DNA Index System (CODIS) and European Standard Set (ESS) standards.

Since COVID-19 has emerged and become a global health issue, an awareness of wearing face masks has been attentive to prevent the spread of the disease. Face masks that have become a part of daily life, may be encountered at crime scenes and serve as a potential source of DNA for human identification. This study developed a rapid method for obtaining abundant DNA from 3-layer disposable face masks. Ten healthy volunteers were recruited to wear the masks for a period of 2h. The optimal method for retrieving DNA was determined to be direct cutting of the middle section of the mask's inner layer. The average DNA concentrations from males and females were 0.127 0 ± 0.233 7ng/μL and 0.069 9 ± 0.107 4ng/μL, respectively, with no significant difference observed between the sexes (P = 0.286). Furthermore, testing a smaller area (28cm2) from the mouth-covering region still yielded sufficient DNA for STR genotyping (average 0.025 5 ± 0.021 8ng/μL). Comparison of the inner and the filter layers revealed that the filter layer contained significantly less DNA (average 0.021 3 ± 0.020 3ng/μL, P < 0.05). A mockup study was conducted on two female volunteers using six commercial brands of medical, carbon, and super 3D mask types. The results showed that the minimum DNA concentration (0.008 3 ± 0.003 1ng/μL) extracted from a carbon type is also plenteous for short tandem repeats (STR) genotyping. Finally, mockup samples with low copy number were genotyped and they produced profiles with >19 autosomal STR loci. This suggests that the method was suitable in DNA analysis when a face mask is found as forensic DNA evidence.

Context Population-specific characteristics play a crucial role in shaping the behavior of human genotypes and phenotypes. Examining genetic relationships between populations provides insight into patterns of genetic change over time. Such comparisons help identify factors that may have influenced the evolution of specific traits, genotypes, and the overall genetic diversity of populations. Despite Nigeria’s ethnic diversity, the genetic diversity of these groups remains largely undefined. Short tandem repeat (STR) markers offer a powerful tool for characterizing such diversity. Aims To determine the genetic relationships between the Igbo, Ibibio, Yoruba, Tiv, and Hausa ethnic groups using nine autosomal short tandem repeat (STR) DNA markers. Settings and Design An observational study involving 250 consenting participants from five major Nigerian ethnic groups. Methods and Materials Participants of Igbo, Yoruba, Hausa, Ibibio, and Tiv ethnic origin were randomly selected from their native communities across Nigeria. DNA Extraction: Performed on whole blood using commercial DNA extraction kits. STR Analysis: Nine autosomal STR loci were amplified using PCR and analyzed via electrophoresis. Allele Interpretation: Allele types and sizes were read, recorded, and scored for each individual across all loci. Statistical Analysis Allele frequencies, population pairwise genetic distances (FST and RST), analysis of molecular variance (AMOVA), and principal component analysis (PCA) were computed using GenAlEx v6.502. Results Fixation index (FST) values ranged from 0.001 to 0.500 across the populations. AMOVA revealed that 99.98% of total genetic variation occurred within individuals, while only 0.10% occurred among populations. PCA identified four heterogeneous genetic clusters, with the first three axes accounting for 32.86% of genetic variation. Conclusions Genetic relationships among the Igbo, Ibibio, Yoruba, Tiv, and Hausa closely mirrored their linguistic classifications. This suggests that language may have influenced historical patterns of interaction, gene flow, and ultimately the genetic structure of these populations.

Short tandem repeat (STR) is a significant genetic marker for the identification of forensic DNA. DNA databases worldwide, including those in China, are established based on STR markers. Length- and sequence-based polymorphism are two features of STR markers. Sequence-based polymorphism includes polymorphisms in both repeat and flanking regions. Traditional capillary electrophoresis-based STR genotyping method can only profile length-based genotypes. However, a deep understanding of the sequence polymorphism of core STR loci is crucial for primer design and DNA identification. Firstly, single nucleotide polymorphisms and insertions/deletions in STR primer binding regions may reduce the affinity between primers and DNA templates, leading to allele dropout or poor interlocus balance, thereby impacting the accuracy of DNA identification. Secondly, sequence-based polymorphism can be unveiled by next-generation sequencing technology, which could significantly enhance the detectable polymorphic information of core STR loci and improve the efficiency of individual identification and kinship analysis. Thirdly, different populations exhibit distinct STR sequence characteristics. Over the past decade, studies on sequence-based polymorphisms of STR loci have increased alongside the application of next-generation sequencing technology, and sequence-based polymorphisms from multiple populations were reported. However, previously studied populations and data were scattered, and different formats of repeat region sequences were used in various studies. These limitations result in the absence of a systematic summary and analysis of sequence polymorphism for core STR loci, hindering its further application in forensic practices. A comprehensive understanding of core STR loci sequence characteristics is crucial for individual identification from trace DNA, deconvolution of mixed samples, and determination of mutation origins in paternity testing. In this review, we focus on 19 autosomal core STRs and systematically review the sequence polymorphisms of these loci based on population data reported in the literature. We summarize variations in repeat regions, analyze variation patterns, present high-frequency variations in flanking regions within the Chinese population, and discuss potential challenges encountered in STR sequence analyses, with the aim to provide a reference for the analyses and application of STR sequence, the identification of rare alleles in criminal case testing, and the development of STR genotyping panel.

This study investigates the cause of a non-Mendelian inheritance pattern observed at short tandem repeat (STR) loci on chromosome 2 in a paternity test and aims to provide a definitive identification opinion. STR genotyping was performed using the AGCU Expressmarker 22, Goldeneye DNA Identification System 22NC, and AGCU X Plus STR kits. Additionally, chromosomal microarray analysis (CMA) was used to assess copy number variations and regions of homozygosity (ROH). The cumulative paternity index based on 21 autosomal STR loci (AGCU Expressmarker 22) was 8.4361E-04. When combined with the 39 STR loci from the Goldeneye System, the index increased to 3.1806E + 04. All discordant loci-D2S441 and D2S1338-were located on chromosome 2, where the alleged father had genotypes 11,11.3 and 17,19, respectively, while the child had 14,14 and 24,24. The TPOX locus (also on chromosome 2) showed homozygosity (8,8) in the child. X-STR analysis confirmed that the alleged father contributed all necessary alleles. CMA revealed a 243.03Mb ROH spanning 2p25-q37.3 in the child, suggesting identity by descent or uniparental disomy. These findings highlight the potential for chromosomal anomalies to mimic false exclusions in paternity testing. Comprehensive genetic analysis is recommended when inconsistencies arise at multiple loci on the same chromosome.

India's genomic diversity is influenced by regional population dynamics and strong endogamy. The present study explores the genetic structure of the Brahmin population in Gujarat, a culturally and genetically preserved community, using autosomal Short Tandem Repeats (STR). Genomic DNA from the blood samples of 819 healthy individuals (562 males and 257 females) were subjected to autosomal STR typing using the GlobalFiler™ Express PCR Amplification Kit. Capillary electrophoresis-based fragment analysis was performed using the ABI 3500 Genetic Analyser, and proceeded to genotype analysis using GeneMapper ID-X. In total, 74 alleles were assessed with observed heterozygosity (Ho) of 0.80 ± 0.01, expected heterozygosity (He) of 0.79 ± 0.02, and unbiased expected heterozygosity (uHe) of 0.79 ± 0.02. Hardy-Weinberg equilibrium was followed by most of the markers except D16S539 and D3S1358. Forensic efficiency measures (PIC = 0.63-0.95; PD = 1; PE = 0.99999) confirmed the significance of these markers for genetic and forensic research. Additionally, comparative genomic analysis using principal component analysis (PCA) and multidimensional scaling (MDS) revealed close genetic affinity between Brahmins of Gujarat and Brahmins from Haryana and Rajasthan. These findings enhance the genetic makeup of the Brahmin population of Gujarat. Additionally, a novel germline-origin Type 2 tri-allelic pattern (8, 9, 11) at the D13S317 locus was identified in a healthy male participant, which was traced to a Type 2(B) tri-allelic variant at the same locus in his biological mother. These findings highlight the need for understanding the tri-allelic pattern for its appropriate interpretation in STR analysis.

Alcohol use disorder (AUD) arises from complex interactions among genetic, cultural, and environmental factors. Baghdad’s diverse population provides an ideal setting to explore these dynamics. We genotyped 23 autosomal short tandem repeat (STR) loci in 200 alcohol‑dependent individuals and 50 controls from Baghdad using the PowerPlex®23 System between January2023 and January2025. Locus PentaE exhibited the highest gene diversity (GD=0.962) and discrimination capacity (DC=0.9310), whereas DYS391 and D10S1248 showed lower polymorphism (GD=0.850). The combined random‑match probability across all loci was 9.66×10⁻²⁶. These results demonstrate that our 23‑locus STR panel delivers robust forensic resolution and establishes a reference dataset for future population‑genetic and AUD‑related research in Iraq.

Analysis of individual single sperm can assist in mixture deconvolution when there are multiple perpetrators to a sexual assault or instances in which limited sperm are available for analysis. This not only allows for reconstitution of the sperm donor's genotype but also allows for microscopic confirmation that the cell typed was a sperm cell. Single sperm analysis can be challenging due to sperm's low DNA template amount (~3.3 pg) and haploid nature, allowing for only half the donor's genotype to be detected in a single sperm cell. This can make clustering single sperm by donor difficult, as each sperm sample results in a different multilocus haplotype; although, some degree of allele sharing is expected between single sperm from the same donor. In the present work, FaSTR™ was validated for single sperm analysis. The database matching and sample-to-sample matching features of FaSTR™ were used to develop a clustering method to group autosomal short tandem repeat (aSTR) genotyped single sperm by donor. Once clustered, probabilistic genotyping replicate analysis (STRmix™) was conducted in a proof-of-concept investigative manner, resulting in highly probative single source 99% component diploid DNA genotypes which could be used for database (e.g., known offender database) searching when a reference is unavailable. This approach was applied to two different 2-, 3-, and 4-person semen mixtures (total of 6 mixtures) resulting in the recovery of highly probative single source genotypes.