Y-STRs are used in forensic genetics to investigate male trace material in cases where no useful autosomal STR profiles can be established. While non-matching suspects can be safely excluded from trace donorship this way, interpreting Y-STR haplotype matches is less straightforward. Equating the corresponding Y-STR match probabilities to haplotype frequencies estimated from population databases is not only common practice, but is also recommended by the International Society for Forensic Genetics. However, this approach ignores that population databases, in principle, cannot be representative of all plausible alternative suspects in each and every case. Therefore, we previously introduced a novel mathematical framework for calculating Y-STR match probabilities drawing upon the suspect's male pedigree, thereby obviating the use of haplotype frequency estimates from population databases for this purpose. Here, we present the implementation of this framework into a publicly available software tool, named MatchY. Expanding the original approach in various ways, the tool can handle any number of single or multi-copy Y-STRs with known mutation rates and allowing both one-step and two-step mutations. MatchY can calculate match probabilities for pedigrees of any size and complexity based upon the haplotype information of its typed members, while simulating haplotypes of untyped ones. In addition to considering all plausible trace donor candidates within the pedigree, the tool can also consider a hypothetical, most closely related candidate from outside the pedigree. The performance of MatchY has been tested using various marker sets and example pedigrees. Together, these features make MatchY a practical and formally correct tool for the interpretation of Y-STR matches by calculating Y-STR match probabilities based on the suspect's male pedigree.

The formal assessment of a genetic match between a suspect and some biological trace material is one of the key tasks of forensic genetics, particularly in cases of sexual offence. The analysis of Y-chromosomal short tandem repeats (Y-STRs) has proven especially useful in this context. For a long time, however, calculating the probability of a perfect Y-STR profile match under the defense hypothesis that the suspect was not the trace donor posed a great challenge. This was due to the inherent uncertainty about the population of alternative donors, the so-called suspect population. We recently proposed to resolve this controversy by systematically favoring the suspect and considering his close male relatives as the suspect population. However, since the mathematical framework developed for this purpose was simulation-based, its practical application turned out increasingly difficult with increasing pedigree size. Here, we present an adaptation of the so-called Elston-Stewart algorithm, originally developed for the linkage analysis of human genetic diseases, to allow calculation of exact match probabilities in a time that scales linearly with pedigree size. The adapted algorithm was implemented in a publicly available software tool, and its correctness was verified by the comparison of its output with the correct, analytical results obtained for selected example pedigrees. The new implementation mostly outperforms the simulation-based solution, albeit with the important exception of Y-STRs present in multiple copies. Given the increasingly prominent role of such multicopy markers in forensic genetics, the complementary use of both approaches appears the most sensible strategy for the time being.

Y-chromosomal short tandem repeats (Y-STRs) with elevated mutation rates are valuable markers for distinguishing male suspects from their paternal male relatives - something that is typically not possible with standard Y-STRs. However, while the 26 rapidly mutating Y-STRs (RM Y-STRs) we identified in our two previous screens substantially improve male relative differentiation compared to standard Y-STRs, many close relatives cannot be separated with these markers. Aiming to further enhance the discrimination power of male relatives, particularly closely related ones, we performed a new chromosome-wide search for Y-STRs with elevated mutation rates by integrating in-silico marker discovery with experimental marker verification. Relative to previous screens, three major advancements were applied: (1) use of the Y-chromosome sequence from the telomere-to-telomere reference genome and other genomes, (2) consideration of all repeat motifs from homopolymers to hexanucleotides, and (3) use of targeted massively parallel sequencing to genotype male relatives for marker verification. To ensure robust allele calling and mutation detection for dinucleotide repeats prone to PCR slippage, we developed and applied a novel curve-fitting approach that accounts for all observed signals: true alleles and stutter products. Overall, we identified 14 novel Y-STRs with previously unreported elevated mutation rates, most of which were dinucleotide repeats. Relative to the 30-marker set of the RMplex tool, this novel set increased the empirical differentiation rates of close relatives separated by 1-4 meioses by 20.0%, 15.6%, 9.9% and 4.1%, respectively. The combined set of 44 novel and previous markers empirically differentiated 46.9%, 80.4%, 86.4%, and 84.8% of close relatives separated by 1-4 meioses and 96.4-100% of distant relatives separated by 5-15 meioses. The differentiation capacities of this 44-marker set, estimated from locus-specific mutation rates, were 50.2%, 75.2%, 87.6%, and 93.8% for these close relatives and 96.9-100% for the distant ones. Provided the development and successful forensic validation of a targeted genotyping tool, we anticipate that this expanded set of 44 Y-STRs with elevated mutation rates will enhance the ability to distinguish a male suspect from his paternal male relatives. This will benefit solving criminal cases where an autosomal STR profile of the male perpetrator cannot be generated and where standard Y-STR profiling yields a haplotype match between the suspect and the trace as well as haplotype sharing between the suspect and his male relative(s).

While widely applied in forensic practice, equating Y-STR match probabilities with haplotype frequencies derived from population databases has been considered inappropriate, as these databases cannot be representative of each and every case-specific suspect population. To address this inherent limitation of the forensic application of Y-STRs, we previously introduced a mathematical framework for calculating Y-STR match probabilities based on the suspect’s male pedigree. Recently, we extended and implemented this framework in the publicly available MatchY software tool, which allows users to calculate match probabilities for any Y-STRs with known mutation rates and for pedigrees of varying size and complexity. Here, we present a comprehensive exploration of marker and pedigree-based factors impacting the Y-STR match probability outcomes obtained with MatchY. Moreover, we introduce another application of MatchY, the trace mode, aimed at forensic intelligence rather than evidential value computation. These insights provide valuable guidance on the practical use of MatchY for calculating Y-STR match probabilities in routine forensic casework for cases both with and without suspects.

For decades, there has been scientific interest in the variation and geographic distribution of paternal lineages associated with the human Y chromosome. However, the relevant data have been dispersed across numerous publications, making it difficult to consolidate. Additionally, understanding the relationships between different variants, and the tools used to analyze them, have evolved over time, further complicating efforts to harmonize this information. The Universal Y-SNP Database (UYSD) marks a substantial advancement by providing a comprehensive and accessible platform for Y-SNP and haplogroup data from populations around the world. UYSD harmonizes diverse datasets into a unified repository, facilitating the exploration of global Y-chromosomal variation. The platform handles data generated with both high- and low-throughput technology and is compatible with the automated analysis software tool, Yleaf v3. Key functionalities include the ability to: i) visualize haplogroup distributions on an interactive world map, ii) estimate haplogroup frequencies in geographic regions with sparse data through interpolation, and iii) display detailed phylogenetic trees of Y-chromosomal haplogroups. Currently, UYSD encompasses data from over 6,600 males across 27 populations. This dataset largely aligns with known global Y-haplogroup patterns, but also reveals unexplored finer-scale geographic variations. While the present dataset is largely European-centered, UYSD is designed for ongoing expansion by the scientific community, aiming to include more global data and higher-resolution population sequencing data. The platform thus offers valuable insights into human genetic diversity and migration patterns, serving several fields of research such as: human population genetics, genetic anthropology, ancient DNA analysis and forensic genetics.

Y-chromosomal short tandem repeat (Y-STR) markers are routinely used in forensic casework to identify male donors of biological traces left at crime scenes, particularly in sexual assault cases. However, the evidential value of a match between the Y-STR profile of a trace and a potential donor, usually a crime suspect, is difficult to quantify, and the common albeit inappropriate practise to equate Y-STR match probabilities with Y-STR profile frequencies estimated from population databases has been subject to scientific debate for decades. As a solution to this long-standing problem, we suggest an alternative approach to the calculation of Y-STR match probabilities that involves splitting the group of potential donors other than the suspect into two: (i) his close male relatives (termed his ‘pedigree’) and (ii) all other males. While an upper limit to the match probability is easily calculated for the second group, it is computationally challenging to derive for the first. We therefore developed a mathematical framework that uses importance sampling to reconstruct and evaluate the Y-STR profiles of untyped members of the suspect’s pedigree by way of simulation. Extensive testing with elementary pedigrees of different structure and complexity confirmed that both, the framework and its Python-based software implementation yield match probability estimates that approximate well the correct analytical results, depending upon the number of simulations performed. Our methodology thus facilitates a more appropriate and valid solution to the long-standing problem of interpreting Y-STR profile matches in forensic casework.

ABSTRACT In patients receiving allogenic hematopoietic stem cell transplantation (aHSCT) complete chimerism is desired. However, this brings a challenge when non‐invasive assessment of recipient germline DNA is required for genetic testing. Here, we aim to create awareness for (pharmaco)genetic sampling in patients following aHSCT based on a case report of a patient following aHSCT in which pharmacogenetic analysis was performed. Six pharmacogenetic genes were analyzed in DNA extracted from peripheral blood (pre‐ and post‐transplant), buccal swab, and hair follicles. To investigate the presence of donor DNA in post‐transplant samples, short tandem repeat and digital droplet PCR analysis were performed. DNA from post‐transplantation peripheral blood and the buccal swab showed identical genotypes: CYP2C9*1/*1, CYP2C19*1/*2, CYP2D6*2/*9, CYP3A4*1/*1, VKORC1‐1639TT, SLCO1B1*1/*5. Pre‐transplant DNA showed different genotypes for CYP2D6 (*1/*2) and SLCO1B1 (*1/*1). Due to the low DNA amount extracted from hair follicles, only CYP2D6 and SLCO1B1 were examined, showing identical genotypes to pre‐transplantation DNA. Short tandem repeat analysis showed that the buccal swab contained DNA from both donor and recipient. It was estimated that the buccal swab contained ~63% donor DNA, while DNA from hair follicles showed 0% donor DNA. In conclusion, pharmacogenetic profiling after allogenic HSCT should be done with consideration. Analysis of pre‐transplant peripheral blood is preferred over buccal swab due to the presence of donor DNA contamination. DNA extracted from hair is also a reliable source; however, application might be restricted due to limited DNA yield.

Y-chromosomal short tandem repeats (Y-STRs) at rapidly mutating (RM) loci have been suggested as tools for differentiating paternally related males. RMplex is a recently developed system that incorporates 26 RM loci and four fast-mutating (FM) loci, targeting 44 male-specific loci. Here, we evaluated the RMplex by estimating Y-STR mutation rates and the overall differentiation rates for 542 Korean father-son pairs, as well as the genetic population values for 409 unrelated males. RMplex performed well, distinguishing 50.7 % of the father-son pairs by at least one mutation, a value 10 times higher than the previously reported differentiation rate achieved using the PowerPlex® Y23 System. Of the 369 mutations, 361 (97.8 %) were single-step mutations, with locus-specific mutation rates varying from 1.8 × 10-3 to 1.1 × 10-1 mutations per generation, and an average mutation rate of 2.3 × 10-2. Gene diversity values ranged from 0.5696 for DYS442 to 0.9970 for DYF1000, and the haplotype discrimination capacity of unrelated males was 100 %. Among the loci studied, DYS712 exhibited the highest mutation rate in this study of the Korean population. Similarly, the mutation rate of this locus is reported to be substantially higher for the Japanese and Chinese populations than for European populations. These findings suggest that DYS712 mutations are relatively frequent in East Asian populations. Although we did not detect significant relationships among the Y-chromosome single nucleotide polymorphism-based haplogroups, allele length was strongly correlated with the mutation rate at DYS712, which is consistent with previous studies. Although the incorporation of multi-copy loci into RMplex contributed significantly to the high mutation rates detected and to its discrimination capacity, this requires careful interpretation, owing to the potential for duplications. Nonetheless, these findings provide evidence regarding the suitability of the RMplex for distinguishing paternally related males in the Korean population.

The principal limitation of forensic Y-STR analysis, which identifies a male lineage rather than an individual man, is being addressed by the discovery and application of rapidly mutating Y-STRs (RM Y-STRs). Due to their higher mutation rates compared to standard Y-STRs used in forensics, RM Y-STRs significantly enhance the ability to differentiate between male relatives. However, some male relatives - particularly closely related ones - remain indistinguishable. Given the design and execution of the two previous RM Y-STR searches that discovered the 26 currently known RM Y-STRs, it is unlikely that future searches will largely increase the number of RM Y-STRs. To address the ongoing forensic challenge of differentiating between male relatives using Y chromosome analysis, this study explorers an alternative approach: Y-chromosomal singe nucleotide variants (Y-SNVs) obtained via whole genome sequencing (WGS). To assess the feasibility of the WGS technology in differentiating closely and distantly related males, we sequenced DNA samples of 24 male individuals belonging to three deep-rooted pedigrees, covering 12 father-son pairs and 72 pairs of distant male relatives separated by 8-15 meioses. Among the 76 meioses analyzed in total, 90 male relative-differentiating Y-SNVs were identified across the approximately 25 Mbp Y chromosome sequence generated per sample. A total of 141 male relative-differentiating Y chromosome mutations were observed when also considering Y-STRs from Yfiler Plus, RMplex, and WGS analyses. Of the 12 father-son pairs, six (50 %) were differentiated by one or more Y-SNVs, and 9 (75 %) with WGS and CE methods combined. All of the 72 pairs of distant male relatives were distinguished both through Y-SNVs and RM Y-STRs. Overall, when compared to RMplex, WGS yielded a 1.7-fold increase in the number of observed mutations in father-son pairs and a 4-fold increase in distantly related males. Our proof-of-principle study demonstrates (i) the feasibility and high value of Y-SNV markers and WGS technology in differentiating both close and distant male relatives; (ii) the superior performance of Y-SNVs from WGS relative to the previously used RM Y-STR markers and RMplex method; and (iii) the enhanced male relative differentiation achieved by combining both marker types and methods. We envision WGS as the method of choice for maximizing male relative differentiation based on Y chromosome information in high-profile criminal cases with male suspects where no autosomal STR profiles are available and where standard Y-STR and RM Y-STR analyses fail to distinguish the suspect from his male paternal relatives.

Although national criminal offender DNA databases (NCODDs) including autosomal short tandem repeats (STRs) have been a successful tool to identify criminals for decades in many countries, yet there are many criminal cases they cannot solve. In cases with mixed male-female samples, particularly sexual assault, expanding NCODDs with Y-chromosomal STR (Y-STR) profiles allows database matching in the absence of autosomal STR profiles. Although Y-STR matches are not individual-specific, this can be largely overcome with rapidly mutating Y-STRs (RM Y-STR) allowing separation of paternally related men. Expanding NCODDs with Y-STR profiles is also beneficial for law enforcement in cases without known suspects via familial searching. Expanding NCODDs with Y-STR profiles may raise concerns about genetic privacy and fundamental human rights. A legal analysis of the European Convention on Human Rights revealed that when primarily for reidentifying convicted sex offenders, it would be in line with the case law of the European Court of Human Rights, while a generalized approach primarily for familial searching and involving all types of offenders may not. This paper aims to stimulate a debate among various stakeholders regarding the benefits and risks of expanding NCODDs with Y-STR profiles that in some countries has already been practically implemented.

Y-chromosome short tandem repeats (Y-STRs) are widely used in various forensic contexts and are considered the genetic tool of choice in case of unbalanced female/male mixtures, particularly in sexual assault cases. The introduction of a class of Y-STRs featuring unusually high mutation rates, named rapidly mutating Y-STRs (RM Y-STRs), has made it possible to significantly increase the discrimination capacity (DC) among both unrelated and related males. In our previous studies, we observed a low DC based on Y-STRs in African populations. Specifically, the analysis of 1369 males from 75 populations of East, North and sub-Saharan Africa resulted in a relatively low discrimination power based on 25 Y-STRs included in the Yfiler Plus PCR Amplification Kit (DC = 0.898), which we strongly increased by the subsequent analysis of additional first-generation RM Y-STRs (DC = 0.958). In the present study, we applied 30 Y-STRs with increased mutation rates (16 of which had not been analysed in our previous studies) included in the recently described RMplex tool to 107 African males who share Y-chromosome haplotypes based on the 34 previously analysed YSTRs. By raising the number of analysed Y-STRs to a total of 50 (including all currently known 24 first and second generation rapidly mutating and 9 fast mutating Y-STRs), we were able to further discriminate 60% of the residual males, thereby increasing the overall DC in the total African dataset to 0.983. Notably, based on the available family information, with a single exception, all remaining males with shared haplotypes (43 out of 1369 that shared 20 haplotypes) were found to be paternal cousins or closer paternal relatives suggesting that the DC approaches the maximum value for unrelated males from the African continent. Exemplified in understudied Africans, our study underlines the power of RM Y-STRs in general, and the RMplex tool in particular, to differentiate among both unrelated and related males, as relevant in forensic and other Y-STR applications.

Y-chromosomal short tandem repeats (Y-STRs) are widely used in forensic, genealogical, and population genetics. With the recent increase in the number of rapidly mutating (RM) Y-STRs, an unprecedented level of male differentiation can be achieved, widening and improving the applications of Y-STRs in various fields, including forensics. The growing complexity of Y-STR data increases the need for automated data analyses, but dedicated software tools are scarce. To address this, we present the Male Pedigree Toolbox (MPT), a software tool for the automated analysis of Y-STR data in the context of patrilineal genealogical relationships. The MPT can estimate mutation rates and male relative differentiation rates from input Y-STR pedigree data. It can aid in determining ancestral haplotypes within a pedigree and visualize the genetic variation within pedigrees in all branches of family trees. Additionally, it can provide probabilistic classifications using machine learning, helping to establish or prove the structure of the pedigree and the level of relatedness between males, even for closely related individuals with highly similar haplotypes. The tool is flexible and easy to use and can be adjusted to any set of Y-STR markers by modifying the intuitive input file formats. We introduce the MPT software tool v1.0 and make it publicly available with the goal of encouraging and supporting forensic, genealogical, and other geneticists in utilizing the full potential of Y-STRs for both research purposes and practical applications, including criminal casework.

Motivation:We introduce SMapper, a novel web and software tool for visualizing spatial prevalence data of all types including those suffering from incomplete geographic coverage and insufficient sample sizes. We demonstrate the benefits of our tool in overcoming interpretational issues with existing tools caused by such data limitations. We exemplify the use of SMapper by applications to human genotype and phenotype data relevant in an epidemiological, anthropological and forensic context.Availability and implementation:A web implementation is available at https://rhodos.ccg.uni-koeln.de/smapper/. A stand-alone version, released under the GNU General Public License version 3 as published by the Free Software Foundation, is available from https://rhodos.ccg.uni-koeln.de/smapper/software-download.php as a Singularity container (https://docs.sylabs.io/guides/latest/user-guide/index.html) and a native Linux Python installation.

Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were suggested for differentiating patrilineally related men as relevant in forensic genetics, anthropological genetics, and genetic genealogy. Empirical data are available for closely related males, while differentiation rates for more distant relatives are scarce. Available RM Y-STR mutation rate estimates are typically based on father-son pair data, while pedigree-based studies for efficient analysis requiring less samples are rare. Here, we present a large-scale pedigree analysis in 9379 pairs of men separated by 1-34 meioses on 30 Y-STRs with increased mutation rates including all known RM Y-STRs (RMplex). For comparison, part of the samples were genotyped at 25 standard Y-STRs mostly with moderate mutation rates (Yfiler Plus). For 43 of the 49 Y-STRs analyzed, pedigree-based mutation rates were similar to previous father-son based estimates, while for six markers significant differences were observed. Male relative differentiation rates from the 30 RMplex Y-STRs were 43%, 84%, 96%, 99%, and 100% for relatives separated by one, four, six, nine, and twelve meioses, respectively, which largely exceeded rates obtained by 25 standard Y-STRs. Machine learning based models for predicting the degree of patrilineal consanguinity yielded accurate and reasonably precise predictions when using RM Y-STRs. Fully matching haplotypes resulted in a 95% confidence interval of 1-6 meioses with RMplex compared to 1-25 with Yfiler Plus. Our comprehensive pedigree study demonstrates the value of RM Y-STRs for differentiating male relatives of various types, in many cases achieving individual identification, thereby overcoming the largest limitation of forensic Y-chromosome analysis.

The discovery of rapidly mutating (RM) Y-STRs started to move the field of forensic Y-STR analysis from male lineage identification towards male individual identification. Previously, the forensic value of RM Y-STRs for differentiating male relatives was limited due to the modest number of 13 identified RM Y-STRs. Recently, new RM Y-STRs were discovered, with strong expectations for significantly improving male relative differentiation; however, empirical evidence is missing yet. More recently, the genotyping method RMplex for efficiently analyzing 30 Y-STRs with high mutation rates, including all 26 currently known RM Y-STRs, was introduced. Here, we applied RMplex as well as the current state-of-the-art commercial Y-STR kit: Yfiler™ Plus PCR Amplification kit, to several hundreds of DNA-confirmed father-son pairs. Newly established estimates confirmed the high mutation rates of novel and previous RM Y-STRs. By combining current with previous data, we provide updated consensus estimates of mutation rates for all 49 Y-STRs targeted with both methods. Based on RMplex, 42% of 499 father-son pairs were differentiated, while 14% of 530 pairs based on Yfiler™ Plus, and 48% of 499 pairs based on both methods combined. Regarding brothers, RMplex also clearly outperformed Yfiler™ Plus, with differentiation rates of 62% and 33%, respectively. By combining both methods 72.9% of the brothers showed at least one mutation. For unrelated males, both methods achieved a discrimination capacity of 99.8% and a haplotype diversity of 0.999991, since all males had different haplotypes, except for two, perhaps indicating a hidden paternal relationship. Overall, this study underlines the value of RM Y-STRs in general and RMplex in particular for differentiating male relatives highly relevant in forensic genetics. It provides the first empirical evidence on the high value of RMplex for differentiating close male relatives, which for father-son pairs was almost 60% higher than with the initial set of 13 RM Y-STRs and three times higher than with Yfiler™ Plus. Based on our results from closely related males, we expect RMplex to also improve the differentiation of more distantly related males significantly, which needs empirical demonstration in future studies. We encourage the forensic community to apply RMplex in all forensic cases where a match with a commercial Y-STR kit was obtained between the male suspect and the evidence material, or to solely use RMplex in such cases, aiming to find out if the male suspect or any of his male paternal relatives left the evidence material at the crime scene.

Rapidly mutating Y chromosomal short tandem repeat markers (RM Y-STRs) -characterized by at least one mutation per 100 generations- are suitable for differentiating both related and unrelated males. The recently introduced multiplex method RMplex allows for the efficient analysis of 30 Y-STRs with increased mutation rates, including all 26 currently known RM Y-STRs. While currently available RM Y-STR mutation rates were established mostly from European individuals, here we applied RMplex to DNA samples of 178 genetically confirmed father-son pairs from East Asia. For several Y-STRs, we found significantly higher mutation rates in Japanese compared to previous estimates. The consequent father-son differentiation rate based on RMplex was significantly higher (52%) in Japanese than previously reported for Europeans (42%), and much higher than with Yfiler Plus in both sample sets (14% and 13%, respectively). Further analysis suggests that the higher mutation and relative differentiation rates in Japanese can in part be explained by on average longer Y-STR alleles relative to Europeans. Moreover, we show that the most striking difference, which was found in DYS712, could be linked to a Y-SNP haplogroup (O1b2-P49) that is common in Japanese and rare in other populations. We encourage the forensic Y-STR community to generate more RMplex data from more population samples of sufficiently large sample size in combination with Y-SNP data to further investigate population effects on mutation and relative differentiation rates. Until more RMplex data from more populations become available, caution shall be placed when applying RM Y-STR mutation rate estimates established in one population, such as Europeans, to forensic casework involving male suspects of paternal origin from other populations, such as nonEuropeans.

It has been advocated before that appearance prediction of unknown suspects from crime scene DNA, in the context of Forensic DNA Phenotyping (FDP), is mostly suitable for single source DNA samples, whereas FDP from DNA mixtures to which more than one person contributed, is viewed challenging.With this report on a murder case, we practically demonstrate the feasibility of appearance DNA prediction of an unknown suspect from a mixed crime scene trace, to which the unknown suspect and the known victim had contributed.From this twoperson DNA mixture, we successfully predicted eye, hair and skin color of the unknown suspect with the HIrisPlex-S system by applying targeted massively parallel sequencing (MPS).We argue that at least three factors benefit appearance DNA prediction of unknown suspects from mixed crime scene traces, which were met in this murder case: i) SNP genotype knowledge from reference DNA analysis for one of the two persons in the mixture (here the known victim), ii) about equal DNA contributions by both donors to the mixed crime scene stain, and iii) the use of MPS allowing quantitative SNP analysis.Moreover, we show that additionally analyzing animal DNA in this mixed crime scene trace provides further investigative information.We envision that the investigative DNA strategy that we applied here for analyzing a two-person mixed crime scene trace in a murder case, will be applied in the future to more criminal cases with two-person DNA mixtures, for instance sexual assault cases.

Y-chromosomal short tandem repeats (Y-STRs) with high mutation rates are recognized as valuable genetic markers for differentiating paternally related men, who typically cannot be separated with standard Y-STRs, and were shown to provide paternal lineage differentiation on a higher resolution level than standard Y-STRs. Both features make Y-STRs with high mutation rates relevant in criminal casework, particularly in sexual assault cases involving highly unbalanced male-female DNA mixtures that often fail autosomal forensic STR profiling for the male donor. Previously, the number of known Y-STRs with mutation rates higher than 10(-2) per locus per generation termed rapidly mutating Y-STRs (RM Y-STRs) was limited to 13, which has recently been overcome by the discovery and characterization of 12 additional RM Y-STRs. Here, we present the development and validation of RMplex, an efficient genotyping system for analyzing 30 Y-STRs with high mutation rates, including all currently known RM Y-STRs, using multiplex PCR with capillary electrophoresis (CE) or massively parallel sequencing (MPS), overall targeting a total of 44 male-specific loci. If previously unavailable, repeat number assignations were provided based on newly generated MPS data. Validation tests based on the CE method demonstrated that the results were both repeatable and reproducible, full profiles were achieved with minimal input DNA of 250 pg for RMplex 1 and 100 pg for RMplex 2, and in the presence of inhibitors, or with a surplus of female DNA, the assays performed reasonably well. Application of RMplex to differentiate between paternally related men was exemplified in 32 males belonging to five different paternal pedigrees. Given further successful forensic validation testing, we envision the future application of RMplex in criminal cases where it is suspected, or cannot be excluded, that the crime scene trace originated from a male relatives of the suspect who is high-lighted with standard Y-STR matching. Other applications of RMplex are in criminal cases without known suspects to differentiate between male relatives highlighted in familial searching based on standard Y-STR matching.

Short tandem repeat polymorphisms on the male-specific part of the human Y-chromosome (Y-STRs) are valuable tools in many areas of human genetics. Although their paternal inheritance and moderate mutation rate (similar to 10(-3) mutations per marker per meiosis) allow detecting paternal relationships, they typically fail to separate male relatives. Previously, we identified 13 Y-STR markers with untypically high mutation rates (>10(-2)), termed rapidly mutating (RM) Y-STRs, and showed that they improved male relative differentiation over standard Y-STRs. By applying a newly developed in silico search approach to the Y-chromosome reference sequence, we identified 27 novel RM Y-STR candidates. Genotyping them in 1,616 DNA-confirmed father-son pairs for mutation rate estimation empirically highlighted 12 novel RM Y-STRs. Their capacity to differentiate males related by 1, 2, and 3 meioses was 27%, 47%, and 61%, respectively, while for all 25 currently known RM Y-STRs, it was 44%, 69%, and 83%. Of the 647 Y-STR mutations observed in total, almost all were single repeat changes, repeat gains, and losses were well balanced; allele length and fathers' age were positively correlated with mutation rate. We expect these new RM Y-STRs, together with the previously known ones, to significantly improving male relative differentiation in future human genetic applications.