Next generation sequencing of Y-STRs in father-son pairs and comparison with traditional capillary electrophoresis.

To evaluate the promising advantages of massively parallel sequencing (MPS) in our casework, we analysed a total of 33 Y-chromosomal short tandem repeats (Y-STRs) with traditional capillary electrophoresis (CE) and 25 Y-STRs using the newer MPS technology. We studied the outcome of both technologies in 64 father-son pairs using stock and custom-designed kits. Current MPS technology confirmed the 13 mutational events observed with CE and improved our understanding of the complex nature of STR mutations. By detecting isometric sequence variants between unrelated males, we show that sequencing Y-STRs using MPS can boost discrimination power.

RMplex reveals population differences in RM Y-STR mutation rates and provides improved father-son differentiation in Japanese.

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 non-Europeans.

Improving the differentiation of closely related males by RMplex analysis of 30 Y-STRs with high mutation rates.

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.

Efficiency analysis of VersaPlex™ 27PY system in Central Indian Population: First report from Indian population.

In the current scenario, DNA typing is the need of forensic science field due to its ability to provide results in much shorter time. In view of advancement of forensic DNA typing and incensement in the number of STRs markers, Promega offered a new VersaPlex™ 27PY system with 27 loci (23 autosomal STR loci, Amelogenin, DYS391 and two rapidly mutating Y-STR loci (DYS570 and DYS576)). In this study, the efficacy of "23 autosomal STR loci" for paternity testing and personal identification was demonstrated in Indian population. For this, 217 central Indians were tested and all the statistical parameters of forensic and population genetic interest were calculated. In addition, sensitivity of the kit was also tested for forensic casework. During investigation with VersaPlex™ 27PY system, allele 11 at locus TPOX was observed to be most frequent with the highest allelic frequency 0.432. Studied 23 loci showed valuable together with highest value of combined power of discrimination (CPD = 1), combined power of exclusion (CPI = 0.9999999989) and lowest value of combined matching probability (CPM = 7.92x10-28).

RMplex: An efficient method for analyzing 30 Y-STRs with high mutation rates.

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 highlighted 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.

Identification and characterization of novel rapidly mutating Y-chromosomal short tandem repeat markers.

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 (~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.

Male individualization using 12 rapidly mutating Y-STRs in Araein ethnic group and shared paternal lineage of Pakistani population.

A multiplex assay has been developed with newly designed primer sets comprising high mutation rate 12 RM Y-STR markers (DYS570, DYF399S1, DYS547, DYS612, DYF387S1, DYS449, DYS576, DYS5626, DYF403S1 (a + b), DYS627, DYS526, and DYF404S1). Rapidly mutating Y-STRs were evaluated in 167 male individuals among 97 were unrelated from Araein ethnic group and 70 belonged to shared paternal lineage including 20 pairs of father-son and 15 pairs of brother-brother relationship collected from Punjabi population of Pakistan. Forensic competency parameters were implemented for each marker and exceptionally significant results found wherein polymorphism information content (PIC) was in range of 0.7494 (DYS576) to 0.8994 (DYS627). Samples were also analyzed with Y-filer kit for comparison and marked differentiations observed. Haplotype discrimination capacity was 100% as no haplotype shared among all the unrelated individuals of same ethnic group as compared to 17 Y-filer loci (78%). While in closely related males, discrimination capacity was 96.4% with haplotype diversity value of 0.98. Resulted high mutation rate 1 × 10-2 to 7.14 × 10-2 as compared to Y-filer (1 × 10-4 to 1 × 10-3) manifested the power of RM Y-STRs for considering absolute individualization of interrelated and unrelated male individuals. However, multiplex assay would be useful for male discrimination in mixed DNA specimen, azoospermic males, and multiple male DNA contributors in sexual assault cases and mass disasters victim's identification as well as anthropological studies.

Improved Y-STR typing for disaster victim identification, missing persons investigations, and historical human skeletal remains.

Bones are a valuable source of DNA in forensic, anthropological, and archaeological investigations. There are a number of scenarios in which the only samples available for testing are highly degraded and/or skeletonized. Often it is necessary to perform more than one type of marker analysis on such samples in order to compile sufficient data for identification. Lineage markers, such as Y-STRs and mitochondrial DNA (mtDNA), represent important systems to complement autosomal DNA markers and anthropological metadata in making associations between unidentified remains and living relatives or for characterization of the remains for historical and archaeological studies. In this comparative study, Y-STR typing with both Yfiler™ and Yfiler™ Plus (Thermo Fisher Scientific, Waltham, MA, USA) was performed on a variety of human skeletal remains, including samples from the American Civil War (1861-1865), the late nineteenth century gold rush era in Deadwood, SD, USA (1874-1877), the Seven Years' War (1756-1763), a seventeenth-century archaeological site in Raspenava, Bohemia (Czech Republic), and World War II (1939-1945). The skeletal remains used for this study were recovered from a wide range of environmental conditions and were extracted using several common methods. Regardless of the DNA extraction method used and the age/condition of the remains, 22 out of 24 bone samples yielded a greater number of alleles using the Yfiler™ Plus kit compared to the Yfiler™ kit using the same quantity of input DNA. There was no discernable correlation with the degradation index values for these samples. Overall, the efficacy of the Yfiler™ Plus assay was demonstrated on degraded DNA from skeletal remains. Yfiler™ Plus increases the discriminatory power over the previous generation multiplex due to the larger set of Y-STR markers available for analysis and buffer modifications with the newer version kit. Increased haplotype resolution is provided to infer or refute putative genetic relationships.

Mutation rates at 42 Y chromosomal short tandem repeats in Chinese Han population in Eastern China.

Mutation analysis of 42 Y chromosomal short tandem repeats (Y-STRs) loci was performed using a sample of 1160 father-son pairs from the Chinese Han population in Eastern China. The results showed that the average mutation rate across the 42 Y-STR loci was 0.0041 (95% CI 0.0036-0.0047) per locus per generation. The locus-specific mutation rates varied from 0.000 to 0.0190. No mutation was found at DYS388, DYS437, DYS448, DYS531, and GATA_H4. DYS627, DYS570, DYS576, and DYS449 could be classified as rapidly mutating Y-STRs, with mutation rates higher than 1.0 × 10-2. DYS458, DYS630, and DYS518 were moderately mutating Y-STRs, with mutation rates ranging from 8 × 10-3 to 1 × 10-2. Although the characteristics of the Y-STR mutations were consistent with those in previous studies, mutation rate differences between our data and previous published data were found at some rapidly mutating Y-STRs. The single-copy loci located on the short arm of the Y chromosome (Yp) showed relatively higher mutation rates more frequently than the multi-copy loci. These results will not only extend the data for Y-STR mutations but also be important for kinship analysis, paternal lineage identification, and family relationship reconstruction in forensic Y-STR analysis.

Rapidly mutating Y-STR analyses of compromised forensic samples.

Rapidly mutating Y-chromosomal short tandem repeats (RM Y-STRs) were identified to improve differentiation of unrelated males and also to enable separating closely and distantly related males in human identity testing in forensic and other applications. RM-Yplex assay was developed as a single multiplex that is capable of simultaneously amplifying all currently known RM Y-STRs, and reproducibility and sensitivity testing were performed on reference samples. Additional analyses are necessary to test its suitability for analysing compromised forensic samples. For this purpose, we applied the RM-Yplex assay to approximately 70-year-old skeletons that were used as a model for poorly preserved, challenging forensic samples. We analysed 57 male skeletal remains (bones and teeth) from 55 skeletons excavated from the Second World War (WWII) mass graves in Slovenia. The RM-Yplex typing was successful in all 57 samples; there were 56% full profiles obtained, and in partial profiles, up to 7 locus drop-outs were observed and they appeared correlated with low DNA quantities and degradation of DNA obtained from WWII bone and tooth samples. The longest loci, DYS403S1b, DYS547, DYS627 and DYS526b, were the most often dropped-out RM Y-STRs. In spite of high frequency of drop-out events, the RM-Yplex typing was successful in all WWII samples, showing the possibility of successful amplification of at least half of the RM Y-STRs even from the most compromised samples analysed.

Differences in urbanization degree and consequences on the diversity of conventional vs. rapidly mutating Y-STRs in five municipalities from a small region of the Tyrolean Alps in Austria.

In this study we set out to test at a micro-geographic scale for the potential effects of differences in urbanization degree on Y-chromosomal diversity and the paternal lineage differentiation of "conventional" and rapidly-mutating (RM) Y-STR markers. To avoid systematic underrepresentation of common lineages, 551 male samples were collected under a sampling regime allowing for the inclusion of paternal relatives. All participants came from a small, topographically highly structured, yet culturally homogeneous settlement area in the Tyrolean Alps of Austria, a region that is characterized by a longstanding coexistence of communities differing considerably in size and connection. The study participants reported provenance in one of the three rural villages Alpbach, Brandenberg, and Wildschönau - all being separated by topographical barriers from each other - or in one of the two more urban-like and better connected municipalities Kitzbühel and St. Johann in Tirol. When compared with the sample pools from the two larger communities, the three small villages showed distinctly higher rates of self-reported patrilocality since the paternal grandfather (85-95% vs. ∼42%), and featured evidence for a considerably higher proportion of close and cryptic paternal relationships among the study participants. We observed marked differences in the Y-SNP haplogroup frequency spectra and statistically significant Y-STR-based FST distances among the municipality samples, suggesting population sub-structuring along municipality borders. While for the two larger settlements a widely used "core" set of 17 conventional Y-STRs (Yfiler) provided reasonably high lineage resolution (H: 0.99515±0.00256, 0.99739±0.00224), a markedly reduced haplotype diversity was seen in samples from the rural villages (H: 0.96126±0.00701-0.98515±0.00278). This difference largely diminished when instead using a set of 13 RM Y-STRs (H: 0.99180±0.00380-0.99922±0.00187, for all groups). Most notably, in the Alpbach sample the number of different haplotypes rose from 42 (Yfiler) to 99 (RM Y-STRs) and the proportion of matching haplotypes dropped from nearly 4% (Yfiler) to about 0.4% (RM Y-STRs) of the pairwise comparisons. Consistent results were obtained with a reduced version of the dataset, being devoid of inferred close male relatives up to the degree of first cousins. Finally, consequences potentially arising from a gain in lineage-resolution for population reference-sample size requirements will be addressed briefly.

Analysis of Rapidly Mutating Y Chromosome Short Tandem Repeats (RM Y-STRs).

Rapidly mutating (RM) Y-STRs were recently identified and can help to differentiate between paternally related males in paternity and forensic casework. Normal Y-STRs are often used in casework due to their paternal inheritance, which can help to resolve kinship cases, and identify male components in male/female mixtures; however, more discriminating profiles are obtained if rapidly mutating Y-STRs are used. Previously two or three multiplex PCRs have been used to amplify 13 RM Y-STRs; here, an assay amplifying these 13 markers in a single multiplex PCR is described. Commercially available male control DNA samples have been genotyped during the validation of this assay, thus providing a tool for calibrating genotyping results. It is expected that the assay will provide a niche tool for genotyping casework samples.

Yfiler(®) Plus amplification kit validation and calculation of forensic parameters for two Austrian populations.

With the new 6-dye AmpFISTR(®) Yfiler(®) Plus amplification kit (Thermo Fisher Scientific, Waltham, MA, USA) a set of 25 Y-chromosomal short tandem repeat loci (Y-STRs), including seven rapidly mutating Y-STRs (RM Y-STRs), is now available for forensic DNA typing. In this study we present our validation data for the AmpFISTR(®) Yfiler(®) Plus amplification kit and show the results of Y-chromosomal typing of 425 unrelated male individuals from two Austrian populations (Salzburg and Upper Austria) with the AmpFISTR(®) Yfiler(®) Plus amplification kit. Forensic parameters were calculated and compared for four Y-STR marker sets. We also typed five brother pairs to evaluate the power of discrimination for related individuals. The AmpFISTR(®) Yfiler(®) Plus (Yfiler Plus) kit appeared to be unimpaired by typical inhibitors such as hematin and humic acid or by large amounts of female components. An upgrade of analyzed markers resulted in increased discrimination capacity that is crucial for forensic trace analysis.

Genetic polymorphisms and mutation rates of 27 Y-chromosomal STRs in a Han population from Guangdong Province, Southern China.

In this study, we collected blood samples from 1033 father-son pairs of a Han population from Guangdong Province, Southern China, of which 1007 fathers were unrelated male individuals. All together, 2040 male individuals were analyzed at 27 Y-chromosomal short tandem repeats (Y-STRs) with Yfiler(®) Plus system. A total of 1003 different haplotypes were observed among 1007 unrelated fathers, with the overall haplotype diversity (HD) 0.999992 and discrimination capacity (DC) 0.996. The gene diversity (GD) values for the 27 Y-STR loci ranged from 0.4400 at DYS438 to 0.9597 at DYS385a/b. 11 off-ladder alleles and 25 copy number variants were detected in 1007 males. Population relationships were analyzed by comparison with 19 other worldwide populations. With 27,920 allele transfers in 1033 father-son pairs, 124 mutation events occurred, of which 118 were one-step mutations and 6 were two-step mutations. Eleven father-son pairs were found to have mutations at two loci, while one pair at three loci. The estimated locus-specific mutation rates varied from 0 to 1.74×10(-2), with an average estimated mutation rate 4.4×10(-3) (95%CI: 3.7×10(-3) to 5.3×10(-3)). Mutations were most frequently observed at three rapidly mutating Y-STRs (RM Y-STRs), DYS576, DYS518 and DYS627. However, at DYS570, DYS449 and DYF387S1 loci, which were also described as RM Y-STRs, the mutation rates in Guangdong Han population were not as high as estimated in other populations.

A novel multiplex assay for simultaneously analysing 13 rapidly mutating Y-STRs.

A multiplex polymerase chain reaction (PCR) assay (RM-Yplex) was developed which is capable of simultaneously amplifying 13 recently introduced rapidly mutating Y-STR markers (RM Y-STRs). This multiplex assay is expected to aid human identity testing in forensic and other applications to improve differentiating unrelated males and allow separating related males. The 13 RM Y-STR markers included in the multiplex are: DYF387S1, DYF399S1, DYF403S1ab, DYF404S1, DYS449, DYS518, DYS526ab, DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. This study reflects the proof of concept to analyse all currently known RM Y-STRs simultaneously and describes the optimization of the multiplex assay. The RM-Yplex assay generated complete RM Y-STR profiles down to 62.5pg of male template DNA, and from male-female DNA mixtures at all ratios tested. We herewith introduce and make available for widespread use in forensic and anthropological studies, an effective and sensitive single multiplex assay for simultaneous genotyping of 13 RM Y-STRs.

Development of an Italian RM Y-STR haplotype database: Results of the 2013 GEFI collaborative exercise.

Recently introduced rapidly mutating Y-chromosomal short tandem repeat (RM Y-STR) loci, displaying a multiple-fold higher mutation rate relative to any other Y-STRs, including those conventionally used in forensic casework, have been demonstrated to improve the resolution of male lineage differentiation and to allow male relative separation usually impossible with standard Y-STRs. However, large and geographically-detailed frequency haplotype databases are required to estimate the statistical weight of RM Y-STR haplotype matches if observed in forensic casework. With this in mind, the Italian Working Group (GEFI) of the International Society for Forensic Genetics launched a collaborative exercise aimed at generating an Italian quality controlled forensic RM Y-STR haplotype database. Overall 1509 male individuals from 13 regional populations covering northern, central and southern areas of the Italian peninsula plus Sicily were collected, including both "rural" and "urban" samples classified according to population density in the sampling area. A subset of individuals was additionally genotyped for Y-STR loci included in the Yfiler and PowerPlex Y23 (PPY23) systems (75% and 62%, respectively), allowing the comparison of RM and conventional Y-STRs. Considering the whole set of 13 RM Y-STRs, 1501 unique haplotypes were observed among the 1509 sampled Italian men with a haplotype diversity of 0.999996, largely superior to Yfiler and PPY23 with 0.999914 and 0.999950, respectively. AMOVA indicated that 99.996% of the haplotype variation was within populations, confirming that genetic-geographic structure is almost undetected by RM Y-STRs. Haplotype sharing among regional Italian populations was not observed at all with the complete set of 13 RM Y-STRs. Haplotype sharing within Italian populations was very rare (0.27% non-unique haplotypes), and lower in urban (0.22%) than rural (0.29%) areas. Additionally, 422 father-son pairs were investigated, and 20.1% of them could be discriminated by the whole set of 13 RM Y-STRs, which was very close to the theoretically expected estimate of 19.5% given the mutation rates of the markers used. Results obtained from a high-coverage Italian haplotype dataset confirm on the regional scale the exceptional ability of RM Y-STRs to resolve male lineages previously observed globally, and attest the unsurpassed value of RM Y-STRs for male-relative differentiation purposes.

"New turns from old STaRs": enhancing the capabilities of forensic short tandem repeat analysis.

The field of research and development of forensic STR genotyping remains active, innovative, and focused on continuous improvements. A series of recent developments including the introduction of a sixth dye have brought expanded STR multiplex sizes while maintaining sensitivity to typical forensic DNA. New supplementary kits complimenting the core STRs have also helped improve analysis of challenging identification cases such as distant pairwise relationships in deficient pedigrees. This article gives an overview of several recent key developments in forensic STR analysis: availability of expanded core STR kits and supplementary STRs, short-amplicon mini-STRs offering practical options for highly degraded DNA, Y-STR enhancements made from the identification of rapidly mutating loci, and enhanced analysis of genetic ancestry by analyzing 32-STR profiles with a Bayesian forensic classifier originally developed for SNP population data. As well as providing scope for genotyping larger numbers of STRs optimized for forensic applications, the launch of compact next-generation sequencing systems provides considerable potential for genotyping the sizeable proportion of nucleotide variation existing in forensic STRs, which currently escapes detection with CE.