Y-STR Profiles Research Articles

Genetic analysis of the human remains of the burials of the 17th–18th centuries of the Corpus Christi Church in Nesvizh (Belarus)

During archaeological excavation in the territory of the Corpus Christi Church in Nesvizh, the regular burials dated to the 17th–18th centuries were discovered. The genetic material extracted from the bones of seven unidentified individuals was analyzed using the forensic genetics approaches, including STR profiling and DNA phenotyping. The genetic examination revealed that the remains of three samples (#1, #2, #6) belonged to women, and the four others (#3, #4, #5, and #7) belonged to men. Autosomal STR-data and Y-chromosomal profiles were obtained for five samples. The kinship analysis excluded that woman #1 and men #3, #4, #5, #7 were first-degree relatives. According to the Y-STR profiles, men #3, #4, #7 referred to the haplogroup R1a, the haplotype of individual #5 corresponded to I2. The both haplogroups are widely represented in Eastern Europe, which, with a high degree of probability, suggests the Slavic origin of the individuals under investigation. To predict eye and hair color, we used the HIrisPlex DNA phenotyping system. The analysis gave the satisfactory results for woman #1 and man #7. In correspondence to the allelic variants of the 24 SNP system, woman #1 had an intermediate type of iris pigmentation and dark blond hair (p = 0.635) with dark shade (0.639), light skin tone, low tendency to sunburn, and a high probability of freckles and pigmented spots of the skin. For male #7, the HIrisPlex model predicted blue eye color with a high probability (p = 0.915), as well as blond hair color (p = 0.915) and light hair color shade (p = 0.962). Our data allow us to conclude that the unknown individuals under investigation have significant genetical and phenotypical similarity with the modern Belarusian population.

Evaluation of Promega PowerSeq™ Auto/Y systems prototype on an admixed sample of Rio de Janeiro, Brazil: Population data, sensitivity, stutter and mixture studies

Forensic DNA typing typically relies on the length-based (LB) separation of PCR products containing short tandem repeat loci (STRs). Massively parallel sequencing (MPS) elucidates an additional level of STR motif and flanking region variation. Also, MPS enables simultaneous analysis of different marker-types - autosomal STRs, SNPs for lineage and identification purposes, reducing both the amount of sample used and the turn-around-time of analysis. Therefore, MPS methodologies are being considered as an additional tool in forensic genetic casework. The PowerSeq™ Auto/Y System (Promega Corp), a multiplex forensic kit for MPS, enables analysis of the 22 autosomal STR markers (plus Amelogenin) from the PowerPlex® Fusion 6C kit and 23 Y-STR markers from the PowerPlex® Y23 kit. Population data were generated from 140 individuals from an admixed sample from Rio de Janeiro, Brazil. All samples were processed according to the manufacturers' recommended protocols. Raw data (FastQ) were generated for each indexed sample and analyzed using STRait Razor v2s and PowerSeqv2.config file. The subsequent population data showed the largest increase in expected heterozygosity (23%), from LB to sequence-based (SB) analyses at the D5S818 locus. Unreported allele was found at the D21S11 locus. The random match probability across all loci decreased from 5.9 × 10-28 to 7.6 × 10-33. Sensitivity studies using 1, 0.25, 0.062 and 0.016 ng of DNA input were analyzed in triplicate. Full Y-STR profiles were detected in all samples, and no autosomal allele drop-out was observed with 62 pg of input DNA. For mixture studies, 1 ng of genomic DNA from a male and female sample at 1:1, 1:4, 1:9, 1:19 and 1:49 proportions were analyzed in triplicate. Clearly resolvable alleles (i.e., no stacking or shared alleles) were obtained at a 1:19 male to female contributor ratio. The minus one stutter (−1) increased with the longest uninterrupted stretch (LUS) allele size reads and according to simple or compound/complex repeats. The haplotype-specific stutter rates add more information for mixed samples interpretation. These data support the use of the PowerSeqTM Auto/Y systems prototype kit (22 autosomal STR loci, 23 Y-STR loci and Amelogenin) for forensic genetics applications.

Genetic polymorphisms and phylogenetic analyses of the Ü-Tsang Tibetan from Lhasa based on 30 slowly and moderately mutated Y-STR loci

As a result of the expansion of old Tibet on the Qinghai-Tibet Plateau, Tibetans diverged into three main branches, Ü-Tsang, Amdo, and Kham Tibetan. Ü-Tsang Tibetans are geographically distributed across the wide central and western portions of the Qinghai-Tibet Plateau while Lhasa is the central gathering place for Tibetan culture. The AGCU Y30, a 6-dye fluorescence kit including 30 slowly and moderately mutated Y-STR loci, has been validated for its stability and sensitivity in different biomaterials and diverse Chinese populations (Han and other minorities), and widely used in the practical work of forensic science. However, the 30 Y-STR profiling of Tibetan, especially for Ü-Tsang Tibetan, were insufficient. We utilized the AGCU Y30 to genotype 577 Ü-Tsang Tibetan unrelated males from Lhasa in the Tibet Autonomous Region of China to fill up the full and accurate Y-STR profiles. A total of 552 haplotypes were observed, 536 (97.10%) of which were unique. One hundred and ninety-four alleles were observed at 26 single copy loci and the allelic frequencies ranged from 0.0017 to 0.8180. For the two multi-copy loci DYS385a/b and DYS527a/b, 64 and 36 allelic combinations were observed, respectively. The gene diversity (GD) values ranged from 0.3079 at DYS391 to 0.9142 at DYS385a/b and the overall haplotype diversity (HD) was 0.9998, and its discrimination capacity (DC) was 0.9567. The population genetic analyses demonstrated that Lhasa Ü-Tsang Tibetan had close relationships with other Tibetan populations from Tibet and Qinghai, especially with Ü-Tsang Tibetan. From the perspective of Y haplogroups, the admixture of the southward Qiang people with dominant haplogroup O-M122 and the northward migrations of the initial settlers of East Asia with haplogroup D-M175 hinted the Sino-Tibetan homologous, thus, we could not ignore the gene flows with other Sino-Tibetan populations, especially for Han Chinese, to characterize the forensic genetic landscape of Tibetan.

Mutational data and population profiling of 23 Y-STRs in three Brazilian populations

Y-chromosomal STRs are important markers in forensic genetics, due to some peculiar characteristics. The absence of recombination makes them a useful tool to infer kinship in complex cases involving distant paternal relatives, or to infer paternal bio-geographic ancestry. The presence of a single copy, being transmitted from father to son, allow tracing mutational events in Y-STRs without ambiguity. For the statistical interpretation of forensic evidences based on Y-STR profiles, it is necessary to have estimates on both mutation rates and haplotype frequencies. In this work, 407 father-son duos from São Paulo and Rio de Janeiro states and 204 unrelated individuals from Manaus were analyzed. Haplotype frequencies and mutation rates for the Y-STRs from the PowerPlex Y23 commercial kit were estimated. Thirty-six mutations were observed in 15 of the 22 Y-STRs analyzed, for an average mutation rate of 3.84 × 10−3 (95 % CI 2.69 × 10−3 to 5.32 × 10−3). All mutations in GAAA repeats occurred in alleles with 13 or more uninterrupted units. Mutations in GATA repeats were observed in alleles with 9–17 uninterrupted units. An analysis carried out in different father's age groups showed an increase of 2.48 times the mutation rate in the age group of 40–50 years, when compared to the 20–30 age group, in agreement with the described for autosomal STRs. A high haplotype diversity was found in the three Brazilian populations. Pairwise genetic distance analysis (FST) showed no significant differences between the three populations in this study, which were also close to populations with strong European influence. The highest distances among the Brazilian populations were with São Gabriel da Cachoeira, which has a high Native American ancestry.

Study for the DNA Profiling of Mixed Samples of Multiple Contributors and Suggested Procedures to Overcome Mixture Troubleshooting in Forensic Investigation

In modern forensic genetics, the analysis of DNA mixtures is a challenge because of the obstacles found in defining the number of contributors, especially when they exceed two. The goal of the present study was to find the most reliable method of detection for profiling the two-male and female-male mixtures obtained. Bloodstains, saliva, and body tissues from various crime scenes were obtained as samples. All samples for DNA profiling have been subjected to DNA extraction, amplification, separation, and detection. Results revealed that DNA profiling showed two-male and female-male mixtures. Results also showed the presence of a major and a minor male contributor in each genetic locus according to peak heights. Differential extraction provided a reliable method for separating female and male contributors from each other in the case of the female-male mixture. The profiling of the male donor in the female-male mixture was furtherly verified using the Y-STR profiling process. Eventually, we can infer that a good approach to identifying the two-male mixture is autosomal STR interpretation centered on major and minor differentiation. In addition, differential separation and Y-STR profiling proved to be the best processes for profiling mixed samples comprising female-male contributors as only the male profile was identified.

Predicting haplogroups using a versatile machine learning program (PredYMaLe) on a new mutationally balanced 32 Y-STR multiplex (CombYplex): Unlocking the full potential of the human STR mutation rate spectrum to estimate forensic parameters

We developed a new mutationally well-balanced 32 Y-STR multiplex (CombYplex) together with a machine learning (ML) program PredYMaLe to assess the impact of STR mutability on haplogourp prediction, while respecting forensic community criteria (high DC/HD). We designed CombYplex around two sub-panels M1 and M2 characterized by average and high-mutation STR panels. Using these two sub-panels, we tested how our program PredYmale reacts to mutability when considering basal branches and, moving down, terminal branches. We tested first the discrimination capacity of CombYplex on 996 human samples using various forensic and statistical parameters and showed that its resolution is sufficient to separate haplogroup classes. In parallel, PredYMaLe was designed and used to test whether a ML approach can predict haplogroup classes from Y-STR profiles. Applied to our kit, SVM and Random Forest classifiers perform very well (average 97 %), better than Neural Network (average 91 %) and Bayesian methods (< 90 %). We observe heterogeneity in haplogroup assignation accuracy among classes, with most haplogroups having high prediction scores (99–100 %) and two (E1b1b and G) having lower scores (67 %). The small sample sizes of these classes explain the high tendency to misclassify the Y-profiles of these haplogroups; results were measurably improved as soon as more training data were added. We provide evidence that our ML approach is a robust method to accurately predict haplogroups when it is combined with a sufficient number of markers, well-balanced mutation rate Y-STR panels, and large ML training sets. Further research on confounding factors (such as CNV-STR or gene conversion) and ideal STR panels in regard to the branches analysed can be developed to help classifiers further optimize prediction scores.

Assessment of autosomal and male DNA extracted from casework samples using Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit for autosomal-STR and Y-STR profiling

Short repetitive regions in autosomal and Y chromosomes known as short tandem repeats (STRs) are currently used for DNA profiling in crime investigations. However, DNA profiling requires a sufficient quality and quantity of DNA template, which is often not obtained from trace evidence or degraded biological samples collected at the scene of a crime. Here, we assessed autosomal and male DNA components extracted from crime scene and mock casework samples using the Casework Direct Kit, Custom and compared the results against those obtained by extraction of matching samples using well-established Maxwell 16 System DNA IQ Casework Pro Kit. The quantity and quality of extracted DNA obtained using both Casework Direct Kit, Custom and Maxwell 16 System DNA IQ Casework Pro Kit were analyzed using PowerQuant Systems followed by autosomal and Y-chromosome STR profiling using GlobalFiler Express PCR Amplification Kit and PowerPlex Y23 System, respectively. Our results showed that the Casework Direct Kit and Maxwell 16 DNA IQ Casework Pro Kit have more or less equal capacity to extract inhibitor free DNA, but that the latter produces slightly better quality and more DNA template and subsequently higher numbers of STR allele calls for autosomal and Y-STR analyses. Nonetheless, the Casework Direct Kit, Custom is the quicker and cheaper option for extraction of good, clean DNA from high content material and might best be used for extraction of reference samples. Such reference DNA samples typically come from buccal swabs or freshly drawn blood. So, in general, they can confidently be expected to have a high nucleic acid content and to be inhibitor-free.

A Study of the Background Levels of Male DNA on Underpants Worn by Females

This study is the first to examine the background level of male DNA on underpants worn by females in the absence of sexual contact. Here, we examined 103 samples from the inside front of underpants from 85 female volunteers. Samples were examined for the presence of male DNA using NGM SElect and PowerPlex Y23 kits. Only five samples gave a "complete" Y-STR profile, even though 83.5% of our volunteers cohabited with a male. In all cases where a partner reference sample was available, the Y-STR profile matched the cohabiting partner. We have demonstrated that a Y-STR profile is not expected on the inside front of underpants worn by females after social contact alone. The results of this study are informative for evaluating the significance of a Y-STR profile on underpants in cases of alleged sexual assault.

Performance comparison of two most recent Y-STR multiplex systems for real forensic casework analysis

Detecting presence of male DNA in a mixture is still a challenge in forensic DNA typing. The study presents performance comparison of the two most recent Y-STR systems available for forensic case work, Y filer plus (YFP) and PowerPlex Y23 (PP Y23) multiplex system. Comparative study was conducted by using full and half reaction volumes of both the systems. On the basis of the work done on more than 200 case samples of sexual assault, PP Y23 multiplex system was found to be more promising than YFP. In the experiments conducted with lesser reaction volumes, it was observed that even 8 μL PCR reaction mix of PP Y23 was able to detect 16 pg of male DNA; however, with YFP it was possible with 100 pg. More balanced Y-STR profiles were observed with low male DNA with PPY23.

Evaluation of usefulness of further Y-STR analysis in sexual assault cases on PSA positive samples resulting in female autosomal STR profiling

Samples from male to female sexual assault cases that are positive for the presumptive prostate specific antigen (PSA) test often do not result in a male autosomal STR profile. Due to highly unequal proportions of female and male DNA in typical sexual assault samples, routine autosomal STR analysis often fails to detect the DNA of the assailant, even after differential extraction of the samples. Previous studies have already shown the value of Y-STR analysis in such cases [1]. In Belgium, forensic DNA laboratories are only allowed to perform Y-STR profiling on sexual assault samples by a specific requisition, after routine autosomal STR analysis has been performed. However, a request for additional Y-STR analysis is rather exceptional.In this study, we evaluated the usefulness of further Y-STR analysis. For 100 PSA positive rinses and swabs from male to female sexual assault cases resulting in female autosomal STR profiling, 7% resulted in a full or partial Y-STR profile useful for comparison, using the 23-loci Y-STR PowerPlex Y23 System (Promega). The success rate raised to 12.5% with a higher DNA input in the PCR mix. In conclusion, these results support the usefulness of performing Y-STRs analysis on the sperm DNA extracts to identify the alleged assailant in sexual assault cases.

Enhancing the sexual assault workflow: Development of a rapid male screening assay incorporating molecular non-microscopic sperm identification

Sexual assault samples are among the most difficult sample types encountered by forensic laboratories. Typically, a sexual assault sample poses multiple challenges including small quantity of male DNA and a relatively high quantity of female DNA. Differential extraction procedures are time-consuming and labor-intensive, particularly with microscopic sperm identification. A rapid upfront screening or triage assay prior to use of differential extraction procedures could ensure that male DNA is present prior to the use of full DNA workflows. Previously reported rapid male screening assays do not provide a confirmation of whether sperm are present and therefore still require the use of microscopic sperm identification. This work was focused on an improved rapid male DNA screening assay with an upfront sperm identification using mRNA profiling.The rapid male screening assay consists of a brief lysis using only a small tip portion of a swab, to obtain both RNA (eluted) and DNA (extraction ‘waste’) followed by a one-step reverse transcription-high resolution melt assay for PRM2, a sperm-specific mRNA. Without additional purification, the DNA ‘waste’ fraction can be quantitated and used to obtain an upfront Y-STR profile of the sperm donor. The assay takes 30 min for lysis followed by ∼2 h for quantitation and sperm identification.Here, we demonstrate the specificity of the assay to detect male DNA in semen samples with no cross reactivity with other body fluids and no PRM2 detection in vasectomized males. RNA and DNA profiling results were obtained with as little as ∼0.15 μl of semen in vaginal-semen admixtures. In all samples tested, Y-STR profiling results were obtained when male DNA was indicated based on quantitation results.

Assessment of the Yfiler® Plus PCR amplification kit for the detection of male DNA in semen-negative sexual assault cases

The ability to detect male epithelial cells deposited during digital penetration or penile penetration without ejaculation is limited by the sensitivity of the Y-STR profiling kit. In this study, the relative profiling success of the Thermofisher Yfiler® Plus kit was compared to its predecessor, AmpFlSTR Yfiler®, for 104 semen-negative sexual assault samples from casework at Forensic Science SA, Adelaide, South Australia. Yfiler Plus generated allele information in 25% more samples than Yfiler and gave a higher recovery of informative alleles in all but two samples where detectable male DNA was present. Where a profile was obtained in both kits, 92% of samples gave a higher percentage of informative loci with Yfiler Plus compared to Yfiler. Yfiler Plus also resolved DNA mixtures in 15 samples as compared to 1 sample with Yfiler. Detection of male DNA with the Quantifiler™ Trio DNA Quantification kit was shown to correlate with a successful profiling outcome with Yfiler Plus. The success of profiling with Yfiler Plus was independent of the time elapsed between the alleged offence and the sample being collected, the type of sexual penetration which occurred, and the anatomical origin of the sample.

Rapidly mutating Y-STRs in rapidly expanding populations: Discrimination power of the Yfiler Plus multiplex in northern Africa

The male-specific northern African genetic pool is characterised by a high frequency of the E-M81 haplogroup, which expanded in very recent times (2–3 kiloyears ago). As a consequence of their recent coalescence, E-M81 chromosomes often cannot be completely distinguished on the basis of their Y-STR profiles, unless rapidly-mutating Y-STRs (RM Y-STRs) are analysed. In this study, we used the Yfiler® Plus kit, which includes 7 RM Y-STRs and 20 standard Y-STR, to analyse 477 unrelated males coming from 11 northern African populations sampled from Morocco, Algeria, Libya and Egypt. The Y chromosomes were assigned to monophyletic lineages after the analysis of 72 stable biallelic polymorphisms and, as expected, we found a high proportion of E-M81 subjects (about 46%), with frequencies decreasing from west to east. We found low intra-population diversity indexes, in particular in the populations that experienced long-term isolation. The AMOVA analysis showed significant differences between the countries and between most of the 11 populations, with a rough differentiation between northwestern Africa and northeastern Africa, where the Egyptians Berbers from Siwa represented an outlier population. The comparison between the Yfiler® and the Yfiler® Plus network of the E-M81 Y chromosomes confirmed the high power of discrimination of the latter kit, thanks to higher variability of the RM Y-STRs: indeed, the number of chromosomes sharing the same haplotype was drastically reduced from 201 to 81 and limited, in the latter case, to subjects from the same population.

Investigating the genetic diversity and affinities of historical populations of Tibet

This study elucidates Y chromosome distribution patterns in the three general provincial populations of historical Tibet, Amdo (n = 88), Dotoe (n = 109) and U-Tsang (n = 153) against the backdrop of 37 Asian reference populations. The central aim of this study is to investigate the genetic affinities of the three historical Tibetan populations among themselves and to neighboring populations. Y-SNP and Y-STR profiles were assessed in these historical populations. Correspondence analyses (CA) were generated with Y-SNP haplogroup data. Y-STR haplotypes were determined and employed to generate multidimensional scaling (MDS) plots based on Rst distances. Frequency contour maps of informative Y haplogroups were constructed to visualize the distributions of specific chromosome types. Network analyses based on Y-STR profiles of individuals under specific Y haplogroups were generated to examine the genetic heterogeneity among populations. Average gene diversity values and other parameters of population genetics interest were estimated to characterize the populations. The Y chromosomal results generated in this study indicate that using two sets of markers (Y-SNP, and Y-STR) the three Tibetan populations are genetically distinct. In addition, U-Tsang displays the highest gene diversity, followed by Amdo and Dotoe. The results of this transcontinental biogeographical investigation also indicate various degrees of paternal genetic affinities among these three Tibetan populations depending on the type of loci (Y-SNP or Y-STR) analyzed. The CA generated with Y-SNP haplogroup data demonstrates that Amdo and U-Tsang are closer to each other than to any neighboring non-Tibetan group. In contrast, the MDS plot based on Y-STR haplotypes displays Rst distances that are much shorter between U-Tsang and its geographic nearby populations of Ladakh, Punjab, Kathmandu and Newar than between it and Amdo. Moreover, although Dotoe is isolated from all other groups using both types of marker systems, it lies nearer to the other Tibetan collections in the Y-SNP CA than in the Y-STR MDS plot. High resolution and shallow evolutionary time frames engendered by Y-STR based analyses may reflect a more recent demographic history than that delineated by the more conserved Y-SNP markers.

Prehistoric migrations through the Mediterranean basin shaped Corsican Y-chromosome diversity.

The rarity of human remains makes it difficult to apprehend the first settlements in Corsica. It is admitted that initial colonization could have occurred during the Mesolithic period when glaciations would have shortened the open water travel distance from the continent. Mesolithic sites in Corsica show relatively short and irregular occupation, and suggest discontinuous settling of very mobile groups probably traveling by boat. Previous genetic studies on Corsican populations showed internal differentiation and a relatively poor genetic relationship with continental populations, despite intense historical contacts, however local Mesolithic-based genetic inheritance has never been properly estimated. The aim of this study was to explore the Corsican genetic profile of Y-chromosomes in order to trace the genetic signatures back to the first migrations to Corsica. This study included 321 samples from men throughout Corsica; samples from Provence and Tuscany were added to the cohort. All samples were typed for 92 Y-SNPs, and Y-STRs were also analyzed. Results revealed highly differentiated haplogroup patterns among Corsican populations. Haplogroup G had the highest frequency in Corsica, mostly displaying a unique Y-STR profile. When compared with Provence and Tuscany, Corsican populations displayed limited genetic proximity. Corsican populations present a remarkable Y-chromosome genetic mixture. Although the Corsican Y-chromosome profile shows similarities with both Provence and to a lesser extent Tuscany, it mainly displays its own specificity. This study confirms the high level of genetic diversity in Corsican populations and backs genetic contributions from prehistoric migrations associated with the Mesolithic, Neolithic and Metal Age eras, rather than from historical movements to Corsica, respectively attested by frequencies and TMRCA of haplogroups G2a-L91 and G2a-P15, J2a-M241 and J2-DYS445 = 6, R1b-U152 and R1b-U106. These results suggest that marine routes to reach the Corsican coast in many different points may have led to such a genetic heterogeneity.

Male DNA under female fingernails after scratching: transfer and persistence evaluation by RT-PCR analysis and Y-STR typing

The collection of biological debris beneath fingernails can be useful in forensic casework when a struggle between the victim and the offender is suspected. In the present study, we set up a controlled scratching experiment in which female volunteers scratched the male volunteers' forearms, simulating a defensive action during an assault. A total of 160 fingernail samples were collected: 80 "control samples" before the scratching, 40 samples immediately after the scratching (t = 0h), and 40 samples 5h after the scratching (t = 5h). The aim was to evaluate, using a real-time PCR approach and Y-STR profiling, the transfer and the persistence of male DNA under female fingernails after scratching. A significant reduction in DNA yield was observed between fingernail samples collected immediately and those collected 5h after scratching, with a corresponding decrease in Y-STR profile quality. Overall, 38/40 (95%) of the fingernail samples collected immediately (t = 0h) and 24/40 (60%) of those collected 5h later (t = 5h) were suitable for comparison and the scratched male volunteers could not be excluded as donors of the foreign DNA from 37 (92.5%) of the t = 0h and from 10 (25%) of the t = 5h profiles. The analysis of male DNA under female fingernails showed that Y-chromosome STR typing may provide extremely valuable genetic information of the male contributor(s), although 5h after scratching the profile of the scratched male was lost in three-quarters of samples.

Likelihood ratio development for mixed Y-STR profiles

In this paper we introduce a new likelihood ratio method for evaluating mixed Y-STR profiles that is based on the premise that, given a haplotype has been seen in the person of interest, the most likely source of a second haplotype, matching at all or most loci, is in an individual with a recent common ancestor. We have called the new method the “Haplotype centred” (HC) method for likelihood ratio derivation. For single source, unambiguous haplotypes the HC method performs identically with the Kappa method proposed by Brenner.When attention is turned to mixtures we are required to assign a probability to many haplotypes seen neither in the database nor any person typed in the case. We derive a likelihood ratio formula in a way that allows a locus by locus approach. We demonstrate the application of the HC method on a series of Y-STR mixtures, originating from two to four individuals, in a manner that is still calculated locus-by-locus in nature.

UK and Irish Y-STR population data—A catalogue of variant alleles

A total of 3128 Y-STR profiles from three UK and one Irish population have been analysed with the PowerPlex Y23 system and are reported here. Instances of haplotype sharing between apparently unrelated individuals were identified and further investigated with the use of the 5 additional markers within the Yfiler Plus kit, resulting in a reduction by 76% in the number of shared haplotypes. Furthermore, Yfiler Plus was also employed to verify locus deletions and duplications observed in Y23 genotypes while inconsistencies between the two kits were sequenced, revealing underlying Y23 primer binding site mutations in loci DYS392 and DYS576. Finally, the mechanism behind a previously reported population specific peak shift observed in DYS481 in South Asian samples has been evaluated and further investigated in a novel case of this phenomenon seen in a Black British individual featuring a different flanking region mutation.

Background levels of male DNA in the vaginal cavity

In alleged sexual assault investigations, the detection of male DNA on female intimate swabs can be established through Y-STR profiling. However, in cases where the defendant lives in the same dwelling as the complainant, the defendant may argue that his DNA was transferred through normal social contact, and dispute that intimate contact occurred. This study aimed to determine whether or not male DNA could be detected in samples collected from the vaginal cavity after everyday activities. A total of 300 samples from eight individuals were subjected to DNA quantification and Y-STR amplification. Three donors reported sexual contact during the time of sampling, hence the detection of male-specific DNA in post-coital samples was concomitantly evaluated. The majority of samples (n=237, 79%) were undetermined for male DNA by real-time PCR quantification. No Y-STR profiles having three or more alleles were obtained in these samples, with the exception of three samples which displayed three alleles each. One sample was collected six days post-coital, one sample likely presented artefacts, and one exhibited two alleles at the same locus. Male-specific quantification values were obtained for the remaining 63 samples (21%), however only 31 samples displayed alleles at three or more loci. Twenty-three of the 63 samples showed inhibition during real-time PCR quantification, and the majority of these exhibited no Y-STRs. A further three samples were excluded from the dataset due to possible contamination. The maximum confirmed time frame for obtaining a full Y-STR profile was six days post-coital, but may be longer in some donors. Overall concordance between DNA quantification and Y-STR amplification was 85.8%. This study demonstrates that obtaining a Y-STR profile comprising three or more alleles from a vaginal swab collected from a woman who has not participated in recent intimate sexual activities is not indicated. This suggests that there is unlikely to be detectable adventitious transfer of DNA from male individuals living in the same household as a female donor. However, approximately one in seven samples may display 1–2 unexpected allelic peaks (<400 RFU). These are unlikely to originate from the male partner or cohabitant of the donor and should be interpreted with extreme caution, and with every effort attempted to replicate results before a conclusion is derived about their relevance. These findings support the significance of male DNA profiles consisting of more than three alleles obtained from female intimate samples during investigations of alleged sexual assault.

Extensive geographical and social structure in the paternal lineages of Saudi Arabia revealed by analysis of 27 Y-STRs

Saudi Arabia’s indigenous population is organized into patrilineal descent groups, but to date, little has been done to characterize its population structure, in particular with respect to the male-specific region of the Y chromosome. We have used the 27-STR Yfiler® Plus kit to generate haplotypes in 597 unrelated Saudi males, classified into five geographical regions (North, South, Central, East and West). Overall, Yfiler® Plus provides a good discrimination capacity of 95.3%, but this is greatly reduced (74.7%) when considering the reduced Yfiler® set of 17 Y-STRs, justifying the use of the expanded set of markers in this population. Comparison of the five geographical divisions reveals striking differences, with low diversity and similar haplotype spectra in the Central and Northern regions, and high diversity and similar haplotype spectra in the East and West. These patterns likely reflect the geographical isolation of the desert heartland of the peninsula, and the proximity to the sea of the Eastern and Western areas, and consequent historical immigration. We predicted haplogroups from Y-STR haplotypes, testing the performance of prediction by using a large independent set of Saudi Arabian Y-STR + Y-SNP data. Prediction indicated predominance (71%) of haplogroup J1, which was significantly more common in Central, Northern and Southern groups than in East and West, and formed a star-like expansion cluster in a median-joining network with an estimated age of ∼2800 years. Most of our 597 participants were sampled within Saudi Arabia itself, but ∼16% were sampled in the UK. Despite matching these two groups by home sub-region, we observed significant differences in haplotype and predicted haplogroup constitutions overall, and for most sub-regions individually. This suggests social structure influencing the probability of leaving Saudi Arabia, correlated with different Y-chromosome compositions. The UK-recruited sample is an inappropriate proxy for Saudi Arabia generally, and caution is needed when considering expatriate groups as representative of country of origin. Our study shows the importance of geographical and social structuring that may affect the utility of forensic databases and the interpretation of Y-STR profiles.