Y-STR Profiles Research Articles

Bed bugs, Cimex lectularius: Undercover agents in forensic investigations.

Insects have long played a role in forensic investigations and can be used to estimate minimum time since death, corpse translocation, and link an individual to a crime scene. Bed bugs (Cimex lectularius) are wingless ectoparasitic insects of potential forensic utility, given that all mobile life stages feed on vertebrate blood. Successful profiling of autosomal short tandem repeats (STRs) from human DNA isolated from bed bugs has been previously reported. This proof-of-concept study looked to expand this work and determine any possible limitations of using bed bugs for both rapid stain identification (RSID™) for human blood and Y-STR profiling. To achieve this, bed bugs were fed either human male only or human pooled (female:male) blood for 30 min and subsequently collected at 12-h intervals up to 108 h post-blood meal (PBM). RSID™ blood testing was successful from the bed bug carcass remaining after DNA isolation, regardless of blood meal type and time of collection PBM. Complete Y-STR profiles were generated from bed bugs <60 h PBM. As the time PBM increased, DNA quantity decreased, while the degradation index increased. Collection of bed bugs at a crime scene could provide a valuable source of human blood for Y STR profiling and be used to link an individual to a crime scene or for potential male suspect exclusion. Future studies should look to replicate the results of this proof-of-concept study with larger numbers of bed bugs, more diverse blood donors, and additional STR profiling kits.

RETRACTION: A convenient guideline to determine if two Y-STR profiles are from the same lineage.

RETRACTION: A convenient guideline to determine if two Y-STR profiles are from the same lineage.

Uncertainty in the number of contributor estimation methods applied to a Y-STR profile

Maximum allele count (MAC) and total allele count (TAC) methods are widely used for estimating the number of contributors (NoC) of autosomal short tandem repeat (STR) profile in many forensic laboratories. In this study, we applied NoC estimation methods to mixed Y-STR profiles and evaluated its uncertainty and performance. For the MAC method, as recent Y-STR typing kits involve single- and multi-copy loci, we defined “MAC-single” for use across only single-copy loci and “MAC-multi” for use across only multi-copy loci. We generated a dataset containing 120,000 Y-STR profiles for a one to six-person mixture in silico based on previously reported haplotype frequencies of 27 Y-STR loci in Yfiler Plus for the U.S. population (reported by NIST) and the Henan Han population. The dataset was randomly split into a training set and a test set. The training set was used to construct a TAC distribution (TAC curve), whereas the test set was used to calculate the performance metrics (accuracy, precision, recall, and F1-score). In addition, the effect of the upper limit of NoC considered for estimation on overall accuracy was evaluated. The overall accuracies of MAC-single, MAC-multi, and TAC methods when the upper limit of NoC was set to six-person were 0.7920, 0.4329, and 0.7877 for the U.S. population and 0.8207, 0.4609, and 0.8385 for the Henan Han population. Our results suggest that the MAC-single and TAC methods can estimate the NoC for mixed Y-STR profiles with high levels of accuracy.

Uncovering the Past: DNA Analysis of Skeletal Remains from the Medieval Bosnian City of Bobovac

Numerous archaeological sites in Bosnia and Herzegovina represent a historical heritage and testify to the rich cultural, social, and political life of medieval Bosnia. Bobovac, the capital of the Bosnian Kingdom after King Tvrtko I's coronation in 1377, featured a royal complex with a palace, church, and fortification. Recent molecular-genetic research on skeletal remains from Bobovac aims to uncover medieval ancestors' customs and genetic origins. Fifteen well-preserved teeth samples from Bobovac were processed. STR amplification employed PowerPlex® Fusion and Investigator® 24plex QS Kits, with Y-STR profiles generated using the PowerPlex® Y23 System. Fourteen partial autosomal STR profiles were obtained, enabling sex determination and kinship analysis. STR amplification success varied due to ancient DNA degradation, with larger loci showing lower amplification rates. Kinship analysis confirmed appropriate marker selection, demonstrating high reliability for determining close relationships. Integrating aDNA analysis with archaeological research enhances our understanding of historical populations, connecting archaeology and forensic genetics to contribute to the broader narrative of human history.

Genetic Diversity Based on the Analysis of 27 Y- Short Tandem Repetition (STR) Loci in Two Populations in the Apuseni Mountains, Romania.

Y chromosome analysis is used in various fields of forensic genetics, genetic genealogy, and evolutionary research, due to its unique characteristics. Short tandem repetitions (STR) are particularly relevant in population genetic studies. The aimof this study is to analyze the genetic profile of two populations in the Apuseni Mountains area, Băița and Roșia Montană, Romania. 27 STR loci of the Y chromosome were analyzed to investigate the genetic profile of two populations from the Apuseni Mountains area. Investigating genetic diversity by analyzing allele frequency, haplotype frequency, calculating forensic parameters, and presenting the main haplogroups identified based on Y-STR markers. Gene diversity in the batch from Băița varies from 0.515 for the DYS393 locus to 0.947 for the DYS385 locus. In the Roșia Montană population, gene diversity ranges from 0.432 for DYS393 to 0.931 for DYS385. The haplotype diversity in Roșia Montană was 0.991, and the haplotype diversity was 1.000 in the population from Băița. A total of nine haplogroups was identified in the batch from Băița, while only seven haplogroups were observed in the batch from Roșia Montană. Both groups are based on the same five major haplogroups (E, G, I, J, and R) and the most common haplogroup is R1b in both populations. In this study, the genetic diversity of two distinct populations was assessed using genetic analyses based on different markers. Analysis of Y-STR profiles revealed significant genetic diversity in both studied groups. All haplogroups identified were similar to those present in other Romanian populations.

Nails as optimal source of DNA for molecular identification of 5 decomposed bodies recovered from seawater: from Y-ancestry to personal identification.

Molecular identification of extremely compromised human remains in forensic field is usually performed from DNA typing of bones, which are a difficult sample to work with. Moreover, autosomal STR profiles do not always result in the identification of the donor due to lack of comparisons or non-hit throughout database searching. An attempt to overcome these issues is represented by fingernails as an alternative DNA source and Y-STRs typing to infer both geographical and familial ancestry of the unknown donor. In this study, we analyzed both 24 autosomal and 27 Y-chromosome STRs from unidentified human remains (UHRs) of five males recovered from the water near the southwestern coast of Sardinia by the Italian Harbor Master's Office. Nail clippings provided an optimal source of autologous DNA for molecular identification in a very short time, producing complete autosomal and Y-STR profiles even under conditions of high body degradation. Unfortunately, no match neither compatibility occurred using autosomal STRs (aSTRs), initially. Upon analyzing the Y-haplotypes, we found out they had already been observed in northern Africa, providing us important investigative leads. This prompted the International Criminal Police Organization (INTERPOL) to provide us with references of alleged relatives that were then confirmed to be related. The use of fingernails represents an excellent DNA source especially for genetic identification of decomposed bodies recovered in seawater environment. Notably, DNA extracted from nails gave high-quality Y-STR haplotypes by which predicting paternal ancestry of the unidentified donors may result fundamental in the forensic investigative context.

Considerations on the application of a mutation model for Y-STR interpretation

If Y-STR profiling is to be more effective in criminal casework, the methods used to evaluate evidential weight require improvement. Many forensic scientists assign an evidential weight by estimating the number of times a Y-STR profile obtained from a questioned sample has been observed in YHRD datasets. More sophisticated models have been suggested but not yet implemented into routine casework, e.g. Andersen & Balding [1]. Mutation is inherent to STR meiosis (or inheritance) and is encountered in practice. We evaluated a mutation model that can be incorporated into a method for assigning evidential weight to Y-STR profiles, an essential part of bringing any method into practice. Since an important part of implementation to casework is communication, the article is written in an accessible format for practitioners as well as statisticians.The mutation component within the MUTEA model by Willems et al. [2] incorporates the potential for multistep mutations and a tendency for alleles to revert towards a central length, reflecting observed mutation data, e.g. [3]. We have estimated the parameters in this model and in a simplified symmetric version of this model, using sequence data from father/son pairs [4] and deep-rooted pedigrees [5]. Both datasets contain multistep mutations, which may have an effect on models based on simulations [1].We introduce Beta-Binomial and Beta-Geometric conjugate analyses for estimating rate and step parameters for the mutation models presented here, which require only summations and multiplications. We proved mathematically that the parameters can be estimated independently. We show the importance of reporting the variability of the parameters and not only a point estimate. The parameters can be easily incorporated into statistical models, and updated sequentially as more data becomes available. We recommend fuller publication of data to enable the development and evaluation of a wider range of mutation models.

Multiple TP53 p.R337H haplotypes and implications for tumor susceptibility

The germline TP53 p.R337H mutation is reported as the most common germline TP53 variant. It exists at a remarkably high frequency in the population of southeast Brazil as founder mutation in two distinct haplotypes with the most frequent co-segregating with the p.E134∗ variant of the XAF1 tumor suppressor and an increased cancer risk. Founder mutations demonstrate linkage disequilibrium with neighboring genetic polymorphic markers that can be used to identify the founder variant in different geographic regions and diverse populations. We report here a shared haplotype among Brazilian, Portuguese, and Spanish families and the existence of three additional distinct TP53 p.R337H alleles. Mitochondrial DNA sequencing and Y-STR profiling of Brazilian carriers of the founder TP53 p.R337H allele reveal an excess of Native American haplogroups in maternal lineages and exclusively European haplogroups in paternal lineages, consistent with communities established through male European settlers with extensive intermarriage with Indigenous women. The identification of founder and independent TP53 p.R337H alleles underlines the importance for considering the haplotype as a functional unit and the additive effects of constitutive polymorphisms and associated variants in modifier genes that can influence the cancer phenotype.

Paternity pseudo-exclusion caused by tetragametic chimerism in a gestational surrogacy case

Chimerism is a condition when an organism is composed of two or more populations of genetically distinct cells. Chimerism often produces curious results of medical and genetic investigations and could be a major cause of false negative conclusions in parentage testing. Here we describe a paternity pseudo-exclusion due to tetragametic chimerism in a gestational surrogacy case originated in a fertility clinic. Initial analysis using a buccal swab from the child and a peripheral blood sample from the father showed paternity exclusion at 6 STR loci. To find out the reason for observed paternal discrepancy father's semen sample used for IVF and samples from other tissues were genotyped. Buccal swabs, semen, hair follicles, nail clippings and cerumen showed identical mixed autosomal STR profiles originated from two genetically different cell lines and for all the 24 informative loci contain paternal obligate alleles. Results of Y-STR profiling of all paternal sample types showed a DNA profile originated from a single man. The mixed profiles obtained for different tissue types suggest that two genetically different cell lines contributed to formation of both the endoderm and the ectoderm of the father. The mesoderm seems to be monoclonal having originated from a genetically homogenous cell line as evidenced by the STR profile of peripheral blood. Such allelic pattern for various tissues suggests that the clonal origin happened at the very early stage of embryonic development. Approaches to minimise the rate of false exclusions in DNA parentage tests due to chimerism are discussed.

Weight of evidence of Y-STR matches computed with the discrete Laplace method: Impact of adding a suspect's profile to a reference database.

The discrete Laplace method is recommended by multiple parties (including the International Society for Forensic Genetics, ISFG) to estimate the weight of evidence in criminal cases when a suspect's Y-STR profile matches the crime scene Y-STR profile. Unfortunately, modelling the distribution of Y-STR profiles in the population reference database is time-consuming and requires expert knowledge. When the suspect's Y-STR profile is added to the database, as would be the protocol in many cases, the parameters of the discrete Laplace model must be re-estimated. We found that the likelihood ratios with and without adding the suspect's Y-STR profile were almost identical with 1,000 or more Y-STR profiles in the database for Y-STR profiles with 8, 12, and 17 loci. Thus, likelihood ratio calculations can be performed in seconds if an established discrete Laplace model based on at least 1,000 Y-STR profiles is used. A match in a population reference database with 17 Y-STR loci from at least 1,000 male individuals results in a likelihood ratio above 10,000 in approximately 94% of the cases, and above 100,000 in approximately 82% of the cases. We offer free software accessible without restrictions to estimate a discrete Laplace model using a Y-STR reference database and subsequently to calculate likelihood ratios.

Generating human STR DNA profiles from blood ingested by leeches

Aquatic crime scenes may include the presence of annelids, such as leeches, which may have ingested human blood and can potentially aid in the identification of a victim and/or suspect. In this research, human blood from one male donor was fed to 35 North American medicinal leeches. These leeches were then euthanized at various periods (0, 1, 2, 4-, 6-, 12-, and 24-hours post-feed). Blood from their midguts (crop) was amplified using three methods. First, the tip of each Copan microFLOQ® Direct Swab was dipped into the midgut of the individual leeches and amplified directly. Second, Copan microFLOQ® Direct Swabs were utilized to sample the crop, concentrating crop blood followed by direct amplification. Third, 4N6 FLOQSwabs® forensic collection devices collected, extracted, and amplified remaining crop blood. The aim was to determine if blood found in the midgut of leeches can be used in revealing human identity. This research can aid in unique forensic cases where annelids might be present at a crime scene. Autosomal STR profiles were generated using PowerPlex Fusion 6 C System and GlobalFiler Express. Y-STR profiles were obtained with PowerPlex® Y23 System and Yfiler® Plus amplification kits. Complete and partial, concordant, and consistent, autosomal, and Y-STR profiles were observed between 420 autosomal and 420 Y-STR profiles. All three methods can be used to generate DNA profiles from blood ingested by leeches when collected within a 24-hour period. The results indicate that blood ingested by annelids can serve as a valuable source of evidence in unique crime scene cases.

Kinship analysis of skeletal remains from the Middle Ages

Medieval cemeteries Klisa-Guca Gora, Alihodze and Glavica-Han Bila located in the Travnik area (Travnik, Bosnia and Herzegovina) were archaeologically examined in the period 2011–2014, revealing human skeletal remains of 11 individuals in total. Archaeological skeletal samples, previously deposited in Travnik Homeland Museum (Travnik, Bosnia and Herzegovina) were subjected to genetic analysis. The aim of this research was to test familiar relationship of 11 individuals excavated from three medieval cemeteries and to predict Y-haplogroup for male individuals. In order to perform molecular-genetic characterisation of collected human skeletal remains, two systems of genetic markers were analysed: autosomal and Y-STR loci. Complete or partial data obtained by autosomal STR typing of 11 individuals were subjected to kinship analysis. Male sex was determined in eight samples out of 11. Direct relatives of the "brother-brother" type were detected in one case with high kinship probability (KP) value of 99.99996 %. Complete or nearly complete and usable Y-STR profiles were obtained for six out of eight male individuals. The presence of identical haplotypes at Y-STR loci and results of Y-haplogroup prediction suggest that all male individuals share the same paternal lineage and belong to J2a haplogroup. Overall, this study emphasises the usefulness, efficiency and sensitivity of STR markers in the molecular-genetic characterisation of old skeletal remains as well as the importance of employing additional markers like Y-STRs in archaeogenetic studies, besides traditionally used autosomal STR markers, in order to get a comprehensive information about close and distant relatives, and ancestry.

Examining the transfer of male DNA onto female underwear in simulated non sexual social interaction versus digital penetration to assist in the evaluation of results in casework scenarios

Experiments have been carried out by the UK and Ireland Association of Forensic Science Providers Body Fluid Forum (AFSP BFF) to determine the levels of male DNA, detected during Y-STR analysis, that may be expected on female underwear from non-sexual social interaction and digital penetration, versus non-sexual social interaction only. The data obtained strongly supports the existing assumptions made: whilst low levels of DNA may be inadvertently transferred to the inside surface of a female’s underwear during social interaction with a male, there is a low expectation of detecting a matching Y-STR profile to that male, which is suitable for statistical evaluation, unless he is a co-habitant of that female.

Y-STR mixture deconvolution by single-cell analysis.

Since Y-STR typing only amplifies male Y chromosomal DNA, it can simplify the interpretation of some DNA mixtures that contain female DNA. However, if there are multiple male contributors, mixed Y-STR DNA profiles will often be obtained. Y-STR mixture analysis cases are particularly challenging though as, currently, there are no validated probabilistic genotyping (PG) software solutions commercially available to aid in their interpretation. One approach to fully deconvoluting these challenging mixtures into their individual donors is to conduct single-cell genotyping by isolating individual cells from a mixture prior to conducting DNA typing. In this work, a physical micromanipulation technique involving a tungsten needle and direct PCR with decreased reaction volume and increased cycle number was applied to equimolar 2- and 3-person buccal cell male DNA mixtures and a mock touch DNA case scenario involving the consecutive firing of a handgun by two males. A consensus DNA profiling approach was then utilized to obtain YFiler™ Plus Y-STR haplotypes. Buccal cells were used to optimize and test the direct single-cell subsampling approach, and 2-3 person male buccal cell mixtures were fully deconvoluted into their individual donor Y-STR haplotypes. Single-cell (or agglomerated cell clump) subsampling from the gun's trigger recovered single-source Y-STR profiles from both individuals who fired the gun, the owner, and the other unrelated male. Only the non-owner's DNA was found in the cells recovered from the handle. In summary, direct single-cell subsampling as described represents a potential simple way to analyze and interpret Y-STR mixtures.

The effects of 21 cytological and nucleic acid binding dyes on autosomal and Y-chromosomal STR markers using new generation STR amplification kits

Before DNA fingerprinting analysis, many biochemical and histopathological testing are conducted on a forensic biological sample. The samples are exposed to cytological and nuclear binding dyes for their visualization under a microscope. The present study assessed the usability of these biological samples exposed to 21 routinely used histological and nuclear binding dyes for forensic DNA examination in a simulated experiment. Wright's stain, Hematoxylin, Coomassie Brilliant Blue, and Evan's Blue did not show any effect on the amplification of autosomal STR markers. Y-STRs showed higher robustness in presence of 10 dyes. Wright's stain showed the least effect on the generation of complete autosomal and Y-STR profiles. Mobility issues in capillary electrophoresis were observed in the presence of Eosin yellowish, Coomassie Brilliant Blue, and Hematoxylin for autosomal STRs and Wright's stain, Orcein, Malachite Green, Nigrosin, Bromophenol blue, and Fast green for Y STRs. Peak heights of the respective Y STR markers showed a mixed result in comparison to the positive control. In autosomal STR analysis, the peak heights showed a sharp decrease in comparison to the positive control.

Genetic Analysis of Father-Daughter Incest Using Multifaceted STR Markers and Study of Inheritance Pattern of Alleles

Background: Two cases involving father-daughter incest, a rare report in the Indian population, have been analyzed in the current study. STR markers on both autosomal and sex chromosomes were employed to expound the cases. Objective: The objective of this study is to confirm the identity of the fetus as a product of father-- daughter incest and to study the inheritance pattern of alleles in such cases. Methods: Autosomal, X, and Y chromosomal analyses were performed to confirm the identity of the fetus. Statistical analyses were carried out to strengthen the evidentiary value of the obtained results. Results: In both cases, the aborted fetus was found to be the product of an incestuous father-daughter relationship. The probability of paternity as well as maternity was found to be &gt;99.9999% in both cases. Analysis of other paternity and forensic parameters also substantiated the inclusion of the alleged individuals. Father-daughter incest had a tremendous effect on the genome as evidenced from the dramatical decrease in unrelated alleles between father/child [16.66% (Case 1), 20% (Case 2)] and mother/child [26.66% (Case 1), 21.66% (Case 2)]. Genetic evidence also suggested an increased biallelic match i.e., 26.66% (Case 1) and 33.33% (Case 2) between mother and fetus, which are at par/ above the normal siblings’ values i.e., 26.66%. Conclusion: A significant increase in the percentage of homozygous alleles (53.33% in both cases) was observed in the product of father-daughter incest. Both daughters share the same X chromosome from the father, which also suggested the case to be of father-daughter incest. Similarly, the same Y-STR profile between the male fetus and alleged father confirmed the correct pattern of inheritance of the Y chromosome in this case.

Haplogroup diversity in the Indian population using 23 Y-STRs

Background A Y-STR polymorphism study is a convenient tool in molecular anthropology and forensic DNA analysis. Aim Through standard ethical procedures, the proposed study explored the genetic scenario in male lineage in Madhya Pradesh, a central Indian state, by Y-STR genotyping and haplogroup studies. Subjects and methods Five hundred and eleven unrelated male blood samples were directly amplified, and fragment separation was done using capillary electrophoresis to generate a Y-STR profile for 23 forensic relevant markers through PowerPlex® Y 23 multiplex system. The different statistical methods were applied for studying the forensic and genetics parameters. Subsequently, population comparison was performed by AMOVA, PCoA, and MDS plot, and Haplogroups were predicted with Whit Athey's haplogroup predictor tool. Conclusion These data represented the potential value of the PowerPlex® Y-23 multiplex system for the forensic and human genetics application in the population of Madhya Pradesh, India. Simultaneously the Haplogroup analysis revealed information about the multi-geographic origin as well as multi-ethnic genetic affinities of the Madhya Pradesh population.

Genetic Genealogy Using Y-STR Markers in Conjunction with Reverse Parentage Testing Aid Efforts in Human Identification and Familial Search Investigation (Case Study)

The personal identification of victim of mass disasters is one of the powers of forensic DNA typing. Genetic genealogy using Y-chromosome STR markers and reverse parentage testing (RPT) aid efforts in familial search investigation. A Dornier aircraft with commandants on board was declared overdue and a month later, skeletal remains found under sea, were subjected to DNA analysis. The case was quite challenging as the skeletal remains exhibited different rates of degradation. The skeletal remains along with reference samples from blood relatives of victims were subjected to different protocols to generate reproducible and typeable, amelogenin, autosomal and Y-STR profiles. Y-chromosome analysis in conjunction with RPT led to establishment of victim identity and rendered fruitful results in familial search investigation.

DNA profiling of single sperm cells after whole genome amplification

Intimate swabs taken for examination in sexual assault cases typically yield mixtures of sperm and epithelial cell types. While powerful, differential extraction protocols to overcome such cell type mixtures by separate lysis of epithelial cells and spermatozoa can still prove ineffective, in particular if only few sperm cells are present or if swabs contain sperm from more than one individual leading to complex low level DNA mixtures. A means to avoid such mixtures consists in the analysis of single micromanipulated sperm cells. However, the quantity of DNA from single sperm cells is not sufficient for conventional STR analysis. Here, we describe a simple method for micromanipulating individual sperm cells from intimate swabs and show that whole genome amplification can generate sufficient amounts of DNA from single cells for subsequent DNA profiling. We recovered over 80% of alleles of haploid autosomal STR profiles from the majority of individual sperm cells. Furthermore, we demonstrate that in mixtures of sperm from two contributors, Y-STR and X-STR profiles of individual sperm cells can be used to sort the haploid autosomal profiles to develop the diploid consensus STR profiles of the individual donors. Finally, by analyzing single sperm cells from mock sexual assault swabs with one or two sperm donors, we showed that our protocols enabled the identification of the unknown male contributors.

Interpretation of DNA data within the context of UK forensic science - evaluation.

Forensic DNA provides a striking contribution to the provision of justice worldwide. It has proven to be crucial in the investigative phase of an unsolved crime where a suspect needs to be identified, e.g. from a DNA database search both nationally and internationally. It is also a powerful tool in the assignment of evidential weight to the comparison of a profile of a person of interest and a crime scene profile. The focus of this document is the evaluation of autosomal profiles for criminal trials in the UK. A separate review covers investigation and evaluation of Y-STR profiles, investigation using autosomal profiles, kinship analysis, body identification and Forensic Genetic Genealogy investigations. In less than 40 years, forensic DNA profiling has developed from a specialist technique to everyday use. Borrowing on advances in genome typing technology, forensic DNA profiling has experienced a substantial increase in its sensitivity and informativeness. Alongside this development, novel interpretation methodologies have also been introduced. This document describes the state of the art and future advances in the interpretation of forensic DNA data.