Y-STR Typing Research Articles

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.

Improving the efficiency of Y-chromosome detection and the quality of STR typing in forensic casework with an in-house made qPCR and HRM system based on SYTO™ 9 chemistry

DNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation. AimTo evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results. Material & methodsBiological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively. ResultsAfter DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CVCt% ≤ 9.55) within the dynamic range analyzed (0.016–50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms. ConclusionWe demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.

Verification of a loss of heterozygosity at the D8S1179 locus in a paternity case by the MiSeq FGx system.

Capillary electrophoresis is widely used to study short tandem repeats (STRs) in forensic genetics. However, next-generation sequencing platforms have become a new strategy for forensic DNA typing. In this study, we report a false four-step STR mutation between an alleged father (AF) and child in a paternity case. A total of 23 autosomal STR loci were evaluated using the Huaxia™ Platinum and Goldeneye™ 20A kits, revealing a single mismatch in D8S1179 between the AF (10/10) and the male child (14/14). Additional Y-STR typing of the AF and child was performed, and the results were consistent with those based on 27 Y-STR loci. To further confirm the experimental results, we sequenced the individuals using the MiSeq FGx system and detected 10/15 unbalanced alleles in the D8S1179 locus of the AF and 14/15 unbalanced alleles in the D8S1179 locus of the child. Sanger sequencing revealed that both the AF and child had the C→G point mutation in the primer binding region of D8S1179 resulting in allelic dropout. Therefore, the verification of STR typing by different sequencing systems is helpful for the interpretation of results in cases of multistep STR mutations.

MPKin-YSTR: Interpretation of Y chromosome STR haplotypes for missing persons cases.

Y chromosome Short Tandem Repeat (STR) haplotypes have been used in assisting forensic investigations primarily for identification and male lineage determination. The current SWGDAM interpretation guidelines for Y-STR typing provide helpful guidance on those purposes but do not address the issue of kinship analysis with Y-STR haplotypes. Because of the high mutation rate of Y-STRs, there are complex missing person cases in which inconsistent Y-STR haplotypes between true paternal lineage relatives will arise and cases with two or more male references in the same lineage and yet differ in their haplotypes. Therefore, more useful methods are needed for interpreting the Y-STR haplotype data. Computational methods and interpretation guidelines have been developed specifically addressing this issue, either using a mismatch-based counting method or a pedigree likelihood ratio method. In this study, a software program, MPKin-YSTR, was developed by implementing those more sophisticated methods. This software should be able to improve the interpretation of complex cases with Y-STR haplotype evidence. Thus, more biological evidence will be interpreted, which in turn will result in more investigation leads to help solve crimes.

Y-chromosomal Short Tandem Repeat Variation in Kurd, Assyrian, and Armenian populations in Iraq Kurdistan

Background: North central Middle Eastern countries Iran, Iraq, Turkey, and Syria all have persistent Kurdish regions. Over thousands of years, several ethnicities have immigrated, settled, or resided in the region, including Turks, Persians, Arabs, Kurds, Armenians, Assyrians, Chechens, and Azeris. Methods: Eleven Y-chromosome STRs were evaluated in a total of 90 unrelated males from the Kurds, Armenians, and Assyrians populations in the Kurdistan region of Iraq (DYS19, DYS390, DYS393, DYS426, DYS437, DYS439, DYS447, DYS460, DYS461, DYS481, and DYS576). Using a DNA extraction kit, total DNA was isolated from leukocytes. PCR products were run on 8% polyacrylamide gel with a 50bp ladder DNA marker to size the bands, and silver staining was used to identify the DNA bands. Power Marker V3.25 software was used to determine variety of genetic parameters, including total allele number, allele frequency, gene diversity, polymorphic information content (PIC), and phylogenetic tree was constructed by MEGA-X software. Results: The total number of alleles identified in the three populations was 380. The sizes of the alleles ranged from 87bp to 275bp.The most diverse loci were DYS447 and DYS576 (GD: 0.949), whereas DYS426 showed the least diversity (GD:0.896). The Phylogenetic tree divided the populations into two main clusters: The Kurdish and Armenian clades in one cluster and the Assyrian in another cluster. Few of dendrogram leaves from the three examined groups were admixed with each other. Conclusions: This study confirms the high-resolution Y-STR typing's ability to discriminate. We conclude that the genetic distance between Kurd and Armenians is less than the genetic distance between the Kurd and the Assyrians, meaning that the Armenians population are genetically closer to the Kurds population.

Y- chromosomal STR Variation in Arab, Soran and Behdinan Kurds population in Kurdistan region of Iraq

Background: Over than eight tenant groups make up Kurdistan region of Iraq local populations. Overall majority of the inhabitants are Muslim Kurds, followed by Yezidi Kurds. Alternative groups including Armenians, Assyrian, Chaldea Syriacs as well as a little minority of Arab and Turkmen individuals. Methods: A total of 60 unrelated males from three population groups in Kurdistan region of Iraq Arabs, Soran and Behdinan kurd were successfully analyzed for ten Y- chromosome STRs (DYS19, DYS390, DYS393, DYS437, DYS439, DYS447, DYS460, DYS461, DYS481, DYS576). Whole DNA has been extracted from the blood samples using DNA extraction kit. PCR products were run on 8% polyacrylamide gel with a 50bp DNA ladder marker to size the bands for each sample. silver staining was used to identify the DNA bands. Power Marker V3.25 software was used successfully to determine a variety of genetic parameters which include total allele number, allele frequency, gene diversity and polymorphic information content (PIC). Phylogenetic tree was constructed by MEGA-X software. Results: The total number of alleles identified in the three populations was 155. The size of the alleles ranged from 112bp to 245bp. The DYS19 had the highest diversity (GD: 0.941), whereas DYS393 locus had the lowest value among all (GD: 0.813). The Dendrogram split the populations into two main clusters: Arabs in one cluster, whereas Soran and Behdinan in another cluster. Conclusions: This study validates that the discrimination potential of high-resolution Y-STR typing and supports the main datasets on the samples from Kurdistan region of Iraq. The comparison of two group of Kurds and Arab datasets offers an intriguing total pattern of Kurd groups. Meaning that Sorans are genetically closer to the Behdinan population.

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.

Obtaining Typeable STR Profile from Highly Decomposed and Mutilated Remains by Organic Extraction

Disaster victims were identified by routine DNA analysis involving standard autosomal, amelogenin, and Y-STR typing elsewhere. However, the remains of one of the victims that were received at the DNA Division of FSD, Chennai were in a highly decomposed and mutilated state, which proved difficult to analyze. A partial DNA profile could only be obtained for the remains using modern-day extraction protocols. The conventional organic extraction protocol is not preferred in routine analysis owing to the time constraint faced by DNA labs. Nevertheless, the organic extraction protocol enabled us to get an improved recovery of DNA, and that in turn paved the way for typeable DNA profiles. Hence, we advocate that the organic DNA extraction protocol, though laborious, can still be used for better results, from difficult samples and it outweighs the kit-based extraction procedure for analysis of degraded bone samples.

Removal effect of DNA contamination by hydrogen peroxide plasma compared to ethylene-oxide gas

We examined the ability of hydrogen peroxide plasma (HPP) to remove DNA contamination, to evaluate whether it is a suitable forensic-grade treatment under ISO 18385. HPP treatment was compared to ethylene-oxide gas (EOG) treatment, which is required by ISO 18385. For the evaluation, commercial control DNA solution and cultured cells sprinkled on Petri dishes were used, and the DNA fragments (214 and 80 bp autosomal DNA fragments and 75 bp Y chromosome fragment) were quantified. HPP treatment was performed up to four times and EOG treatment was performed once. Performing HPP treatment three times was as effective as EOG treatment, with all fragments decreasing to below 1/1,000 in DNA solution. With STR and Y-STR typing, no alleles were detected for three HPP treatments of control DNA using the original amount, i.e., 1 ng. Therefore, HPP appears useful for removing DNA contamination. For cells sprinkled on Petri dishes, the DNA degradation abilities of the HPP and EOG were comparable. However, less DNA was degraded with the HPP and EOG and neither met the ISO criteria. Although the current version of ISO 18385 recommends an evaluation method using cultured cells sprinkled on Petri dishes, it needs to be revised. These findings should be considered when revising ISO 18385.

Heating Up Cold Cases: An Interview with Bruce Budowle on Human Identification

Heating Up Cold Cases: An Interview with Bruce Budowle on Human Identification

Development and validation of a novel 29-plex Y-STR typing system for forensic application

Short tandem repeat within the male-specific part of the human Y chromosome (Y-STR) is an effective forensic tool in mixture identification, patrilineal relationship evaluation, and familial searches. Despite their usefulness, current Y-STR-based genotyping systems often lack the discriminatory power to resolve genetic relationships between distant relatives or within patrilocal populations. In this study, we developed a novel Y-STR 29-plex typing system, which combined the 17 Y-STR loci used in the AmpFLSTR® Yfiler® PCR Amplification Kit (Yfiler), eight Y-STR loci with a low–medium mutation rate, and four rapidly mutating Y-STR loci. The system was generated to achieve greater discriminatory power between male subjects and improved ability to infer haplogroup classifications. The system was extensively tested on data from 752 individuals for its sensitivity, male specificity, species specificity, mixture resolution, reproducibility, concordance, stutter and size accuracy, precision, and population genetics, following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. The results demonstrated that the Y-STR 29-plex typing system was time-efficient, reproducible, accurate, sensitive, and robust to familial searching and paternal biogeographic ancestry inference.

Detection of prostate specific antigen and salivary amylase in vaginal swabs using SERATEC® immunochromatographic assays

Immunochromatographic assays are used by crime laboratories to conduct simple and quick analyses of bodily fluids. These streamlined tests are ideal for decreasing the sexual assault kit backlog in the United States. A large-scale analysis of the frequency of positive results of amylase and prostate specific antigen (PSA) endogenously found in the vaginal cavity was conducted using the SERATEC PSA Semiquant and Amylase tests. Vaginal swabs were self-collected by participants after 7–10 days of no oral contact or male ejaculation. In this study of 50 participants, 98% were negative for PSA and 92% were negative for amylase. Positive results were confirmed to contain no exogenous DNA by male-specific quantitation, short tandem repeat (STR) typing, and Y-STR typing. These results can be used by crime laboratories to help guide interpretation of immunochromatographic test results from vaginal swabs and aid in decision-making in downstream DNA testing.

Analysis of Detection of Mixed Semen Stains with Different Immunological Test Methods and Case Applications.

Objective To perform the separation and confirmation of mixed semen stains with immunological test method, and find a more effective method for the detection of mixed semen stains. Methods The semens of three volunteers were mixed. The mixed semen stains were processed and tested with prostate-specific antigen （PSA） colloidal gold immunoassay strip method, immunomagnetic beads method and laser capture microdissection, respectively. Statistics of the results of STR were gathered and compared with those of a single semen stain. Results After PSA colloidal gold immunoassay strip method testing, the samples showed a purplish red line in the test area and the control area. The results obtained with the immunomagnetic beads method showed a more complete and effective short tandem repeat （STR） sequence. The mixed semen stains were processed with laser capture microdissection and low volume amplified. The results were summarized and superimposed to obtain a complete single typing, which matched the single semen stain typing, with a typing success rate of 84.00%. Single suspect Y-STR typing was obtained with the application of the method above in actual cases, which provided evidence basis for rapid solving of the case. Conclusion The combination of PSA colloidal gold immunoassay strip method, immunomagnetic beads method and laser capture microdissection can be used to separate and confirm the mixed semen stains.

Genetic kinship and admixture in Iron Age Scytho-Siberians.

Scythians are known from written sources as horse-riding nomadic peoples who dominated the Eurasian steppe throughout the Iron Age. However, their origins and the exact nature of their social organization remain debated. Three hypotheses prevail regarding their origins that can be summarized as a "western origin", an "eastern origin" and a "multi-regional origin". In this work, we first aimed to address the question of the familial and social organization of some Scythian groups (Scytho-Siberians) by testing genetic kinship and, second, to add new elements on their origins through phylogeographical analyses. Twenty-eight Scythian individuals from 5 archeological sites in the Tuva Republic (Russia) were analyzed using autosomal Short Tandem Repeats (STR), Y-STR and Y-SNP typing as well as whole mitochondrial (mtDNA) genome sequencing. Familial relationships were assessed using the Likelihood Ratio (LR) method. Thirteen of the 28 individuals tested were linked by first-degree relationships. When related, the individuals were buried together, except for one adult woman, buried separately from her mother and young sister. Y-chromosome analysis revealed a burial pattern linked to paternal lineages, with men bearing closely related Y-haplotypes buried on the same sites. Inversely, various mtDNA lineages can be found on each site. Y-chromosomal and mtDNA haplogroups were almost equally distributed between Western and Eastern Eurasian haplogroups. These results suggest that Siberian Scythians were organized in patrilocal and patrilineal societies with burial practices linked to both kinship and paternal lineages. It also appears that the group analyzed shared a greater genetic link with Asian populations than Western Scythians did.

English

English

Application of Y-STR typing in solving a forensic case: A case study

Application of Y-STR typing in solving a forensic case: A case study

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.

A study of the genetic diversity in the Heze Han population using a novel genotyping system based on 24 Y-chromosomal STR loci

We established a 24-plex Y-STR typing system by including the 13 Y-STR loci used in AmpFLSTR® Yfiler® PCR Amplification Kit (Applied Biosystems, Foster City, CA) and 11 additionally selected Y-STR loci that significantly increased power of discrimiation. We applied the system to genotype 195 unrelated Chinese Han males, resulting in 195 distinct haplotypes. The diversity observed across loci ranged from 0.0885 (DYS645) to 0.9646 (DYS385a/b)., while the haplotype diversity was 1 based on the 24 Y-STR loci. The ultra-high differentiation power of the genotyping system has great potential in forensic applications.

Analysis of degraded casework DNA by redesigning a mini Y-STR multiplex

As partial genetic profile from degraded DNA samples of a case limiting the application of Y-STRs in forensic sciences, a new multiplex mini Y-STR typing was developed to obtain more useful information and complement current commercially available multiplex kits. The multiplex mini Y-STR amplification including three Y-STR loci (DYS385a/b, DYS389 II, and DYS392) was performed to generate amplicons smaller than those of PowerPlex® Y system over 140bp by redesigning primers. Allelic ladders for three mini Y-STR loci were created using a dilution of allelic ladders from the PowerPlex® Y system. A sensitivity test using serially diluted standard 9948 male DNA showed that three mini Y-STR loci were reliable at template concentrations as low as 0.25ng. By comparing of the multiplex mini Y-STR amplification and PowerPlex® Y system with referenced DNA samples, the results indicated stable concordance in genotyping. And then the successful analysis for the degraded DNA samples from the forensic case produced complete genetic profiles with these three loci. So the multiplex mini Y-STR typing combining the aforementioned commercially available kit increased chance of gaining of 16 Y-STR loci profiles within some core loci in Y Chromosome Haplotype Reference Database from forensic casework sample. It is an alternative strategy for recovering locus-specific information from degraded to redesign the suitable mini Y-STR complement besides new commercial Y-STR kits.

The finding of disaccord in haplogroup prediction by online software in a father-son pair

The Y-chromosome haplogroup usually determined by the patterns of single nucleotide polymorphism (SNP) refers the geographic or ethnic origin of unknown samples. Recent studies have showed that the haplotypes defined by Y short tandem repeats (STRs) may also be used to predict haplogroup status. The development of online software for Y-haplogroup predictions provides numerous benefits to the forensic application. When samples from a father-son pair in a case were genotyped using the AmpFLSTR® Yfiler® Plus system, mutation at DYS385ab locus (13,14–13,15) was observed. Then haplogroup prediction based on their different haplotypes was conducted with Whit Atheys’ haplogroup predictor (http://www.hprg.com/hapest5/hapest5b/hapest5.htm), and disagreement between the father and the son (Q and O3, respectively) was detected. After performing necessary validation using SNP analysis, consistent haplogroup of the father-son pair was assigned. Although Y-STR typing seems more widely used in forensic casework and convenient to predict haplogroup with online tools, our finding suggested that the occurrence of errors in Y-chromosome haplogroup prediction should be cautious. As increasing the number of STRs employed to predict the haplogroup, correcting inaccurate prediction due to mutation is required by means of precise Y-SNP phylogenetic data.