Analysis of non-suspect samples lacking visually identifiable sperm using a Y-STR 10-plex

Y-STRs are valuable in the investigation of sexual assaults in which autosomal STR genotype interpretation is challenging. To detect male DNA from compromised sexual assault evidence, 45 non-suspect samples were differentially extracted and analyzed with 10 Y-STRs. These samples were positive for the presence of human seminal fluid, but were negative for spermatozoa by microscopic examination. Y-STR data were obtained in approximately 86.2% of the epithelial or sperm fractions. On samples yielding incomplete profiles, results were obtained on an average of 5 loci per sample. The inability to obtain results may be due to insufficient amplifiable male DNA, PCR inhibition, or unfounded accusations of sexual assault. This study indicates that it is possible to obtain a male STR profile even in the absence of visually identifiable spermatozoa. Furthermore, Y-STR loci should become components of CODIS if they are to be used in solving non-suspect sexual assaults.     

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.     

Novel Y-STR typing strategies reveal the genetic profile of the semen donor in extended interval post-coital cervicovaginal samples

For a variety of reasons, some victims of sexual assault provide vaginal samples more than 24-36 h after the incident. In these cases, the ability to obtain an autosomal STR profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended. We have used a number of carefully selected Y-STR loci in a variety of multiplex or monoplex formats to extend the post-coital interval from which a genetic profile of the semen donor can be obtained. The proposed Y-STR typing strategies enable the routine detection of the male donor Y-STR haplotype in cervicovaginal samples recovered up to 4 days post-coitus. We attribute our success to a number of factors that significantly improve the sensitivity and specificity of the analysis. Firstly, we utilize a subset of Y-STR loci that have been carefully selected for their superior performance under stressed conditions in both multiplex and monoplex formats. Specifically these loci function with low copy number templates in the presence of a vast excess of potentially confounding female DNA. Secondly, sperm and non-sperm DNA is co-extracted without a differential extraction process to prevent the unnecessary loss of the small number of structurally fragile sperm remaining in the cervicovaginal tract several days after intercourse. Thirdly, low copy number detection is facilitated by increasing the cycle number to 34-35 cycles and by the ability to input up to 450 ng of co-extracted sperm/non-sperm DNA into the PCR reaction without the appearance of confounding female artifacts. Lastly, the proper collection of post-coital cervicovaginal samples, instead of the lower or mid-vaginal tract samples often taken, is required for optimal recovery of sperm for analysis. In this report we demonstrate that our previously described 19 Y-STR loci systems (MPI and MPII) permit a reliable high resolution haplotype determination of the semen donor in cervicovaginal samples taken up to 48 h after intercourse. However, as the post-coital interval is extended further, dramatic loss of signal is observed and haplotype determination of the male donor is no longer possible with MPI and MPII. Nonetheless, subsets of these 19 loci (MPA and MPB) have been developed specifically to detect the male haplotype in samples recovered 4 days after intercourse. Thus, it is possible to derive an 11-19 locus Y-STR profile of the semen donor in cervicovaginal samples recovered 2-4 days after intercourse.     

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.     

Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault

In cases of sexual assault involving an azoospermic assailant, vaginal swabs taken from the victim may fail to provide an autosomal DNA profile with which to search a suspect database, as the signal from any male cells present would be masked by that from the overwhelming number of female cells collected on the swab. Here, we describe a method of visually identifying diploid male cells in such samples using fluorescence in situ hybridisation, and selectively harvesting them by means of laser microdissection. This combination of techniques was tested on 26 post-coital vaginal swabs taken at a range of times after intercourse; the collected cells were then subjected to a simple lysis procedure and DNA was amplified using the AmpFlSTR^® SGMPlus^® multiplex under low copy number conditions. Useful DNA profiles were generated from samples taken up to 24 h after intercourse.     

Y-STR DNA amplification as biological evidence in sexually assaulted female victims with no cytological detection of spermatozoa

Identification of spermatozoa is the biological evidence most often sought in specimens from rape victims. Absence of spermatozoa usually terminates biological investigations, and the victim's testimony can be contested. We assessed the utility and reliability of PCR amplification using Y-chromosomal STR polymorphisms in specimens from female victims of sexual assault with negative cytology.One hundred and four swabs without spermatozoa detected by cytology were collected from 79 alleged sexually assaulted female victims and amplification of Y-STR and of amelogenin was performed.Overall, Y-chromosome was detected and evidenced sexual penetration in 28.8% of swabs. In the population of victims examined more than 48 h after the sexual assault, Y-STR were still evidenced in 30% of the cases. These results show that swabs should be taken from victims for Y-chromosome DNA typing even after long delays between sexual assault and medical examination.     

Y-haplotype screening of local patrilineages followed by autosomal STR typing can detect likely perpetrators in some populations

The male-specific, human Y-chromosomal short tandem repeats (Y-STRs) are very useful in forensic analysis and human evolution studies. The authors report two sexual crime cases in which the perpetrators were successfully traced using Y-haplotype screening of local patrilineages followed by autosomal STR typing. First, several main local patrilineages from local cases were investigated using Y-STR haplotyping, aimed to find the pedigrees whose haplotypes were identical or similar to those of the crime scene samples. Then, several key suspects were defined from the screened pedigrees, and autosomal STR typing was performed to identify the perpetrator of the crime. The application of Y-haplotype screening of local patrilineages followed by autosomal STR typing in these two cases demonstrates its usefulness for solving sexually related crimes in certain populations.     

Isolation and identification of female DNA on postcoital penile swabs

After sexual assault, cells originating from the assailant may be recovered from the victim. Through polymerase chain reaction (PCR)-based technology, positive scientific identification of the assailant may be made from these cells. Described is a prospective study describing a method for positively identifying cells from a female sex partner obtained from postcoital swabs of the penis of the male sex partner. Swabs were taken from the penis of a man at 1- to 24-hour intervals after coitus. DNA was isolated from each swab through standard organic extraction methods. The presence of female DNA was detected using the gender-specific amelogenin marker. Extracted DNA was amplified for eight different genetic loci using the Promega PowerPlex kit (Promega) and Amplitaq Gold (Perkin Elmer). Amplified samples were electrophoresed on precast sequencing gels (Hitachi) and were analyzed fluorescently using Hitachi's FMBIO 2 fluorescent scanner and software. Each sample obtained from a penile swab or condom was compared to male and female buccal controls. Female DNA was isolated from all postcoital penile swabs as determined by exclusive amplification of the X-chromosome specific 212 base pair amelogenin marker. In all cases, scientific identification of the female DNA from the swabs was determined by coamplification of eight STR loci (PowerPlex) and was compared to female and male control profiles. Cells shed from a female victim during sexual intercourse can be retrieved from the penis of a male offender after sexual intercourse during a 1- to 24-hour postcoital interval. DNA can be extracted from these cells and can be used to scientifically identify the female sexual participant through PCR-based technology. It is suggested that penile swabs be taken from alleged perpetrators of sexual assaults to associate them with a female victim.     

Applicability of the ParaDNA® Screening System to Seminal Samples

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA^® Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework.     

Assessing the performance of quantity and quality metrics using the QIAGEN Investigator® Quantiplex® pro RGQ kit

The Quantiplex® Pro RGQ kit quantifies DNA in a sample, supports the detection of mixtures and assesses the extent of DNA degradation based on relative ratios of amplified autosomal and male markers. Data show no significant difference in the accuracy and sensitivity of quantification between this and the Promega PowerQuant® System, both detecting the lowest amount of DNA tested, 4 pg. Laboratory controlled mixed male:female DNA samples together with mock sexual assault samples were quantified across a range of mixture ratios. Analysis software detected mixed DNA samples across all ratios for both quantification kits. Subsequent STR analysis using the Investigator® 24Plex QS Kit was able to corroborate mixture detection down to 1:25 male:female DNA ratios, past which point mixtures appeared identical to single-source female samples. Analysis software also detected laboratory degraded DNA samples, with data showing a positive trend between the Degradation Index (DI) and length of time of sonication. When used on ancient remains the assay was able to triage samples for further analysis, and STR profiles were concordant with DNA quantification results in all instances. STR analyses of laboratory-controlled sensitivity, mixture, and degradation studies supports the quality metric obtained from quantification. These data support the use of the Quantiplex® Pro RGQ kit for sample screening and quantification in forensic casework and ancient DNA studies.     

Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.     

STR Genotyping from a Dry‐Cleaned Skirt in a Sexual Assault Case

In this sexual assault case, the standard preliminary semen examinations could not confirm physically or biochemically whether the accused's semen had stained the victim's skirt because the skirt had been dry‐cleaned for stain removal and had been worn for more than a year after the assault. Fortunately, however, a photograph taken just after the assault was found in the court records that showed white stains on the checkered skirt. The locations of the stains were estimated based on the checkered pattern of the fabric, and microscopic examination using Baecchi's staining revealed the presence of spermatozoa. Further analysis indicated the male DNA profile generated from the sperm cells was consistent with the suspect's DNA using three multiplex STR typing systems for a total of 21 autosomal and 17 Y chromosomal short tandem repeats (STRs). Ultimately, the result of the DNA profile played a very useful role as additional evidence.     

What is she doing here? Klinefelter syndrome in forensic casework

In this case report, we describe a sexual assault incident in which the male victim’s seminal fluid contained no sperm cells, as indicated by sperm cell staining and microscopic screening, and DNA profiling results from the non-sperm cell fraction showed a major/minor DNA mixture that could be interpreted as female and male. DNA profiling of a sample from a disposable drinking cup used by the victim at the crime scene provided a single source profile, and showed a 2:1 imbalance between the heights of the X and Y chromosomes, respectively. The victim’s DNA reference sample showed a similar imbalance of the X and Y chromosomes. These observations suggested that the victim might suffer from Klinefelter syndrome, a genetic disorder related to the sex chromosomes.Here, we describe the first reported use of the QIAGEN Investigator® Argus X-12 kit for characterization of X-chromosomal STR loci to potentially identify a case of Klinefelter syndrome. This commercially available kit is primarily used in forensic laboratories to investigate kinship relations and for paternity testing in alleged father/daughter cases. Results of the X chromosome DNA profiling from the victim’s disposable drinking cup and reference samples revealed two alleles at various X-chromosomal STR loci. Moreover, this kit can also amplify a Y chromosome specific sequence (AMEL-Y), and the results indicated that this sample actually originated from a male. Evidence of two X chromosomes in the victim's DNA suggested that he was likely to have Klinefelter syndrome. In this case report, we propose the use of the QIAGEN Investigator® Argus X-12 kit as a practical forensic tool for the detection of potential genetic syndromes related to the sex chromosomes, which can affect test results and, at times, make them difficult to interpret. We also aim to increase awareness within the forensic science community regarding the existence of genetic anomalies, which should be considered when analyzing DNA profiles.     

The efficiency of Y-chromosome markers in forensic trace analysis and their inclusion in the Austrian National DNA Database

Y-STR markers are a valuable tool in the analysis of biological traces in which a mixture of male and female trace material is to be expected. It is possible to generate a Y-chromosome DNA profile, even if all the prior sperm tests are negative and no sign of any male component is found in amelogenin. In 38 of a total of 239 sexual offences a perpetrator trace was identified solely using Y-STR analysis. Based on these findings, the Austrian National DNA Database was expanded to include Y-STRs in 2012 with the primary objective to identify serial sexual offences.     

Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR interpretation

Y-chromosome short tandem repeat (Y-STR) markers are being used as potential tools for distinguishing low levels of male DNA in the presence of excess female DNA as is present in many sexual assault samples. Usually single copy Y-STR loci produce a single amplicon in single source samples, and thus the observation of multiple peaks at such a locus could suggest to an analyst that a mixture of more than one male contributor is present in the tested sample. However, many regions of the Y-chromosome are duplicated or even triplicated in some individuals and this fact can thus complicate potential mixture interpretation. Reasons for the presence of duplications at multiple loci within a single sample are explored in the context of Y-STR marker location along the chromosome. True male-male mixtures commonly exhibit more than one locus-specific PCR product across multiple Y-STR loci that are not adjacent to one another on the Y-chromosome. In addition, duplicated loci typically possess alleles that differ by only a single repeat unit and possess similar peak heights.     

Single source DNA profile recovery from single cells isolated from skin and fabric from touch DNA mixtures in mock physical assaults

The ability to obtain DNA profiles from trace biological evidence is routinely demonstrated with so-called ‘touch DNA evidence’, which is generally perceived to be the result of DNA obtained from shed skin cells transferred from a donor's hands to an object or person during direct physical contact. Current methods for the recovery of trace DNA employ swabs or adhesive tape to sample an area of interest. While of practical utility, such ‘blind-swabbing’ approaches will necessarily co-sample cellular material from the different individuals whose cells are present on the item, even though the individuals' cells are principally located in topographically dispersed, but distinct, locations on the item. Thus the act of swabbing itself artifactually creates some of the DNA mixtures encountered in touch DNA samples. In some instances involving transient contact between an assailant and victim, the victim's DNA may be found in such significant excess as to preclude the detection and typing of the perpetrator's DNA. In order to circumvent the challenges with standard recovery and analysis methods for touch DNA evidence, we reported previously the development of a ‘smart analysis’ single cell recovery and DNA analysis method that results in enhanced genetic analysis of touch DNA evidence. Here we use the smart single cell analysis method to recover probative single source profiles from individual and agglomerated cells from various touched objects and clothing items belonging to known donors. We then use the same approach for the detection of single source male donor DNA in simulated physical contact/assault mixture samples (i.e. male ‘assailant’ grabbing the wrist, neck or clothing from the female ‘victim’, or being in transient contact with bedding from the ‘victim’). DNA profiles attributable to the male or female known donors were obtained from 31% and 35% of the single and agglomerated bio-particles (putative cells) tested. The known male donor ‘assailant’ DNA profile was identified in the cell sampling from every mixture type tested. The results of this work demonstrate the efficacy of an alternative strategy to recover single source perpetrator DNA profiles in physical contact/assault cases involving trace perpetrator/victim cellular admixtures.     

Latent profiles among sexual assault survivors: implications for defensive coping and resistance

Rape resistance trainings need to prepare women to recognize and resist sexual assault across a range of experiences and contexts. To help address this need, this research used an investigation of 415 college women who completed a survey about their situational responding to an experience of acquaintance sexual assault. A previously established person-centered analysis model of four distinct risk and protective factor profiles was used to investigate the variability in women's responding. The profiling factors included women's prior victimization, alcohol consumption, relationship expectancies of the assailant, and assertive precautionary habits. Multivariate ANOVA was used to test for differences among the four profile groups on their cognitive, emotional, and behavioral responding. Results showed that the profile groups significantly differed in their assault responses. The findings highlight the utility of holistic, multivariate analyses for understanding women's sexual assault responses and help inform tailored resistance and empowerment trainings.     

Results of a collaborative study of the EDNAP group regarding the reproducibility and robustness of the Y-chromosome STRs DYS19, DYS389 I and II, DYS390 and DYS393 in a PCR pentaplex format

A collaborative exercise was carried out by the European DNA Profiling Group (EDNAP) in the frame work of the STADNAP program, i.e. standardization of DNA profiling in Europe, in order to evaluate the performance of a Y-chromosome STR pentaplex, which includes the loci DYS19, DYS389 I and II, DYS390 and DYS393 and to determine whether uniformity of results could be achieved among different European laboratories.Laboratories were asked to analyze the five Y-STRs using singleplex and multiplex conditions in three bloodstains and one mixed stain (95% female and 5% male).All the laboratories reported the same results even for the mixed stain included in the exercise. This demonstrates the reproducibility and robustness of Y-chromosome STR typing even with multiplex formats and proves the usefulness of Y-STR systems for analyzing mixed stains with a male component.A total of 930 male samples from 10 different populations from Europe were also analysed for all the loci included in the pentaplex. Eight of these ten populations also included haplotype data.As for single gene analysis, haplotype diversity was higher in Germany and Italy and lower in Western European countries and Finland.Pairwise haplotype analysis shows the Finnish departure from the rest of the populations and a relatively homogeneity in the other European populations with F(ST) estimates lower than 0.05.UPGMA analysis shows an association of Western European population (Ireland, UK, Portugal and Galicia) on the one hand and central European populations on the other.