Mixed DNA Profiles Research Articles

Importance of Y- STR profiling in sexual assault cases with mixed DNA profile

Importance of Y- STR profiling in sexual assault cases with mixed DNA profile

DNA profiling of sperm cells by using micromanipulation and whole genome amplification

The differential extraction method is based on separate lysis of vaginal cells and spermatozoa and was designed to prevent mixed DNA profiles from intimate swabs. However, DNA from the victim can still be present in the sperm fraction, and the suspect’s DNA cannot be identified when only minute amounts of spermatozoa are present. Moreover, differential extraction is not effective when swabs contain sperms from more than one individual. Mixed profiles could ideally be overcome by analysing single spermatozoa. However, current multiplex STR kits are not yet sensitive enough to generate DNA profiles from single cells.The aim of this study was to develop a method that enables DNA profiling of up to a single sperm cell. Spermatozoa were isolated through micromanipulation. Spermatozoa were lysed and their DNA was pre-amplified by whole genome amplification (WGA) to generate sufficient template for PCR. To these ends, several WGA methods were first tested on different amounts of genomic DNA (gDNA) and assessed for allele recovery, allele drop-out (ADO) and allele drop-in (ADI). The best WGA method was selected for use on cell material.The REPLI-g method turned out as the only protocol increasing the sensitivity of DNA profiling. Results of WGA performance on gDNA as well as multiple and single cells will be presented.

Forensic Database Platform for Searching and Interpreting Mixed DNA Profiles

범죄현장 증거물의 DNA 분석에서 2인 이상의 혼합된 DNA 프로필이 종종 검출된다. 법유전학 분야에서는 이러한 혼합 DNA를 분석하고 해석하려는 연구가 지속적으로 이루어지며 관련된 분석 프로그램도 꾸준히 개발되고 있다. 2010년 ‘DNA 신원확인정보의 이용 및 보호에 관한 법률’이 시행되면서 우리나라는 국가 DNA 데이터베이스를 운영하기 시작했다. 그러나 혼합형으로 분석된 DNA 데이터는 시스템의 부재로 검색 및 관리가 효율적으로 이루어지지 않고 있다. 이에 따라 본 연구에서는 이러한 혼합 DNA를 체계적으로 관리하고 검색할 수 있는 시스템인 Forensic Mixed DNA Database Platform (FMDD)를 개발하였다. FMDD는 혼합 DNA 프로필과 용의자 대조 DNA 프로필간의 상호 검색이 가능하고, 일치 결과에 대한 random match probability (RMP), combined probability of exclusion/inclusion (CPE/CPI), 그리 고 likelihood ratio (LR)의 통계 결과를 제공한다. FMDD에서 제공되는 다양한 기능은 혼합 DNA 프로필 로 분석된 사건을 해결하는데 도움을 줄 뿐만 아니라 신뢰성 있는 결과를 제공할 수 있게 될 것이다DNA analysis of crime evidence often involves mixed DNA profiles from several individuals. The mixed DNA interpretation has been continually studied in forensic genetics, with many programs developed for DNA mixture analysis. Although most of these are freely available for performing required calculations, some of them do not provide a database wherein mixed DNA profiles can be searched and managed. The ‘DNA law’ was enacted in Korea, and the forensic DNA database is in operation except for mixed DNA profile. In this study, we developed a web-based system-the Forensic Mixed DNA Database (FMDD)-that allows searches of DNA profiles as well as statistical interpretation for matches. With the FMDD, a suspect’s DNA profile can be searched against a database of mixed DNA profiles and vice versa. Moreover, the FMDD provides different probabilities including the random match probability (RMP), combined probability of exclusion/inclusion (CPE/CPI), and like-lihood ratio (LR). The LR of mixed DNA profiles from two individuals can be computed to obtain customized statistical results using various options available in the program. The multifunctionality of the FMDD make it an essential tool for the crime investigation that is expected to enhance the value of mixed DNA profile analyses.

Validation of SmartRank: A likelihood ratio software for searching national DNA databases with complex DNA profiles

Searching a national DNA database with complex and incomplete profiles usually yields very large numbers of possible matches that can present many candidate suspects to be further investigated by the forensic scientist and/or police. Current practice in most forensic laboratories consists of ordering these ‘hits’ based on the number of matching alleles with the searched profile. Thus, candidate profiles that share the same number of matching alleles are not differentiated and due to the lack of other ranking criteria for the candidate list it may be difficult to discern a true match from the false positives or notice that all candidates are in fact false positives. SmartRank was developed to put forward only relevant candidates and rank them accordingly. The SmartRank software computes a likelihood ratio (LR) for the searched profile and each profile in the DNA database and ranks database entries above a defined LR threshold according to the calculated LR. In this study, we examined for mixed DNA profiles of variable complexity whether the true donors are retrieved, what the number of false positives above an LR threshold is and the ranking position of the true donors. Using 343 mixed DNA profiles over 750 SmartRank searches were performed. In addition, the performance of SmartRank and CODIS were compared regarding DNA database searches and SmartRank was found complementary to CODIS. We also describe the applicable domain of SmartRank and provide guidelines. The SmartRank software is open-source and freely available. Using the best practice guidelines, SmartRank enables obtaining investigative leads in criminal cases lacking a suspect.

A forensic multiplex of nine novel pentameric-repeat STRs

Pentameric-repeat short tandem repeats (STRs), consisting of loci with repeat units of five base-pairs, have the advantage of reduced stutter products compared to their tetrameric-repeat STR counterparts. This characteristic potentially helps the interpretation of mixed DNA profiles when minor component alleles may coincide with stutter peaks from the major components. To develop a simple but informative forensic multiplex with the capability to aid mixture interpretation, we designed an 11-plex assay of nine pentameric STRs new to forensic analysis plus two male- specific markers: DYS391 and the Y-Indel rs2032678 used in GlobalFiler™ (Life Technologies). East Asian-specific variation in the recently adopted Y-Indel rs2032678 is reported in this study for the first time in its forensic use as a sex marker. We estimated the levels of variation observed in the nine pentameric STRs in three of the major population groups sampled in the HGDP-CEPH human genome diversity panel: African, European and East Asian (combining individual populations as their sample sizes were too small for STR allele frequency estimations); and we include genotype data from a population sample of Northwest Spain. From this data, forensic informativeness metrics were estimated when applying the nine novel STRs in identification or kinship analyses. The assay was assessed for forensic sensitivity and ability to successfully genotype highly degraded DNA. In the profiles from the 11-plex assay we observed an average 2.15% stutter ratio in all the pentameric loci compared to 7.32% across equivalently-sized tetrameric STRs in the Promega Powerplex® ESX-17 kit.

Internal validation of STRmix™ for the interpretation of single source and mixed DNA profiles

The interpretation of DNA evidence can entail analysis of challenging STR typing results. Genotypes inferred from low quality or quantity specimens, or mixed DNA samples originating from multiple contributors, can result in weak or inconclusive match probabilities when a binary interpretation method and necessary thresholds (such as a stochastic threshold) are employed. Probabilistic genotyping approaches, such as fully continuous methods that incorporate empirically determined biological parameter models, enable usage of more of the profile information and reduce subjectivity in interpretation. As a result, software-based probabilistic analyses tend to produce more consistent and more informative results regarding potential contributors to DNA evidence. Studies to assess and internally validate the probabilistic genotyping software STRmix™ for casework usage at the Federal Bureau of Investigation Laboratory were conducted using lab-specific parameters and more than 300 single-source and mixed contributor profiles. Simulated forensic specimens, including constructed mixtures that included DNA from two to five donors across a broad range of template amounts and contributor proportions, were used to examine the sensitivity and specificity of the system via more than 60,000 tests comparing hundreds of known contributors and non-contributors to the specimens. Conditioned analyses, concurrent interpretation of amplification replicates, and application of an incorrect contributor number were also performed to further investigate software performance and probe the limitations of the system. In addition, the results from manual and probabilistic interpretation of both prepared and evidentiary mixtures were compared.The findings support that STRmix™ is sufficiently robust for implementation in forensic laboratories, offering numerous advantages over historical methods of DNA profile analysis and greater statistical power for the estimation of evidentiary weight, and can be used reliably in human identification testing. With few exceptions, likelihood ratio results reflected intuitively correct estimates of the weight of the genotype possibilities and known contributor genotypes. This comprehensive evaluation provides a model in accordance with SWGDAM recommendations for internal validation of a probabilistic genotyping system for DNA evidence interpretation

Results of an inter and intra laboratory exercise on the assessment of complex autosomal DNA profiles

The interpretation of complex DNA profiles may differ between laboratories and reporting officers, which can lead to discrepancies in the final reports. In this study, we assessed the intra and inter laboratory variation in DNA mixture interpretation for three European ISO17025-accredited laboratories. To this aim, 26 reporting officers analyzed five sets of DNA profiles. Three main aspects were considered: 1) whether the mixed DNA profiles met the criteria for comparison to a reference profile, 2) the actual result of the comparison between references and DNA profiling data and 3) whether the weight of the DNA evidence could be assessed. Similarity in answers depended mostly on the complexity of the tasks. This study showed less variation within laboratories than between laboratories which could be the result of differences between internal laboratory guidelines and methods and tools available. Results show the profile types for which the three laboratories report differently, which informs indirectly on the complexity threshold the laboratories employ. Largest differences between laboratories were caused by the methods available to assess the weight of the DNA evidence. This exercise aids in training forensic scientists, refining laboratory guidelines and explaining differences between laboratories in court. Undertaking more collaborative exercises in future may stimulate dialog and consensus regarding interpretation. For training purposes, DNA profiles of the mixed stains and questioned references are made available.

Cell type determination and association with the DNA donor

In forensic casework, evidence regarding the type of cell material contained in a stain can be crucial in determining what happened. For example, a DNA match in a sexual offense can become substantially more incriminating when there is evidence supporting that semen cells are present.Besides the question which cell types are present in a sample, also the question who donated what (association) is very relevant. This question is surprisingly difficult, even for stains with a single donor. The evidential value of a DNA profile needs to be combined with knowledge regarding the specificity and sensitivity of cell type tests. This, together with prior probabilities for the different donor-cell type combinations, determines the most likely combination.We present a Bayesian network that can assist in associating donors and cell types. A literature overview on the sensitivity and specificity of three cell type tests (PSA test for seminal fluid, RSID saliva and RSID semen) is helpful in assigning conditional probabilities. The Bayesian network is linked with a software package for interpreting mixed DNA profiles. This allows for a sensitivity analysis that shows to what extent the conclusion depends on the quantity of available research data. This can aid in making decisions regarding further research.It is shown that the common assumption that an individual (e.g. the victim) is one of the donors in a mixed DNA profile can have unwanted consequences for the association between donors and cell types.

Evaluation of low-template DNA profiles using peak heights.

In recent years statistical models for the analysis of complex (low-template and/or mixed) DNA profiles have moved from using only presence/absence information about allelic peaks in an electropherogram, to quantitative use of peak heights. This is challenging because peak heights are very variable and affected by a number of factors. We present a new peak-height model with important novel features, including over- and double-stutter, and a new approach to dropin. Our model is incorporated in open-source R code likeLTD. We apply it to 108 laboratory-generated crime-scene profiles and demonstrate techniques of model validation that are novel in the field. We use the results to explore the benefits of modeling peak heights, finding that it is not always advantageous, and to assess the merits of pre-extraction replication. We also introduce an approximation that can reduce computational complexity when there are multiple low-level contributors who are not of interest to the investigation, and we present a simple approximate adjustment for linkage between loci, making it possible to accommodate linkage when evaluating complex DNA profiles.

Shedding light on the relative DNA contribution of two persons handling the same object

Traces collected on crime scene objects frequently result in challenging DNA mixtures from several contributors in different DNA proportions. Understanding how the relative proportion of DNA deposited by different persons who handled the same object evolves through time has important bearings. For instance, this information may help determine whether the major contributor in a mixed DNA profile is more likely to correspond to the object owner or to the person who may have stolen this object. In this perspective, a simulation-based protocol was designed where randomly paired participants were asked to act either as first (object owner) or second (last) users. The first user was asked to handle/wear 9 different plastic-, metal-, nitrile- and fabric-made objects, commonly found at burglary/robbery crime scenes, for a minimum of 20min during 8 or 10 consecutive days. The second user subsequently used them for 5, 30 or 120min in three distinct simulation sessions. The analysis of the relative DNA contribution on the resulting 234 mock DNA traces revealed a large variability in the contribution depending on the time, substrate and pairs of participants. Despite this, a progressive increase of the second user’s DNA contribution, relative to the first user, was observed over time in 93% of the traces. The second user was shown to become the major contributor in approximately 15%, 33% and 55% of the traces recovered from objects used for 5, 30 and 120min, respectively. Single-source DNA profiles were shown to represent only 1% of the traces. In addition, the DNA profiles of 165 out of 234 (71%) simulated traces displayed extra alleles. Most of these occurred in the minor fraction of mixed DNA profiles and were interpreted as artefacts. Nevertheless, DNA profiles of known participants either involved or not in the simulations were observed in 9 cases (4%). This confirms that indirect DNA transfer should be taken into account when interpreting “touch” DNA evidence.

Developmental validation of STRmix™, expert software for the interpretation of forensic DNA profiles

In 2015 the Scientific Working Group on DNA Analysis Methods published the SWGDAM Guidelines for the Validation of Probabilistic Genotyping Systems [1]. STRmix™ is probabilistic genotyping software that employs a continuous model of DNA profile interpretation. This paper describes the developmental validation activities of STRmix™ following the SWGDAM guidelines. It addresses the underlying scientific principles, and the performance of the models with respect to sensitivity, specificity and precision and results of interpretation of casework type samples. This work demonstrates that STRmix™ is suitable for its intended use for the interpretation of single source and mixed DNA profiles.

Differential reporting of mixed DNA profiles and its impact on jurists’ evaluation of evidence. An international analysis

While DNA analysis is considered by many the gold standard in forensic science, there is ample room for variation in interpretation and reporting. This seems especially the case when working with (complex) mixed DNA profiles. Two consecutive studies on differential DNA reporting were conducted. In Study 1, we first examined type and magnitude of differences when forensic DNA experts across institutes and jurisdictions are handed an identical forensic case with mixed profiles. In Study 2, we explore the impact of the observed differential reporting on jurists’ evaluation of the DNA evidence.19 DNA expert reports from forensic institutes across Western jurisdictions were obtained. Differences between the reports were many and include extensiveness of the reports, explanations of technical issues, use of explanatory appendices, level of reporting, use of context information, and, most markedly, type and substantive content of the conclusions. In Study 2, a group of criminal law students judged a selection of these reports in a quasi experimental study design. Findings show that these differing reports have quite different evidentiary value for jurists, depending on which expert authored the report. It is argued that the impact of differential reporting on jurists’ evaluation was so fundamental and substantive that it is seems reasonable to claim that in an actual court case it could make the difference between acquittal and conviction.

Stabbing simulations and DNA transfer

Technical developments have made it possible to analyze very low amounts of DNA. This has many advantages, but the drawback of this technological progress is that interpretation of the results becomes increasingly complex: the number of mixed DNA profiles increased relatively to single source DNA profiles and stochastic effects in the DNA profile, such as drop-in and drop-out, are more frequently observed. Moreover, the relevance of low template DNA material regarding the activities alleged is not as straightforward as it was a few years ago, when for example large quantities of blood were recovered. The possibility of secondary and tertiary transfer is now becoming an issue.The purpose of this research is twofold: first, to study the transfer of DNA from the handler and secondly, to observe if handlers would transfer DNA from persons closely connected to them. We chose to mimic cases where the offender would attack a person with a knife. As a first approach, we envisaged that the defense would not give an alternative explanation for the origin of the DNA.In our transfer experiments (4 donors, 16 experiments each, 64 traces), 3% of the traces were single DNA profiles. Most of the time, the DNA profile of the person handling the knife was present as the major profile: in 83% of the traces the major contributor profile corresponded to the stabber’s DNA profile (in single stains and mixtures). Mixture with no clear major/minor fraction (12%) were observed. 5% of the traces were considered of insufficient quality (more than 3 contributors, presence of a few minor peaks). In that case, we considered that the stabber’s DNA was absent.In our experiments, no traces allowed excluding the stabber, however it must be noted that precautions were taken to minimize background DNA as knives were cleaned before the experiments. DNA profiles of the stabber’s colleagues were not observed.We hope that this study will allow for a better understanding of the transfer mechanism and of how to assess and describe results given activity level propositions. In this preliminary research, we have focused on the transfer of DNA on the hand of the person. Besides, more research is needed to assign the probability of the results given an alternative activity proposed by the defense, for instance when the source of the DNA is not contested, but that the activities are.

Does the use of probabilistic genotyping change the way we should view sub-threshold data?

The sensitivity and resolution of modern DNA profiling hardware is such that forensic laboratories generate more data than they have resources to analyse. One coping mechanism is to set a threshold, above the minimum required by instrument noise, so that weak peaks are screened out. In binary interpretations of forensic profiles, the impact of this threshold (sometimes called an analytical threshold – AT) was minimal as interpretations were often limited to a clear major component. With the introduction of continuous typing systems, the interpretation of weak minor components of mixed DNA profiles is possible and consequently the consideration of peaks just above or just below the analytical threshold becomes relevant. We investigate here the occurrence of low-level DNA profile information, specifically that which falls below the analytical threshold. We investigate how it can be dealt with and the consequences of each choice in the framework of continuous DNA profile interpretation systems. Where appropriate we illustrate how these can be implemented using the probabilistic interpretation software STRmix. We demonstrate a feature of STRmix that allows the analyst to guide the software, using human observation that there is a low-level contributor present, through user-designated prior distributions for contributor mixture proportions.

Forward-scatter and side-scatter dataset for epithelial cells from touch samples analyzed by flow cytometry.

‘Touch’ or trace biological samples submitted to caseworking labs as evidence often contain biological material from multiple individuals which can result in mixed DNA profiles. These mixture profiles are difficult to interpret and may cause analytical bottlenecks for forensic laboratories. The data in this brief reports the variation in the relative abundance of intact epithelial cells deposited by four different donors across nine days. Touch samples were generated each day by rubbing a polypropylene tube with both hands for five minutes. Forward-scatter area (FSC-A) and side-scatter area (SSC-A) data was acquired with the BD FACSCanto™ II Analyzer. The relative abundance of different sub-populations within the FSC-A and SSC-A plots was calculated against the total number of events analyzed in each sample. Mean and standard deviation values were calculated for each donor.

Complex DNA mixture analysis: Report of two cases

During the last years, DNA profiling techniques have become more sensitive and mixtures with multiple contributors occur often. Nevertheless, interpretation of mixture profiles is the most complex task in the forensic field. The ISFG published recommendations for the interpretation of mixed DNA profiles that may have allelic drop-out or drop-in. The DNA commission of the ISFG has supported the use of likelihood ratios (LRs) as preferable to other methods. LR can be calculated by probabilistic programs, using semi and fully continuous (probabilistic) methods. These methods increase the efficiency of forensic laboratories and improve the consistency and transparency of the reported results. LRmix is an open-source system dedicated to the interpretation of forensic DNA profiles with a particular focus on complex DNA mixtures. This software is programmed on a LR model and facilitates the calculation of LR for complex mixtures, with partial profiles with known and unknown contributors. Here, we report the re-analysis of two old complex cases processed in our laboratory in the last years. In both cases, we used the software LRmix Studio with interesting consequences: in one case we solved the crime, in the other one, the results allowed the case to be reopened.

From sub-source to source: Interpreting results of biological trace investigations using probabilistic models

The current method of reporting a putative cell type is based on a non-probabilistic assessment of test results by the forensic practitioner. Additionally, the association between donor and cell type in mixed DNA profiles can be exceedingly complex. We present a probabilistic model for interpretation of cell typing results. This Bayesian network can be used by forensic practitioners to form a substantiated opinion about the cellular material(s) that a sample is composed of and about the attribution of DNA donor to cell type.

Routine analysis of sexual assault cases in Brasília, Brazil, using 23 Y chromosomal markers

In sexual-assault cases, the use of DNA databases may be decisive in identifying perpetrators. Particularly, the analysis of Y chromosome STRs may play a crucial role in the study of the male portion in (i) male/female cell mixtures, (ii) mixed DNA profiles from more than one perpetrator, or from perpetrators and the sexual partners (iii) and azoospermic or oligospermic perpetrators. The analysis of Y chromosome specific markers can be also important in the study of the geographic ancestry of male lineages. The aim of this study was to genotype samples collected from sexual-assault cases in Brasília, Brazil, using the PowerPlex Y-23 System and to construct a Y-STR database of these profiles in the Instituto de Pesquisa de DNA Forense da Polícia Civil do Distrito Federal (Forensic DNA Research Institute of the Federal District Civil Police) in Brasília, in order to aid forensic casework. A total of 650 evidentiary samples were previously typed for autosomal STRs and, using a DNA database of STRs profiles of biological evidence from sexual assaults, we found 78 serial rapists who attacked 223 women in Brasília. In this work, we analyse 420 complete 23 Y-STR profiles (haplotypes) and we present the results of Y-STR analysis in sexual-assault cases. To assess the substructure of Brazilian populations we conducted Analysis of Molecular Variance (AMOVA) on different Brazilian population groups. The use of a 23 Y-STR DNA database and of an autosomal STR DNA database of samples from sexual assaults in a dual approach of a combined autosomal/Y chromosomal STR analysis played an important role in identifying perpetrators and serial rapists, highlighting the importance of its use in the investigation of sexual assaults.

Crime scene DNA sampling by wet-vacuum applying M-Vac

A possible alternative to conventional stain recovery by swabbing, taping or cutting, is the M-Vac wet-vacuum instrument (M-Vac Systems Inc.). We have evaluated M-Vac for sampling of dried saliva on porous and non-porous surfaces, shed cells on clothes and touch DNA. M-Vac gave significantly higher DNA yields for dried saliva stains on laminated wood, compared with cotton swabs (average DNA concentrations 1.14 vs. 0.57ng/μL, p=0.02). For stains on glass, M-Vac and cotton swabs gave comparable DNA yields. Additionally, M-Vac retrieved three times as much DNA from saliva stains on cotton fabric (T-shirt) compared with saliva on towels (terry cloth), showing that the absorption properties of the surface affect wet-vacuum sampling. M-Vac was also applied for retrieving wearer DNA from clothes, enabling generation of complete DNA profiles from denim jeans, leggings and cotton T-shirt. A mixed DNA profile was retrieved from an “aggressor” pressing a hand against the shoulder area of a worn T-shirt. Since the major component of the obtained mixed DNA profile was from the wearer, M-Vac may not be ideal for touch DNA sampling of clothes. Wet-vacuum sampling requires a fairly large instrument, trained users and DNA extraction procedures handling large sample volumes. The complexity of M-Vac sampling prevents it from being extensively used, but in specific and important cases it can be a valuable sampling tool.

Uncertainty in the number of contributors in the proposed new CODIS set

The probability that multiple contributors are detected within a forensic DNA profile improves as more highly polymorphic loci are analysed. The assignment of the correct number of contributors to a profile is important when interpreting the DNA profiles. In this work we investigate the probability of a mixed DNA profile appearing as having originated from a fewer number of contributors for the African American, Asian, Caucasian and Hispanic US populations. We investigate a range of locus configurations from the proposed new CODIS set. These theoretical calculations are based on allele frequencies only and ignore peak heights. We show that the probability of a higher order mixture (five or six contributors) appearing as having originated from one less individual is high. This probability decreases as the number of loci tested increases.