Uncertainty in estimating the number of contributors from simulated DNA mixture profiles, with and without allele dropout, from Chinese, Malay, Indian, and Caucasian ethnic populations.

Determining the number of contributors (NOC) accurately in a forensic DNA mixture profile can be challenging. To address this issue, there have been various studies that examined the uncertainty in estimating the NOC in a DNA mixture profile. However, the focus of these studies lies primarily on dominant populations residing within Europe and North America. Thus, there is limited representation of Asian populations in these studies. Further, the effects of allele dropout on the NOC estimation has not been explored. As such, this study assesses the uncertainty of NOC in simulated DNA mixture profiles of Chinese, Malay, and Indian populations, which are the predominant ethnic populations in Asia. The Caucasian ethnic population was also included to provide a basis of comparison with other similar studies. Our results showed that without considering allele dropout, the NOC from DNA mixture profiles derived from up to four contributors of the same ethnic population could be estimated with confidence in the Chinese, Malay, Indian and Caucasian populations. The same results can be observed on DNA mixture profiles originating from a combination of differing ethnic populations. The inclusion of an overall 30% allele dropout rate increased the probability (risk) of underestimating the NOC in a DNA mixture profile; even a 3-person DNA mixture profile has a > 99% risk of underestimating the NOC as two or fewer contributors. However, such risks could be mitigated when the highly polymorphic SE33 locus was included in the dataset. Lastly there was a negligible level of risk in misinterpreting the NOC in a mixture profile as deriving from a single source profile. In summary, our studies showcased novel results representative of the Chinese, Malay, and Indian ethnic populations when examining the uncertainty in NOC estimation in a DNA mixture profile. Our results would be useful in the estimation of NOC in a DNA mixture profile in the Asian context.

Human identification: a review of methods employed within an Australian coronial death investigation system.

Whilst many identification methods have been widely described and discussed in the literature, and considered in disaster and humanitarian contexts, there has been limited reporting and evaluation of the identification methods used in domestic medico-legal death investigation contexts. The aim of this study was to evaluate the identification methods utilised at the Victorian Institute of Forensic Medicine (VIFM), which forms part of a coronial medico-legal death investigation system. The method of identification and time taken to complete the identification were reviewed for all cases admitted to the VIFM over a five-year period from 1 July 2015 to 30 June 2020. The majority, 91%, of individuals admitted to the VIFM were visually identified. The remaining 9% of cases required identification by primary methods (i.e. fingerprints, DNA or dental) or, when those methods were not possible, by secondary methods (i.e. circumstantial). Visual identifications were the timeliest, taking an average of 1.5 days, whilst primary identification methods required an average of 5 days to complete. The triaging of identification methods, dependent on the case context, body preservation, availability of ante-mortem data, legal requirements and admissibility of the method, are determined by identification coordinators within the Human Identification Service (HIS) to ensure the most appropriate and timely method is employed. This review of human identification methods provides the foundation for future analyses to compare workflow processes and improve identification methods utilised in domestic medico-legal contexts.

A State Census of Unsubmitted Sexual Assault Kits: Comparing Forensic DNA Testing Outcomes by Geographic and Population Density Characteristics.

A growing number of U.S. cities and states have large numbers of unsubmitted sexual assault kits (SAKs) in police property facilities. Prior research conducted in large urban cities has found that testing these kits yields a sizable number of DNA profiles that meet FBI eligibility for upload to the national criminal DNA database CODIS (Combined DNA Index System) and uploaded profiles return a substantial number of matches to existing criminal profiles in CODIS. It is unknown whether these findings are unique to large urban cities with high crime rates. The purpose of current study was to document forensic testing outcomes from a state census of previously unsubmitted SAKs, which included large urban-suburban centers, as well as smaller cities and rural counties. We inventoried all previously unsubmitted SAKs in Michigan (N = 3422 SAKs) and submitted all kits for forensic DNA testing. A total of n = 1239 SAKs had a DNA profile that met eligibility for upload into CODIS (36.2% unconditional, 56.5% conditional CODIS eligible rate) and n = 585 SAKs yielded a CODIS Hit (17.1% unconditional, 47.2% conditional CODIS hit rate). These rates are consistent with studies from urban areas suggesting approximately half of SAKs tested yield a CODIS profile and approximately half of those uploaded profiles yield a hit. We compared SAK forensic testing outcomes by geographic and population density characteristics, and although rates were often higher in larger metropolitan areas, the obtained rates in micropolitan and rural areas suggest testing is warranted in smaller jurisdictions as well.

Likelihood ratios for evaluating DNA matches obtained from a database search when there is substructure in the population.

DNA database searches are frequently conducted to identify the suspects of crimes. When a match is obtained from such a database search, the evidence must be evaluated. Existing methods for assessment of the DNA evidence in this scenario require assumption of stochastic independence between the profiles in the database. However, when there is substructure in the population, this assumption is violated. The problem of how to account for population substructure in the database search scenario is analyzed and a solution in the form of a bounded estimate for the likelihood ratio is presented. The implications of these methods are investigated in a realistic scenario using published forensic allele frequencies to simulate ten-locus DNA profiles. In the simulated example, it is observed that the strength of the evidence can be inflated by more than a factor of 10 in 11.6% of database search cases if mild population substructure is ignored. With these methods, the magnitude of the subpopulation effect in the database search scenario can be quantified and the weight of the evidence of a DNA match more accurately assessed.

Genetic variation among the major Pakistani populations based on 15 autosomal STR markers.

Pakistan is located at an important cross-road of human history and has been a passageway for many invaders and dynasties in the past. The historic human migrations across this country have resulted in a blend of ancient civilizations, which are still reflected in the current socio-cultural fabrication of this population. This makes Pakistan an ideal country to study the genetic differentiation and various other genomic aspects of a human population.

Factors associated with trace evidence analyses and DNA findings among police reported cases of rape.

OBJECTIVE: The aim of this study was to examine the association between victim, suspect and assault characteristics and (1) forensic analysis of trace evidence, (2) detection of spermatozoa and (3) DNA match in police-reported cases of rape/attempted rape. In addition, we explored whether DNA findings were associated with legal outcome. METHODS: We conducted a retrospective, descriptive study based on police-reported rapes and attempted rapes of women ≥16 years of age in Sør-Trøndelag Police District throughout 1997-2010. Police data were merged with information from the Sexual Assault Centre (SAC) at St. Olavs University Hospital, Trondheim, Norway. We used binary and multivariable logistic regression for the comparisons. RESULTS: We identified 324 victims (mean age 24 years). The police requested analysis in 135 (45%) of the 299 collected victim samples. The police decision to analyze was after adjustment associated with the victim being employed or under education, and a public venue, but not with interval from assault to sampling. Spermatozoa were detected in 79 (61%) of the analyzed cases, of which 71 were collected from victims within 24h. Interval from assault being <24h and reporting a penetrative assault remained associated with the findings of spermatozoa after adjustments. Forensic analyses of trace evidence collected from victim, suspect and/or venue disclosed matching DNA profiles in 57 (40%) of a total of 143 analyzed cases. Matching DNA profiles were associated with suspect being known to the victim and with the venue being private. A higher proportion of cases with a DNA match were prosecuted in court: 20 of the 29 cases prosecuted. However, despite a DNA match 35 cases were anyway dismissed because of insufficient evidence. CONCLUSIONS: Although many of the associations in our study were expected, it is still important to report the actual numbers to gain insight into the importance of a DNA match in legal proceedings. A substantial proportion of cases with DNA match was dismissed because of insufficient evidence. To strengthen the justice response to sexual assault, it is essential to generate knowledge about the role of medico-legal evidence in such cases, and there are obviously other non-medical factors influencing the legal decisions.

A retrospective study of sexual offences in Zambia.

Zambia has recently reported high incidences of sexual abuse against women and children. Zambian law categorises sexual offences into rape, defilement, incest and others, with defilement constituting the majority of the reported cases (>89%). Between 2010 and 2012, convictions of defilement cases were achieved in only 13% of cases reported to the police. DNA evidence has shown prominence in resolving crimes, specifically as an identification tool in sexual offences. Currently there is no empirical evidence describing the role of forensic evidence in sexual crimes in Zambia; as such a retrospective study was conducted to evaluate this between 2007 and 2014 (n = 1154). Only 14 (0.1%) of the cases had forensic samples collected in the form of a vaginal swab for semen analysis. In all cases where a suspect was identified (60%), identification was based on the witness/victim testimonies, and in no case, was forensic DNA evidence used to assist in identification or corroborate the testimonies. Overall, 28.1% of cases were taken to court and the conviction rate was 12.4%. These findings support the use of employing DNA evidence in sexual offence cases to aid the identification of suspects, which is hypothesised to increase the number of cases prosecuted in Zambia.

Development and validation of open-source software for DNA mixture interpretation based on a quantitative continuous model.

In criminal investigations, forensic scientists need to evaluate DNA mixtures. The estimation of the number of contributors and evaluation of the contribution of a person of interest (POI) from these samples are challenging. In this study, we developed a new open-source software "Kongoh" for interpreting DNA mixture based on a quantitative continuous model. The model uses quantitative information of peak heights in the DNA profile and considers the effect of artifacts and allelic drop-out. By using this software, the likelihoods of 1-4 persons' contributions are calculated, and the most optimal number of contributors is automatically determined; this differs from other open-source software. Therefore, we can eliminate the need to manually determine the number of contributors before the analysis. Kongoh also considers allele- or locus-specific effects of biological parameters based on the experimental data. We then validated Kongoh by calculating the likelihood ratio (LR) of a POI's contribution in true contributors and non-contributors by using 2-4 person mixtures analyzed through a 15 short tandem repeat typing system. Most LR values obtained from Kongoh during true-contributor testing strongly supported the POI's contribution even for small amounts or degraded DNA samples. Kongoh correctly rejected a false hypothesis in the non-contributor testing, generated reproducible LR values, and demonstrated higher accuracy of the estimated number of contributors than another software based on the quantitative continuous model. Therefore, Kongoh is useful in accurately interpreting DNA evidence like mixtures and small amounts or degraded DNA samples.

Probabilistically determining the cellular source of DNA derived from differential extractions in sexual assault scenarios.

Sexual assault cases are the type of case that often produces questions about the cellular source of DNA. In these cases multiple findings of microscopy, DNA profiling and presumptive testing need to be considered when addressing source level propositions. In this work, I consider a line of questioning that has been raised a number of times in the recent past, where in court it was disputed that low levels of sperm seen on a microscope slide were the cellular source of the male DNA profile component generated from the sperm fraction of a differential DNA extraction. I demonstrate how the cell scoring results and DNA profiling results can be considered together, in helping address this source level question through the use of Bayesian Networks.

Exploring the efficacy of paternity and kinship testing based on single nucleotide polymorphisms.

Short tandem repeats (STRs) are conventional genetic markers typically used for paternity and kinship testing. As supplementary markers of STRs, single nucleotide polymorphisms (SNPs) have less discrimination power but broader applicability to degraded samples. The rapid improvement of next-generation sequencing (NGS) and multiplex amplification technologies also make it possible now to simultaneously identify dozens or even hundreds of SNP loci in a single pool. However, few studies have been endeavored to kinship testing based on SNP loci. In this study, we genotyped 90 autosomal human identity SNP loci with NGS, and investigated their testing efficacies based on the likelihood ratio model in eight pedigree scenarios involving paternity, half/full-sibling, uncle/nephew, and first-cousin relationships. We found that these SNPs might be sufficient to discriminate paternity and full-sibling, but impractical for more distant relatives such as uncle and cousin. Furthermore, we conducted an in silico study to obtain the theoretical tendency of how testing efficacy varied with increasing number of SNP loci. For each testing battery in a given pedigree scenario, we obtained distributions of logarithmic likelihood ratio for both simulated relatives and unrelated controls. The proportion of the overlapping area between the two distributions was defined as a false testing level (FTL) to evaluate the testing efficacy. We estimated that 85, 127, 491, and 1,858 putative SNP loci were required to discriminate paternity, full-sibling, half-sibling/uncle-nephew, and first-cousin (FTL, 0.1%), respectively. To test a half-sibling or nephew, an additional uncle relative could be included to decrease the required number of putative SNP loci to ∼320 (FTL, 0.1%). As a systematic computation of paternity and kinship testing based only on SNPs, our results could be informative for further studies and applications on paternity and kinship testing using SNP loci.

An assessment of the information content of likelihood ratios derived from complex mixtures.

With the increasing sensitivity of DNA typing methodologies, as well as increasing awareness by law enforcement of the perceived capabilities of DNA typing, complex mixtures consisting of DNA from two or more contributors are increasingly being encountered. However, insufficient research has been conducted to characterize the ability to distinguish a true contributor (TC) from a known non-contributor (KNC) in these complex samples, and under what specific conditions. In order to investigate this question, sets of six 15-locus Caucasian genotype profiles were simulated and used to create mixtures containing 2-5 contributors. Likelihood ratios were computed for various situations, including varying numbers of contributors and unknowns in the evidence profile, as well as comparisons of the evidence profile to TCs and KNCs. This work was intended to illustrate the best-case scenario, in which all alleles from the TC were detected in the simulated evidence samples. Therefore the possibility of drop-out was not modeled in this study. The computer program DNAMIX was then used to compute LRs comparing the evidence profile to TCs and KNCs. This resulted in 140,000 LRs for each of the two scenarios. These complex mixture simulations show that, even when all alleles are detected (i.e. no drop-out), TCs can generate LRs less than 1 across a 15-locus profile. However, this outcome was rare, 7 of 140,000 replicates (0.005%), and associated only with mixtures comprising 5 contributors in which the numerator hypothesis includes one or more unknown contributors. For KNCs, LRs were found to be greater than 1 in a small number of replicates (75 of 140,000 replicates, or 0.05%). These replicates were limited to 4 and 5 person mixtures with 1 or more unknowns in the numerator. Only 5 of these 75 replicates (0.004%) yielded an LR greater than 1,000. Thus, overall, these results imply that the weight of evidence that can be derived from complex mixtures containing up to 5 contributors, under a scenario in which no drop-out is required to explain any of the contributors, is remarkably high. This is a useful benchmark result on top of which to layer the effects of additional factors, such as drop-out, peak height, and other variables.

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.

Distinguishing between donors and their relatives in complex DNA mixtures with binary models.

While likelihood ratio calculations were until the recent past limited to the evaluation of mixtures in which all alleles of all donors are present in the DNA mixture profile, more recent methods are able to deal with allelic dropout and drop-in. This opens up the possibility to obtain likelihood ratios for mixtures where this was not previously possible, but it also means that a full match between the alleged contributor and the crime stain is no longer necessary. We investigate in this article what the consequences are for relatives of the actual donors, because they typically share more alleles with the true donor than an unrelated individual. We do this with a semi-continuous binary approach, where the likelihood ratios are based on the observed alleles and the dropout probabilities for each donor, but not on the peak heights themselves. These models are widespread in the forensic community. Since in many cases a simple model is used where a uniform dropout probability is assumed for all (or for all unknown) contributors, we explore the extent to which this alters the false positive probabilities for relatives of donors, compared to what would have been obtained with the correct probabilities of dropout for each donor.

Application of mixsep software package: Performance verification of male-mixed DNA analysis.

An experimental model of male-mixed DNA (n=297) was constructed according to the mixed DNA construction principle. This comprised the use of the Applied Biosystems (ABI) 7500 quantitative polymerase chain reaction system, with scientific validation of mixture proportion (Mx; root-mean-square error ≤ 0.02). Statistical analysis was performed on locus separation accuracy using mixsep, a DNA mixture separation R-package, and the analytical performance of mixsep was assessed by examining the data distribution pattern of different mixed gradients, short tandem repeat (STR) loci and mixed DNA types. The results showed that locus separation accuracy had a negative linear correlation with the mixed gradient (R(2)=-0.7121). With increasing mixed gradient imbalance, locus separation accuracy first increased and then decreased, with the highest value detected at a gradient of 1:3 (≥ 90%). The mixed gradient, which is the theoretical Mx, was one of the primary factors that influenced the success of mixed DNA analysis. Among the 16 STR loci detected by Identifiler®, the separation accuracy was relatively high (>88%) for loci D5S818, D8S1179 and FGA, whereas the median separation accuracy value was lowest for the D7S820 locus. STR loci with relatively large numbers of allelic drop-out (ADO; >15) were all located in the yellow and red channels, including loci D18S51, D19S433, FGA, TPOX and vWA. These five loci featured low allele peak heights, which was consistent with the low sensitivity of the ABI 3130xl Genetic Analyzer to yellow and red fluorescence. The locus separation accuracy of the mixsep package was substantially different with and without the inclusion of ADO loci; inclusion of ADO significantly reduced the analytical performance of the mixsep package, which was consistent with the lack of an ADO functional module in this software. The present study demonstrated that the mixsep software had a number of advantages and was recommended for analysis of mixed DNA. This software was easy to operate and produced understandable results with a degree of controllability.

DNA in the Criminal Justice System: The DNA Success Story in Perspective.

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high-volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high-volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high-volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high-volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool.

Toward male individualization with rapidly mutating y-chromosomal short tandem repeats.

Relevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve individuals and populations completely. Here, 52 centers generated quality-controlled data of 13 rapidly mutating (RM) Y-STRs in 14,644 related and unrelated males from 111 worldwide populations. Strikingly, >99% of the 12,272 unrelated males were completely individualized. Haplotype diversity was extremely high (global: 0.9999985, regional: 0.99836-0.9999988). Haplotype sharing between populations was almost absent except for six (0.05%) of the 12,156 haplotypes. Haplotype sharing within populations was generally rare (0.8% nonunique haplotypes), significantly lower in urban (0.9%) than rural (2.1%) and highest in endogamous groups (14.3%). Analysis of molecular variance revealed 99.98% of variation within populations, 0.018% among populations within groups, and 0.002% among groups. Of the 2,372 newly and 156 previously typed male relative pairs, 29% were differentiated including 27% of the 2,378 father-son pairs. Relative to Yfiler, haplotype diversity was increased in 86% of the populations tested and overall male relative differentiation was raised by 23.5%. Our study demonstrates the value of RM Y-STRs in identifying and separating unrelated and related males and provides a reference database.

Derechos fundamentales afectados en la toma de muestras biológicas para la obtención de medios de pruebas de ADN válidos y eficaces, desde el punto de vista del Derecho interno y del Derecho de la Unión Europea / Fundamental rights affected by biological sampling to obtain valid and efficient DNA evidence from the point of view of national law and European Union law

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The interpretation of single source and mixed DNA profiles.

A method for interpreting autosomal mixed DNA profiles based on continuous modelling of peak heights is described. MCMC is applied with a model for allelic and stutter heights to produce a probability for the data given a specified genotype combination. The theory extends to handle any number of contributors and replicates, although practical implementation limits analyses to four contributors. The probability of the peak data given a genotype combination has proven to be a highly intuitive probability that may be assessed subjectively by experienced caseworkers. Whilst caseworkers will not assess the probabilities per se, they can broadly judge genotypes that fit the observed data well, and those that fit relatively less well. These probabilities are used when calculating a subsequent likelihood ratio. The method has been trialled on a number of mixed DNA profiles constructed from known contributors. The results have been assessed against a binary approach and also compared with the subjective judgement of an analyst.

Evaluating forensic DNA profiles using peak heights, allowing for multiple donors, allelic dropout and stutters.

Increases in the sensitivity of DNA profiling technology now allow profiles to be obtained from smaller and more degraded DNA samples than was previously possible. The resulting profiles can be highly informative, but the subjective elements in the interpretation make it problematic to achieve the valid and efficient evaluation of evidential strength required in criminal cases. The problems arise from stochastic phenomena such as "dropout" (absence of an allele in the profile that is present in the underlying DNA) and experimental artefacts such as "stutter" that can generate peaks of ambiguous allelic status. Currently in the UK, evidential strength evaluation uses an approach in which the complex signals in the DNA profiles are interpreted in a semi-manual fashion by trained experts aided by a set of guidelines, but also relying substantially on professional judgment. We introduce a statistical model to calculate likelihood ratios for evaluating DNA evidence arising from multiple known and unknown contributors that allows for such stochastic phenomena by incorporating peak heights. Efficient use of peak heights allows for more crime scene profiles to be reported to courts than is currently possible. The model parameters are estimated from experimental data incorporating multiple sources of variability in the profiling system. We report and analyse experimental results from the SGMPlus system, run at 28 amplification cycles with no enhancements, currently used in the UK. Our methods are readily adapted to other DNA profiling systems provided that the experimental data for the parameter estimation is available.