Forensic DNA Profiling Research Articles

The Importance of Critically Examining the Level of Propositions When Evaluating Forensic DNA Results.

The proposal of a discussion about the use of software to help assign likelihood ratios for forensic DNA profiling results, and the use of their output in the legal process, is both timely and important (see also related contributions elsewhere in Frontiers, e.g., Biedermann et al., 2014). Ever since their introduction in forensic science, DNA profiling analyses have been accompanied with the results of calculations of various sorts. Their scope is well illustrated and documented in several reference monographs (e.g., Evett and Weir, 1998; Buckleton et al., 2005; Balding and Steele, 2015). This solid body of scholarly research and established practice has contributed to the widely held view among scientists and recipients of expert information that eliciting the probative strength of forensic DNA profiling results is per se a numerical task. In this commentary, we intend—in a first part—to make the point that although calculations are, by virtue, an integral part of the quantification of probative strength, it is equally important at the outset to be clear about the question “Why are we doing a calculation?” (Buckleton et al., 2005, p. 151). We will argue that this is not a question that statistics can answer. Stated otherwise, we will contend that, as much it is important to be clear in any instance about what a particular computation exactly purports to do, it is essential to define the questions that are of interest in a particular case at hand. In a second part, we will emphasize on the extent to which, why and how recently issued guidelines (e.g., ENFSI, 2015) encourage such thinking about cases prior to conducting calculations, if any.

How far does it get?—The effect of shooting distance and type of firearm on the simultaneous analysis of DNA and RNA from backspatter recovered from inside and outside surfaces of firearms

When a firearm projectile hits a biological target a spray of biological material (e.g. blood and tissue) is ejected from the entrance wound and propelled back into the direction of the firearm. This phenomenon has been termed ‘backspatter’ and if backspattered biological material reaches the firearm on its backward trajectory it may persist on and be recovered from the firearm's inside surfaces. Molecular genetic analysis of backspatter generated by contact shots and shots from very short distances has already been demonstrated to critically contribute to victim identification and the reconstruction of firearm-related crimes.It is not known, however, up to what shooting distance can backspatter be found on firearms’ inside surfaces and what influence the weapon's type and caliber has on backspatter attributes (e.g. reach, amount and distribution).Therefore, the present pilot study investigated the effect of serval combinations of shooting distances and types of firearms and ammunitions on the analyzability of co-extracted DNA and micro-RNA in samples of backspatter collected from interior and exterior surfaces of the firearms after experimental shootings employing standardized ballistic models.We demonstrate the limiting effect of shooting distance and the type of firearm on the yield of nucleic acids recovered from backspatter and the success rates of forensic DNA profiling and RNA based body-fluid and organ tissue identification in experimental shootings.

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.

On the effect of shooting distance, ballistic model construction, doping and weapon type on the simultaneous analysis of DNA and RNA from backspatter recovered from inside surfaces of firearms

Investigations at crime scenes after criminal acts involving gunshot injuries have occurred often encompass the analysis of traces of blood and so-called backspatter. Molecular genetic analysis of backspatter generated by contact shots and shots from very short distances has already been demonstrated to critically contribute to victim identification and the reconstruction of firearm-related crimes.Herein, we investigated the effect of several combinations of shooting distances and types of firearms on backspatter generation and co-extraction and simultaneous analysis of DNA and RNA isolated from traces of backspatter. Additionally, we assessed whether ‘triple contrast’ doping of ballistic models interferes with forensic analysis of DNA, mtDNA and co-extracted mRNA and miRNA from backspatter collected from inside parts of firearms generated by experimental shootings.We show the effect of shooting distance and the type of firearm in experimental shootings on the yields of DNA and RNA co-extracted from backspatter and the success rates of forensic DNA profiling and RNA based organ identification. Furthermore, we demonstrate that ‘triple contrast’ stained biological samples collected from inside surfaces of firearms are amenable to forensic DNA profiling and permit analysis of the entire mtDNA D-loop even for ‘low template’ DNA amounts that preclude standard short tandem repeat DNA analysis.

Logical Framework of Forensic Identification: Ability to Resist Fabricated DNA.

Over the past 30years, DNA analysis has revolutionized forensic science and has become the most useful single tool in the multifaceted fight against crime. Today, DNA profiling with sets of highly polymorphic autosomal short tandem repeat markers is widely employed and accepted in the courts due to its high discriminating power and reliability. However, an artificial bloodstain purposefully created using molecular biology techniques succeeded in tricking a leading forensic DNA laboratory. The disturbing possibility that a forensic DNA profile can be faked shocked the general public and the mass media, and generated serious discussion about the credibility of DNA evidence. Herein, we present two exemplary assays based on tissue-specific methylation patterns and cell-specific mRNA expression, respectively. These two assays can be integrated into the DNA analysis pipelines without consumption of additional samples. We show that the two assays can not only distinguish between artificial and genuine samples, but also provide information on tissue origin. The two assays were tested on natural and artificial bloodstains (generated by polymerase chain reaction and whole genome amplification technique) and the results illustrated that the logical framework of forensic identification is still useful for forensic identification with the high credibility.

A novel forensic DNA profiling technique for protected species

Geographic assignment of an unknown sample is a key aim in wildlife forensic science. In the course of examining 28 hypervariable STR loci for this purpose, a locus (MsF14) was observed that routinely exhibited variable amplification efficacy, appearing heritable in a Mendelian manner and reproducible. A correlation was seen between efficacy of amplification and geographic origin using 155 samples from across the species’ native range. We propose that this STR locus shows potential to assist in the geographical assignment of an unknown python sample.

Lab Retriever: a software tool for calculating likelihood ratios incorporating a probability of drop-out for forensic DNA profiles

BackgroundTechnological advances have enabled the analysis of very small amounts of DNA in forensic cases. However, the DNA profiles from such evidence are frequently incomplete and can contain contributions from multiple individuals. The complexity of such samples confounds the assessment of the statistical weight of such evidence. One approach to account for this uncertainty is to use a likelihood ratio framework to compare the probability of the evidence profile under different scenarios. While researchers favor the likelihood ratio framework, few open-source software solutions with a graphical user interface implementing these calculations are available for practicing forensic scientists.ResultsTo address this need, we developed Lab Retriever, an open-source, freely available program that forensic scientists can use to calculate likelihood ratios for complex DNA profiles. Lab Retriever adds a graphical user interface, written primarily in JavaScript, on top of a C++ implementation of the previously published R code of Balding. We redesigned parts of the original Balding algorithm to improve computational speed. In addition to incorporating a probability of allelic drop-out and other critical parameters, Lab Retriever computes likelihood ratios for hypotheses that can include up to four unknown contributors to a mixed sample. These computations are completed nearly instantaneously on a modern PC or Mac computer.Conclusions Lab Retriever provides a practical software solution to forensic scientists who wish to assess the statistical weight of evidence for complex DNA profiles. Executable versions of the program are freely available for Mac OSX and Windows operating systems.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0740-8) contains supplementary material, which is available to authorized users.

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.

Genotyping and interpretation of STR-DNA: Low-template, mixtures and database matches—Twenty years of research and development

The introduction of Short Tandem Repeat (STR) DNA was a revolution within a revolution that transformed forensic DNA profiling into a tool that could be used, for the first time, to create National DNA databases. This transformation would not have been possible without the concurrent development of fluorescent automated sequencers, combined with the ability to multiplex several loci together. Use of the polymerase chain reaction (PCR) increased the sensitivity of the method to enable the analysis of a handful of cells. The first multiplexes were simple: ‘the quad’, introduced by the defunct UK Forensic Science Service (FSS) in 1994, rapidly followed by a more discriminating ‘six-plex’ (Second Generation Multiplex) in 1995 that was used to create the world's first national DNA database. The success of the database rapidly outgrew the functionality of the original system – by the year 2000 a new multiplex of ten-loci was introduced to reduce the chance of adventitious matches. The technology was adopted world-wide, albeit with different loci. The political requirement to introduce pan-European databases encouraged standardisation – the development of European Standard Set (ESS) of markers comprising twelve-loci is the latest iteration. Although development has been impressive, the methods used to interpret evidence have lagged behind. For example, the theory to interpret complex DNA profiles (low-level mixtures), had been developed fifteen years ago, but only in the past year or so, are the concepts starting to be widely adopted. A plethora of different models (some commercial and others non-commercial) have appeared. This has led to a confusing ‘debate’ about the ‘best’ to use. The different models available are described along with their advantages and disadvantages. A section discusses the development of national DNA databases, along with details of an associated controversy to estimate the strength of evidence of matches. Current methodology is limited to searches of complete profiles – another example where the interpretation of matches has not kept pace with development of theory. STRs have also transformed the area of Disaster Victim Identification (DVI) which frequently requires kinship analysis. However, genotyping efficiency is complicated by complex, degraded DNA profiles. Finally, there is now a detailed understanding of the causes of stochastic effects that cause DNA profiles to exhibit the phenomena of drop-out and drop-in, along with artefacts such as stutters. The phenomena discussed include: heterozygote balance; stutter; degradation; the effect of decreasing quantities of DNA; the dilution effect.

Development and validation of a rapid PCR method for the PowerPlex® 16 HS system for forensic DNA identification.

Currently, the amplification step of most forensic DNA profiling systems takes 3-4 h to complete. A decrease in the amplification time would allow for increased laboratory throughput, which may help reduce backlog when is due to limited cycling capacity. By using the SpeedSTAR™ HS Polymerase (Takara Bio, Otsu, Japan) in combination with the Veriti® (Applied Biosystems, Foster City, CA, USA) rapid thermal cycler, the amplification time for the PowerPlex® 16 HS (Promega, Madison, WI, USA) kit was reduced by 66% (1 h). The sensitivity of this fast method was comparable to the standard system (0.13 ng). Although this rapid protocol showed an increase in average stutter ratios (2.8%) and a decrease in average peak height ratios (7%) across all loci when compared with standard conditions, it was able to consistently generate reliable DNA profiles. The results of this study indicate that the rapid protocol could be implemented in forensic laboratories with an optimal range of 0.25-2 ng of input DNA using appropriate analytical interpretation guidelines.

Choice of population database for forensic DNA profile analysis.

When evaluating the weight of evidence (WoE) for an individual to be a contributor to a DNA sample, an allele frequency database is required. The allele frequencies are needed to inform about genotype probabilities for unknown contributors of DNA to the sample. Typically databases are available from several populations, and a common practice is to evaluate the WoE using each available database for each unknown contributor. Often the most conservative WoE (most favourable to the defence) is the one reported to the court. However the number of human populations that could be considered is essentially unlimited and the number of contributors to a sample can be large, making it impractical to perform every possible WoE calculation, particularly for complex crime scene profiles. We propose instead the use of only the database that best matches the ancestry of the queried contributor, together with a substantial FST adjustment. To investigate the degree of conservativeness of this approach, we performed extensive simulations of one- and two-contributor crime scene profiles, in the latter case with, and without, the profile of the second contributor available for the analysis. The genotypes were simulated using five population databases, which were also available for the analysis, and evaluations of WoE using our heuristic rule were compared with several alternative calculations using different databases. Using FST = 0.03, we found that our heuristic gave WoE more favourable to the defence than alternative calculations in well over 99% of the comparisons we considered; on average the difference in WoE was just under 0.2 bans (orders of magnitude) per locus. The degree of conservativeness of the heuristic rule can be adjusted through the FST value. We propose the use of this heuristic for DNA profile WoE calculations, due to its ease of implementation, and efficient use of the evidence while allowing a flexible degree of conservativeness.

The variability in likelihood ratios due to different mechanisms.

Recently there has been a drive towards standardisation of forensic DNA interpretation methods resulting in the uptake of probabilistic interpretation software. Some of these software solutions utilise Markov chain Monte Carlo techniques (MCMC). They will not produce an identical answer after repeat interpretations of the same evidence profile because of the Monte Carlo aspect. This is a new source of variability within the forensic DNA analysis process. In this paper we explore the size of the MCMC variability within the interpretation software STRmix™ compared to other sources of variability in forensic DNA profiling including PCR, capillary electrophoresis load and injection, and the makeup of allele frequency databases. The MCMC variability within STRmix™ was shown to be the smallest source of variability in this process.

Enhanced low-template DNA analysis conditions and investigation of allele dropout patterns

Forensic DNA analysis applying PCR enables profiling of minute biological samples. Enhanced analysis conditions can be applied to further push the limit of detection, coming with the risk of visualising artefacts and allele imbalances. We have evaluated the consecutive increase of PCR cycles from 30 to 35 to investigate the limitations of low-template (LT) DNA analysis, applying the short tandem repeat (STR) analysis kit PowerPlex ESX 16. Mock crime scene DNA extracts of four different quantities (from around 8-84 pg) were tested. All PCR products were analysed using 5, 10 and 20 capillary electrophoresis (CE) injection seconds. Bayesian models describing allele dropout patterns, allele peak heights and heterozygote balance were developed to assess the overall improvements in EPG quality with altered PCR/CE settings. The models were also used to evaluate the impact of amplicon length, STR marker and fluorescent label on the risk for allele dropout. The allele dropout probability decreased for each PCR cycle increment from 30 to 33 PCR cycles. Irrespective of DNA amount, the dropout probability was not affected by further increasing the number of PCR cycles. For the 42 and 84 pg samples, mainly complete DNA profiles were generated applying 32 PCR cycles. For the 8 and 17 pg samples, the allele dropouts decreased from 100% using 30 cycles to about 75% and 20%, respectively. The results for 33, 34 and 35 PCR cycles indicated that heterozygote balance and stutter ratio were mainly affected by DNA amount, and not directly by PCR cycle number and CE injection settings. We found 32 and 33 PCR cycles with 10 CE injection seconds to be optimal, as 34 and 35 PCR cycles did not improve allele detection and also included CE saturation problems. We find allele dropout probability differences between several STR markers. Markers labelled with the fluorescent dyes CXR-ET (red in electropherogram) and TMR-ET (shown as black) generally have higher dropout risks compared with those labelled with JOE (green) and fluorescein (blue). Overall, the marker D10S1248 has the lowest allele dropout probability and D8S1179 the highest. The marker effect is mainly pronounced for 30-32 PCR cycles. Such effects would not be expected if the amplification efficiencies were identical for all markers. Understanding allele dropout risks and the variability in peak heights and balances is important for correct interpretation of forensic DNA profiles.

Forensic DNA profiling: state of the art

Forensic DNA profiling: state of the art

Investigation into stutter ratio variability between different laboratories.

The determination of parameters such as stutter ratio is important to inform a laboratory's forensic DNA profile interpretation strategy. As part of a large data analysis project to implement a continuous model of DNA profile interpretation we analysed stutter ratio data from eight different forensic laboratories for the Promega PowerPlex(®) 21 multiplex. This allowed a comparison of inter laboratory variation. The maximum difference for any one laboratory from the average of the best fit determined by the model was 0.31%. These results indicate that stutter ratios calculated from samples analysed using the same profiling kit are not expected to differ between laboratories, even those using different capillary electrophoresis platforms. A common set of laboratory parameters are able to be generated and used for profile interpretation at all laboratories using the same multiplex and cycle number, potentially reducing the need for individual laboratories to determine stutter ratios.

Verifying likelihoods for low template DNA profiles using multiple replicates.

To date there is no generally accepted method to test the validity of algorithms used to compute likelihood ratios (LR) evaluating forensic DNA profiles from low-template and/or degraded samples. An upper bound on the LR is provided by the inverse of the match probability, which is the usual measure of weight of evidence for standard DNA profiles not subject to the stochastic effects that are the hallmark of low-template profiles. However, even for low-template profiles the LR in favour of a true prosecution hypothesis should approach this bound as the number of profiling replicates increases, provided that the queried contributor is the major contributor. Moreover, for sufficiently many replicates the standard LR for mixtures is often surpassed by the low-template LR. It follows that multiple LTDNA replicates can provide stronger evidence for a contributor to a mixture than a standard analysis of a good-quality profile. Here, we examine the performance of the likeLTD software for up to eight replicate profiling runs. We consider simulated and laboratory-generated replicates as well as resampling replicates from a real crime case. We show that LRs generated by likeLTD usually do exceed the mixture LR given sufficient replicates, are bounded above by the inverse match probability and do approach this bound closely when this is expected. We also show good performance of likeLTD even when a large majority of alleles are designated as uncertain, and suggest that there can be advantages to using different profiling sensitivities for different replicates. Overall, our results support both the validity of the underlying mathematical model and its correct implementation in the likeLTD software.

Retrospective study of the impact of miniSTRs on forensic DNA profiling of touch DNA samples.

The theoretical advantages of miniSTRs are undeniable. Several studies show that miniSTRs are more sensitive and robust in the analysis of low template and degraded DNA. In this study we want to show the overall benefit of using miniSTRs in real forensic casework samples and show the percentage of samples that benefit from analysis with additional miniSTR loci in terms of resulting in a useful profile. The considered samples were 3064 touch DNA samples, analyzed in our accredited routine forensic DNA profiling laboratory between mid 2009 and mid 2013. Of these 3064 samples, 618 samples were analyzed using 13 loci, 532 samples using 15 loci and 1914 samples using 20 loci of which 5 were the mini- and midi-STR loci that were added to the extended European Standard Set (ESS). The retrospective results show a small increased success rate after implementation of extra loci and an even smaller increase after the implementation of the mini- and midi-STR analysis. The percentage of touch DNA samples that benefit from the analysis of additional mini- and midi-STR loci is limited.

Forensic DNA databases–Ethical and legal standards: A global review

BackgroundThe Forensic Genetics Policy Initiative (www.dnapolicyinitiative.org) is a civil society-led project which aims to set human rights standards for DNA databases around the world, by establishing best practice and involving experts, policy makers and members of the public in open debate. The authors have collected a comprehensive data set of information on the state of forensic DNA profiling and the development of DNA databases for policing purposes in more than 100 countries. The information is available in wiki which can be expanded, updated or corrected by interested persons (http://wiki.dnapolicyinitiative.org). ResultsA summary of the current global situation and issues for debate highlights: (1) a growing global consensus on the need for legislative provisions for the destruction of biological samples and deletion of innocent people’s DNA profiles, following the European Court of Human Rights’ judgement on this issue in 2008; (2) emerging best practice on scientific standards and standards for the use of DNA in court which are necessary to prevent miscarriages of justice; (3) ongoing debate regarding the appropriate safeguards for DNA collection from suspects; restrictions on access, use and data sharing across borders; and data protection standards. ConclusionThere is an ongoing need for greater public and policy debate as DNA databases expand around the world. Some safeguards are implemented at the national or regional level, but there is an ongoing lack of global standards and a need for more societal engagement and debate.

Analysis of allelic drop-out using the Identifiler(®) and PowerPlex(®) 16 forensic STR typing systems.

Low-template (LT) DNA profiles continue to present interpretational challenges to the forensic community. Whether the LT contribution comprises the main profile, or whether it is present as the minor component of a mixture, ambiguity arises from the possibility that alleles present in the biological sample may not be detected in the resulting DNA profile. This phenomenon is known as allelic drop-out. This ambiguity complicates both the assessment of the potential number of contributors and estimation of the weight of the DNA evidence for or against specific propositions. One solution to estimating the weight of the evidence is to use a likelihood ratio (LR) that incorporates the probability of allelic drop-out P(DO) estimated for the specific evidence sample under consideration. However, although a vast repository of data exists, few empirical studies to determine allelic drop-out probabilities have been performed to date. Here we characterized patterns of allelic drop-out in single-source samples using both universal and run-specific analytical thresholds. Not surprisingly, we found fewer instances of apparent drop-out when using a lower (run-specific) detection threshold. Also, unsurprisingly, a positive correlation exists between allele drop-out and allele length, even in good quality samples. We used logistic regression to model the fraction of alleles that dropped out of a profile as a function of the average height of the detected peaks. The equation derived from the logistic regression model allowed us to estimate the expected drop-out probability for an evidentiary sample based on the average peak height of the profile. We show that the LRs calculated using the estimated drop-out probabilities were similar to those calculated using the benchmark drop-out probabilities, suggesting that the estimates of the drop-out probability are accurate and useful. This trend holds even when using the data from the PowerPlex(®) 16 typing system to estimate the drop-out probability for an Identifiler(®) profile, and vice versa. Thus we demonstrate that use of a LR that incorporates empirically estimated allelic drop-out probabilities provides a reliable means for extracting additional information from LT forensic DNA profiles.

An investigation of the potential of DIP-STR markers for DNA mixture analyses.

The genetic characterization of unbalanced mixed stains remains an important area where improvement is imperative. In fact, using the standard tools of forensic DNA profiling (i.e., STR markers), the profile of the minor contributor in mixed DNA stains cannot be successfully detected if its quantitative share of DNA is less than 10% of the mixed trace. This is due to the fact that the major contributor's profile "masks" that of the minor contributor. Besides known remedies to this problem, such as Y-STR analysis, a new compound genetic marker that consists of a Deletion/Insertion Polymorphism (DIP) linked to a Short Tandem Repeat (STR) polymorphism, has recently been developed and proposed. These novel markers are called DIP-STR markers. This paper compares, from a statistical and forensic perspective, the potential usefulness of these novel DIP-STR markers (i) with traditional STR markers in cases of moderately unbalanced mixtures, and (ii) with Y-STR markers in cases of female-male mixtures. This is done through a comparison of the distribution of 100,000 likelihood ratio values obtained using each method on simulated mixtures. This procedure is performed assuming, in turn, the prosecution's and the defence's point of view.