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Massively Parallel Sequencing (MPS) has gained interest in the forensic community over the past decade. Most of the published MPS methods focus on specialty applications intended for use in a limited number of samples with protocols that are relatively laborious. Recent developments using Reverse-Complement PCR enable an efficient MPS protocol suited for routine analysis of high numbers of samples. This method is implemented in the IDseek® OmniSTR™ Global Autosomal STR Profiling kit (Nimagen) for sequencing 28 of the most commonly used forensic autosomal STRs, one Y-chromosomal STR and Amelogenin. This study describes the validation of this kit and focuses on sensitivity, inhibitor tolerance, sequence variation detection and performance with mixtures up to 5 contributors. Results are compared to a Capillary Electrophoresis method (the PowerPlex® Fusion 6 C system, Promega) and the first commercial forensic MPS kit (ForenSeq™ DNA Signature prep, Qiagen) and for a concordance study with data from the Powerseq® MPS kit as well. Analysis settings in FDSTools are deduced and discussed, and an almost completely automated analysis is achieved. Using FDSTools noise correction, contributions in a mixture down to a level of 1.5 % of the major allele of a marker can be detected., Competing Interests: Declaration of Competing Interest The authors declare that the experiments and analyses have been performed without competing financial interests at the NFI as the collaboration with NimaGen (part of the authors that do have a financial interest) served to optimise the assay with input from the NFI and provide early information for performing data analysis., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: The field of forensic DNA analysis has undergone rapid advancements in recent decades. The integration of massively parallel sequencing (MPS) has notably expanded the forensic toolkit, moving beyond identity matching to predicting phenotypic traits and biogeographical ancestry. This shift is of particular significance in cases where conventional DNA profiling fails to identify a single suspect. Supplementing forensic analyses with estimated biological age may be valuable but involves a complex and time-consuming DNA methylation analysis. This study explores and validates the performance of a comprehensive forensic third-generation sequencing assay utilizing Oxford Nanopore Technologies (ONT) in an adaptive and direct sequencing approach. We incorporated the most widely used forensic markers, i.e., STRs, SNPs, InDels, mitochondrial DNA (mtDNA), and two methylation-based clock classifiers, thereby combining forensic genetic and epigenetic analysis in one single workflow., Methods and Results: In our investigation, DNA from six anonymous individuals was sequenced using the ONT standard adaptive direct sequencing approach, reaching a mean percentage of on-target reads ranging from 6.6 % to 7.7 % per sample. ONT data was compared to standard MPS data and Illumina EPIC DNA methylation profiles. Basecalling employed recommended ONT software packages. TREAT was used for ONT-based analysis of autosomal and Y-chromosome STRs, achieving 90-92 % correct calls depending on allelic read depth thresholds. InDel analyses for two lower-quality samples proved challenging due to inadequate read depth, while the remaining four samples significantly contributed to the observed percentage markers (60.9 %) and correct calls (97.8 %). SNP analysis achieved a 98 % call rate, with only two mismatches and two missed alleles. ONT-generated DNA methylation data demonstrated Pearson's correlation coefficients with EPIC data ranging from 0.67 to 0.97 for Horvath's clock. Additional age-associated markers exhibited Pearson's correlation coefficients with chronological age between 0.14 (ELOVL2) and 0.96 (FHL2) at read depths of <30 and <20, respectively. Despite excluding mtDNA from our targeted sequencing approach, adaptive proof-reading fragments covered the complete mtDNA with an average read depth of 21-72, showing 100 % concordance with reference data., Discussion: Our exploratory study using ONT adaptive sequencing for conventional forensic and age associated DNA methylation markers showed high sequencing accuracy for a significant number of markers, showcasing ONT as a promising (epi)genetic forensic method. Future studies must address three critical aspects: determining clear quantity and quality measures and detection thresholds for accuracy, optimizing input DNA quantity for forensic casework expectations, and addressing ethical considerations associated with phenotype and ancestry analysis to prevent ethnic biases., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Searching a DNA Database with a DNA profile from an evidentiary trace can provide investigative leads in a forensic case. Various searching approaches exist such as conventional methods based on matching alleles or more advanced methods computing likelihood ratios (LR) while considering drop-in and drop-out. Here we examine the potential of using a quantitative LR model (EuroForMix model incorporated in ProbRank method) that takes peak heights into account in comparison to a qualitative LR model (LRmix model implemented in SmartRank method). Both methods present DNA database candidates in order of decreasing LR. Especially regarding minor contributors in complex mixtures, the method using the quantitative model outperforms the method using the qualitative model in terms of sensitivity and specificity as more true donors and less adventitious matches are retrieved. ProbRank is to be implemented in DNAStatistX and is sufficiently fast for daily use., Competing Interests: Declaration of Competing Interest We have no conflict of interest. We confirm that neither the manuscript nor any parts of its content are under consideration or published in another journal., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Tobacco smoking is a frequent habit sustained by > 1.3 billion people in 2020 and the leading preventable factor for health risk and premature mortality worldwide. In the forensic context, predicting smoking habits from biological samples may allow broadening DNA phenotyping. In this study, we aimed to implement previously published smoking habit classification models based on blood DNA methylation at 13 CpGs. First, we developed a matching lab tool based on bisulfite conversion and multiplex PCR followed by amplification-free library preparation and targeted paired-end massively parallel sequencing (MPS). Analysis of six technical duplicates revealed high reproducibility of methylation measurements (Pearson correlation of 0.983). Artificially methylated standards uncovered marker-specific amplification bias, which we corrected via bi-exponential models. We then applied our MPS tool to 232 blood samples from Europeans of a wide age range, of which 90 were current, 71 former and 71 never smokers. On average, we obtained 189,000 reads/sample and 15,000 reads/CpG, without marker drop-out. Methylation distributions per smoking category roughly corresponded to previous microarray analysis, showcasing large inter-individual variation but with technology-driven bias. Methylation at 11 out of 13 smoking-CpGs correlated with daily cigarettes in current smokers, while solely one was weakly correlated with time since cessation in former smokers. Interestingly, eight smoking-CpGs correlated with age, and one displayed weak but significant sex-associated methylation differences. Using bias-uncorrected MPS data, smoking habits were relatively accurately predicted using both two- (current/non-current) and three- (never/former/current) category model, but bias correction resulted in worse prediction performance for both models. Finally, to account for technology-driven variation, we built new, joint models with inter-technology corrections, which resulted in improved prediction results for both models, with or without PCR bias correction (e.g. MPS cross-validation F 1 -score > 0.8; 2-categories). Overall, our novel assay takes us one step closer towards the forensic application of viable smoking habit prediction from blood traces. However, future research is needed towards forensically validating the assay, especially in terms of sensitivity. We also need to further shed light on the employed biomarkers, particularly on the mechanistics, tissue specificity and putative confounders of smoking epigenetic signatures., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The introduction of massively parallel sequencing in forensic analysis has been facilitated with typing kits, analysis software and allele naming tools such as the ForenSeq DNA Signature Prep (DSP) kit, FDSTools and STRNaming respectively. Here we describe how FDSTools 2.0 with integrated and refined STRNaming nomenclature was validated for implementation under ISO 17025 accreditation for the ForenSeq DSP kit. Newly-added options result in efficient automatic allele calling for the majority of markers while specific settings are applied for 'novel' sequence variants to avoid the calling of remaining variable noise observed in samples sequenced with the ForenSeq DSP kit that seem to arise in the PCR. Genome-wide built-in reference data allows for greatly simplified configuration of allele naming for human targets., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The possibility of providing investigative leads when conventional DNA identification methods fail to solve a case can be of extreme relevance to law enforcement. Therefore, the forensic genetics community has focused research towards the broadened use of DNA, particularly for prediction of appearance traits, bio-geographical ancestry and age. The VISible Attributes through GEnomics (VISAGE) Consortium expanded the use of DNA phenotyping by developing new molecular and statistical tools for appearance, age and ancestry prediction. The VISAGE basic tool for appearance (EVC) and ancestry (BGA) prediction was initially developed using Ampliseq chemistry, but here is being evaluated using ForenSeq chemistry. The VISAGE basic tool offers a total of 41 EVC and 115 BGA SNPs and thus provides more predictions, i.e., skin color, than achieved with the ForenSeq DNA Signature Prep kit that is based on 24 EVC and 56 BGA SNPs. Five VISAGE laboratories participated in collaborative experiments to provide foreground for developmental validation of the assay. Assessment of assay performance and quality metrics, reproducibility, sensitivity, inhibitor tolerance and species specificity are described. Furthermore, the assay was tested using challenging samples such as mock casework samples and artificially degraded DNA. Two different analysis strategies were applied for this study and output on genotype calls and read depth was compared. Overall, inter-laboratory, inter-method and concordance with publicly available data were analysed and compared. Finally, the results showed a reliable and robust tool, which can be easily applied for laboratories already using a MiSeq FGx with ForenSeq reagents., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

The analysis of DNA methylation has become an established method for chronological age estimation. This has triggered interest in the forensic community to develop new methods for age estimation from biological crime scene material. Various assays are available for age estimation from somatic tissues, the majority from blood. Age prediction from semen requires different DNA methylation markers and the only assays currently developed for forensic analysis are based on SNaPshot or pyrosequencing. Here, we describe a new assay using massively parallel sequencing to analyse 13 candidate CpG sites targeted in two multiplex PCRs. The assay has been validated by five consortium laboratories of the VISible Attributes through GEnomics (VISAGE) project within a collaborative exercise and was tested for reproducible quantification of DNA methylation levels and sensitivity with DNA methylation controls. Furthermore, DNA extracts and stains on Whatman FTA cards from two semen samples were used to evaluate concordance and mimic casework samples. Overall, the assay yielded high read depths (> 1000 reads) at all 13 marker positions. The methylation values obtained indicated robust quantification with an average standard deviation of 2.8% at the expected methylation level of 50% across the 13 markers and a good performance with 50 ng DNA input into bisulfite conversion. The absolute difference of quantifications from one participating laboratory to the mean quantifications of concordance and semen stains of remaining laboratories was approximately 1%. These results demonstrated the assay to be robust and suitable for age estimation from semen in forensic investigations. In addition to the 13-marker assay, a more streamlined protocol combining only five age markers in one multiplex PCR was developed. Preliminary results showed no substantial differences in DNA methylation quantification between the two assays, indicating its applicability with the VISAGE age model for semen developed with data from the complete 13-marker tool., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Y-chromosomal short tandem repeats (Y-STRs) with high mutation rates are recognized as valuable genetic markers for differentiating paternally related men, who typically cannot be separated with standard Y-STRs, and were shown to provide paternal lineage differentiation on a higher resolution level than standard Y-STRs. Both features make Y-STRs with high mutation rates relevant in criminal casework, particularly in sexual assault cases involving highly unbalanced male-female DNA mixtures that often fail autosomal forensic STR profiling for the male donor. Previously, the number of known Y-STRs with mutation rates higher than 10 -2 per locus per generation termed rapidly mutating Y-STRs (RM Y-STRs) was limited to 13, which has recently been overcome by the discovery and characterization of 12 additional RM Y-STRs. Here, we present the development and validation of RMplex, an efficient genotyping system for analyzing 30 Y-STRs with high mutation rates, including all currently known RM Y-STRs, using multiplex PCR with capillary electrophoresis (CE) or massively parallel sequencing (MPS), overall targeting a total of 44 male-specific loci. If previously unavailable, repeat number assignations were provided based on newly generated MPS data. Validation tests based on the CE method demonstrated that the results were both repeatable and reproducible, full profiles were achieved with minimal input DNA of 250 pg for RMplex 1 and 100 pg for RMplex 2, and in the presence of inhibitors, or with a surplus of female DNA, the assays performed reasonably well. Application of RMplex to differentiate between paternally related men was exemplified in 32 males belonging to five different paternal pedigrees. Given further successful forensic validation testing, we envision the future application of RMplex in criminal cases where it is suspected, or cannot be excluded, that the crime scene trace originated from a male relatives of the suspect who is highlighted with standard Y-STR matching. Other applications of RMplex are in criminal cases without known suspects to differentiate between male relatives highlighted in familial searching based on standard Y-STR matching., (Copyright © 2021. Published by Elsevier B.V.)

The interpretation of short tandem repeat (STR) profiles can be challenging when, for example, alleles are masked due to allele sharing among contributors and/or when they are subject to drop-out, for instance from sample degradation. Mixture interpretation can be improved by increasing the number of STRs and/or loci with a higher discriminatory power. Both capillary electrophoresis (CE, 6-dye) and massively parallel sequencing (MPS) provide a platform for analysing relatively large numbers of autosomal STRs. In addition, MPS enables distinguishing between sequence variants, resulting in enlarged discriminatory power. Also, MPS allows for small amplicon sizes for all loci as spacing is not an issue, which is beneficial with degraded DNA. Altogether, MPS has the potential to increase the weights of evidence for true contributors to (complex) DNA profiles. In this study, likelihood ratio (LR) calculations were performed using STR profiles obtained with two different MPS systems and analysed using different settings: 1) MPS PowerSeq™ Auto System profiles analysed using FDSTools equipped with optimized settings such as noise correction, 2) ForenSeq™ DNA Signature Prep Kit profiles analysed using the default settings in the Universal Analysis Software (UAS), and 3) ForenSeq™ DNA Signature Prep Kit profiles analysed using FDSTools empirically adapted to cope with one-directional reads and provisional, basic settings. The LR calculations used genotyping data for two- to four-person mixtures varying for mixture proportion, level of drop-out and allele sharing and were generated with the continuous model EuroForMix. The LR results for the over 2000 sets of propositions were affected by the variation for the number of markers and analysis settings used in the three approaches. Nevertheless, trends for true and non-contributors, effects of replicates, assigned number of contributors, and model validation results were comparable for the three MPS approaches and alike the trends known for CE data. Based on this analogy, we regard the probabilistic interpretation of MPS STR data fit for forensic DNA casework. In addition, guidelines were derived on when to apply LR calculations to MPS autosomal STR data and report the corresponding results., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The introduction of Massively Parallel Sequencing in the forensic domain has exposed the need for comprehensive nomenclature of sequenced Short Tandem Repeat (STR) alleles. In general, three strategies are at hand: 1) the full sequence mapped to the human genome reference sequence, which ensures exact data exchange; 2) shortened, human-readable formats for forensic reporting and data presentation and 3) very short codes that enable compact figures and tables but do not convey any sequence information. Here, we describe an algorithm of the second type: STRNaming, which generates human-readable names for sequenced STR alleles. STRNaming is guided by a reference sequence at each locus and then functions independently to automatically assign a unique, sequence-descriptive name that also includes the capillary electrophoresis allele number. STRNaming settings were established based on preferences that were surveyed internationally in the forensic community. These settings ensure that a small change in the sequence corresponds to a small change in the allele name, which is helpful for recognising for instance stutter products. Sequence variants outside of the repeat units are indicated as simple variant calls. Since the STR name is sequence-descriptive, the sequence can be traced back from the allele name. Because STRNaming is fully guided by an assignable reference sequence, no central coordination or configuration is required and the method will work for any STR locus, be it autosomal, Y-, X-chromosomal in current or future use. The algorithm is publicly available online and offline., (Copyright © 2021 Elsevier B.V. All rights reserved.)

In recent years, forensic mRNA profiling has increasingly been used to identify the origin of human body fluids. By now, several laboratories have implemented mRNA profiling and also use it in criminal casework. In 2018 the FoRNAP (Forensic RNA Profiling) group was established among a number of these laboratories with the aim of sharing experiences, discussing optimization potential, identifying challenges and suggesting solutions with regards to mRNA profiling and casework. To compare mRNA profiling methods and results a collaborative exercise was organized within the FoRNAP group. Seven laboratories from four countries received 16 stains, comprising six pure body fluid / tissue stains and ten mock casework samples. The laboratories were asked to analyze the provided stains with their in-house method (PCR/CE or MPS) and markers of choice. Five laboratories used a DNA/RNA co-extraction strategy. Overall, up to 11 mRNA markers per body fluid were analyzed. We found that mRNA profiling using different extraction and analysis methods as well as different multiplexes can be applied to casework-like samples. In general, high input samples were typed with high accuracy by all laboratories, regardless of the method used. Irrespective of the analysis strategy, samples of low input or mixed stains were more challenging to analyze and interpret since, alike to DNA profiling, a higher number of markers dropped out and/or additional unexpected markers not consistent with the cell type in question were detected. It could be shown that a plethora of different but valid analysis and interpretation strategies exist and are successfully applied in the Forensic Genetics community. Nevertheless, efforts aiming at optimizing and harmonizing interpretation approaches in order to achieve a higher consistency between laboratories might be desirable in the future. The simultaneous extraction of DNA alongside RNA showed to be an effective approach to identify not only the body fluid present but also to identify the donor(s) of the stain. This allows investigators to gain valuable information about the origin of crime scene samples and the course of events in a crime case., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

This study describes a multi-laboratory validation of DNAxs, a DNA eXpert System for the data management and probabilistic interpretation of DNA profiles [1], and its statistical library DNAStatistX to which, besides the organising laboratory, four laboratories participated. The software was modified to read multiple data formats and the study was performed prior to the release of the software to the forensic community. The first exercise explored all main functionalities of DNAxs with feedback on user-friendliness, installation and general performance. Next, every laboratory performed likelihood ratio (LR) calculations using their own dataset and a dataset provided by the organising laboratory. The organising laboratory performed LR calculations using all datasets. The datasets were generated with different STR typing kits or analysis systems and consisted of samples varying in DNA amounts, mixture ratios, number of contributors and drop-out level. Hypothesis sets had the correct, under- and over-assigned number of contributors and true and false donors as person of interest. When comparing the results between laboratories, the LRs were foremost within one unit on log10 scale. The few LR results that deviated more had differences for the parameters estimated by the optimizer within DNAStatistX. Some of these were indicated by failed iteration results, others by a failed model validation, since unrealistic hypotheses were included. When these results that do not meet the quality criteria were excluded, as is in accordance with interpretation guidelines, none of the analyses in the different laboratories yielded a different statement in the casework report. Nonetheless, changes in software parameters were sought that minimized differences in outcomes, which made the DNAStatistX module more robust. Overall, the software was found intuitive, user-friendly and valid for use in multiple laboratories., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The value of the evidence depends critically on propositions. In the second of two papers intended to provide advice to the community on difficult aspects of evaluation and the formulation of propositions, we focus primarily on activity level propositions. This helps the court address the question of "How did an individual's cell material get there?". In order to do this, we expand the framework outlined in the first companion paper. First, it is important not to conflate results and propositions. Statements given activity level propositions aim to help address issues of indirect vs direct transfer, and the time of the activity, but it is important to avoid use of the word 'transfer' in propositions. This is because propositions are assessed by the Court, but DNA transfer is a factor that scientists need to take into account for the interpretation of their results. Suitable activity level propositions are ideally set before knowledge of the results and address issues like: X stabbed Y vs. an unknown person stabbed Y but X met Y the day before. The scientist assigns the probability of the evidence, if each of the alternate propositions is true, to derive a likelihood ratio. To do this, the scientist asks: a) "what are the expectations if each of the propositions is true?" b) "What data are available to assist in the evaluation of the results given the propositions?" When presenting evidence, scientists work within the hierarchy of propositions framework. The value of evidence calculated for a DNA profile cannot be carried over to higher levels in the hierarchy - the calculations given sub-source, source and activity level propositions are all separate. A number of examples are provided to illustrate the principles espoused, and the criteria that such assessments should meet. Ideally in order to assign probabilities, the analyst should have/collect data that are relevant to the case in question. These data must be relevant to the case at hand and we encourage further research and collection of data to form knowledge bases. Bayesian Networks are extremely useful to help us think about a problem, because they force us to consider all relevant possibilities in a logical way. An example is provided., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Forensic DNA Phenotyping (FDP) provides the ability to predict externally visible characteristics from minute amounts of crime scene DNA, which can help find unknown perpetrators who are typically unidentifiable via conventional forensic DNA profiling. Fundamental human genetics research has led to a better understanding of the specific DNA variants responsible for physical appearance characteristics, particularly eye, hair, and skin color. Recently, we introduced the HIrisPlex-S system for the simultaneous prediction of eye, hair, and skin color based on 41 DNA variants generated from two forensically validated SNaPshot multiplex assays using capillary electrophoresis (CE). Here we introduce massively parallel sequencing (MPS) solutions for the HIrisPlex-S (HPS) system on two MPS platforms commonly used in forensics, Ion Torrent and MiSeq, that cover all 41 DNA variants in a single assay, respectively. Additionally, we present the forensic developmental validation of the two HPS-MPS assays. The Ion Torrent MPS assay, based on Ion AmpliSeq technology, illustrated the successful generation of full HIrisPlex-S genotypic profiles from 100 pg of input control DNA, while the MiSeq MPS assay based on an in-house design yielded complete profiles from 250 pg of input DNA. Assessing simulated forensic casework samples such as saliva, hair (bulb), blood, semen, and low quantity touch DNA, as well as artificially damaged DNA samples, concordance testing, and samples from numerous species, all illustrated the ability of both versions of the HIrisPlex-S MPS assay to produce results that motivate forensic applications. By also providing an integrated bioinformatics analysis pipeline, MPS data can now be analyzed and a file generated for upload to the publically accessible HIrisPlex online webtool (https://hirisplex.erasmusmc.nl). In addition, we updated the website to accept VCF input data for those with genome sequence data. We thus provide a user-friendly and semi-automated MPS workflow from DNA sample to individual eye, hair, and skin color prediction probabilities. Furthermore, we present a 2-person mixture separation tool that not only assesses genotype reliability with regards genotyping confidence but also provides the most fitting mixture scenario for both minor and major contributors, including profile separation. We envision this MPS implementation of the HIrisPlex-S system for eye, hair, and skin color prediction from DNA as a starting point for further expanding MPS-based forensic DNA phenotyping. This may include the future addition of SNPs predictive for more externally visible characteristics, as well as SNPs for bio-geographic ancestry inference, provided the statistical framework for DNA prediction of these traits is in place., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The data management, interpretation and comparison of sets of DNA profiles can be complex, time-consuming and error-prone when performed manually. This, combined with the growing numbers of genetic markers in forensic identification systems calls for expert systems that can automatically compare genotyping results within (large) sets of DNA profiles and assist in profile interpretation. To that aim, we developed a user-friendly software program or DNA eXpert System that is denoted DNAxs. This software includes features to view, infer and match autosomal short tandem repeat profiles with connectivity to up and downstream software programs. Furthermore, DNAxs has imbedded the 'DNAStatistX' module, a statistical library that contains a probabilistic algorithm to calculate likelihood ratios (LRs). This algorithm is largely based on the source code of the quantitative probabilistic genotyping system EuroForMix [1]. The statistical library, DNAStatistX, supports parallel computing which can be delegated to a computer cluster and enables automated queuing of requested LR calculations. DNAStatistX is written in Java and is accessible separately or via DNAxs. Using true and non-contributors to DNA profiles with up to four contributors, the DNAStatistX accuracy and precision were assessed by comparing the DNAStatistX results to those of EuroForMix. Results were the same up to rare differences that could be attributed to the different optimizers used in both software programs. Implementation of dye specific detection thresholds resulted in larger likelihood values and thus a better explanation of the data used in this study. Furthermore, processing time, robustness of DNAStatistX results and the circumstances under which model validations failed were examined. Finally, guidelines for application of the software are shared as an example. The DNAxs software is future-proof as it applies a modular approach by which novel functionalities can be incorporated., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Continuous probabilistic genotyping software enables the interpretation of highly complex DNA profiles that are prone to stochastic effects and/or consist of multiple contributions. The process of introducing probabilistic genotyping into an accredited forensic laboratory requires testing, validation, documentation and training. Documents that include guidelines and/or requirements have been published in order to guide forensic laboratories through this extensive process and there has been encouragements to share the results obtained from internal laboratory studies. To this end, we present the results obtained from the quantitative probabilistic genotyping system EuroForMix applied to mixed DNA profiles with known contributions mixed in known proportions, levels of allele sharing and levels of allelic drop-out. The mixtures were profiled using the PowerPlex® Fusion 6C (PPF6C) kit. Using these mixtures, 427 Hp-true tests and 408 Hd-true tests were performed. In the Hd-true tests, non-contributors were selected deliberately to a have large overlap with the alleles within the mixture and worst-case scenarios were examined in which a simulated relative of one of the true donors was considered as the person of interest under the prosecution hypothesis. The effects of selecting different EuroForMix modelling options, the use of PCR replicates, allelic drop-out, and varying the assigned number of contributors were examined. Instances of Type I and Type II errors are discussed. In addition 330 likelihood ratio results from EuroForMix are compared to the semi-continuous model LRmix Studio. Results demonstrate the performance and trends of EuroForMix when applied to PPF6C profiles., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Human head hair shape, commonly classified as straight, wavy, curly or frizzy, is an attractive target for Forensic DNA Phenotyping and other applications of human appearance prediction from DNA such as in paleogenetics. The genetic knowledge underlying head hair shape variation was recently improved by the outcome of a series of genome-wide association and replication studies in a total of 26,964 subjects, highlighting 12 loci of which 8 were novel and introducing a prediction model for Europeans based on 14 SNPs. In the present study, we evaluated the capacity of DNA-based head hair shape prediction by investigating an extended set of candidate SNP predictors and by using an independent set of samples for model validation. Prediction model building was carried out in 9674 subjects (6068 from Europe, 2899 from Asia and 707 of admixed European and Asian ancestries), used previously, by considering a novel list of 90 candidate SNPs. For model validation, genotype and phenotype data were newly collected in 2415 independent subjects (2138 Europeans and 277 non-Europeans) by applying two targeted massively parallel sequencing platforms, Ion Torrent PGM and MiSeq, or the MassARRAY platform. A binomial model was developed to predict straight vs. non-straight hair based on 32 SNPs from 26 genetic loci we identified as significantly contributing to the model. This model achieved prediction accuracies, expressed as AUC, of 0.664 in Europeans and 0.789 in non-Europeans; the statistically significant difference was explained mostly by the effect of one EDAR SNP in non-Europeans. Considering sex and age, in addition to the SNPs, slightly and insignificantly increased the prediction accuracies (AUC of 0.680 and 0.800, respectively). Based on the sample size and candidate DNA markers investigated, this study provides the most robust, validated, and accurate statistical prediction models and SNP predictor marker sets currently available for predicting head hair shape from DNA, providing the next step towards broadening Forensic DNA Phenotyping beyond pigmentation traits., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The interpretation of evidence continues to be one of the biggest challenges facing the forensic community. This is the first of two papers intended to provide advice on difficult aspects of evaluation and in particular on the formulation of propositions. The scientist has a dual role: investigator (crime-focused), where often there is no suspect available and a database search may be required; evaluator (suspect-focused), where the strength of evidence is assessed in the context of the case. In investigative mode, generally the aim is to produce leads regarding the source of the DNA. Sub-source level propositions will be adequate to help identify potential suspects who can be further investigated by the authorities. Once in evaluative mode, given the defence version of events of the person of interest, it may become necessary to consider alternatives that go beyond the source of the DNA (i.e., to consider activity level propositions). In the evaluation phase, it is crucial that formulation of propositions allows the assessment of all the results that will help with the issue at hand. Propositions should therefore be precise (indication of the number of contributors, information on the relevant population etc.), be about causes, not effects (e.g. a 'matching' DNA profile) and to avoid bias, must not be findings-led. This means that ideally, propositions should be decided based on the case information and before the results of the comparisons are known. This paper primarily reflects upon what has been coined as "sub-source level propositions". These are restricted to the evaluation of the DNA profiles themselves, and help answer the issue regarding the source of the DNA. It is to be emphasised that likelihood ratios given sub-source level propositions cannot be carried over to a different level - for example, activity level propositions, where the DNA evidence is put into the context of the alleged activities. This would be highly misleading and could give rise to miscarriages of justice; this will be discussed in a second paper. The value of forensic results depends not only on propositions, but also on the type of results (e.g. allelic designations, peak heights, replicates) and upon the model used: it is therefore important to discuss these aspects. Finally, since communication is key to help understanding by courts, we will explore how to convey the value of the results and explain the importance of avoiding the practice of transposing the conditional., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as concordance testing, demonstrated the robustness, efficiency, and forensic suitability of the new 17-plex assay, as previously shown for the 24-plex assay. Finally, we provide an update to the publically available HIrisPlex website https://hirisplex.erasmusmc.nl/, now allowing the estimation of individual probabilities for 3 eye, 4 hair, and 5 skin colour categories from HIrisPlex-S input genotypes. The HIrisPlex-S DNA test represents the first forensically validated tool for skin colour prediction, and reflects the first forensically validated tool for simultaneous eye, hair and skin colour prediction from DNA., (Copyright © 2018 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Massively parallel sequencing (MPS) is on the advent of a broad scale application in forensic research and casework. The improved capabilities to analyse evidentiary traces representing unbalanced mixtures is often mentioned as one of the major advantages of this technique. However, most of the available software packages that analyse forensic short tandem repeat (STR) sequencing data are not well suited for high throughput analysis of such mixed traces. The largest challenge is the presence of stutter artefacts in STR amplifications, which are not readily discerned from minor contributions. FDSTools is an open-source software solution developed for this purpose. The level of stutter formation is influenced by various aspects of the sequence, such as the length of the longest uninterrupted stretch occurring in an STR. When MPS is used, STRs are evaluated as sequence variants that each have particular stutter characteristics which can be precisely determined. FDSTools uses a database of reference samples to determine stutter and other systemic PCR or sequencing artefacts for each individual allele. In addition, stutter models are created for each repeating element in order to predict stutter artefacts for alleles that are not included in the reference set. This information is subsequently used to recognise and compensate for the noise in a sequence profile. The result is a better representation of the true composition of a sample. Using Promega Powerseq™ Auto System data from 450 reference samples and 31 two-person mixtures, we show that the FDSTools correction module decreases stutter ratios above 20% to below 3%. Consequently, much lower levels of contributions in the mixed traces are detected. FDSTools contains modules to visualise the data in an interactive format allowing users to filter data with their own preferred thresholds., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

In forensics, DNA profiling is used for the identification of the donor of a trace, while messenger RNA (mRNA) profiling can be applied to identify the cellular origin such as body fluids or organ tissues. The presence of male cell material can be readily assessed by the incorporation of Y-chromosomal markers in quantitation or STR profiling systems. However, no forensic marker exists to positively identify female cell material; merely the presence of female DNA is deduced from the absence of a Y peak, or unbalanced X-Y signals at the Amelogenin locus or unbalanced response of the total and Y-specific quantifier. The presence of two X-chromosomes in female cells invokes dosage compensation, which is achieved through inactivation of one of the X-chromosomes in females. Since this process involves specific RNA molecules, identification of female cellular material may be possible through RNA profiling. Additionally, male material may be identified through RNAs expressed from the Y-chromosome. RNAs preferentially expressed in either sex were assessed for their potential to act as sex markers in forensic RNA assays. To confirm sex-specificity, body fluids and organ tissues of multiple donors of either sex were tested. Additionally, sensitivity of the markers and the suitability of positively identifying male-female mixtures were assessed and degraded samples were used to assess performance of the markers in forensic settings. The addition of sex-specific markers is of added informative value in any RNA profiling system and both markers were incorporated into existing RNA assays that either target body fluids or organs. These are the first forensic assays that enable positive identification of female cellular material., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

A collaborative European DNA Profiling (EDNAP) Group exercise was undertaken to assess the performance of an earlier described SNaPshot™-based screening assay (denoted mini-mtSNaPshot) (Weiler et al., 2016) [1] that targets 18 single nucleotide polymorphism (SNP) positions in the mitochondrial (mt) DNA control region and allows for discrimination of major European mtDNA haplogroups. Besides the organising laboratory, 14 forensic genetics laboratories were involved in the analysis of 13 samples, which were centrally prepared and thoroughly tested prior to shipment. The samples had a variable complexity and comprised straightforward single-source samples, samples with dropout or altered peak sizing, a point heteroplasmy and two-component mixtures resulting in one to five bi-allelic calls. The overall success rate in obtaining useful results was high (97.6%) given that some of the participating laboratories had no previous experience with the typing technology and/or mtDNA analysis. The majority of the participants proceeded to haplotype inference to assess the feasibility of assigning a haplogroup and checking phylogenetic consistency when only 18 SNPs are typed. To mimic casework procedures, the participants compared the SNP typing data of all 13 samples to a set of eight mtDNA reference profiles that were described according to standard nomenclature (Parson et al., 2014) [2], and indicated whether these references matched each sample or not. Incorrect scorings were obtained for 2% of the comparisons and derived from a subset of the participants, indicating a need for training and guidelines regarding mini-mtSNaPshot data interpretation., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Mitochondrial DNA (mtDNA) analysis is regularly applied to forensic DNA samples with limited amounts of nuclear DNA (nDNA), such as hair shafts and bones. Generally, this mtDNA analysis involves examination of the hypervariable control region by Sanger sequencing of amplified products. When samples are severely degraded, small-sized amplicons can be applied and an earlier described mini-mtDNA method by Eichmann et al. [1] that accommodates ten mini amplicons in two multiplexes is found to be a very robust approach. However, in cases with large numbers of samples, like when searching for hairs with an mtDNA profile deviant from that of the victim, the method is time (and cost) consuming. Previously, Chemale et al. [2] described a SNaPshot™-based screening tool for a Brazilian population that uses standard-size amplicons for HVS-I and HVS-II. Here, we describe a similar tool adapted to the full control region and compatible with mini-mtDNA amplicons. Eighteen single nucleotide polymorphisms (SNPs) were selected based on their relative frequencies in a European population. They showed a high discriminatory power in a Dutch population (97.2%). The 18 SNPs are assessed in two SNaPshot™ multiplexes that pair to the two mini-mtDNA amplification multiplexes. Degenerate bases are included to limit allele dropout due to SNPs at primer binding site positions. Three SNPs provide haplogroup information. Reliability testing showed no differences with Sanger sequencing results. Since mini-mtSNaPshot screening uses only a small portion of the same PCR products used for Sanger sequencing, no additional DNA extract is consumed, which is forensically advantageous., (Copyright © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.)

Especially when minute evidentiary traces are analysed, background cell material unrelated to the crime may contribute to detectable levels in the genetic analyses. To gain understanding on the composition of human cell material residing on surfaces contributing to background traces, we performed DNA and mRNA profiling on samplings of various items. Samples were selected by considering events contributing to cell material deposits in exemplary activities (e.g. dragging a person by the trouser ankles), and can be grouped as public objects, private samples, transfer-related samples and washing machine experiments. Results show that high DNA yields do not necessarily relate to an increased number of contributors or to the detection of other cell types than skin. Background cellular material may be found on any type of public or private item. When a major contributor can be deduced in DNA profiles from private items, this can be a different person than the owner of the item. Also when a specific activity is performed and the areas of physical contact are analysed, the "perpetrator" does not necessarily represent the major contributor in the STR profile. Washing machine experiments show that transfer and persistence during laundry is limited for DNA and cell type dependent for RNA. Skin conditions such as the presence of sebum or sweat can promote DNA transfer. Results of this study, which encompasses 549 samples, increase our understanding regarding the prevalence of human cell material in background and activity scenarios., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The forensic identification of human body fluids and tissues by means of messenger RNA (mRNA) profiling is a long studied methodology that is increasingly applied to casework samples. Previously, we have described an mRNA multiplex system that targets blood, saliva, semen, menstrual secretion, vaginal mucosa and skin (Lindenbergh et al. and van den Berge et al.). In this study we consider various topics to improve this mRNA profiling system or its use and adapt the method accordingly. Bodily secretions that may be encountered at a crime scene whilst not targeted by the multiplex-id est nasal mucosa, sweat, tears, faeces and urine-were examined for false positive signals. The results prompted us to identify a nasal mucosa marker that allows the discrimination of nasal mucosa from saliva or vaginal mucosa and nosebleed blood from peripheral blood. An updated version of the multiplex was prepared to which the nasal mucosa marker was added and in which markers for semen, vaginal mucosa and blood were replaced. Lactobacillus markers were regarded unsuitable as replacement for vaginal mucosa mRNA markers because of background signals on penile swabs that appeared devoid of female DNA. Furthermore, we provide approaches to deal with highly unbalanced mixtures. First, a differential extraction protocol was incorporated into a co-extraction protocol to allow DNA and RNA analysis of separated non-sperm and sperm fractions. In a second approach, besides the standard multiplex, a customized multiplex is used which excludes markers for prevailing cell types. This allows the use of lower cDNA inputs for the prevailing cell types and higher inputs for cell types that appear masked. Additionally, we assessed the relation between the percentage of alleles or markers detected in DNA or RNA profiles when decreasing sample amounts are analysed. While blood, saliva, semen and menstrual secretion show the trend that DNA profiling is more sensitive than RNA profiling, the reverse is seen for skin and variable results occur for vaginal and nasal mucosa. Lastly, we show that replicates are useful for interpretation of RNA data, as variations can be found even for true technical replicates. Increased numbers of replicates (over four) do, however, not cancel out the impact of this variation on data interpretation. Overall, the results of this study further forensic RNA profiling., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

There is increasing interest in forensic ancestry tests, which are part of a growing number of DNA analyses that can enhance routine profiling by obtaining additional genetic information about unidentified DNA donors. Nearly all ancestry tests use single nucleotide polymorphisms (SNPs), but these currently rely on SNaPshot single base extension chemistry that can fail to detect mixed DNA. Insertion-deletion polymorphism (Indel) tests have been developed using dye-labeled primers that allow direct capillary electrophoresis detection of PCR products (PCR-to-CE). PCR-to-CE maintains the direct relationship between input DNA and signal strength as each marker is detected with a single dye, so mixed DNA is more reliably detected. We report the results of a collaborative inter-laboratory exercise of 19 participants (15 from the EDNAP European DNA Profiling group) that assessed a 34-plex SNP test using SNaPshot and a 46-plex Indel test using PCR-to-CE. Laboratories were asked to type five samples with different ancestries and detect an additional mixed DNA sample. Statistical inference of ancestry was made by participants using the Snipper online Bayes analysis portal plus an optional PCA module that analyzes the genotype data alongside calculation of Bayes likelihood ratios. Exercise results indicated consistent genotyping performance from both tests, reaching a particularly high level of reliability for the Indel test. SNP genotyping gave 93.5% concordance (compared to the organizing laboratory's data) that rose to 97.3% excluding one laboratory with a large number of miscalled genotypes. Indel genotyping gave a higher concordance rate of 99.8% and a reduced no-call rate compared to SNP analysis. All participants detected the mixture from their Indel peak height data and successfully assigned the correct ancestry to the other samples using Snipper, with the exception of one laboratory with SNP miscalls that incorrectly assigned ancestry of two samples and did not obtain informative likelihood ratios for a third. Therefore, successful ancestry assignments were achieved by participants in 92 of 95 Snipper analyses. This exercise demonstrates that ancestry inference tests based on binary marker sets can be readily adopted by laboratories that already have well-established CE regimes in place. The Indel test proved to be easy to use and allowed all exercise participants to detect the DNA mixture as well as achieving complete and concordant profiles in nearly all cases. Lastly, two participants successfully ran parallel next-generation sequencing analyses (each using different systems) and achieved high levels of genotyping concordance using the exercise PCR primer mixes unmodified., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

DNA-based prediction of hair morphology, defined as straight, curly or wavy hair, could contribute to an improved description of an unknown offender and allow more accurate forensic reconstructions of physical appearance in the field of forensic DNA phenotyping. Differences in scalp hair morphology are significant at the worldwide scale and within Europe. The only genome-wide association study made to date revealed the Trichohyalin gene (TCHH) to be significantly associated with hair morphology in Europeans and reported weaker associations for WNT10A and FRAS1 genes. We conducted a study that centered on six SNPs located in these three genes with a sample of 528 individuals from Poland. The predictive capacity of the candidate DNA variants was evaluated using logistic regression; classification and regression trees; and neural networks, by applying a 10-fold cross validation procedure. Additionally, an independent test set of 142 males from six European populations was used to verify performance of the developed prediction models. Our study confirmed association of rs11803731 (TCHH), rs7349332 (WNT10A) and rs1268789 (FRAS1) SNPs with hair morphology. The combined genotype risk score for straight hair had an odds ratio of 2.7 and these predictors explained ∼ 8.2% of the total variance. The selected three SNPs were found to predict straight hair with a high sensitivity but low specificity when a 10-fold cross validation procedure was applied and the best results were obtained using the neural networks approach (AUC=0.688, sensitivity=91.2%, specificity=23.0%). Application of the neural networks model with 65% probability threshold on an additional test set gave high sensitivity (81.4%) and improved specificity (50.0%) with a total of 78.7% correct calls, but a high non-classification rate (66.9%). The combined TTGGGG SNP genotype for rs11803731, rs7349332, rs1268789 (European frequency=4.5%) of all six straight hair-associated alleles was identified as the best predictor, giving >80% probability of straight hair. Finally, association testing of 44 SNPs previously identified to be associated with male pattern baldness revealed a suggestive association with hair morphology for rs4679955 on 3q25.1. The study results reported provide the starting point for the development of a predictive test for hair morphology in Europeans. More studies are now needed to discover additional determinants of hair morphology to improve the predictive accuracy of this trait in forensic analysis., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Interpretation of DNA mixtures with three or more contributors, defined here as high order mixtures, is difficult because of the inevitability of allele sharing. Allele sharing complicates the estimation of the number of contributors, which is an important parameter to assess the probative value. Consequently, these mixtures may not be deemed suitable for interpretation and reporting. In this study, we generated three-, four- and five-person mixtures with little or no drop-out and with varying levels of allele sharing. For these DNA mixtures we computed likelihood ratios (LRs) using the LRmix model, and always using persons of interest that are true contributors. We assessed the influence of different scenarios on the LR, and used (1) the true or an incorrect number of contributors, (2) zero, one or two anchored individuals and (3) an equal number of contributors under Hp and Hd or an extra contributor under Hd. It was shown that the LR varied considerably when the hypotheses used an incorrect number of contributors, especially when individuals were anchored under the hypotheses. Overall, when analysing high order mixtures, there may occur a transition from LR greater than one to less than one if an incorrect number of contributors is conditioned. This is a result of allele sharing among the multiple contributors rather than allele drop-out, since this study only utilised samples with little or no drop-out., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Minute amounts of DNA representing only few diploid cells, may be interrogated using enhanced DNA profiling, which will be accompanied by stochastic amplification effects. Notwithstanding, a weight of evidence statistic may be calculated using current interpretation software. In this study, we profiled single donor, two- and three-person samples having only 3 pg to 12 pg of DNA per contributor using both standard and enhanced capillary electrophoresis (CE) injection settings. Likelihood ratios (LRs) were computed using LRmix Studio, compared for both types of profiles and examined in relation to the amount of DNA, drop-out level, number of detected alleles, peak heights and reproducibility of alleles. Especially for DNA profiles that were generated using enhanced CE, the obtained LRs could indicate strong evidence in favour of the prosecution (log10(LR)>6), also when the amount of DNA represented about half of a diploid cell equivalent in the amplification. These results illustrate that an assessment of the criminalistic relevance of a sample carrying minute amounts of DNA is essential prior to applying enhanced interrogation techniques and/or calculating a weight of evidence statistic., (Copyright © 2015 The Chartered Society of Forensic Sciences. Published by Elsevier Ireland Ltd. All rights reserved.)

Human biological traces have the potential to present strong evidence for placing a suspect at a crime scene. In cases, the activity that led to deposition of an individual's cellular material is increasingly disputed, for which the identification of cell types could be crucial. This review aims to give an overview of the possibilities of the employment of mRNA, miRNA, DNA methylation and microbial markers for tissue identification in a forensic context. The biological background that renders these markers tissue-specificity is considered, as this can affect data interpretation. Furthermore, the forensic relevance of inferring certain cell types is discussed, as are the various methodologies that can be applied. Forensic stains can carry minute amounts of cell material that may be degraded or polluted and most likely cell material of multiple sources will be present. The interpretational challenges that are imposed by this compromised state will be discussed as well., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The European DNA profiling group (EDNAP) organized a sixth collaborative exercise on RNA/DNA co-analysis for body fluid/tissue identification and STR profiling. The task was to identify skin samples/contact traces using specific RNA biomarkers and test three housekeeping genes for their suitability as reference genes. Eight stains, a skin RNA dilution series and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 22 participating laboratories using RNA extraction or RNA/DNA co-extraction methods. Two sets of previously described skin-specific markers were used: skin1 pentaplex (LCE1C, LCE1D, LCE2D, IL1F7 and CCL27) and skin2 triplex (LOR, KRT9 and CDSN) in conjunction with a housekeeping gene, HKG, triplex (B2M, UBC and UCE). The laboratories used different chemistries and instrumentation. All laboratories were able to successfully isolate and detect mRNA in contact traces (e.g., human skin, palm-, hand- and fingerprints, clothing, car interiors, computer accessories and electronic devices). The simultaneous extraction of RNA and DNA provides an opportunity for positive identification of the tissue source of origin by mRNA profiling as well as a simultaneous identification of the body fluid donor by STR profiling. The skin markers LCE1C and LOR and the housekeeping gene marker B2M were detected in the majority of contact traces. Detection of the other markers was inconsistent, possibly due to the low amounts and/or poor quality of the genetic material present in shed skin cells. The results of this and the previous collaborative RNA exercises support RNA profiling as a reliable body fluid/tissue identification method that can easily be combined with current STR typing technology., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The interpretation of mixed DNA profiles obtained from low template DNA samples has proven to be a particularly difficult task in forensic casework. Newly developed likelihood ratio (LR) models that account for PCR-related stochastic effects, such as allelic drop-out, drop-in and stutters, have enabled the analysis of complex cases that would otherwise have been reported as inconclusive. In such samples, there are uncertainties about the number of contributors, and the correct sets of propositions to consider. Using experimental samples, where the genotypes of the donors are known, we evaluated the feasibility and the relevance of the interpretation of high order mixtures, of three, four and five donors. The relative risks of analyzing high order mixtures of three, four, and five donors, were established by comparison of a 'gold standard' LR, to the LR that would be obtained in casework. The 'gold standard' LR is the ideal LR: since the genotypes and number of contributors are known, it follows that the parameters needed to compute the LR can be determined per contributor. The 'casework LR' was calculated as used in standard practice, where unknown donors are assumed; the parameters were estimated from the available data. Both LRs were calculated using the basic standard model, also termed the drop-out/drop-in model, implemented in the LRmix module of the R package Forensim. We show how our results furthered the understanding of the relevance of analyzing high order mixtures in a forensic context. Limitations are highlighted, and it is illustrated how our study serves as a guide to implement likelihood ratio interpretation of complex DNA profiles in forensic casework., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The genotypes of 36 Y-chromosomal short tandem repeat (Y-STR) marker units were analysed in a Dutch population sample of 2085 males. Profiling results were compared for several partially overlapping kits, i.e. PowerPlex Y, Yfiler, PowerPlex Y23, and two in-house designed multiplexes with rapidly mutating Y-STRs. Nineteen Y-STR marker units, of which two are rapidly mutating, reside in at least two of these multiplexes, and for these markers concordance testing was performed. Two samples showed discordant genotyping results and the probable causative base change was revealed by Sanger sequencing. In addition, we encountered concordant, but aberrant genotyping results including one allele with low peak height and several null alleles. For 12 samples, this involved a null allele in two adjacent loci suggesting a large and recurrent deletion as the samples represent three distinct haplogroups. For each marker unit, the allele counts and frequencies are presented, as are the haplotype counts and haplotype diversities for several combinations of markers., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Mitochondrial DNA (mtDNA) can be used for matrilineal biogeographic ancestry prediction and can thus provide investigative leads towards identifying unknown suspects, when conventional autosomal short tandem repeat (STR) profiling fails to provide a match. Recently, six multiplex genotyping assays targeting 62 ancestry-informative mitochondrial single nucleotide polymorphisms (mt-SNPs) were developed. This hierarchical system of assays allows detection of the major haplogroups present in Africa, America, Western Eurasia, Eastern Eurasia, Australia and Oceania, thus revealing the broad geographic region of matrilineal origin of a DNA donor. Here, we provide a forensic developmental validation study of five multiplex assays targeting all the 62 ancestry-informative mt-SNPs following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines. We demonstrate that the assays are highly sensitive; being able to produce full profiles at input DNA amounts of as little as 1pg. The assays were shown to be highly robust and efficient in providing information from degraded samples and from simulated casework samples of different substrates such as blood, semen, hair, saliva and trace DNA samples. Reproducible results were successfully achieved from concordance testing across three independent laboratories depicting the ease and reliability of these assays. Overall, our results demonstrate the suitability of these five mt-SNP assays for application to forensic casework and other purposes aiming to establish an individual's matrilineal genetic ancestry. With this validated tool, it is now possible to determine the matrilineal biogeographic origin of unknown individuals on the level of continental resolution from forensic DNA samples to provide investigative leads in criminal and missing person cases where autosomal STR profiling is uninformative., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

When dealing with mixed DNA profiles where contributors have donated DNA in unequal amounts, it is often useful to deduce the genotype of the major contributor. Inference of a major contributor's genotype empowers storage of the DNA profile in a DNA database (DDB), which is especially of interest in cases without a suspect. When a major contributor's genotype cannot be inferred straightforwardly, for instance because low level components are present, replicate analyses can be prepared and combined into a consensus profile. Here we describe an automated and freely available tool to deduce the major component's alleles in mixed consensus DNA profiles. In these consensus profiles, theoretical peak heights (PHs) are assigned to the alleles using the sum of the PHs in the individual amplifications. The LoCIM-tool (Locus Classification & Inference of the Major-tool) uses these PHs plus parameters on the stochastic threshold, heterozygote balance (HB) and major to minor(s) ratio to classify every locus as a type 1, type 2 or type 3 locus, which represent classes of increasing complexity. Based on the type of locus, the LoCIM-tool applies an inclusion percentage to deduce the alleles for the major contributor. Using the LoCIM-tool, 99.9% of all type 1 loci and 96.7% of all type 2 loci were inferred correctly from a large set of consensus DNA profiles that were generated from mixtures varying for the mixture ratio, amount of DNA per contributor, number of contributors, quality of DNA, and allele sharing among the contributors. For type 3 loci, we aimed at inferring the major contributor's alleles and possibly extra alleles, which occurred for 87.2% of all type 3 loci analysed using the LoCIM-tool. When compared to the overall results of manual inference by a group of forensic scientists, the LoCIM-tool obtains a higher percentage of correctly inferred loci. From our results, we conclude that the LoCIM-tool presents an objective, uniform and fast method to reliably deduce alleles of a major component., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

The European Forensic Genetics Network of Excellence (EUROFORGEN-NoE) undertook a collaborative project on mRNA-based body fluid/skin typing and the interpretation of the resulting RNA and DNA data. Although both body fluids and skin are composed of a variety of cell types with different functions and gene expression profiles, we refer to the procedure as 'cell type inference'. Nine laboratories participated in the project and used a 20-marker multiplex to analyse samples that were centrally prepared and thoroughly tested prior to shipment. Specimens of increasing complexity were assessed that ranged from reference PCR products, cDNAs of indicated or unnamed cell type source(s), to challenging mock casework stains. From this specimen set, information on the overall sensitivity and specificity of the various markers was obtained. In addition, the reliability of a scoring system for inference of cell types was assessed. This scoring system builds on replicate RNA analyses and the ratio observed/possible peaks for each cell type [1]. The results of the exercise support the usefulness of this scoring system. When interpreting the data obtained from the analysis of the mock casework stains, the participating laboratories were asked to integrate the DNA and RNA results and associate donor and cell type where possible. A large variation for the integrated interpretations of the DNA and RNA data was obtained including correct interpretations. We infer that with expertise in analysing RNA profiles, clear guidelines for data interpretation and awareness regarding potential pitfalls in associating donors and cell types, mRNA-based cell type inference can be implemented for forensic casework., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

Regularly, STR results obtained with different PCR amplification kits are compared, for instance with old cases, when revisiting cold cases or when addressing cross-border crimes. It is known that differences in primer design for the same loci in different kits may give rise to null alleles or shifted alleles. In this study, the genotyping results of 2085 Dutch male samples were compared for six autosomal STR kits (Promega's PowerPlex(®) 16, ESX-16 and ESI-17 Systems, Qiagen's Investigator(®) ESSplex Kit and Applied Biosystems' AmpFlSTR(®) Identifiler and NGM PCR Amplification Kits). A total of 19 discordant autosomal genotyping results were obtained that were examined by sequence analysis using Roche-454 next generation sequencing and/or Sanger sequencing. A further 25 discordances were found and sequenced for the Amelogenin locus. The 24 samples showing the same primer binding site mutation at the Amelogenin locus were subjected to X-STR analysis in order to assess whether they could share a common origin, which appeared not to be the case. Based on the sequencing results, we set the final genotypes and determined the allele frequencies of 23 autosomal STRs for the Dutch reference database., (Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.)

Forensic DNA Phenotyping or 'DNA intelligence' tools are expected to aid police investigations and find unknown individuals by providing information on externally visible characteristics of unknown suspects, perpetrators and missing persons from biological samples. This is especially useful in cases where conventional DNA profiling or other means remain non-informative. Recently, we introduced the HIrisPlex system, capable of predicting both eye and hair colour from DNA. In the present developmental validation study, we demonstrate that the HIrisPlex assay performs in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines providing an essential prerequisite for future HIrisPlex applications to forensic casework. The HIrisPlex assay produces complete profiles down to only 63 pg of DNA. Species testing revealed human specificity for a complete HIrisPlex profile, while only non-human primates showed the closest full profile at 20 out of the 24 DNA markers, in all animals tested. Rigorous testing of simulated forensic casework samples such as blood, semen, saliva stains, hairs with roots as well as extremely low quantity touch (trace) DNA samples, produced complete profiles in 88% of cases. Concordance testing performed between five independent forensic laboratories displayed consistent reproducible results on varying types of DNA samples. Due to its design, the assay caters for degraded samples, underlined here by results from artificially degraded DNA and from simulated casework samples of degraded DNA. This aspect was also demonstrated previously on DNA samples from human remains up to several hundreds of years old. With this paper, we also introduce enhanced eye and hair colour prediction models based on enlarged underlying databases of HIrisPlex genotypes and eye/hair colour phenotypes (eye colour: N = 9188 and hair colour: N = 1601). Furthermore, we present an online web-based system for individual eye and hair colour prediction from full and partial HIrisPlex DNA profiles. By demonstrating that the HIrisPlex assay is fully compatible with the SWGDAM guidelines, we provide the first forensically validated DNA test system for parallel eye and hair colour prediction now available to forensic laboratories for immediate casework application, including missing person cases. Given the robustness and sensitivity described here and in previous work, the HIrisPlex system is also suitable for analysing old and ancient DNA in anthropological and evolutionary studies., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Forensic samples regularly involve mixtures, which are readily recognised in forensic analyses. Combined DNA and mRNA profiling is an upcoming forensic practice to examine donors and cell types from the exact same sample. From DNA profiles individual genotypes may be deconvoluted, but to date no studies have established whether the cell types identified in corresponding RNA profiles can be associated with individual donors. Although RNA expression levels hold many variables from which an association may not be expected, proof of concept is important to forensic experts who may be cross examined about this possible correlation in court settings. Clearly, the gender-specificity of certain body fluids (semen, vaginal mucosa, menstrual secretion) can be instructive. However, when donors of the same gender or gender-neutral cell types are involved, alternatives are needed. Here we analyse basic two-component mixtures (two cell types provided by different donors) composed of six different cell types, and assess whether the heights of DNA and RNA peaks may guide association of donor and cell type. Divergent results were obtained; for some mixtures RNA peak heights followed the DNA results, but for others the major DNA component did not present higher RNA peaks. Also, variation in mixture ratios was observed for RNA profiling replicates and when different donor couples gave the same two body fluids. As sample degradation may affect the two nucleic acids and/or distinct cell types differently (and thus influence donor and cell type association), mixtures were subjected to elevated temperature or UV-light. Variation in DNA and RNA stability was observed both between and within cell types and depended on the method inducing degradation. Taken together, we discourage to associate cell types and donors from peak heights when performing RNA and DNA profiling., (Copyright © 2013 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.)

A third collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Twenty saliva and semen stains, four dilution series (10-0.01 μl saliva, 5-0.01 μl semen) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by 20 participating laboratories using an RNA extraction or RNA/DNA co-extraction method. Two novel mRNA multiplexes were used: a saliva triplex (HTN3, STATH and MUC7) and a semen pentaplex (PRM1, PRM2, PSA, SEMG1 and TGM4). The laboratories used different chemistries and instrumentation and a majority (16/20) were able to successfully isolate and detect mRNA in dried stains. The simultaneous extraction of RNA and DNA from individual stains not only permitted a confirmation of the presence of saliva/semen (i.e. tissue/fluid source of origin), but allowed an STR profile of the stain donor to be obtained as well. The method proved to be reproducible and sensitive, with as little as 0.05 μl saliva or semen, using different analysis strategies. Additionally, we demonstrated the ability to positively identify the presence of saliva and semen, as well as obtain high quality DNA profiles, from old and compromised casework samples. The results of this collaborative exercise involving an RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of saliva and semen in forensic casework that is compatible with current DNA analysis methodologies., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

An essential aspect for forensic methods is the prevention of cognitive (confirmation, expectation or motivational) bias. While implementing RNA profiling in casework, we developed a stepwise procedure for unbiased assessment in which: (1) the RNA researcher who generates DNA/RNA fractions and performs RNA profiling, remains uninformed about the context of the case and (2) presents RNA profiling results that are derived by clear guidelines in a results table that uses six different scoring categories, (3) the DNA fractions are processed and analysed by DNA analysts following the standard routine after which (4) reporting officers interpret the DNA profiles and establish the relation to the RNA results which is succeeded by (5) collating all generated results in the case and formulating conclusions in expert reports. The scoring guidelines and results table have a general purpose and can apply to any RNA multiplex. This procedure was applied in a comparative study encompassing seven mock cases designed to be especially interesting for body fluid identification by RNA profiling. Samples were prepared in duplicates and subjected to either presumptive testing combined with standard DNA typing or RNA/DNA co-extraction followed by RNA and DNA profiling. For all cases, the results from presumptive testing and RNA profiling agreed to the level of details the tests can give and concordant DNA results were obtained. RNA profiling was especially useful when (1) menstrual secretion and peripheral blood needed to be distinguished, (2) presence of vaginal mucosa was questioned or (3) presence of skin cells was informative. For forensic reports, we propose to use sets of hypotheses evaluated by the conclusions obtained with DNA and RNA analyses., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Complex DNA mixtures with low template (LT) components provide the most challenging cases to interpret and report. In this study, we designed such mixtures and we describe how reporting officers (ROs) at the Netherlands Forensic Institute (NFI) assess these when embedded in a mock case setting. DNA mixtures containing LT DNA from two to four contributors, sporadic contamination (mimicked by adding 6pg of DNA, which represents once cell equivalent) and/or DNA of relatives (brothers), were amplified four-fold using the AmpFlSTR(®) NGM™ PCR Amplification Kit. Consensus profiles were then generated which included the alleles detected in at least half of the replicates. Four mock cases were created by including reference profiles of a hypothetical victim and suspect. The mock cases were assessed by eight ROs following the stepwise interpretation approach currently in use at the NFI. With this approach, the results of the comparisons between the DNA profiles of the evidentiary trace and the reference profiles are classified into four categories of evidential value [1]. The interpretations by the ROs were compared to the likelihood ratios (LRs) obtained from a probabilistic model that allows a calculation of LRs to assist the interpretation of LT DNA evidence and both were compared to the true composition of the designed mixtures., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

The AmpFlSTR(®) NGM™ kit shows an increased sensitivity compared to previous AmpFlSTR(®) kits, and the addition of a 29th PCR cycle was found to be the major cause for this. During in-house validation, we evaluated whether the increased sensitivity requires elevation of the stochastic threshold (below which alleles are prone to drop out due to low template amplification effects). To determine the stochastic threshold, over 500 false homozygotes were examined and the threshold was set at the rfu value where 99% of the alleles had a peak height below this value. Using 2085 Dutch reference samples, locus-specific stutter ratios were empirically determined and compared with the ones provided by Applied Biosystems. Application of sharp stutter filters is especially important for the analysis of unequal mixtures. To prevent allele calling of 99% of the -1 repeat unit stutters, thirteen stutter ratio filters could be lowered by up to 1.79% and for two loci the stutter ratio filters had to be elevated slightly with a maximum of 0.06%. At all loci +1 repeat stutters were visible for the higher DNA inputs and for lower inputs at the tri-nucleotide repeat locus D22S1045 as well. The overall +1 stutter ratio filter was set to 2.50% and for D22S1045 it was determined to be 7.27%. To find the optimal strategy to sensitise genotyping for low template DNA samples, a comparison was made between enhancing the capillary electrophoresis settings (9kV for 10s) and increasing the number of PCR cycles (29+5 cycles)., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

In current forensic practice, information about the possible biological origin of forensic traces is mostly determined using protein-based presumptive testing. Recently, messenger RNA-profiling has emerged as an alternative strategy to examine the biological origin. Here we describe the development of a single multiplex mRNA-based system for the discrimination of the most common forensic body fluids as well as skin cells. A DNA/RNA co-isolation protocol was established that results in DNA yields equivalent to our standard in-house validated DNA extraction procedure which uses silica-based columns. An endpoint RT-PCR assay was developed that simultaneously amplifies 19 (m)RNA markers. This multiplex assay analyses three housekeeping, three blood, two saliva, two semen, two menstrual secretion, two vaginal mucosa, three general mucosa and two skin markers. The assay has good sensitivity as full RNA profiles for blood, semen and saliva were obtained when using ≥0.05 μL body fluid starting material whereas full DNA profiles were obtained with ≥0.1 μL. We investigated the specificity of the markers by analysing 15 different sets of each type of body fluid and skin with each set consisting of 8 individuals. Since skin markers have not been incorporated in multiplex endpoint PCR assays previously, we analysed these markers in more detail. Interestingly, both skin markers gave a positive result in samplings of the hands, feet, back and lips but negative in tongue samplings. Positive identification (regarding both DNA- and RNA-profiling) was obtained for specimens stored for many years, e.g. blood (28 years-old), semen (28 years-old), saliva (6 years-old), skin (10 years-old) and menstrual secretion (4 years-old). The described approach of combined DNA- and RNA-profiling of body fluids and contact traces assists in the interpretation of forensic stains by providing information about not only the donor(s) that contributed to the stain but also by indicating which cell types are present., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Forensic DNA typing involves a multi-step workflow. Steps include DNA isolation, quantification, amplification of a set of short tandem repeat (STR) markers, separation of polymerase chain reaction (PCR) products by capillary electrophoresis (CE) and DNA profile analysis and interpretation. With that, the process takes around 10-12h. For several scenarios it may be very valuable to speed up this process and obtain an interpretable DNA profile, suited to search a DNA database, within a few hours. For instance in cases of national security, abduction with danger of life, risk of repetition by a serial perpetrator or when custody time of suspects is limited. By a direct and rapid PCR approach we reduced the total DNA profiling time to 2-3h after which genotyping information for the 10 STR markers plus the amelogenin (AMEL) marker present in the commercially available AmpFℓSTR(®) SGM Plus™ (SGM+) profiling kit is obtained. This reduction in time is achieved by using the following elements: (1) the inhibitor tolerant, highly processive Phusion(®) Flash DNA polymerase; (2) a modified, non-adenylated allelic ladder; (3) the quick PIKO(®) thermal cycler system with ultra-thin walled reaction tubes; (4) profile interpretation guidelines with an increased allele calling threshold, modified stutter ratios and marked low-level artefact peaks and (5) regulation of sample input by the use of mini-tapes that lift a limited amount of cell material from swabs or fabrics. The procedure is specifically effective for high level DNA, single source samples such as samples containing saliva, blood, semen and hair roots. Success rates, defined as a complete DNA profile, depend on stain type and surface. Due to the use of tape lifting as the sampling technique, the swab or fabric remains dry and intact and can be analyzed at a later stage using regular procedures. Validation experiments were performed which showed that the protocol effectively instructs researchers unfamiliar with the procedure. We have incorporated direct and rapid PCR in a "DNA-6h" service that can assist police investigations by rapidly deriving DNA information from trace evidence left by a perpetrator, searching the STR profile against a DNA database and reporting the outcomes to police or prosecution., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

After manual strangulation, epithelial cells originating from the offender can often be found on the skin of the victim. In order to obtain a conclusive DNA profile, it is important to secure as many epithelial cells from the offender and as few epithelial cells from the victim as possible. In this study, two methods for securing offender DNA were compared: the double swab method and an adapted tape-lifting method, so-called stubbing. 50 male volunteers were asked to simulate manual strangulation on the forearm of a female volunteer. After securing the epithelial material, DNA profiles were generated. The contribution of both donors to the samples was determined from the number of detected alleles, specific for each donor, and the average peak height of the donor-specific alleles. For the offender, in all cases except one, partial or full profiles were obtained and no difference between the double swab and the stubbing method was observed. For the victim, fewer alleles were detected by means of double swab than by means of stubbing. In conclusion, the double swab method performs slightly better than the stubbing method. However, from a practical point of view, the stubbing method may be preferred over the double swab technique., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

A second collaborative exercise on RNA/DNA co-analysis for body fluid identification and STR profiling was organized by the European DNA Profiling Group (EDNAP). Six human blood stains, two blood dilution series (5-0.001 μl blood) and, optionally, bona fide or mock casework samples of human or non-human origin were analyzed by the participating laboratories using a RNA/DNA co-extraction or solely RNA extraction method. Two novel mRNA multiplexes were used for the identification of blood: a highly sensitive duplex (HBA, HBB) and a moderately sensitive pentaplex (ALAS2, CD3G, ANK1, SPTB and PBGD). The laboratories used different chemistries and instrumentation. All of the 18 participating laboratories were able to successfully isolate and detect mRNA in dried blood stains. Thirteen laboratories simultaneously extracted RNA and DNA from individual stains and were able to utilize mRNA profiling to confirm the presence of blood and to obtain autosomal STR profiles from the blood stain donors. The positive identification of blood and good quality DNA profiles were also obtained from old and compromised casework samples. The method proved to be reproducible and sensitive using different analysis strategies. The results of this collaborative exercise involving a RNA/DNA co-extraction strategy support the potential use of an mRNA based system for the identification of blood in forensic casework that is compatible with current DNA analysis methodology., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Forensic laboratories employ various approaches to obtain short tandem repeat (STR) profiles from minimal traces (<100 pg DNA input). Most approaches aim to sensitize DNA profiling by increasing the amplification level by a higher cycle number or enlarging the amount of PCR products analyzed during capillary electrophoresis. These methods have limitations when unequal mixtures are genotyped, since the major component will be over-amplified or over-loaded. This study explores an alternative strategy for improved detection of the minor components in low template (LT) DNA typing that may be better suited for the detection of the minor component in mixtures. The strategy increases the PCR amplification efficiency by extending the primer annealing time several folds. When the AmpFℓSTR(®) Identifiler(®) amplification parameters are changed to an annealing time of 20 min during all 28 cycles, the drop-out frequency is reduced for both pristine DNA and single or multiple donor mock case work samples. In addition, increased peak heights and slightly more drop-ins are observed while the heterozygous peak balance remains similar as with the conventional Identifiler protocol. By this extended protocol, full DNA profiles were obtained from only 12 sperm heads (which corresponds to 36 pg of DNA) that were collected by laser micro dissection. Notwithstanding the improved detection, allele drop-outs do persist, albeit in lower frequencies. Thus a LT interpretation strategy such as deducing consensus profiles from multiple independent amplifications is appropriate. The use of extended PCR conditions represents a general approach to improve detection of unequal mixtures as shown using four commercially available kits (AmpFℓSTR(®) Identifiler, SEfiler Plus, NGM and Yfiler). The extended PCR protocol seems to amplify more of the molecules in LT samples during PCR, which results in a lower drop-out frequency., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)