Casework Laboratories Research Articles

Differentiation of vaginal cells from epidermal cells using morphological and autofluorescence properties: Implications for sexual assault casework involving digital penetration

Analysis of DNA mixtures from sexual assault evidence is an ongoing challenge for DNA casework laboratories. To assist the forensic scientist address source and activity level propositions there is a significant need for new techniques that can provide information as to the source of DNA, particularly for sexual assault samples that do not involve semen. The goal of this study was to develop a new biological signature system that provides additional probative value to samples comprised of mixtures of epidermal and vaginal cells, as may be observed in cases involving digital penetration. Signatures were based on morphological and autofluorescence properties of individual cells collected through Imaging Flow Cytometry (IFC). Comparisons to reference cell populations from vaginal tissue and epidermal cells collected from hands showed strong multivariate differences across > 80 cellular measurements. These differences were used to build a predictive framework for classifying unknown cell populations as originating from epithelial cells associated with digital penetration or epidermal tissue. As part of the classification scheme, posterior probabilities of specific tissue group membership were calculated for each cell, along with multivariate similarity to that tissue type. We tested this approach on cell populations from reference tissue as well as mock casework samples involving hand swabbings following digital vaginal penetration. Many more cells classifying as non-epidermal tissue were detected in digital penetration hand swab samples than control hand swabbings. Minimum interpretation thresholds were developed to minimize false positives; these thresholds were also effective when screening licked hands, indicating the potential utility of this method for a variety of biological mixture types and depositional events relevant to forensic casework. Results showed that samples collected subsequent to digital penetration possessed markedly higher numbers of cells classifying as vaginal tissue as well as higher posterior probabilities for vaginal tissue (≥ 0.90) compared to cell populations collected from hands without prior contact with vaginal tissue. Additionally, digital penetration cell populations may be resolved from saliva cell populations and other non-target tissue types.

Effect of swabbing technique and duration on forensic DNA recovery

Various factors have been shown to affect performance of the conventional wet-dry double and single wet swabbing techniques to recover DNA, such as pressure and angle of application, volume and type of wetting agent, and swab type. However, casework laboratories in some jurisdictions have recently adopted different swabbing techniques that include wet-moist double swabbing and moist-dry single swabbing. Factors affecting the effectiveness of these recent techniques in maximising DNA recovery therefore need to be investigated. Here, the performance of traditional and recent swabbing techniques was compared and the impact of swabbing duration on DNA recovery was investigated. Ten µl aliquots of a known concentration of DNA extracted from human blood were deposited on pre-cleaned DNA-free cotton swatches (porous) and porcelain tiles (non-porous). Five swabbing techniques were used, of which three were double swabbing techniques: wet-moist, wet-wet and wet-dry, and two were single swabbing techniques: wet and moist-dry. For a ‘wet’ or ‘moist’ swab, 100 or 50 µL water was added, respectively. For a moist-dry swab, water was applied to one side of the swab, leaving the other side drier. Each swabbing technique was applied for two durations, 15 and 30 s per swab, with 5 reps of each combination (n = 100 plus controls). All samples were extracted and quantified, and a sub-set was profiled. The results showed that the wet-moist double swabbing technique with a swabbing duration of 30 s maximised DNA recovery from cotton. From tile, a single wet or moist-dry swab maximised DNA recovery, but increasing swabbing duration from 15 to 30 s had no impact. These data can be used to inform standardisation of DNA collection protocols across casework laboratories.

Portable near infrared spectroscopy for the isomeric differentiation of new psychoactive substances

Rapid and efficient identification of the precise isomeric form of new psychoactive substances (NPS) by forensic casework laboratories is a relevant challenge in the forensic field. Differences in legal status occur for ring-isomeric species of the same class, thus leading to different penalties and judicial control. Portable systems such as near-infrared (NIR) spectroscopy recently emerged as suitable techniques for the on-scene identification of common drugs of abuse such as cocaine, MDMA and amphetamine. This way, the overall forensic process becomes more efficient as relevant information on substance identity becomes available directly at the scene of crime. Currently, no NIR-based applications exist for the rapid, on-scene detection of NPS isomers. Herein, we present the differentiation of cathinone and phenethylamine-type NPS analogues based on their NIR spectrum recorded in 2 seconds on a portable 1350 – 2600 nm spectrometer. A prior developed data analysis model was found suitable for the identification of the methylmethcathinone (MMC) isomers 2-MMC, 3-MMC and 4-MMC. In 51 mixtures and 22 seized casework samples, the correct isomeric form was detected in all cases except for a few mixtures with an active ingredient content of 10 wt%. These results show the feasibility of on-site NPS detection as presumptive test performed directly at the scene of crime with a small size NIR-spectrometer. Additionally, in the illicit drug analysis laboratory the combination of NIR and GC–MS analysis might be suitable for robust identification of NPS isomers and analogues.

Evaluation of the performance of the beta version of the ForenSeq DNA signature Prep Kit on the MiSeq FGx forensic genomics system

Massively Parallel Sequencing (MPS) are innovative approaches of analysis of genetic markers, supported by the forensic community. The Illumina Beta Version of the ForenSeq DNA Signature Prep Kit is a high-throughput, multiplexing kit allowing simultaneous, high resolution sequencing of 153 identity informative markers (including 27 autosomal STRs, 7 X-chromosomal, 24 Y-chromosomal haplotype markers and 94 SNPs) plus 56 biogeographical ancestry and 22 phenotypic informative SNPs. Implementation of MPS technology in forensic casework laboratories needs comprehensive, internal validation according to SWGDAM guidelines. The aim of this study was to determine with ForenSeq DNA Signature Prep Kit, method’s precision/accuracy, sensitivity as well as possibility of analysis of two persons mixtures. Analysis of challenging casework samples coming from various tissues were performed and compared to results obtained with capillary electrophoresis (CE). In order to evaluate the quality and reliability of the acquired results, informative metrics of STR and SNP markers including depth of coverage (DoC), allele coverage ratio (ACR), and sequence coverage ratios (SCRs) were assessed.

Establishing STR and identity SNP analysis thresholds for reliable interpretation and practical implementation of MPS gDNA casework

Massively parallel sequencing (MPS) evaluation by the global forensic community has demonstrated immediate advantages and forthcoming, versatile capabilities to aid missing persons and criminal investigations. As a result, an increased number of operational casework laboratories are executing validation geared towards informing MPS interpretation guidelines and SOPs for routine implementation. As with CE-based data, establishing analysis thresholds that enable reliable interpretation within the limitations of a system is a pivotal decision point. In this article, we present an empirical data-driven approach for determining analytical and stochastic thresholds (AT, ST) for the 152 STR (auto, Y, and X) and identity-informative SNP (iiSNP) loci targeted in the ForenSeq™ DNA Signature assay. While the methods utilized mimic CE threshold determination, fundamental distinctions specific to MPS data are addressed. Key considerations include relative and fixed read count values as well as interlocus balance expectations when increasing numbers of markers and/or marker types are multiplexed together in a single amplification. Internal data as well as data from collaborating laboratories will be presented. For AT assessment, sensitivity data (1 ng to 6 pg gDNA input) were evaluated with a combination of percent-based and static minimum read count AT values. Comparison of varying AT values, their effect on true allele call rates, and unanticipated read detection were assessed to achieve an ideal balance of data quality and quantity. Additionally, 4 theoretical options for ST application will be offered which include a detailed review of the stepwise methods, calculations, and resulting data that support a locus “grouping” approach based on locus performance, or intensity groups, within each STR and iiSNP marker set.

Automation of library preparation using Illumina ForenSeq kit for routine sequencing of casework samples

Massively Parallel Sequencing (MPS) technologies are called upon to play a major role in forensic genomics in the next few years. However, library preparation protocols contain numerous steps which can complexify casework analysis in routine. Implementation of a MPS analytical workflow, in a casework laboratory, requires automation to guarantee a full sample tracking and to minimize contamination risks. In this study, we present the development and validation of a fully automated workflow using Illumina ForenSeq DNA signature Prep Kit on a NGS STARlet HID Illumina platform. Automation greatly helps with library preparation reproducibility and significantly reduces hands-on time. We are confident that automation of MPS technologies will enhance forensic laboratory to adopt these powerful analytical tools determinant for the future of the forensic genomics field.

Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results.

Assessing the impact of common forensic presumptive tests on the ability to obtain results using a novel rapid DNA platform

The rise of DNA evidence to the forefront of forensic science has led to high sample numbers being submitted for profiling by investigators to casework laboratories: bottleneck effects are often seen resulting in slow turnaround times and sample backlog. The ParaDNA® Screening and Intelligence Tests have been designed to guide investigators on the viability of potential sources of DNA allowing them to determine which samples should be sent for full DNA analysis. Both tests are designed to augment the arsenal of available forensic tests for end users and be used concurrently to those commonly available. Therefore, assessing the impact that common forensic tests have on such novel technology is important to measure. The systems were tested against various potential inhibitors to which samples may be exposed as part of the investigative process. Presumptive test agents for biological materials (blood, semen and saliva) and those used as fingerprint enhancement agents were both used. The Screening Test showed a drop in performance following application of aluminium powder and cyanoacrylate (CNA) on fingerprints samples; however this drop in performance was not replicated with high template DNA. No significant effect was observed for any agent using the Intelligence Test. Therefore, both tests stand up well to the chemical agents applied and can be used by investigators with confidence that system performance will be maintained.

Facile semi-automated forensic body fluid identification by multiplex solution hybridization of NanoString® barcode probes to specific mRNA targets.

A DNA profile from the perpetrator does not reveal, per se, the circumstances by which it was transferred. Body fluid identification by mRNA profiling may allow extraction of contextual 'activity level' information from forensic samples. Here we describe the development of a prototype multiplex digital gene expression (DGE) method for forensic body fluid/tissue identification based upon solution hybridization of color-coded NanoString(®) probes to 23 mRNA targets. The method identifies peripheral blood, semen, saliva, vaginal secretions, menstrual blood and skin. We showed that a simple 5 min room temperature cellular lysis protocol gave equivalent results to standard RNA isolation from the same source material, greatly enhancing the ease-of-use of this method in forensic sample processing. We first describe a model for gene expression in a sample from a single body fluid and then extend that model to mixtures of body fluids. We then describe calculation of maximum likelihood estimates (MLEs) of body fluid quantities in a sample, and we describe the use of likelihood ratios to test for the presence of each body fluid in a sample. Known single source samples of blood, semen, vaginal secretions, menstrual blood and skin all demonstrated the expected tissue-specific gene expression for at least two of the chosen biomarkers. Saliva samples were more problematic, with their previously identified characteristic genes exhibiting poor specificity. Nonetheless the most specific saliva biomarker, HTN3, was expressed at a higher level in saliva than in any of the other tissues. Crucially, our algorithm produced zero false positives across this study's 89 unique samples. As a preliminary indication of the ability of the method to discern admixtures of body fluids, five mixtures were prepared. The identities of the component fluids were evident from the gene expression profiles of four of the five mixtures. Further optimization of the biomarker 'CodeSet' will be required before it can be used in casework, particularly with respect to increasing the signal-to-noise ratio of the saliva biomarkers. With suitable modifications, this simplified protocol with minimal hands on requirement should facilitate routine use of mRNA profiling in casework laboratories.

Development and validation of InnoQuant™, a sensitive human DNA quantitation and degradation assessment method for forensic samples using high copy number mobile elements Alu and SVA.

There is a constant need in forensic casework laboratories for an improved way to increase the first-pass success rate of forensic samples. The recent advances in mini STR analysis, SNP, and Alu marker systems have now made it possible to analyze highly compromised samples, yet few tools are available that can simultaneously provide an assessment of quantity, inhibition, and degradation in a sample prior to genotyping. Currently there are several different approaches used for fluorescence-based quantification assays which provide a measure of quantity and inhibition. However, a system which can also assess the extent of degradation in a forensic sample will be a useful tool for DNA analysts. Possessing this information prior to genotyping will allow an analyst to more informatively make downstream decisions for the successful typing of a forensic sample without unnecessarily consuming DNA extract. Real-time PCR provides a reliable method for determining the amount and quality of amplifiable DNA in a biological sample. Alu are Short Interspersed Elements (SINE), approximately 300bp insertions which are distributed throughout the human genome in large copy number. The use of an internal primer to amplify a segment of an Alu element allows for human specificity as well as high sensitivity when compared to a single copy target. The advantage of an Alu system is the presence of a large number (>1000) of fixed insertions in every human genome, which minimizes the individual specific variation possible when using a multi-copy target quantification system. This study utilizes two independent retrotransposon genomic targets to obtain quantification of an 80bp "short" DNA fragment and a 207bp "long" DNA fragment in a degraded DNA sample in the multiplex system InnoQuant™. The ratio of the two quantitation values provides a "Degradation Index", or a qualitative measure of a sample's extent of degradation. The Degradation Index was found to be predictive of the observed loss of STR markers and alleles as degradation increases. Use of a synthetic target as an internal positive control (IPC) provides an additional assessment for the presence of PCR inhibitors in the test sample. In conclusion, a DNA based qualitative/quantitative/inhibition assessment system that accurately predicts the status of a biological sample, will be a valuable tool for deciding which DNA test kit to utilize and how much target DNA to use, when processing compromised forensic samples for DNA testing.

Assessing the potential of next generation sequencing technologies for missing persons identification efforts

To assess the utility of next generation sequencing (NGS) technologies for missing persons applications, we have recently initiated a study of various platforms and target enrichment strategies for sample types regularly encountered in our large-scale identification efforts. Specific laboratory workflows based on target marker enrichment and NGS platform are being considered for different sample types, and the overall effort is being undertaken with a strong emphasis on both raw data and final consensus sequence quality. The current study is first and foremost a general evaluation of these data and technologies from the standpoint of forensic application, yet the strategy we are pursuing is ultimately intended to facilitate NGS integration into standard casework laboratories. We are, therefore, evaluating NGS workflows and data for the typical nuclear and mitochondrial DNA markers used in forensics, while still allowing for future work that may take greater advantage of the strengths of these technologies. Here, we present an overview of our NGS strategy.

Forensic Science Laboratory/Forensic Science Program Cooperation and Relationships: The View from the Forensic Science Laboratory

Abstract Some areas of common interest and potential cooperation between casework forensic science laboratories and university and college based forensic science programs are discussed. These include internships, joint research, the use of laboratory personnel as instructors in programs, and the use of faculty as consultants for the casework laboratory.