Expanded and mega-plex STR panels as a tool for presumptive population assignment

Historically, forensic STR panels have been unsuccessful for population assignment due to the limited ancestry information that can be derived from the non-coding STR loci and the low number of loci included in the panel. However, given the recent adoption of expanded (16+ loci) and ‘mega-plex’ (23+ loci) STR panels, the ability to identify source population groups may be improved. This study assessed the impact of increasing locus number on population assignment under different analysis conditions using a published US population dataset comprised of individuals from the African American, Caucasian, Hispanic and Asian populations. The Bayesian clustering programme STRUCTURE was used to assess first, whether increasing the number of loci and the inclusion of known sample population data enabled greater resolution between the four populations in the dataset, and second, the utility for population assignment using criteria based on inferred ancestry scores. Results suggest that increasing the number of loci and including population of origin data allowed the identification of more distinct populations, with three primary populations being observed; African American, Asian, and Caucasian/Hispanic. The close grouping of the Caucasian and Hispanic populations is supported by their recently common ancestry from Western Europe. The ability of the programme to support population assignment to each of the four existing populations was assessed through the application of population and panel specific assignment thresholds based on the inferred ancestry scores obtained from the analysis programme. Predictive accuracy based on a training dataset of 984 individuals suggest that assignment accuracy is > 96% across the four populations and can reach 100% under some test conditions. The accuracy was > 90% when blind testing was performed on 40 ‘unknown’ individuals. As such, the approach described is considered within the acceptable range for a presumptive test and can be performed using data already collected as part of routine forensic investigations

Enhanced 3-epi-25-hydroxyvitamin D3 signal leads to overestimation of its concentration and amplifies interference in 25-hydroxyvitamin D LC-MS/MS assays

Background 3-epi-25-hydroxyvitamin D3 (3-epi-25OHD3) interferes in most liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays for 25-hydroxyvitamin D (25OHD). The clinical significance of this is unclear, with concentrations from undetectable to 230 nmol/L reported. Many studies have quantified 3-epi-25OHD3 based on 25OHD3 calibrators or other indirect methods, and we speculated that this contributes to the observed variability in reported 3-epi-25OHD3 concentrations. Methods We compared continuous MS/MS infusions of 3-epi-25OHD3 and 25OHD3 solutions, spiked both analytes into the same serum matrix and analysed patient samples to assess the effect of three different quantitation methods on 3-epi-25OHD3 concentration. Experiments were performed on an LC-MS/MS system using a phenyl column which does not resolve 3-epi-25OHD3, and a modified method utilizing a Zorbax SB-CN column that chromatographically resolves 3-epi-25OHD3 from 25OHD3. Results A greater 3-epi-25OHD3 signal, compared with 25OHD3, was observed during equimolar post-column continuous infusion of analyte solutions, and following analysis of a serum pool spiked with both analytes. 3-epi-25OHD3 signal enhancement was dependent on mobile phase composition. Compared with 3-epi-25OHD3 calibrators, indirect quantitation methods resulted in up to 10 times as many samples having 3-epi-25OHD3 concentrations ≥ 10 nmol/L, and an approximately fourfold increase in the maximum observed 3-epi-25OHD3 concentration to 95 nmol/L. Conclusions Enhanced 3-epi-25OHD3 signal leads to overestimation of its concentrations in the indirect quantitation methods used in many previous studies. The enhanced signal may contribute to greater interference in some 25OHD LC-MS/MS assays than others. We highlight that equimolar responses cannot be assumed in LC-MS/MS systems, even if two molecules are structurally similar

The ParaDNA® Screening System - a case study in bringing forensic R&D to market.

The creation of new technologies and their application to forensic science is key to the field's development. Rapid DNA profiling is one such area of research which has grown in response to a desire from enforcement authorities for in-house forensic DNA processing and rapid access to forensic genetic intelligence. However, introducing novel technologies into the forensics market must be carefully monitored and controlled as the success or failure of any technology ultimately has long standing implications for victims, suspects, and also to Police and forensic practitioners. This article outlines the research, development, validation and implementation of the ParaDNA® Screening System as a case study in taking forensic research and development to market

Defining end user requirements for a field-based molecular detection system for wildlife forensic investigations

The increasing use of non-laboratory-based DNA and protein detection methods promise to provide rapid investigative intelligence and support sample prioritisation. Primarily developed for human forensic or medical applications, current systems may also show utility in the field of wildlife forensic science. However, it is currently unknown whether the requirements of the wildlife forensic community can be met by current non-laboratory based tools. Given the diverse array of stakeholders and sample types commonly encountered, it is necessary to first identify the needs of the community and then try and map their needs to current instrumentation. By using a market research style questionnaire, this study identified key requirements for a non-laboratory-based system following feedback from the wildlife forensic community. Data showed that there is strong support for field-based detection methods while highlighting concerns including contamination risks and reduced quality assurance associated with non-laboratory testing. Key species and applications were identified alongside hurdles to implementation and adoption. Broadly, the requirements align with many of the developmental drivers that have led to the rise of in-field portable detection instrumentation, specifically rapid detection within one hour, ease-of-use, and ≥95% accuracy. Several existing platforms exist that met some of the identified requirements but not all. With further collaboration between industry partners and the wildlife forensic community it is possible that new field-based systems can be developed and applied routinely

Identification of a serum biomarker panel for the differential diagnosis of cholangiocarcinoma and primary sclerosing cholagnitis

The non-invasive differentiation of malignant and benign biliary disease is a clinical challenge. Carbohydrate antigen 19-9 (CA19-9), leucine-rich α2-glycoprotein (LRG1), interleukin 6 (IL6), pyruvate kinase M2 (PKM2), cytokeratin 19 fragment (CYFRA21.1) and mucin 5AC (MUC5AC) have reported utility for differentiating cholangiocarcinoma (CCA) from benign biliary disease. Herein, serum levels of these markers were tested in 66 cases of CCA and 62 cases of primary sclerosing cholangitis (PSC) and compared with markers of liver function and inflammation. Markers panels were assessed for their ability to discriminate malignant and benign disease. Several of the markers were also assessed in pre-diagnosis biliary tract cancer (BTC) samples with performances evaluated at different times prior to diagnosis. We show that LRG1 and IL6 were unable to accurately distinguish CCA from PSC, whereas CA19-9, PKM2, CYFRA21.1 and MUC5AC were significantly elevated in malignancy. Area under the receiver operating characteristic curves for these individual markers ranged from 0.73–0.84, with the best single marker (PKM2) providing 61% sensitivity at 90% specificity. A panel combining PKM2, CYFRA21.1 and MUC5AC gave 76% sensitivity at 90% specificity, which increased to 82% sensitivity by adding gamma-glutamyltransferase (GGT). In the pre-diagnosis setting, LRG1, IL6 and PKM2 were poor predictors of BTC, whilst CA19-9 and C-reactive protein were elevated up to 2 years before diagnosis. In conclusion, LRG1, IL6 and PKM2 were not useful for early detection of BTC, whilst a model combining PKM2, CYFRA21.1, MUC5AC and GGT was beneficial in differentiating malignant from benign biliary disease, warranting validation in a prospective trial

Species detection using HyBeacon(®) probe technology: Working towards rapid onsite testing in non-human forensic and food authentication applications.

Identifying individual species or determining species' composition in an unknown sample is important for a variety of forensic applications. Food authentication, monitoring illegal trade in endangered species, forensic entomology, sexual assault case work and counter terrorism are just some of the fields that can require the detection of the biological species present. Traditional laboratory based approaches employ a wide variety of tools and technologies and exploit a number of different species specific traits including morphology, molecular differences and immuno-chemical analyses. A large number of these approaches require laboratory based apparatus and results can take a number of days to be returned to investigating authorities. Having a presumptive test for rapid identification could lead to savings in terms of cost and time and allow sample prioritisation if confirmatory testing in a laboratory is required later. This model study describes the development of an assay using a single HyBeacon(®) probe and melt curve analyses allowing rapid screening and authentication of food products labelled as Atlantic cod (Gadus morhua). Exploiting melt curve detection of species specific SNP sites on the COI gene the test allows detection of a target species (Atlantic cod) and closely related species which may be used as substitutes. The assay has been designed for use with the Field Portable ParaDNA system, a molecular detection platform for non-expert users. The entire process from sampling to result takes approximately 75min. Validation studies were performed on both single source genomic DNA, mixed genomic DNA and commercial samples. Data suggests the assay has a lower limit of detection of 31 pg DNA. The specificity of the assay to Atlantic cod was measured by testing highly processed food samples including frozen, defrosted and cooked fish fillets as well as fish fingers, battered fish fillet and fish pie. Ninety-six (92.7%) of all Atlantic cod food products, tested, provided a correct single species result with the remaining samples erroneously identified as containing non-target species. The data shows that the assay was quick to design and characterise and is also capable of yielding results that would be beneficial in a variety of fields, not least the authentication of food

Growth and characterization of semiconductor nanoparticles in porous sol-gel films

Published versio

Applicability of the ParaDNA(®) Screening System to Seminal Samples.

Seminal fluid represents a common biological material recovered from sexual assault crime scenes. Such samples can be prescreened using different techniques to determine cell type and relative amount before submitting for full STR profiling. The ParaDNA(®) Screening System is a novel forensic test which identifies the presence of DNA through amplification and detection of two common STR loci (D16S539 and TH01) and the Amelogenin marker. The detection of the Y allele in samples could provide a useful tool in the triage and submission of sexual assault samples by enforcement authorities. Male template material was detected on a range of common sexual assault evidence items including cotton pillow cases, condoms, swab heads and glass surfaces and shows a detection limit of 1 in 1000 dilution of neat semen. These data indicate this technology has the potential to be a useful tool for the detection of male donor DNA in sexual assault casework

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

Convergence of multiple markers and analysis methods defines the genetic distinctiveness of cryptic pitvipers

Using multiple markers and multiple analytical approaches is critical for establishing species boundaries reliably, especially so in the case of cryptic species. Despite development of new and powerful analytical methods, most studies continue to adopt a few, with the choice often being subjective. One such example is routine analysis of Amplified Fragment Length Polymorphism (AFLP) data using population genetic models despite disparity between method assumptions and data properties. The application of newly developed methods for analyzing this dominant marker may not be entirely clear in the context of species delimitation. In this study, we use AFLPs and mtDNA to investigate cryptic speciation in the Trimeresurus macrops complex that belongs to a taxonomically difficult lineage of Asian pitvipers. We analyze AFLPs using population genetic, phylogenetic, multivariate statistical, and Bayes Factor Delimitation methods. A gene tree from three mtDNA markers provided additional evidence. Our results show that the inferences about species boundaries that can be derived from population genetic analysis of AFLPs have certain limitations. In contrast, four multivariate statistical analyses produced clear clusters that are consistent with each other, as well as with Bayes Factor Delimitation results, and with mtDNA and total evidence phylogenies. Furthermore, our results concur with allopatric distributions and patterns of variation in individual morphological characters previously identified in the three proposed species: T. macrops sensu stricto, T. cardamomensis, and T. rubeus. Our study provides evidence for reproductive isolation and genetic distinctiveness that define these taxa as full species. In addition, we re-emphasize the importance of examining congruence of results from multiple methods of AFLP analysis for inferring species diversity