Interpretation and reporting of mixed DNA profiles by seven forensic laboratories in the UK and Ireland.

In 2014, the UK Forensic Science Regulator (FSR) commissioned a collaborative trial to assess the methods used by forensic service providers (FSPs) in the UK and Ireland for analysis, interpretation and reporting of mixed DNA profiles. Five different mixed samples of varying complexity with supporting mock case circumstances were tested using SGMPlus™ and the newly introduced DNA-17(+) multiplexes and reported by participating laboratories. The results demonstrated a high degree of consistency in analytical methods and allele designations, but some variation in the statistical evaluation and reporting of results. Some of the differences noted were attributable to the major technology change to 17(+)-STR systems which had recently been implemented across the UK at that time. The FSR made recommendations based on the trial outcomes which were intended to produce a more consistent approach to mixtures analysis, interpretation and reporting. Four years later, the Association of Forensic Science Providers (AFSP) repeated the trial, with all major UK and Ireland FSPs (both public sector and private companies) again participating. This second trial used the same mixture set as the 2014 trial but was focussed on the methods for interpretation and evaluation. Since 2014, all UK and Ireland FSPs have implemented probabilistic statistical software using continuous models enabling statistical evaluation of more complex mixtures than was possible in 2014. The trial was therefore aimed at investigating the value of these improved capabilities and also to investigate if there appeared to be marked differences between the different software tools in use in the UK. The results demonstrate a high degree of concordance within and between FSPs and across different evaluation models, and will provide important support for the use of such models in evaluation of mixed DNA profiles.

Combining current knowledge on DNA methylation-based age estimation towards the development of a superior forensic DNA intelligence tool.

The estimation of chronological age from biological fluids has been an important quest for forensic scientists worldwide, with recent approaches exploiting the variability of DNA methylation patterns with age in order to develop the next generation of forensic 'DNA intelligence' tools for this application. Drawing from the conclusions of previous work utilising massively parallel sequencing (MPS) for this analysis, this work introduces a DNA methylation-based age estimation method for blood that exhibits the best combination of prediction accuracy and sensitivity reported to date. Statistical evaluation of markers from 51 studies using microarray data from over 4000 individuals, followed by validation using in-house generated MPS data, revealed a final set of 11 markers with the greatest potential for accurate age estimation from minimal DNA material. Utilising an algorithm based on support vector machines, the proposed model achieved an average error (MAE) of 3.3 years, with this level of accuracy retained down to 5 ng of starting DNA input (~ 1 ng PCR input). The accuracy of the model was retained (MAE = 3.8 years) in a separate test set of 88 samples of Spanish origin, while predictions for donors of greater forensic interest (< 55 years of age) displayed even higher accuracy (MAE = 2.6 years). Finally, no sex-related bias was observed for this model, while there were also no signs of variation observed between control and disease-associated populations for schizophrenia, rheumatoid arthritis, frontal temporal dementia and progressive supranuclear palsy in microarray data relating to the 11 markers.

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.

Persistence of DNA from laundered semen stains: Implications for child sex trafficking cases.

In sexual assault cases, particularly those involving internal child sex trafficking (ICST), victims often hide their semen-stained clothing. This can result in a lag time of several months before the items are laundered and subsequently seized during a criminal investigation. Although it has been demonstrated previously that DNA can be recovered from clothing washed immediately after semen deposition, laundered items of clothing are not routinely examined in ICST cases, due to the assumption that the time delay and washing would result in no detectable DNA. The aim of this study was to examine whether viable DNA profiles could be recovered from laundered semen stains where there has been a significant lag time between semen deposition from one or more individuals and one or more washes of the stained clothing. Items of UK school uniform (T-shirts, trousers, tights) were stained with fresh semen (either from a single donor or a 1:1 mixture from two donors) and stored in a wardrobe for eight months. Stained and unstained items (socks) were then washed at 30 °C or 60 °C and with non-biological or biological detergent. DNA samples extracted from the semen-stained sites and from the unstained socks were quantified and profiled. High quantities of DNA, (6-18 µg) matching the DNA profiles of the semen donors, were recovered from all semen-stained clothing that had been laundered once, irrespective of wash conditions. This quantity,and profile quality,did not decline significantly with multiple washes. The two donor semen samples yielded ∼ 10-fold more DNA from the T-shirts than from the trousers. This disparity resulted in the T-shirts yielding a ∼ 1:1 mixture of DNA from the two donors, whereas the trousers yielded a major DNA profile matching only that of the second donor. The quantities of DNA recovered from the unstained socks were an order of magnitude lower, with most of the DNA being attributable to the donor of the semen on the stained clothing within the same wash, demonstrating the transfer of semen-derived DNA among items of clothing in the washing machine. This study demonstrates that complete DNA profiles can be obtained from laundered semen stains on school uniform-type clothing, with an eight-month lag time between semen deposition and laundering, despite multiple washes and stains from two semen donors. These data emphasise the need to recover and examine the clothing of victims for semen and DNA evidence, even if the clothing has been stored for several months or washed multiple times since the sexual offence took place.

A global analysis of Y-chromosomal haplotype diversity for 23 STR loci.

In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci (DYS19, DYS389I, DYS389II, DYS390, DYS391, DYS392, DYS393, DYS385ab, DYS437, DYS438, DYS439, DYS448, DYS456, DYS458, DYS635, GATAH4, DYS481, DYS533, DYS549, DYS570, DYS576, and DYS643) and using the PowerPlex Y23 System (PPY23, Promega Corporation, Madison, WI). Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent.

Worldwide F(ST) estimates relative to five continental-scale populations.

We estimate the population genetics parameter FST (also referred to as the fixation index) from short tandem repeat (STR) allele frequencies, comparing many worldwide human subpopulations at approximately the national level with continental-scale populations. FST is commonly used to measure population differentiation, and is important in forensic DNA analysis to account for remote shared ancestry between a suspect and an alternative source of the DNA. We estimate FST comparing subpopulations with a hypothetical ancestral population, which is the approach most widely used in population genetics, and also compare a subpopulation with a sampled reference population, which is more appropriate for forensic applications. Both estimation methods are likelihood-based, in which FST is related to the variance of the multinomial-Dirichlet distribution for allele counts. Overall, we find low FST values, with posterior 97.5 percentiles < 3% when comparing a subpopulation with the most appropriate population, and even for inter-population comparisons we find FST < 5%. These are much smaller than single nucleotide polymorphism-based inter-continental FST estimates, and are also about half the magnitude of STR-based estimates from population genetics surveys that focus on distinct ethnic groups rather than a general population. Our findings support the use of FST up to 3% in forensic calculations, which corresponds to some current practice.

Forensic DNA methylation profiling--potential opportunities and challenges.

Investigating the DNA sequence is the most powerful tool that can be employed in forensic genetics for the identification of an individual, or to determine specific ethnic and phenotypic characteristics. However, there are also other heritable changes in gene function or cellular phenotype which are caused by mechanisms other than differences in the DNA sequence itself. Over the last decade it has become evident that epigenetic markers can be of substantial forensic significance. The determination of possible alterations in DNA methylation patterns could aid various forensic investigations, such as differentiating monozygotic twins, identifying the tissue source or determining the age of tissue donors. This review aims to give a brief overview of the possible advantages of forensic DNA methylation profiling and sheds light on the limitations of this approach.