PCR in Forensic Science: A Critical Review.

The polymerase chain reaction (PCR) has played a fundamental role in our understanding of the world, and has applications across a broad range of disciplines. The introduction of PCR into forensic science marked the beginning of a new era of DNA profiling. This era has pushed PCR to its limits and allowed genetic data to be generated from trace DNA. Trace samples contain very small amounts of degraded DNA associated with inhibitory compounds and ions. Despite significant development in the PCR process since it was first introduced, the challenges of profiling inhibited and degraded samples remain. This review examines the evolution of the PCR from its inception in the 1980s, through to its current application in forensic science. The driving factors behind PCR evolution for DNA profiling are discussed along with a critical comparison of cycling conditions used in commercial PCR kits. Newer PCR methods that are currently used in forensic practice and beyond are examined, and possible future directions of PCR for DNA profiling are evaluated.

Predicting probative levels of touch DNA on tapelifts using Diamond™ Nucleic Acid Dye.

Tapelifting is a common strategy to recover touch DNA deposits from porous exhibits in forensic DNA casework. However, it is known that only about 30 % of tapelifts submitted for DNA analysis in operational forensic laboratories yield profiles suitable for comparison or upload to a searchable database. A reliable means to identify and remove non-probative tapelifts from the workflow would reduce sample backlogs and provide significant cost savings. We investigated whether the amount of macroscopic or microscopic fluorescence on a tapelift following staining with Diamond Nucleic Acid Dye (DD), determined using a Polilight and Dino Lite microscope respectively, could predict the DNA yield and/or the DNA profiling outcome using controlled (saliva), semi-controlled (finger mark) and uncontrolled (clothing) samples. Both macroscopic and microscopic DD fluorescence could predict DNA yield and profiling outcome for all sample types, however the predictive power deteriorated as the samples became less controlled. For tapelifts of clothing, which are operationally relevant, Polilight fluorescence scores were significantly impacted by clothing fibres and other non-cellular debris and could not be used to identify non-probative samples. The presence of less than 500 cells on a clothing tapelift using microscopic counting of stained corneocytes was identified as a potential threshold for a non-probative DNA profiling outcome. A broader examination of the reliability of this threshold using a casework trial is recommended. Due to the labour intensiveness of microscopic cell counting, and the increased risk of inadvertent contamination, automation of this process using image software in conjunction with artificial neural networks (ANN) should be explored.

Up in the air: Presence and collection of DNA from air and air conditioner units.

Biological material is routinely collected at crime scenes and from exhibits and is a key type of evidence during criminal investigations. Touch or trace DNA samples from surfaces and objects deemed to have been contacted are frequently collected. However, a person of interest may not leave any traces on contacted surfaces, for example, if wearing gloves. A novel means of sampling human DNA from air offers additional avenues for DNA collection. In the present study, we report on the results of a pilot study into the prevalence and persistence of human DNA in the air. The first aspect of the pilot study investigates air conditioner units that circulate air around a room, by sampling units located in four offices and four houses at different time frames post-cleaning. The second aspect investigates the ability to collect human DNA from the air in rooms, with and without people, for different periods of time and with different types of collection filters. Results of this pilot study show that human DNA can be collected on air conditioner unit surfaces and from the air, with air samples representing the more recent occupation while air conditioner units showing historic use of the room.

Emerging use of air eDNA and its application to forensic investigations - A review.

Biological material is routinely collected at crime scenes and from exhibits and is a key type of evidence during criminal investigations. Improvements in DNA technologies allow collection and profiling of trace samples, comprised of few cells, significantly expanding the types of exhibits targeted for DNA analysis to include touched surfaces. However, success rates from trace and touch DNA samples tend to be poorer compared to other biological materials such as blood. Simultaneously, there have been recent advances in the utility of environmental DNA collection (eDNA) in identification and tracking of different biological organisms and species from bacteria to naked mole rats in different environments, including, soil, ice, snow, air and aquatic. This paper examines the emerging methods and research into eDNA collection, with a special emphasis on the potential forensic applications of human DNA collection from air including challenges and further studies required to progress implementation.

A practical treatment of sensitivity analyses in activity level evaluations.

Evaluations of forensic observations considering activity level propositions are becoming more common place in forensic institutions. A measure that can be taken to interrogate the evaluation for robustness is called sensitivity analysis. A sensitivity analysis explores the sensitivity of the evaluation to the data used when assigning probabilities, or to the level of uncertainty surrounding a probability assignment, or to the choice of various assumptions within the model. There have been a number of publications that describe sensitivity analysis in technical terms, and demonstrate their use, but limited literature on how that theory can be applied in practice. In this work we provide some simplified examples of how sensitivity analyses can be carried out, when they are likely to show that the evaluation is sensitive to underlying data, knowledge or assumptions, how to interpret the results of sensitivity analysis, and how the outcome can be reported. We also provide access to an application to conduct sensitivity analysis.

A diagnosis of the primary difference between EuroForMix and STRmix™.

There is interest in comparing the output, principally the likelihood ratio, from the two probabilistic genotyping software EuroForMix (EFM) and STRmix™. Many of these comparison studies are descriptive and make little or no effort to diagnose the cause of difference. There are fundamental differences between EFM and STRmix™ that are causative of the largest set of likelihood ratio differences. This set of differences is for false donors where there are many instances of LRs just above or below 1 for EFM that give much lower LRs in STRmix™. This is caused by the separate estimation of parameters such as allele height variance and mixture proportion using MLE under Hp and Ha for EFM. This can result in very different estimations of these parameters under Hp and Ha . It results in a departure from calibration for EFM in the region of LRs just above and below 1.

Estimation of population specific values of theta for sequence-based STR profiles.

We describe the estimation of θ (theta) values from autosomal STR sequencing data for five metapopulations. The data were compiled from 20 publications and included 39 datasets comprising a total of 7005 samples. The estimates are suitable for use within the calculation of match probabilities in forensic casework. We also have constructed a phylogenetic tree using this data that aligns with our understanding of human evolution.

Determining the number and size of background samples derived from an area adjacent to the target sample that provide the greatest support for a POI in a target sample.

When sampling an item or surface for DNA originating from an action of interest, one is likely to collect DNA unrelated to the action of interest (background DNA). While adding to the complexity of a generated DNA profile, background DNA has been shown to aid in resolving the genotypes of contributors in a targeted sample, and where references of donors to the background DNA are not available, strengthen the LR supporting a person of interest contributing to the targeted sample. This is possible thanks to advances in probabilistic genotyping, where forensic labs are able to deconvolute complex DNA profiles to obtain lists of genotypes and their associated weights. Coupled with DBLR™, one can then compare multiple evidentiary profiles to each other to determine the contribution of common, but unknown, contributors. Here, we consider factors associated with taking background samples and whether one should collect multiple background samples that all relate to a single target sample, or if one should collect larger background samples rather than smaller samples. Background samples consisted of DNA accumulated on the items primarily by one or both occupants of a single household, while targeted samples were generated from touch deposits, or saliva deposits that had been left to air dry. Samples were collected from areas of various sizes, consisting of only the background, the target and the background directly beneath it, and the target and additional surrounding background. A broad range of DNA quantities were recovered, with larger background samples (400 cm2) yielding significantly more DNA than smaller background samples (30 cm2). Significant differences in DNA quantities between target samples were not observed. Generated DNA profiles were interpreted using STRmix™ and DBLR™, and where there was support for a common donor between the background and target sample, pairwise comparisons were performed to observe the effect on the LR supporting the target DNA donor contributing to the targeted sample when conditioning on one (or two) common donor between the targeted sample and 1-8 background samples. Multiple background samples gave significantly higher LRs compared to a single background sample, the larger sampled background area resulted in larger LR gains than the smaller areas, and four or more background samples reduced LR variability considerably. Here we provide recommendations for the minimum and ideal number of additional background samples that should be collected, and that several smaller samples may be more beneficial than a single larger sample.

The secret hidden in dust: Assessing the potential to use biological and chemical properties of the airborne fraction of soil for provenance assignment and forensic casework.

The airborne fraction of soil (dust) is both ubiquitous in nature and contains localised biological and chemical signatures, making it a potential medium for forensic intelligence. Metabarcoding of dust can yield biological communities unique to the site of interest, similarly, geochemical analyses can uncover elements and minerals within dust that can be matched to a geographic location. Combining these analyses presents multiple lines of evidence as to the origin of dust collected from items of interest. In this work, we investigated whether bacterial and fungal communities in dust change through time and whether they are comparable to soil samples of the same site. We integrated dust metabarcoding into a framework amenable to forensic casework, (i.e., using calibrated log-likelihood ratios) to predict the origin of dust samples using models constructed from both dust samples and soil samples from the same site. Furthermore, we tested whether both metabarcoding and geochemical/mineralogical analyses could be conducted on a single swabbed sample, for situations where sampling is limited. We found both analyses could generate results from a single swabbed sample and found biological and chemical signatures unique to sites. However, we did find significant variation within sites, where this did not always correlate with time but was a random effect of sampling. This variation within sites was not greater than between sites and so did not influence site discrimination. When modelling bacterial and fungal diversity using calibrated log-likelihood ratios, we found samples were correctly predicted using dust 67% and 56% of the time and using soil 56% and 22% of the time for bacteria and fungi communities respectively. Incorrect predictions were related to within site variability, highlighting limitations to assigning dust provenance using metabarcoding of soil.

Addressing uncertain assumptions in DNA evidence evaluation.

Evidential value of DNA mixtures is typically expressed by a likelihood ratio. However, selecting appropriate propositions can be contentious, because assumptions may need to be made around, for example, the contribution of a complainant's profile, or relatedness between contributors. A choice made one way or another disregards any uncertainty that may be present about such an assumption. To address this, a complex proposition that considers multiple sub-propositions with different assumptions may be more appropriate. While the use of complex propositions has been advocated in the literature, the uptake in casework has been limited. We provide a mathematical framework for evaluating DNA evidence given complex propositions and discuss its implementation in the DBLR™ software. The software simultaneously handles multiple mixed samples, reference profiles and relationships as described by a pedigree, which unlocks a variety of applications. We provide several examples to illustrate how complex propositions can efficiently evaluate DNA evidence. The addition of this feature to DBLR™ provides a tool to approach the long-accepted, but often impractical suggestion that propositions should be exhaustive within a case context.

Carrying out common DNA donor analysis using DBLR™ on two or five-cell mini-mixture subsamples for improved discrimination power in complex DNA mixtures.

Probabilistic genotyping systems are able to analyse complex mixed DNA profiles and show good power to discriminate contributors from non-contributors. However, the abilities of the statistical analyses are still unavoidably bound by the quality of information being analysed. If a profile has a high number of contributors, or a contributor that is present in trace amounts, then the amount of information about those individuals in the DNA profile is limited. Recent work has shown the ability to gain better resolution of the genotypes of contributors to complex profiles using cell subsampling. This is the process of taking many sets of a limited number of cells and individually profiling each set. These 'mini-mixtures' can provide greater information about the genotypes of underlying contributors. In our work we take the resulting profiles from multiple subsamplings of complex DNA profiles in equal amounts and show how testing for, and then assuming, a common DNA donor can further improve the ability to resolve the genotypes of contributors. Using direct cell sub-sampling and statistical analysis software DBLR™, we were able to recover single source profiles of uploadable quality from five out of the six contributors of an equally proportioned mixture. Through the analysis of mixtures in this work we provide a template for carrying out common donor analysis for maximum effect.

A lights-out forensic DNA analysis workflow for no-suspect crime.

An automated system of DNA profile processing (termed a 'lights-out' workflow) was trialled for no-suspect cases over a three-month period at Forensic Science SA (FSSA). The lights-out workflow utilised automated DNA profile reading using the neural network reading feature in FaSTR™ DNA with no analytical threshold. The profile information from FaSTR™ DNA was then processed in STRmix™ using a top-down analysis and automatically compared to a de-identified South Australian searchable DNA database. Computer scripts were used to generate link reports and upload reports and these were compared to the links and uploads that were obtained for the cases during their standard processing within the laboratory. The results of the lights-out workflow was an increase in both uploads and links compared to the standard workflow, with minimal adventitious links or erroneous uploads. Overall, the proof-of-concept study shows the potential for using automated DNA profile reading and top-down analysis to improve workflow efficiency in a no-suspect workflow.

Extending the discrete Laplace method: incorporating multi-copy loci, partial repeats and null alleles.

The discrete Laplace method can be used to estimate the frequency of a Y-chromosomal STR haplotype using a random sample from the population. Two limitations of the method are the assumptions that each profile has exactly one allele at every locus and that this allele has an integer repeat number. We relax these assumptions to allow for multi-copy loci, partial repeats and null alleles. We show how the parameters to the extension of the model can be estimated by numerical optimisation using an off-the-shelf solver. Concordance with the discrete Laplace method is obtained when the data satisfy the more stringent assumptions of the original method. We also investigate the performance of the (extended) discrete Laplace method when used to assign match probabilities for haplotypes. A simulation study shows that as more loci are used, match probabilities are underestimated more severely. This is consistent with the hypothesis that the discrete Laplace method cannot model the matches that arise by being identical by descent (IBD). As the number of loci increases the fraction of matches that are IBD increases. Simulation provides support that the discrete Laplace can model those matches that arise from identity by state (IBS) only.

Exploring how the LR of a POI in a target sample is impacted by awareness of the profile of the background derived from an area adjacent to the target sample.

DNA unrelated to an action of interest (background DNA) is routinely collected when sampling an area for DNA that may have originated from an action of interest. Background DNA can add to the complexity of a recovered DNA profile and could impact the discrimination power when comparing it to the reference profile of a person of interest. Recent advances in probabilistic genotyping and the development of new tools, now allow for the comparison of multiple evidentiary profiles to query for a common DNA donor. Here, we explore the additional discrimination power that can be gained by having an awareness of the background DNA present on a surface prior to the deposition of target DNA. Samples with varying number of contributors and DNA quantities were generated on cleaned plastic pipes (where ground truth was known) and items used by occupants of a single household (where ground truth was not known). The background consisted of deposits made by hands (touch) while target deposits were both touch and saliva. Samples were collected from areas consisting of only the background (A), the target and the background directly beneath it (B), and the target and additional surrounding background (B+C). Samples B and B+C yielded similar DNA amounts when the target consisted of saliva, but when the target consisted of touch, significantly more DNA was recovered from B+C. Subsequently generated DNA profiles were interpreted using STRmix™ and DBLR™. The first approach involved no conditioning while the second approach involved conditioning on the reference profiles of the known background DNA donors. The third approach involved conditioning on one common DNA donor between A and B or A and B+C. The fourth and final approach involved conditioning on two common DNA donors between A and B or A and B+C. As more information was applied to the analysis, the greater the increase in the LR for the comparison of the target sample to the POI. Conditioning on two common donors between the target and the background provided almost the same amount of information as conditioning on the references of the known background DNA donors. This resulted in an increase in the LR that was over 10 orders of magnitude for known donors in the target sample. Here we have demonstrated the value in collecting additional background samples from an area adjacent to a targeted sample, and that this has the potential to improve discrimination power.

DNA accumulation and transfer within an operational forensic exhibit storeroom.

The increased sensitivity of current DNA profiling technologies allows the detection of trace amounts of DNA. With these advancements, there is an increased probability of detecting trace levels of DNA from contamination. Studies which investigate the accumulation and transfer of DNA within forensic laboratories provide insight into the possible mechanisms which may result in the contamination of exhibits. To gain a greater understanding of the level of DNA transfer between exhibit packaging and forensic workspaces, the accumulation of DNA within an operational forensic exhibit storeroom was investigated. Samples were collected from previously cleaned forensic exhibit storeroom shelves at various time points over a 14-week period. To determine the source of accumulating DNA, profiles generated from shelf samples were compared to the laboratory staff elimination database and the profiles generated from exhibits stored on each of the shelves sampled over the course of the study. Additionally, all samples were compared using STRmix™ mixture-to-mixture profile analysis, to identify the presence of common non-staff DNA donors and DNA from exhibits stored on the shelves sampled. As sampling time intervals increased, there was a significant increase in DNA quantity (ng) and number of profile contributors. The shelf height was also observed to influence the number of profile contributors, with higher numbers of contributors being found on lower shelves. DNA profiles generated from the shelf samples were matched to DNA from forensic staff members who enter the storeroom and police employees, who do not enter the storeroom. There were three instances where a common DNA profile contributor was identified between a shelf sample and the profile generated from an exhibit.This study provides insight into whether current exhibit storage procedures are still adequate given the highly sensitive DNA profiling systems currently used.

Combining artificial neural network classification with fully continuous probabilistic genotyping to remove the need for an analytical threshold and electropherogram reading.

Standard processing of electrophoretic data within a forensic DNA laboratory is for one (or two) analysts to designate peaks as either artefactual or non-artefactual in a process commonly referred to as profile 'reading'. Recently, FaSTR™ DNA has been developed to use artificial neural networks to automatically classify fluorescence within an electropherogram as baseline, allele, stutter or pull-up. These classifications are based on probabilities assigned to each timepoint (scan) within the electropherogram. Instead of using the probabilities to assign fluorescence into a category they can be used directly in the profile analysis. This has a number of advantages; increased objectivity in DNA profile processing, the removal for the need for analysts to read profiles, the removal for the need of an analytical threshold. Models within STRmix™ were extended to incorporate the peak label probabilities assigned by FaSTR™ DNA. The performance of the model extensions was tested on a DNA mixture dataset, comprising 2-4 person samples. This dataset was processed in a 'standard' manner using an analytical threshold of 50rfu, analyst peak designations and STRmix™ V2.9 models. The same dataset was then processed in an automated manner using no analytical threshold, no analysts reading the profile and using the STRmix™ models extended to incorporate peak label probabilities. Both datasets were compared to the known DNA donors and a set of non-donors. The result between the two processes was a very close performance, but with a large efficiency gain in the 0rfu process. Utilising peak label probabilities opens up the possibility for a range of workflow process efficiency gains, but beyond this allows full use of all data within an electropherogram.

Developmental validation of STRmix™ NGS, a probabilistic genotyping tool for the interpretation of autosomal STRs from forensic profiles generated using NGS.

We describe the developmental validation of the probabilistic genotyping software - STRmix™ NGS - developed for the interpretation of forensic DNA profiles containing autosomal STRs generated using next generation sequencing (NGS) also known as massively parallel sequencing (MPS) technologies. Developmental validation was carried out in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) Guidelines for the Validation of Probabilistic Genotyping Systems and the International Society for Forensic Genetics (ISFG) recommendations and included sensitivity and specificity testing, accuracy, precision, and the interpretation of case-types samples. The results of developmental validation demonstrate the appropriateness of the software for the interpretation of profiles developed using NGS technology.

Pedicled Superficial Circumflex Iliac Artery Perforator (SCIP) flap for perineo-scrotal reconstruction following Fournier's gangrene.

BACKGROUND: Fournier's gangrene is a form of necrotising fasciitis involving perineo-scrotal skin. It is treated with radical debridement, infection control and often leaves a large anatomical defect that is challenging to reconstruct. The anatomical location of the defect leads to faecal contamination, difficulties when mobilizing, and negative psychological impact. Traditional approaches for managing such defects have relied on either healing by secondary intention or skin grafting. There are few reported cases in the literature to cover such defects with a flap. METHODS: Pedicled Superficial Circumflex Iliac Artery Perforator (SCIP) flap reconstruction was performed in three patients who had perineo-scrotal defects following debridement for Fournier's gangrene. RESULTS: All flaps survived with no significant postoperative complications and good functional and aesthetic outcomes were achieved. The mean age of patient was 52 years and the largest defect measured 22 × 10 cm. CONCLUSION: The reconstruction of perineo-scrotal defects is difficult despite a range of reconstructive options. The pedicled SCIP flap offers many advantages over standard techniques. This flap is thin, pliable, and has a consistent anatomy. With continued experience, we feel that this flap could be considered the gold standard of treatment for such defects.