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.

The potential for investigator-mediated contamination to occur during routine search activities.

The sensitivity and discrimination power of modern DNA profiling systems means that very small amounts of DNA from an individual can be detected on an item leading to large inclusionary statistics for that person. The sensitivity of these systems has significant benefits in the investigation of crime but also can be highly sensitive to contamination of exhibits or crime scenes. It becomes critical to distinguish between deposition during commission of a crime or deposition via some other method unrelated to the crime. This study investigates methodologies used in crime scene examination and the potential for them to cause non-crime-related transfer of DNA. Factors assessed include the source of DNA, the handling time, the amount of movement during contact, and the substrate type. The amount of movement and the number of transfer steps are the most critical in determining whether, and how much, DNA is transferred. This study provides information for crime scene examiners and also scientists assessing transfer scenarios.

Using big data from probabilistic genotyping to solve crime.

Forensic Science South Australia (FSSA) has been using STRmix™ software to deconvolute all reported DNA mixtures since 2012. Almost a decade of deconvolutions had led to a substantial repository of analysed profile data that can be interrogated to observe trends in case type, location or occurrence. In addition, deconvolutions can be compared in order to identify common DNA donors and reveal new intelligence information in cases where DNA profiling has previously provided no investigative information. As a proof of concept all samples deconvoluted as part of criminal casework (suspect or no-suspect) were interrogated and compared to each other using the mixture-to-mixture comparison feature in STRmix™. Within the Adelaide region there were 32 groups of cases that had evidence samples linked by a common DNA donor with LR > 1 million which was in addition to direct links and mixture searching links identified previously. These groups of cases can then be interrogated to reveal additional information to inform Police intelligence gathering. Our paper reports on the findings of this proof-of-concept study.

Validation of a top-down DNA profile analysis for database searching using a fully continuous probabilistic genotyping model.

Slooten described a method of targeting major contributors in mixed DNA profiles and comparing them to individuals on a DNA database. The method worked by taking incrementally more peak information from the profile (based on the peak contribution), and using a semi-continuous model, calculating likelihood ratios for the comparison to database individuals. We describe the performance of this "top down approach" to profile interpretation within probabilistic genotyping software employing a fully continuous model. We interpret both complex constructed profiles where ground truth is known and casework profiles from non-suspect crimes. The interpretation of constructed four- and five- person mixtures demonstrated good discrimination power between contributors and non-contributors to the mixtures. Not all known contributors linked, and this is expected, particularly for minor contributors of DNA to the profile, or when the DNA from contributors was in relatively equal contributions. This finding was also reported by Slooten for the semi-continuous application of the approach. The maximum observed LR was shown to not exceed the LR obtained after a standard interpretation approach outside of that expected due to Monte Carlo variation. The interpretation of 91 complex profiles from no-suspect casework demonstrated that approximately 75% of profiles returned a link to someone on a database of known individuals. With a yearly average of 110 no-suspect cases that fall into this too-complex category at Forensic Science SA, the top down analysis, if applied to all such profiles, would represent an increase of 83 links per year of investigative information that could be provided to investigators.

A risk-based approach to cognitive bias in forensic science.

Over the past decade, the potential impact of cognitive bias in forensic science has instigated much discussion and debate between academics, scientists and those in the justice sector. Evidence of bias influencing subjective decision-making across a range of forensic disciplines has been described in the literature. Forensic service organisations are being urged to address cognitive bias in subjective decision-making by designing processes or procedures to limit access to (irrelevant) contextual information or reduce dependence on cognitive functions. Although some laboratories have implemented bias mitigating strategies, with varying impact on operational efficiency, there has been no systematic assessment of the risk posed by cognitive bias. Forensic Science SA assessed the potential impact of bias on forensic interpretations across multiple disciplines, using a risk management framework. This process proved useful in assessing the effectiveness of existing bias mitigating strategies and identified the latent level of risk posed. While all forensic organisations should seek to implement bias limiting measures that are simple, cost-effective and do not adversely impact efficiency, using a risk-based approach has contextualised the limited benefit of introducing resource hungry measures, as postulated in the literature. That is not to suggest that forensic organisations should dismiss the potential influence of cognitive bias but they need to strike an appropriate balance between risk and return, as they do with any business risk.

Inter-sample contamination detection using mixture deconvolution comparison.

A recent publication has provided the ability to compare two mixed DNA profiles and consider their probability of occurrence if they do, compared to if they do not, have a common contributor. This ability has applications to both quality assurance (to test for sample to sample contamination) and for intelligence gathering purposes (did the same unknown offender donate DNA to multiple samples). We use a mixture to mixture comparison tool to investigate the prevalence of sample to sample contamination that could occur from two laboratory mechanisms, one during DNA extraction and one during electrophoresis. By carrying out pairwise comparisons of all samples (deconvoluted using probabilistic genotyping software STRmix™) within extraction or run batches we identify any potential common DNA donors and investigate these with respect to their risk of contamination from the two proposed mechanisms. While not identifying any contamination, we inadvertently find a potential intelligence link between samples, showing the use of a mixture to mixture comparison tool for investigative purposes.

Likelihood ratio formulae for disputed parentage when the product of conception is trisomic.

We present here the derivation of paternity index formulae that covers situations of a disputed paternity trio with a trisomic product of conception. We consider six possible mechanisms for trisomy to occur: dispermy, dieggy, paternal meiosis I or II, and maternal meiosis I or II in the calculation. We also provide a biological explanation for how each of the mechanisms could give rise to a trisomy. The paper is set out in a general manner so that the tables presented can be used on any instance of trisomic offspring. This work is motivated by a case of disputed paternity where the product of conception was trisomic, i.e. the electropherogram of the product of conception possessed three alleles at each locus. The outcome was extremely strong support for the alleged father's paternity of the product of conception.

Developmental validation of STRmix™, expert software for the interpretation of forensic DNA profiles.

In 2015 the Scientific Working Group on DNA Analysis Methods published the SWGDAM Guidelines for the Validation of Probabilistic Genotyping Systems [1]. STRmix™ is probabilistic genotyping software that employs a continuous model of DNA profile interpretation. This paper describes the developmental validation activities of STRmix™ following the SWGDAM guidelines. It addresses the underlying scientific principles, and the performance of the models with respect to sensitivity, specificity and precision and results of interpretation of casework type samples. This work demonstrates that STRmix™ is suitable for its intended use for the interpretation of single source and mixed DNA profiles.

Observations of DNA transfer within an operational Forensic Biology Laboratory.

Advances in technology to both generate and interpret DNA profiles has seen the expansion of the ability to provide opinions about results obtained from very low levels of starting biological material. The response in court has been to question the mode by which the DNA came to be on an item, rather than questioning its presence. This brings into play a number of real-world aspects such as transfer of biological material, persistence of biological material on items, shedding ability of individuals, just to name a few. There have been a number of studies that investigate different aspects relating the mode of DNA deposition and transfer, mostly under tightly controlled conditions. We add to this knowledge pool by investigating the extent to which individuals at Forensic Science SA (FSSA) deposit their DNA on objects throughout the floor of the building where DNA examinations take place. We find that the results obtained in our minimally controlled study allow us to comment on a number of published concepts.

Evaluating forensic biology results given source level propositions.

The evaluation of forensic evidence can occur at any level within the hierarchy of propositions depending on the question being asked and the amount and type of information that is taken into account within the evaluation. Commonly DNA evidence is reported given propositions that deal with the sub-source level in the hierarchy, which deals only with the possibility that a nominated individual is a source of DNA in a trace (or contributor to the DNA in the case of a mixed DNA trace). We explore the use of information obtained from examinations, presumptive and discriminating tests for body fluids, DNA concentrations and some case circumstances within a Bayesian network in order to provide assistance to the Courts that have to consider propositions at source level. We use a scenario in which the presence of blood is of interest as an exemplar and consider how DNA profiling results and the potential for laboratory error can be taken into account. We finish with examples of how the results of these reports could be presented in court using either numerical values or verbal descriptions of the results.

Random whole metagenomic sequencing for forensic discrimination of soils.

Here we assess the ability of random whole metagenomic sequencing approaches to discriminate between similar soils from two geographically distinct urban sites for application in forensic science. Repeat samples from two parklands in residential areas separated by approximately 3 km were collected and the DNA was extracted. Shotgun, whole genome amplification (WGA) and single arbitrarily primed DNA amplification (AP-PCR) based sequencing techniques were then used to generate soil metagenomic profiles. Full and subsampled metagenomic datasets were then annotated against M5NR/M5RNA (taxonomic classification) and SEED Subsystems (metabolic classification) databases. Further comparative analyses were performed using a number of statistical tools including: hierarchical agglomerative clustering (CLUSTER); similarity profile analysis (SIMPROF); non-metric multidimensional scaling (NMDS); and canonical analysis of principal coordinates (CAP) at all major levels of taxonomic and metabolic classification. Our data showed that shotgun and WGA-based approaches generated highly similar metagenomic profiles for the soil samples such that the soil samples could not be distinguished accurately. An AP-PCR based approach was shown to be successful at obtaining reproducible site-specific metagenomic DNA profiles, which in turn were employed for successful discrimination of visually similar soil samples collected from two different locations.

Successful direct amplification of nuclear markers from a single hair follicle.

We report on successful amplification of DNA profiles from a single hair. Direct amplification was used on the root tip of both anagen and telogen hairs using a kit to amplify 15 STR loci. All 30 anagen hairs tested from five different people gave full DNA profiles after 29 cycles with no allelic drop-in or heterozygous imbalance. Six of the 30 telogen hairs tested resulted in a full DNA profile, and a further four telogen hair samples tested produced a DNA profile of five or more complete loci that could be up-loaded to the National DNA Database (Australia). A full DNA profile was also obtained from the shaft of an anagen hair. Current practice for many laboratories is that a single hair may not be subjected to DNA testing as there is little chance of success, hence this 100 % success rate from anagen hairs is a significant advancement. A full DNA profile was obtained from a 5 year-old single hair illustrating the success when using direct PCR rather than attempting an extraction prior to the amplification step. The process described deliberately uses current DNA profiling methods with no increase in cycle number, such that the methodology can be incorporated readily into operational practice. For the first time in the field of human identification, single hairs can be analyzed with confidence that a meaningful DNA profile will be generated and the data accepted by the criminal justice system.

Is urinary indolyl-3-acryloylglycine a biomarker for autism with gastrointestinal symptoms?

An autism spectrum disorder (ASD) diagnosis is based on clinical behaviours as there are no validated biological diagnostic tools. Indolyl-3-acryloylglycine (IAG) is a chemical produced by gut microflora and there are conflicting reports as to whether urinary levels are elevated in children with ASD compared with controls. Urinary IAG levels in morning urine samples were statistically significantly higher in children with ASD whose caregivers reported the presence of chronic gastrointestinal (GI) disturbance than children with ASD without chronic GI disturbance. Urinary IAG, however, was not statistically significantly higher in children with ASD, compared with siblings or unrelated controls without ASD.