Developing a Machine-Learning 'Smart' PCR Thermocycler, Part 1: Construction of a Theoretical Framework.

The use of PCR is widespread in biological fields. Some fields, such as forensic biology, push PCR to its limits as DNA profiling may be required in short timeframes, may be produced from minute amounts of starting material, and may be required to perform in the presence of inhibitory compounds. Due to the extreme high-throughput of samples using PCR in forensic science, any small improvement in the ability of PCR to address these challenges can have dramatic effects for the community. At least part of the improvement in PCR performance could potentially come by altering PCR cycling conditions. These alterations could be general, in that they are applied to all samples, or they could be tailored to individual samples for maximum targeted effect. Further to this, there may be the ability to respond in real time to the conditions of PCR for a sample and make cycling parameters change on the fly. Such a goal would require both a means to track the conditions of the PCR in real time, and the knowledge of how cycling parameters should be altered, given the current conditions. In Part 1 of our work, we carry out the theoretical groundwork for the ambitious goal of creating a smart PCR system that can respond appropriately to features within individual samples in real time. We approach this task using an open qPCR instrument to provide real-time feedback and machine learning to identify what a successful PCR 'looks like' at different stages of the process. We describe the fundamental steps to set up a real-time feedback system, devise a method of controlling PCR cycling conditions from cycle to cycle, and to develop a system of defining PCR goals, scoring the performance of the system towards achieving those goals. We then present three proof-of-concept studies that prove the feasibility of this overall method. In a later Part 2 of our work, we demonstrate the performance of the theory outlined in this paper on a large-scale PCR cycling condition alteration experiment. The aim is to utilise machine learning so that throughout the process of PCR automatic adjustments can be made to best alter cycling conditions towards a user-defined goal. The realisation of smart PCR systems will have large-scale ramifications for biological fields that utilise PCR.

Developing a Machine Learning 'Smart' Polymerase Chain Reaction Thermocycler Part 2: Putting the Theoretical Framework into Practice.

The introduction of PCR into forensic science and the rapid increases in the sensitivity, specificity and discrimination power of DNA profiling that followed have been fundamental in shaping the field of forensic biology. Despite these developments, the challenges associated with the DNA profiling of trace, inhibited and degraded samples remain. Thus, any improvement to the performance of sub-optimal samples in DNA profiling would be of great value to the forensic community. The potential exists to optimise the PCR performance of samples by altering the cycling conditions used. If the effects of changing cycling conditions upon the quality of a DNA profile can be well understood, then the PCR process can be manipulated to achieve a specific goal. This work is a proof-of-concept study for the development of a smart PCR system, the theoretical foundations of which are outlined in part 1 of this publication. The first steps needed to demonstrate the performance of our smart PCR goal involved the manual alteration of cycling conditions and assessment of the DNA profiles produced. In this study, the timing and temperature of the denaturation and annealing stages of the PCR were manually altered to achieve the goal of reducing PCR runtime while maintaining an acceptable quality and quantity of DNA product. A real-time feedback system was also trialled using an STR PCR and qPCR reaction mix, and the DNA profiles generated were compared to profiles produced using the standard STR PCR kits. The aim of this work was to leverage machine learning to enable real-time adjustments during a PCR, allowing optimisation of cycling conditions towards predefined user goals. A set of parameters was found that yielded similar results to the standard endpoint PCR methodology but was completed 30 min faster. The development of an intelligent system would have significant implications for the various biological disciplines that are reliant on PCR technology.

Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 µL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

A systematic approach to the analysis of illicit drugs for DNA with an overview of the problems encountered.

Due to the restricted nature of illicit drugs, it is difficult to conduct research surrounding the analysis of this drug material for any potential DNA in sufficient quantities acceptable for high numbers of replicates. Therefore, the current research available in peer reviewed journals thus far regarding analysing illicit drugs for DNA has been performed under varying experimental conditions, often using surrogate chemicals in place of illicit drugs. The data presented within this study originated from the analysis of genuine illicit drugs prepared both in controlled environments and those seized at the Australian border (and therefore from an uncontrolled environment) to determine if DNA can be obtained from this type of material. This study has been separated into three main parts (total n=114 samples): firstly, methamphetamine synthesised within a controlled environment was spiked with both saliva and trace DNA to determine the yield following DNA extraction; secondly, methamphetamine also synthesised in a controlled environment but on a larger scale was tested for the amount of DNA added incidentally throughout the synthesis, including the additional steps of recrystallising, homogenising and "cutting" the drug material to simulate preparation for distribution; and thirdly, the detection of human DNA within samples of cocaine and heroin seized at the Australian border. The DNA Fast Flow Microcon Device was utilised to concentrate all replicates from the same source into one combined extract to improve the DNA profiles for the samples where no DNA spiking occurred. Full STR profiles were successfully obtained from drug samples spiked with both saliva and trace DNA. Methamphetamine was present in the final DNA extracts and caused incompatibilities with the quantification of DNA using Qubit. The yields of DNA from drugs not spiked with DNA sources were much lower, resulting in 36 % of samples yielding alleles where all others did not. These results were not unexpected given these were realistic drug samples where the history of the drug material was unknown. This is the first study to obtain DNA profiles from genuine illicit drug material in both controlled and uncontrolled environments and indicates that the analysis of illicit drugs for DNA is an avenue worth pursuing to provide information which can in turn assist with disrupting the supply of these drugs. Given that DNA profiling is carried out worldwide using essentially the same systems as described within this study, the potential for impact is on a national and international scale.

Shedding more light on shedders.

We report on testing 100 individuals for their shedder status with the aim of demonstrating whether the process of cell staining is reproducible when testing a large number of people. A previous report using the same method was based on 11 donors and indicated that there may be a continuum of shedder types within this small sample set. In this report we also expand the time points post-handwashing to 0, 15, 30, 60, and 180 min. Triplicate samples were collected from both the right and left thumbs. Samples were collected by donors placing a thumb on a clean glass slide and then adding a DNA binding dye. The number of cells were recorded within three separate square millimetre areas (cells/mm2) at 220x magnification. The experiments were conducted in triplicate on three different days, giving a total of 72 thumbprints per individual. Finally, there were 3438 observed frames in the entire dataset. Of the 100 donors, 98 gave consistent and reproducible cell number deposition. There was no difference between the cells deposited by the left and right thumbs in 13 of 15 tested. Males tended to deposit more cells than females. If applying arbitrary boundary to a cell count to definitively determine shedder status, then many of the donors fell within two categories. This study based on 100 individuals strongly suggests that shedder status is a continuum phenomenon.

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.

Cell counting to monitor swab efficiency.

Plastic bags, such as ziplock bags, have been used to transport illicit materials worldwide; however, very few studies have tried to optimize the recovery of DNA from these items. This study reports on the best combination of swabs and moistening solution for the greatest recovery of cellular material from ziplock bags. Five swabs, two different variations of Copan Diagnostics nylon 4N6FLOQSwabs, one Medical Wire rayon DRYSWAB, one IsoHelix rayon swab, and one Livingstone cotton swab, were evaluated with two moistening solutions, Triton X-100 in either distilled water or isopropanol. Fingermarks were deposited on ziplock bags and stained with Diamond™ Nucleic Acid Dye to allow visualization of the cells pre- and post-swabbing to determine the number of cells recovered. Based on cell counting data, swabs moistened with Triton X-100 in distilled water performed better than those moistened with isopropanol. Livingstone cotton swabs had the worst recovery of cellular material, while the other swabs tested had no significant difference in their respective solutions. A comparison of the best three swabs for cellular recovery yielded no differences in the DNA concentration extracted. A linear relationship was observed between the log number of cells recovered by swabbing and the DNA concentration following extraction and quantification. The process of monitoring cell collection using fluorescence microscopy on ziplock bags allowed evaluation of swabbing efficacy. Additionally, this study highlights the ability to evaluate cellular recovery independently of traditional extraction, quantification, or profiling techniques which may unequally affect samples.

Visualisation and detection of latent DNA deposited by pangolin scales onto plastic packaging materials.

We report on the detection and visualisation of latent DNA from pangolin scales deposited onto a plastic packaging material through the use of a nucleic acid staining dye. This latent DNA deposited by pangolin scales was subsequently isolated and analysed using DNA barcoding method. Pangolins are the most illegally traded mammalian species due to the demand for their scales and meat. The demand for their scales were mostly fuelled by its use in traditional medicines. The scales are usually packed into bags and transported globally via sea routes. This is the first report detailing the detection of trace latent DNA from processed wildlife products, on surfaces of bags that they were packaged in. Prior to this report, it was not known if the dried pangolin scales contained transferable quantities of biological material for DNA analyses. To address this, scales were removed from a roadkill Sunda pangolin (Manis javanica), processed by drying and packaged into one of five plastic bags. The presence of pangolin latent DNA was detected on the surface of the plastic bags and visualised using Diamond™ nucleic acid dye. Swabs were then used to recover the stained biological material from various locations in the five bags. The DNA was isolated and quantified using a newly designed quantitative PCR (qPCR) specific to M. javanica to amplify a fragment of the mitochondrial DNA cytochrome b gene. There was a positive correlation between the number of stained particles and DNA quantity, and a greater number of stained particles were found at the bottom of the bag than were found at the top. Conventional PCR targeting part of the cyt b gene amplified a product from all 30 samples taken from the bags and in all cases, sequence data generated matched that of the Sunda pangolin, as expected. All negative controls yielded no results. The method described here is the very first use of a nucleic acid staining dye to detect latent DNA from a mammalian species, other than humans, and highlights the opportunity for further use of Diamond™ nucleic acid dye in wildlife forensic science. It is anticipated that this method will be invaluable in retrieving latent DNA deposited by wildlife products from the environment in which they were contained, to determine the presence of these illegal wildlife products even when previously hidden, inaccessible, or no longer present physically. Further research is required to understand if the use on non-human mammalian wildlife species is feasible.

DNA identification of monozygotic twins.

This study details the differentiation of identical twins based on single mutational base differences. There were three pairs of male monozygotic (MZ) twins in this study. DNA samples from blood, a buccal swab or saliva from each individual were all initially genotyped using 22 autosomal STR and 27 Y-STR loci. Preliminary screening confirmed there were no differences in the STR data between each pair of MZ twins. Whole Genome Sequence (WGS) data were generated from DNA extracted from the three body fluids from each individual. Kinship coefficients with 0.4254, 0.4557 and 0.4543 from 3 twins were generated based on WGS data to further confirm that their relationship was that of MZ twins. The fastq data generated by the Illumina Hiseq 2000 between MZ twins were then treated as "normal" as opposed to "tumor" using commercially available software tools to identify mutational single base changes. Sanger DNA sequencing confirmed there were 1, 5 and 9 single base changes found in WGS data from each of the three MZ twin sets. There was individual variation in the mutational base changes when comparing data from the three body fluids. The methods used in this study to differentiate MZ twins based on WGS data can readily be performed in many operational forensic DNA laboratories using user friendly software.

DNA identification from dental pulp and cementum.

Teeth are one of the body tissues remaining after severe decomposition from which a DNA profile can be obtained to aid in human identification. Currently, the standard approach to isolate DNA from teeth requires pulverizing the entire tooth. This destructive approach compromises any further morphological or anthropological study. We report on two methods of DNA isolation that minimizes destruction of the tooth when accessing the DNA within pulp and cementum. Forty-nine teeth, removed as part of normal dental procedures, were buried for up to 92 days, with a further nine teeth acting as unburied controls. Additionally, four teeth samples collected during a forensic examination were included in this study. The two processes were: using a fine drill to access the pulp from the crown and then using endodontic files to collect the biological material; and using a sterile blade to scrape the cementum. It was found that the samples collected from the cementum had greater DNA quality compared to those samples obtained from the pulp. Microbial activity was found to play a role in the degradation of the nuclear material, reducing DNA yields from pulp. DNA profiling data from 24 loci, including 22 STR markers, indicated that multi-rooted teeth provided better DNA quantity and quality than those with a single root. The DNA quantity obtained from pulp samples of teeth which exhibited cavities was adversely affected, although this DNA loss was not from samples collected from the cementum of teeth in similar condition. Obtaining samples from DNA profiling from the cementum was found to be ideal if the morphological preservation of the tooth is required. Obtaining pathogen DNA is of interest when an occlusal approach to retrieve pulp may serve as a good alternative to prepare DNA without destruction of the tooth structure.

Comparison of three DNA extraction methods tested on illicit drug-related powders.

The detection of human DNA on and within illicit drug preparations is novel and a focus of current research. Previous studies have indicated that certain drug-related powders present in illicit drug preparations can interfere with downstream DNA analysis when directly added to the PCR. Therefore, it is important to determine if these drug-related powders are effectively removed during the DNA extraction or whether traces of powder remain to interfere with DNA processing. Three extraction methods were selected to assess their efficiency at removing drug-related powders for downstream processes using DNA from both saliva and touch depositions. This is the first study to compare efficiencies of DNA extraction methods from drug-related powders. The extraction methods compared were the DNA IQ™ System, the QIAamp® DNA Investigator Kit and the combination of a simple lysis step followed by use of the Microcon® DNA Fast Flow device. Saliva was added to dimethylsulfone (DMS), nitrostyrene and PROSOLV® tablet mixture to determine the effect of powder type (based on solubility). Saliva was also added to 0, 50, 200 and 400 mg of DMS to determine the effect of an increase in DMS quantity. Trace DNA was deposited onto DMS using a worn glove approach. These samples were re-tested six months post-DNA deposition and profiled for further comparisons. Ten replicates were conducted for each condition with five replicates of saliva positive controls per method (n = 255 samples). A subset of samples was chemically analysed to determine if DMS was present in the final DNA eluant. The readily soluble DMS did not interfere with any of the extraction methods at lower amounts, however increasing the DMS to 400 mg reduced the relative DNA yields using the Microcon® and Investigator methods. The tablet mixture reduced the relative DNA yield of all three methods, however the nitrostyrene (which was relatively insoluble) only reduced the relative DNA yield of the DNA IQ™. The Investigator method performed the best with the trace samples, followed by the Microcon® method and then the DNA IQ™. DMS was detected in all extracts chemically analysed from the DNA IQ™ and Microcon®, whereas only one sample tested from the Investigator kit contained DMS in the extract and was in a relatively low amount compared to the other samples. Not one kit outperformed the others in all comparisons, however the Investigator kit was the most efficient overall at optimising the DNA yield whilst also removing the powders more effectively.

Detection of Latent DNA Using a DNA Binding Dye.

Latent DNA can be deposited every time a person holds or touches an item. This "touch DNA" can be crucial evidence if the item is of forensic significance. Until very recently, there were no means to visualize this DNA. The advent of using a dye that binds to DNA has opened up this possibility. The application of the dye is simple to perform, and a mobile microscope allows rapid visualization of the cellular material, even in ambient light. The dye can be applied in a solution of either 75% ethanol or water. As this is a solution-based dye, the application works best on non-absorbent surfaces.DNA within cellular material, such as dead skin cells, appears as green dots under 50X magnification; zooming to 220X magnification confirms that these are cells. The location and number of these cells can be photographed allowing a record of the presence of otherwise latent DNA.This chapter details the processes involved in the detection of latent DNA using Diamond™ Nucleic Acid Dye with both control samples (that act as very effective training samples) and the staining of evidential items. By developing skills in determining cell locations, a targeted approach to crime scene collection is now possible.

Persistence of cellular material after exposure to water.

Disposing of items of forensic relevance in bodies of water is one countermeasure offenders can use to avoid detection. The impact of immersion in water has been explored for blood, saliva, and semen; however, few studies have assessed touch DNA. Here we report on the effect of exposure to water on the persistence of touch DNA over prolonged periods of time. To evaluate the persistence of cells from touch DNA, after water exposure, three substrates and two water types were tested: plastic, metal, and ceramic, submerged into seawater or tap water. Diamond™ Nucleic Acid Dye was used to stain cells deposited by touch. Cell counts before and after water exposure were compared to investigate cell loss over time, ranging from 6 hours to 5 days. A logarithmic increase in the percent of cells lost was observed over time when the data for substrate and water type conditions were combined. Substrate type influenced the persistence of cells, with the metal substrate retaining cells longer than plastic or ceramic. The influence of water type appeared dependent on the substrate, with varied cell persistence on metal whereas plastic and ceramic recorded similar cell loss over time between water types. The ability to visualize cells after exposure to water could assist in triaging evidence within operational forensic laboratories and allow for targeted sampling. This proof-of-concept study demonstrated that greater than 50% of cells can persist on various items submerged in aqueous environments for at least 5 days, highlighting the possibility for downstream DNA testing.

Diagnostic models to predict nuclear DNA and mitochondrial DNA recovery from incinerated teeth.

Teeth are frequently used for human identification from burnt remains, as the structure of a tooth is resilient against heat exposure. The intricate composition of hydroxyapatite (HA) mineral and collagen in teeth favours DNA preservation compared to soft tissues. Regardless of the durability, the integrity of the DNA structure in teeth can still be disrupted when exposed to heat. Poor DNA quality can negatively affect the success of DNA analysis towards human identification. The process of isolating DNA from biological samples is arduous and costly. Thus, an informative pre-screening method that could aid in selecting samples that can potentially yield amplifiable DNA would be of excellent value. A multiple linear regression model to predict the DNA content in incinerated pig teeth was developed based on the colourimetry, HA crystallite size and quantified nuclear and mitochondrial DNA. The chromaticity a* was found to be a significant predictor of the regression model. This study outlines a method to predict the viability of extracting nuclear and mitochondrial DNA from pig teeth that were exposed to a wide range of temperatures (27 to 1000 °C) with high accuracy (99.5-99.7%).

Persistence of touch DNA on commonly encountered substrates in different storage conditions.

The persistence of touch DNA deposited after realistic handling of items typically encountered in forensic investigations has been the subject of few studies. Understanding the long-term persistence of touch DNA on different substrates in varying conditions can be central to the effective triage of samples for further processing. As the time between an alleged incident and collection of evidence may vary from a few days to years after an alleged event, this study assessed three different common substrates for the persistence of touch DNA over a time span up to 9 months. These substrates included fabric, steel, and rubber, each of which were handled in a way to imitate what may happen during a criminal act. The three substrates were exposed to two different environments for up to 9 months: inside a dark cupboard with no traffic to act as a control and an outside semi-exposed environment. Ten replicates from each of the 3 substrates were tested at 5 time points to create 300 samples. All samples were processed using a standard operating workflow to provide genotype data after exposure to different environments. It was found that the fabric samples produced informative STR profiles (defined here as 12 or more alleles) up to the 9 month timepoint for either environment. The rubber and steel substrates for the inside condition produced informative STR profiles up to the 9 month timepoint, but only generated informative STR profiles for the outside condition up to 3 and 6 months, respectively. These data add to our understanding of the external factors that affect DNA persistence.

Improvements, factors, and influences on DNA recovery from firearms.

Touch DNA recovery from firearms can be central to many criminal investigations, yet the generation of DNA profiles from these items remains poor. Currently in Australia, published casework data highlights extremely poor DNA success from samples recovered from firearms. Only between 5% and 25% of samples result in useful DNA data and therefore increasing the success of DNA recovered from firearms is highly important but has not yet been explored in-depth. This study focused on increasing the recovery of DNA from ten firearm components that were held for 15 s. Multiple recovery methods were used, and the resulting genetic data compared. DNA evidence may be deliberately removed from firearms after discharge to hamper forensic investigations, therefore this study examined the effect of wiping down the components or handling them with gloves. A standard double swab and rinse swab recovery method resulted in an average of 73% cellular recovery. A cumulative swab process had the highest average recovery at 86%, although it was found that increasing the DNA yield led to an increase in mixture complexity. Wiping over the components was observed to remove on average 69% of cellular material, compared with 33% when handed with gloves. However, the size and texture of the components affected the efficiency of cellular material removal. The results from this study allow for prioritisation of areas to sample on firearms, as well as suggesting techniques that can be applied for the optimum process of cellular recovery and subsequent generation of STR DNA data.

Recording and analysing DNA from osteocytes in resin-embedded bone samples.

We report on a process to record the presence and the location of osteocyte nuclei using two nucleic staining dyes, Diamond™ Nucleic Acids Dye (DD) and DAPI (4',6-diamidino-2-phenylindole). Knowledge of the presence and number of osteocytes is key to any success in subsequent DNA profiling. Osteocytes are most numerous cells and thus the main source of DNA in bone samples, which can be preserved for histological analyses. Archived samples are either fixed in formalin or preserved in ethanol prior to embedding in resin. These resin-embedded samples are potentially used as ante mortem reference samples. Cases of a missing person investigation are one example where this type of preserved reference material may be of value. When resin is required for sample preservation it represents a problem for subsequent DNA profiling, if needed as a reference sample in human identification. It is essential therefore to remove the resin prior to DNA analyses as resin is a known inhibitor of DNA profiling. Current methods of resin removal are lengthy and require toxic chemicals. This report describes a simplified process to remove resin and visualise the location of nucleated osteocytes. Eight sections of bone samples at 5-µm thickness were stained with DD and DAPI. A further three samples were processed using a formalin-fixed method and three additional samples treated following an ethanol-preserved method (11 samples for both the formalin-fixed and 11 for the ethanol-preserved with eight in common). The location and number of nuclei could be recorded clearly due to the fluorescence created by the dye binding to DNA. The number of stained nuclei correlated with the mass of DNA isolated from the sections (r = 0.873, p = 1.21 × 10-10). A significant difference between the degradation indices of two groups (p = 8.505 × 10-5) showed that ethanol preservation is a preferred method to yield DNA of the quality needed for subsequent short tandem repeats (STR) profiling. Ten of the 11 samples isolated using the ethanol-preserved process recorded a complete STR profile (30/30 alleles), whereas eight of the formalin-fixed samples generated full profiles, and only one of the 11 samples amplified less than 23 alleles. Both the ethanol-preserved and formalin-fixed methods are an improvement on current methods by removing the need for strong solutes in resin removal, and the method leads to STR profiles from resin-embedded bone samples within 24 h.

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.