The effect of the anti-coagulant EDTA on the deposition and adhesion of whole blood deposits on non-porous substrates.

An investigation into whether the addition of a commonly used anti-coagulant agent like ethylenediaminetetraacetic acid (EDTA) has an impact on the adhesion potential of blood to non-porous substrates was conducted. Two non-porous substrates (aluminum and polypropylene) exhibiting six different surface roughness categories (R1-R6) were used as test substrates upon which either whole blood or blood treated with EDTA was deposited. Samples were exposed to different drying periods (24 hours, 48 hours, and 1 week) before undergoing a tapping agitation experiment in order to evaluate the adhesion to the surface. Clear differences in adhesion potential were observed between whole blood and blood treated with EDTA. Blood treated with EDTA displayed a stronger adhesion strength to aluminum after a drying time of 24 h pre-agitation, while whole blood presented with a stronger adhesion strength at the drying time of 48 h and 1 week. Both EDTA-treated and EDTA-untreated blood was shown to dislodge less easily on polypropylene with the only difference observed on smooth surfaces (0.51-1.50 µm surface roughness). Thus, when conducting transfer studies using smooth hydrophobic substrates like polypropylene or considering the likelihood of transfer given specific case scenarios, differences in adhesion strength of blood due to hydrophobic substrate characteristics and a decreased surface area need to be considered. Overall, whole blood displayed a better adhesion strength to aluminum, emphasizing that indirect transfer probability experiments using EDTA blood on substrates like aluminum should take an increased dislodgment tendency into account in their transfer estimations.

The impact of substrate characteristics on the collection and persistence of biological materials, and their implications for forensic casework.

This study assessed the level of nucleic acid persistence on the substrate pre-, and post-swabbing, in order to assess whether biological materials (touch, saliva, semen, and blood) are collected differently depending on the substrate characteristics. A total of 48 samples per deposit and substrate variety (n = 384) were assessed by tracking the persistence of nucleic acid using Diamond™ Nucleic Acid Dye (DD) staining and Polilight photography. The number of DD nucleic acid fluorescent complexes formed post-staining were counted (fluorescent count) and in conjunction with the fluorescence signal intensity (DD nucleic acid complex accumulation) used to estimate the level of nucleic acid persistence on substrates. Touch deposits have shown to be the most persistent deposit with strong adhesion capabilities on both substrate verities. Saliva displayed a higher persistence than semen and/or blood. Semen displayed a high collection efficiency as well as a high fluorescence signal intensity. Blood displayed a low persistence on both substrates with a superior collection efficiency that may also indicate a higher probability to become dislodged from surfaces given a particular activity. Our research has shown that the persistence and recovery of biological deposits is not only measurable but more importantly, may have the potential to be estimated, as such, may build an understanding that can provide valuable guidance for collection efficiency evaluations, and the assessing of the probability of particular profiles, given alternate propositions of means of transfer occurring.

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.

How the physicochemical substrate properties can influence the deposition of blood and seminal deposits.

A comprehensive investigation into the impact of the physical and chemical variables of a substrate on the deposition was conducted to aid in the estimation of the subsequent transfer probabilities of blood and semen. The study focussed on surface roughness, topography, surface free energy (SFE), wettability, and the capacity for protein adsorption. Conjointly, evaluations of the physical and chemical characteristics of blood and seminal deposits were conducted, to assess the fluid dynamics of these non-Newtonian fluids and their adhesion potential to aluminium and polypropylene. A linear range of surface roughness parameters (0.5 - 3.5 µm) were assessed for their impact on the deposit deposition spread and adhesion height, to gather insight into the change in fluid dynamics of non-Newtonian fluids. Blood has shown to produce a uniform adhesion coverage on aluminium across all roughness categories while blood deposited on polypropylene exhibited a strong hydrophobic response from a surface roughness of 2.0 µm and beyond. Interestingly, the deposition height of blood resulted in near identical values, whether deposited onto the hydrophobic polypropylene or the hydrophilic aluminium substrate, illustrating the potential influence of a heightened fibrinogen adsorption effect. Semen deposited on aluminium resulted in concentrated localised deposition regions after reaching a surface roughness of 2.0 µm, highlighting the development of crystal formations afforded by the sodium ion concentration in the seminal fluid. The semen deposited on polypropylene conformed to the substrate contours producing a deposition film that was smoother than the substrate itself, underlining the effects of thixotropic fluid dynamics. Variables identified here establish the complexity observed for non-Newtonian fluids, and the effect protein adsorption may have on the deposition behaviour of blood and seminal deposits and inform questions in relation to the adhesion strength of said deposits and their ability to dislodge (becoming detached upon the application of an external force) from the substrate surface during a potential transfer event.

Presence of Human DNA on Household Dogs and Its Bi-Directional Transfer.

Awareness of the factors surrounding the transfer of DNA from a person, item, or surface to another person, item, or surface is highly relevant during investigations of alleged criminal activity. Animals in domestic environments could be a victim, offender, or innocent party associated with a crime. There is, however, very limited knowledge of human DNA transfer, persistence, prevalence, and recovery (DNA TPPR) associated with domestic animals. This pilot study aimed to improve our understanding of DNA TPPR associated with domestic dogs by collecting and analysing samples from various external areas of dogs of various breeds, interactions with humans, and living arrangements, and conducting a series of tests to investigate the possibility of dogs being vectors for the indirect transfer of human DNA. Reference DNA profiles from the dog owners and others living in the same residence were acquired to assist interpretation of the findings. The findings show that human DNA is prevalent on dogs, and in the majority of samples, two-person mixtures are present. Dogs were also found to be vectors for the transfer of human DNA, with DNA transferred from the dog to a gloved hand during patting and a sheet while walking.

Effect of swabbing technique and duration on forensic DNA recovery.

Various factors have been shown to affect performance of the conventional wet-dry double and single wet swabbing techniques to recover DNA, such as pressure and angle of application, volume and type of wetting agent, and swab type. However, casework laboratories in some jurisdictions have recently adopted different swabbing techniques that include wet-moist double swabbing and moist-dry single swabbing. Factors affecting the effectiveness of these recent techniques in maximising DNA recovery therefore need to be investigated. Here, the performance of traditional and recent swabbing techniques was compared and the impact of swabbing duration on DNA recovery was investigated. Ten µl aliquots of a known concentration of DNA extracted from human blood were deposited on pre-cleaned DNA-free cotton swatches (porous) and porcelain tiles (non-porous). Five swabbing techniques were used, of which three were double swabbing techniques: wet-moist, wet-wet and wet-dry, and two were single swabbing techniques: wet and moist-dry. For a 'wet' or 'moist' swab, 100 or 50 µL water was added, respectively. For a moist-dry swab, water was applied to one side of the swab, leaving the other side drier. Each swabbing technique was applied for two durations, 15 and 30 s per swab, with 5 reps of each combination (n = 100 plus controls). All samples were extracted and quantified, and a sub-set was profiled. The results showed that the wet-moist double swabbing technique with a swabbing duration of 30 s maximised DNA recovery from cotton. From tile, a single wet or moist-dry swab maximised DNA recovery, but increasing swabbing duration from 15 to 30 s had no impact. These data can be used to inform standardisation of DNA collection protocols across casework laboratories.

DNA transfer to placed, stored, and handled drug packaging and knives in houses.

Forensic laboratories often sample weapons and clip-seal plastic bags (CSPB) used to package illicit material for the purpose of identifying the handler(s). However, there may be other explanations as to how a person's DNA was transferred to such items. This may include an individual storing the item among their personal belongings for somebody else or the item being stored among their belongings without their knowledge. Here we investigate the direct transfer of DNA to knives and CSPB during handling and explore two feasible alternative explanations related to the indirect transfer of DNA to these items in residential environments. The handling of DNA-free items was performed by 10 individuals who were instructed, on separate occasions, to cut a foam board in half and fill a CSPB with a drug substitute. To explore indirect transfer, sets of these items were (a) placed on kitchen benches and coffee/dining tables for ∼1 min, or (b) stored for two days in kitchen and bedroom drawers within the homes of 10 individuals. After each of the three scenarios, samples were collected from the knife handle and blade, the body and seal of the CSPB, and the surface the items were placed on, the latter as a measure to gain insight into the presence of prevalent and/or background DNA. DNA transfer was observed under all three scenarios, though more frequently when items were handled or stored for 2 days, compared to when placed on a surface for ∼1 min. Under the latter scenario, DNA, if present, was below the level of detection in many samples and produced no profile, suggesting that detectable DNA transfer occurs to a lesser degree from static brief contacts. The study results and associated probabilities will assist forensic examiners with their interpretation of case circumstances regarding the transfer and recovery of DNA from these items.

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.

How changes to the substrate's physical characteristics can influence the deposition of touch and salivary deposits.

An in-depth study into the physical substrate characteristics such as substrate surface roughness, topography, and physicochemical characteristics like wettability and surface free energy (SFE) was conducted to investigate the impact on the deposition and adherence of touch and salivary deposits on aluminium and polypropylene. A robust protocol was established to generate a set of substrates with a controlled linear surface roughness range (0.5-3.5 µm) in order to identify the impact of surface roughness on DNA transfer, persistence, prevalence, and recovery (DNA-TPPR). The polypropylene substrate was shown to produce fibres when artificially roughened, becoming more prominent at a higher surface roughness range, and has shown to have a direct impact on the distribution of salivary and touch deposits. At the low to moderate surface roughness range 0.5-2.0 µm, salivary and touch deposits have generally shown to follow the topographical features of the substrate they were deposited on, before a plateau of the surface roughness measure on the deposit was observed, indicating that a saturation point was reached and the grooves in the substrate were beginning to fill. Touch deposits have shown to maintain a consistent deposition height pre-surface roughness threshold, irrespective of substrate surface roughness while the deposition height of salivary deposits was heavily influenced by substrate surface roughness and topography. The substrate SFE, wettability, hydrophobicity, and the surface tension of the deposit was shown to drive the adhesion properties of the saliva and touch deposits on the respective substrates, and it was observed that this may be of importance for the improvement of the current DNA-TPPR understanding, DNA sampling protocols, and DNA transfer considerations within casework.

DNA transfer between worn clothing and flooring surfaces with known histories of use.

DNA samples recovered from items of clothing are often attributed to the wearer and one or more individuals who may have contacted the item during an alleged criminal activity. Another scenario often proposed by defence counsel is that DNA was transferred from a previously contacted item/surface unrelated to the activity of interest onto the item of clothing. Under such scenarios, DNA may also be transferred from the clothing to the item/surface with which it comes into contact. One such surface is flooring, upon which clothing may be placed while not being worn or may be contacted during wearing, such as falling or being forced to the ground. This study investigates the transfer of DNA to and from clothing and flooring when different contacts are applied between the two surfaces in an environment representative of what investigators would encounter in routine casework, a residential environment. Participants were provided with two sets of new and unused upper and lower garments to wash then wear for ~8 h inside their own home before storing them in paper evidence bags. The two sets of clothing were taken to a home occupied by unrelated individuals, where one set was placed on the floor ('passive') by the researcher while the other was worn by the participant who laid with their back on the floor, rolled to one side and back, then stood up ('active'). Within the houses sampled, the main bedroom was targeted as flooring types and histories of use were more consistent across houses and less variation in DNA profile composition was previously observed for samples collected in the same room. Samples were collected from predetermined areas of the clothing and flooring where contact did and did not occur. Reference profiles were obtained from wearers and individuals they lived with, as well as occupants of the home. DNA transfer was observed from clothing to flooring and from flooring to clothing in both 'active' and 'passive' situations, though greater where a situation involved the application of pressure and friction ('active'), and only where contact between clothing and flooring occurred. Results from this study inform on the composition of DNA profiles one is likely to obtain from an item of clothing or a flooring surface following a similar contact event between the two substrates and will aid investigators when interpreting DNA evidence recovered in a domestic environment and the activities leading to its transfer and subsequent recovery.

"Technical Note:" Optimisation of Diamond™ Nucleic Acid Dye preparation, application, and visualisation, for latent DNA detection.

A targeted sampling approach of latent DNA, deposited when a person makes contact with a surface, can prove challenging during crime scene or evidence processing, with the sampling of latent DNA often relying on the expert judgement from crime scene officers and forensic examiners. As such, the ability to use the quick and robust screening tool Diamond™ Nucleic Acid Dye (DD) was explored, with a focus on the visualisation of latent DNA on non-porous substrates, namely polypropylene, acrylic, aluminium, PVC composite material, glass, and crystalline silicon. The application of DD was performed according to methods reported in literature, where 10 µL of the dye solution (20-fold dilution of DD in 75% EtOH) was applied onto a variety of non-porous substrates via a micropipette and then subsequently visualised using a portable fluorescence microscope. It was discovered that there was scope for improvement in the reported methods due to the observation of crystal formations on all test substrates upon drying of the DD, resulting in the impaired visualisation of latent DNA and fingerprint detail. Thus, changes to the EtOH water ratio of the dye solution, and changes to the mode of dye application from a micropipette to a spray application, were explored to improve the drying time of the dye and mitigate the formation of crystals. While changes to the EtOH water ratio did not improve the overall drying time, the mode of dye application enhanced visualisation, with a spray application eliminating the formation of crystals no matter the EtOH water ratio. Visualisation with a portable Dino-Lite and Zeiss Widefield fluorescence microscope were also explored, with the Zeiss Widefield fluorescence microscope proving to be useful in whole print imaging and a more efficient imaging tool in a laboratory setting.

DNA Transfer in Forensic Science: Recent Progress towards Meeting Challenges.

Understanding the factors that may impact the transfer, persistence, prevalence and recovery of DNA (DNA-TPPR), and the availability of data to assign probabilities to DNA quantities and profile types being obtained given particular scenarios and circumstances, is paramount when performing, and giving guidance on, evaluations of DNA findings given activity level propositions (activity level evaluations). In late 2018 and early 2019, three major reviews were published on aspects of DNA-TPPR, with each advocating the need for further research and other actions to support the conduct of DNA-related activity level evaluations. Here, we look at how challenges are being met, primarily by providing a synopsis of DNA-TPPR-related articles published since the conduct of these reviews and briefly exploring some of the actions taken by industry stakeholders towards addressing identified gaps. Much has been carried out in recent years, and efforts continue, to meet the challenges to continually improve the capacity of forensic experts to provide the guidance sought by the judiciary with respect to the transfer of DNA.

Identifying background microbiomes in an evidence recovery laboratory: A preliminary study.

16S rRNA profiling of bacterial communities may have forensic utility in the identification or association of individuals involved with criminal activities. Microbial profiling of evidence may, in the future, be performed within environments currently utilised for human DNA recovery, such as a forensic biology laboratory. It would be important to establish the background microbiome of such an environment to determine the potential presence of human or environmental microbial signatures to assist forensic scientists in the appropriate interpretation of target microbial communities. This study sampled various surfaces of an Evidence Recovery Laboratory (ERL) on three occasions including (a) before a monthly deep-clean, (b) immediately following the deep-clean, and (c) immediately after the laboratory's use by a single participant for the purposes of routine item examinations. Microbial profiles were also generated for the involved participant and researcher for comparison purposes. Additionally, human nuclear DNA was profiled for each of the samples collected, using standard forensic profiling techniques, to provide a prospective link to the presence or absence of a background microbial signature within the ERL after its use. Taxonomic distributions across ERL samples revealed no consistent signature of any of the items sampled over time, however, major phyla noted within all ERL samples across the three timepoints were consistent with those found in human skin microbiomes. PCoA plots based on the Unweighted Unifrac metric revealed some clustering between participant microbial reference samples and surfaces of the ERL after use, suggesting that despite a lack of direct contact, and adherence to standard operating procedures (SOPs) suitable for human DNA recovery, microbiomes may be deposited into a forensic setting over time. The reference samples collected from the involved participant and researcher generated full STR profiles. Human DNA was observed to varying degrees in samples taken from the ERL across each of the sampling timepoints. There was no correlation observed between samples that contained or did not contain detectable quantities of human nuclear DNA and microbial profile outputs.

Investigation into the presence and transfer of microbiomes within a forensic laboratory setting.

Microbial profiling within forensic science is an emerging field that may have applications in the identification of individuals using microbial signatures. It is important to determine if microbial transfer may occur within a forensic laboratory setting using current standard operating procedures (SOPs) for nuclear DNA recovery, to assess the suitability of such procedures for microbial profiling and establish the potential limitations of microbial profiling for forensic purposes. This preliminary study investigated the presence and potential transfer of human-associated microbiomes within a forensic laboratory. Swabs of laboratory surfaces, external surfaces of personal protective equipment (PPE) and equipment were taken before and after mock examinations of cotton swatches, which harboured microbiota transferred from direct hand-contact. Microbial profiles obtained from these samples were compared to reference profiles obtained from the participants, cotton swatches and the researcher to detect microbial transfer from the individuals and determine potential source contributions. The results revealed an apparent transfer of microbiota to the examined swatches, laboratory equipment and surfaces from the participants and/or researcher following the mock examinations, highlighting potential contamination issues regarding microbial profiling when using current laboratory SOPs for nuclear DNA recovery, and cleaning.

Investigation into the prevalence of background DNA on flooring within houses and its transfer to a contacting surface.

When sampling an item or surface for DNA, the collection of 'background' DNA (bDNA) from previous use poses an issue as it may impact the detectability of 'target' DNA and the interpretation of the DNA results given alleged activities. This study investigates the prevalence and transferability of bDNA on flooring surfaces within occupied houses under conditions similar to those that are encountered in casework. To assess bDNA presence and transferability, and the impact of how and who contacts the surface, areas used frequently and infrequently were targeted in the kitchen, living room, bedroom and bathroom of five houses, and two samples taken from each area; one directly from the floor and another from a cotton surface after contacting the floor. DNA was detected in 97 % (of 39) of samples collected directly from flooring, with 92 % providing interpretable profiles. DNA was detected in 85 % (of 39) samples collected from cotton swatches after contacting the floors, with 79 % providing interpretable profiles. The overall quantity, number of contributors, and likelihood of observing a major contributor was greater for samples obtained directly from the floor compared to the cotton. In 80 % of samples recovered from cotton, the quantity of DNA recovered was less than 20 % of that which was recovered directly from the floor. Overall, no trend was observed between the level of reported activity by occupants within areas of the same room and the quantity of DNA recovered directly from the flooring, the quantity of DNA transferred to and recovered from the cotton, or the number of contributors in resulting DNA profiles. In contrast, greater quantities of DNA were generally obtained from houses with a greater number of occupants. Profile composition was similar for samples collected from different areas of the same room, irrespective of the level of activity and from where the sample was obtained (i.e. directly from the floor or contacting surface). Occupants were often not detected in DNA profiles collected from rooms they were known to use and could be observed in profiles collected from rooms they reportedly did not use. The findings of this preliminary investigation provide an understanding of the complexities of transfer, persistence, prevalence and recovery of DNA traces in houses occupied by multiple people and highlights the need to consider how and who uses a space, in the investigation of criminal activities where DNA traces are recovered from, or have been in contact with, flooring.

Challenges in Human Skin Microbial Profiling for Forensic Science: A Review.

The human microbiome is comprised of the microbes that live on and within an individual, as well as immediately surrounding them. Microbial profiling may have forensic utility in the identification or association of individuals with criminal activities, using microbial signatures derived from a personal microbiome. This review highlights some important aspects of recent studies, many of which have revealed issues involving the effect of contamination of microbial samples from both technical and environmental sources and their impacts on microbiome research and the potential forensic applications of microbial profiling. It is imperative that these challenges be discussed and evaluated within a forensic context to better understand the future directions and potential applications of microbial profiling for human identification. It is necessary that the limitations identified be resolved prior to the adoption of microbial profiling, or, at a minimum, acknowledged by those applying this new approach.

DNA transfer to worn upper garments during different activities and contacts: An inter-laboratory study.

Cellular material derived from contact traces can be transferred via many direct and indirect routes, with the manner of contact and the time of transfer (in relation to the alleged crime-event) having an impact on whether DNA is recovered from the surface and a reportable profile generated. In an effort to acquire information on the transfer and recovery of DNA traces from clothing items worn during scenarios commonly encountered in casework, upper garments were worn during a normal working day before individuals were paired to embrace one another ('contact'), go on an outing together ('close proximity'), or individually asked to spend a day in another person's environment ('physical absence'). Each prescribed activity was repeated by sixteen individuals across four countries, and was the last activity performed before the garment was removed. Samples were collected from several areas of the upper garments and processed from DNA extraction through to profiling within the laboratory of the country in which the individual resided. Activities relating to the garment prior to and during wearing, including the prescribed activity, were recorded by the participant and considered during the interpretation of results. In addition to obtaining reference profiles from the wearer and their activity partner, DNA profiles from the wearers' close associates identified in the questionnaire were obtained to assess the impact of background DNA transferred prior to the prescribed activity. The wearer was typically, but not always, observed as the major contributor to the profiles obtained. DNA from the activity partner was observed on several areas of the garment following the embrace and after temporarily occupying another person's space. Particular areas of the garment were more prone to acquiring the hugging partner or office owner's DNA than others, and whether they were observed as the major or minor component was activity dependent. For each of the pairs, no DNA from the activity partner was acquired by the garments during the outing, even though both participants were in close proximity. This study provides empirical data on the transfer, persistence, prevalence and recovery of DNA from clothing items, and enables a better understanding of the mechanisms which lead to the transfer and detectability of DNA traces in different scenarios.

Investigation of direct and indirect transfer of microbiomes between individuals.

The human microbiome encompasses the fungi, bacteria and viruses that live on, within, and immediately surrounding the body. Microbiomes have potential utility in forensic science as an evidentiary tool to link or exclude persons of interest associated with criminal activities. Research has shown the microbiome is individualised, and that personal microbial signatures can be recovered from surfaces such as phones, shoes and fabrics. Before the human microbiome may be used as an investigative tool, further research is required to investigate the utility and potential limitations surrounding microbial profiling. This includes the detectability of microbial transfer between individuals or items, the associated risks (such as contamination events) and the applicability of microbial profiling for forensic purposes. This research aimed to identify whether an individual's distinguishable microbiome could be transferred to another individual and onto substrates, and vice versa. Paper, cotton, and glass surfaces were chosen to represent a range of substrate matrices. The study involved six participants placed into three pairs; participants took part in two modes of transfer. Transfer Mode 1 involved the pair shaking hands, followed by rubbing a substrate in their right hand. Transfer Mode 2 involved individuals rubbing a substrate in their left hand, swapping substrates with their partner and then rubbing the swapped substrate in their left hand. 16S rRNA sequencing was performed on the extracted microbial DNA from participant and substrate samples. Quantitative Insights into Microbial Ecology 2 (QIIME 2) was used for sequence quality control and beta (between-sample) diversity analyses and taxonomic assignment. Principal Coordinate Analysis (PCoA) based on Jaccard distances was visualised through Emperor software to determine the phylogenetic similarity of bacterial communities between participants and among participant pairs. Statistical testing through PERMANOVA revealed significant differences in the Jaccard distances between each participant pair (P < 0.001), highlighting not only the potential distinguishability of skin microbiomes among individuals, but also the clustering effect observed between participant pairs due to the potential transfer of hand-associated microbiomes between individuals. The study demonstrated that transfer of the human skin microbiome had occurred between all participant pairs, regardless of substrate type or mode of transfer.

DNA detection of a temporary and original user of an office space.

There is a need to improve our awareness of the transfer, persistence, prevalence and recovery of DNA (DNA-TPPR) from items/surfaces, and within different spaces and circumstances, to assist sample targeting during collection and activity level assessments. Here we investigate DNA-TPPR within office spaces. Specifically, to what extent DNA, left by a temporary user of an office space that has been occupied by a regular user for an extended period, is detectable when the duration of their temporary occupancy and their general activities are known. Also, how readily the DNA of the regular user is still detectable after a known period of occupancy by another person, and to what extent DNA of others is present. Samples were collected from 18 core items/surfaces within eight single use office spaces that had been used temporarily by another occupant for 2.5-7 h. Four of these offices were within one forensic laboratory and four within another. Each lab collected and processed the samples to generate DNA profiles using their own set of methodologies. The owner/regular user of an office space was found to be the major/majority contributor to profiles from most items within the space, even after temporary use by another person. The detectability of the temporary occupier of an office space varied among offices and items. The temporary occupier was not observed on all items touched. In most instances, when detected, the temporary occupier was known to have touched the surface at some stage. Therefore, where one is seeking to collect samples that may detect a temporary user of a space, it is advisable to target several potentially touched sites. A difference in methodologies applied from collection through to profiling appears to impact DNA yields and profile types. Ascertaining the impact of using different methodologies on the profiles generated from collected samples, requires further research. More research is also needed to generate data to help determine frequency estimates for different types of profiles given different user histories of an item or space.