Assessing eDNA capture method from aquatic environment to optimise recovery of human mt-eDNA.

Previous studies have shown that environmental DNA (eDNA) from human sources can be recovered from natural bodies of water, and the generation of DNA profiles from such environmental samples may assist in forensic investigations. However, fundamental knowledge gaps exist around the factors influencing the probability of detecting human eDNA and the design of optimal sampling protocols. One of these is understanding the particle sizes eDNA signals are most strongly associated with and the most appropriate filter size needed for efficiently capturing eDNA particles. This study assessed the amount of mitochondrial eDNA associated with different particle sizes from human blood and skin cells recovered from freshwater samples. Samples (300 mL) were taken from experimental 10 L tanks of freshwater spiked with 50 µL of human blood or skin cells deposited by vigorously rubbing hands together for two minutes in freshwater. Subsamples were collected by passing 250 mL of experimental water sample through six different filter pore sizes (from 0.1 to 8 µm). This process was repeated at four time intervals after spiking over 72 hours to assess if the particle size of the amount of eDNA recovered changes as the eDNA degrades. Using a human-specific quantitative polymerase chain reaction (qPCR) assay targeting the HV1 mitochondrial gene region, the total amount of mitochondrial eDNA associated with different particle size fractions was determined. In the case of human blood, at 0 h, the 0.45 µm filter pore size captured the greatest amount of mitochondrial eDNA, capturing 42 % of the eDNA detected. The pattern then changed after 48 h, with the 5 µm filter pore size capturing the greatest amount of eDNA (67 %), and 81 % of eDNA at 72 h. Notably, a ten-fold dilution proved to be a valuable strategy for enhancing eDNA recovery from the 8 µm filter at all time points, primarily due to the PCR inhibition observed in hemoglobin. For human skin cells, the greatest amounts of eDNA were recovered from the 8 µm filter pore size and were consistent through time (capturing 37 %, 56 %, and 88 % of eDNA at 0 hours, 48 hours, and 72 hours respectively). There is a clear variation in the amount of eDNA recovered between different cell types, and in some forensic scenarios, there is likely to be a mix of cell types present. These results suggest it would be best to use a 5 µm filter pore size to capture human blood and an 8 µm filter pore size to capture human skin cells to maximize DNA recovery from freshwater samples. Depending on the cell type contributing to the eDNA, a combination of different filter pore sizes may be employed to optimize the recovery of human DNA from water samples. This study provides the groundwork for optimizing a strategy for the efficient recovery of human eDNA from aquatic environments, paving the way for its broader application in forensic and environmental sciences.

Recovery of DNA from acetaminophen exploring physical state and sampling methods.

Research into the recovery of DNA from illicit drug samples has shown it is possible to get forensically useful profiles from such substrates. However, it is not yet known if the different physical states that drugs can be found in influences the quantity and quality of DNA that can be recovered or what is the best sampling method to adopt for powdered samples. This research used acetaminophen in four different states - large crystalline, powder, in solution, or residue - to determine the efficacy of current DNA technology in recovery and analysis of the resulting sample. Five replicates of each were prepared. Human blood was deposited on or mixed with the drug and left for 1 hour. The surface of the drug was sampled by wet/dry swabbing (where appropriate), or the entire sample was deposited in a tube, and the DNA then extracted using DNA-IQ™. The amount of DNA recovered (ng), degradation index, number of PCR cycles (Ct) required for the IPC to reach threshold, number of alleles in the DNA profile and average peak height (APH) were assessed. All samples, irrespective of the physical state they were collected from, returned full DNA profiles that corresponded to the DNA profile of the blood donor, with no degradation or inhibition detected. It was also found the wet/dry swabbing method returned higher levels of DNA than inclusion of the entire sample into the tube for powdered acetaminophen and the appropriate method to use will be dependent on casework circumstances. The findings of this research further develops our understanding of the recovery of DNA from drugs, and supports the need for further investigation to understand under what conditions DNA can be recovered from illicit substances.

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.

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.

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.

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.

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.

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.

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.

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.

Preliminary investigation into isolation and extraction of DNA recovered from drug residues.

It is a commonly held belief that drug residues may affect the integrity of DNA and/or interfere with DNA analysis, and therefore DNA on drug paraphernalia and the associated drugs may be overlooked as a source of evidence. This study investigated whether DNA could be isolated from a drug residue-bearing surface to ascertain whether a forensically useful DNA profile could be obtained. Human blood and pre-extracted "naked" DNA were deposited on samples of acetaminophen, codeine, morphine, oxycodone, ketamine, and synthetic cannabinoids and left for an hour before DNA extraction using DNA-IQ™. To investigate DNA integrity, the absolute amount of DNA recovered, degradation index, and number of PCR cycles required for the IPC to reach threshold (Ct), number of reportable alleles and average peak height (APH) in the DNA profile, were examined. The samples were also qualitatively analysed using LC:MS to determine if any residual drugs were present in the samples post-DNA extraction. Overall, the drugs had no to minimal degradation or inhibitory effects on the DNA with sufficient DNA recovered to generate a partial or full DNA profile in 80% of naked DNA samples and 100 % of blood samples. The amount of DNA collected was sufficient for further analysis in 86% of naked DNA samples, and 100% of blood samples, with all median APH values being over the 175 RFU standard. Chemical analysis showed that traces of the drug were still present in the samples after DNA extraction was performed. Therefore, this study demonstrates forensically useful DNA can be recovered from surfaces bearing drug residues, even when sampling directly from the samples of drugs.

Assessing the use of environmental DNA (eDNA) as a tool in the detection of human DNA in water.

Environmental DNA (eDNA) is a highly sensitive and cost-effective tool that is increasingly being applied to studies of biodiversity and species detection. This non-invasive method relies on the collection of environmental samples that contain genetic material being shed into surrounding environment by the target organism/s. While forensic science has a long history of using molecular tools for collecting DNA from the environment, the detection of human DNA from environmental water samples has been limited. This study investigated the detection and degradation rates of human eDNA in water samples under controlled laboratory conditions. Using a human-specific qPCR assay targeting the ND1 region of human mitochondrial DNA, eDNA degradation over time in water spiked with human blood was assessed. Recovery of nuclear DNA was investigated by determining if routine DNA short tandem repeat (STR) profiles of the blood source could be generated. Results demonstrated that human eDNA remains detectable for up to 11 days under laboratory conditions in environmental water and up to 35 days in distilled water. Partial STR profiles could be recovered from environmental water only up to 24 h, while, in distilled water, partial profiles continued to be recovered up to 840 h. These findings demonstrate that sampling human eDNA from aquatic samples can provide reliable human DNA detection within relatively short time windows, assisting law enforcement agencies by providing information about the potential time an individual may have been present in an area or assisting in the detection and location of a body or remains in aquatic environments.

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.

Touch DNA recovery from unfired and fired cartridges: Comparison of swabbing, tape lifting and soaking.

Over the recent few years, several DNA collection techniques and methodologies have been published for the recovery of DNA from fired cartridge cases. In this study, swabbing, the DNA collection technique currently used in our jurisdiction (NSW, Australia), was compared with tape lifting and soaking to assess DNA recovery rates, DNA quality and profile quality. Brass .22LR and 9mmP cartridges were used as they are the most commonly encountered in our jurisdiction. The cartridges (n = 107) were loaded into cleaned firearm magazines by three volunteers of unknown shedder status, to mimic routine casework sample types. Half of the handled cartridges were fired whilst the other half were kept unfired. STR genotypes were produced at both 29 and 30 PCR cycles to evaluate which improved handler allele detection. DNA recovery rates showed that swabbing recovered significantly less DNA than tape lifting and soaking. Whilst there were no significant differences between tape lifting and soaking, tape lifting, on average, yielded more DNA than soaking. The calibre of ammunition had no influence on DNA recovery and in line with expectations, firing was found to decrease DNA recovery for all three sampling techniques. Assessment of DNA quality showed no evidence of PCR inhibition in any of the samples for this study. However, degradation indices showed that most samples were slightly to moderately degraded. Fewer handler alleles were detected from both fired tape lifted and soaked cartridges than unfired cartridges. Whilst 30 amplification cycles allowed for the detection of slightly more handler alleles, no statistically significant differences were found between 29 and 30 PCR cycles. Nonetheless, 50% of the profiles from unfired soaked cartridges that were non-uploadable after 29 cycles were uploadable after 30 cycles. Furthermore, 83% of profiles from unfired cartridges that were tape lifted were uploadable onto our jurisdiction's database at both 29 and 30 PCR cycles. All magazine controls, despite cleaning, contained some level of background DNA. Furthermore, increasing the number of PCR cycles to 30 also increased the detection of non-handler alleles in DNA profiles. Our results suggest tape lifting yields more uploadable profiles from unfired and fired cartridge cases than swabbing but also more adventitious (non-handler) alleles. However additional research will be needed to evaluate the full potential of this method.

Transfer of DNA without contact from used clothing, pillowcases and towels by shaking agitation.

DNA is frequently retrieved from commonly used objects or surfaces with no apparent biological stains. This DNA may have come from one or more individuals who directly deposited their DNA, or indirectly transferred the DNA of others, when physically contacting the sampled object or surface. Furthermore, contactless indirect DNA transfer of this 'touch DNA' from fabric substrates was recently demonstrated to be possible in a controlled laboratory environment. The circumstances and extent to which this form of contactless DNA transfer occurs are largely unknown. This study investigated indirect DNA transfer without contact by applying a gentle shaking agitation to used clothing, pillowcases and towels, with known usage and history, of 10 volunteers above the collection zone of the secondary surface. DNA transfer frequently occurred and was possible from all three investigated items. It occurred at levels that often produced informative profiles where transferred profiles closely resembled the profiles generated from the primary item. The outcomes of this study contribute to expanding the understanding of indirect DNA transfer without contact. However, this field would benefit from investigating a wider range of agitations and/or item types with various histories of use to determine the level of transfer and its detectability under different conditions.

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.