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.

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.

The role of cats in human DNA transfer.

Domestic animals, such as cats and dogs, are present in the majority of Australian households. Recently, questions regarding the possibility that domestic animals can serve as silent witnesses, from whom evidence can be collected, or act as vectors of contamination and transfer, have started to be raised. Yet, little is known regarding the transfer and prevalence of human DNA to and from cats. This study investigated if cats are reservoirs and vectors for human DNA transfer. Twenty cats from 15 households were sampled from 4 different areas (head (fur), back (fur), left (skin) and right (fur)) to obtain information on the background DNA that may be found on an animal. Further, transfer of human DNA to and from an animal, after a short patting contact, was tested. Human DNA was found to be prevalent on all cats. Of the areas sampled, most DNA was collected from the top of the fur from the back followed by the head and right/fur. No or very low quantities of human DNA was recovered from the left (skin) area. Most of the human DNA originated from the owners, but DNA from others was also often present (47 % of samples). Further, the transfer tests demonstrated that human DNA transferred readily to (detected in 45 % of samples) and from (detected in 80 % of samples) cats during patting. These results show that animals can act as reservoirs of human DNA and vectors for human DNA transfer that may need to be considered during evaluative DNA reporting. Furthermore, if an interaction between an animal and a perpetrator is suspected, consideration should be given to collecting DNA evidence from suspected contact areas on an animal.

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

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

Transfer and persistence of intruder DNA within an office after reuse by owner.

The heightened sensitivity of DNA typing techniques, paired with the extensive use of trace DNA in forensic investigations, has resulted in an increased need to understand how and when DNA is deposited on surfaces of interest. This study focussed on the transfer, persistence, and prevalence of trace DNA in a single occupation of an office space by an intruder, when all contacts made during occupation and for the two hours prior and post occupation were known. The extent to which DNA could be recovered from contacted/not contacted surfaces was investigated. This study investigates the impacts of these movements and use of an office space when the duration of occupancy, surface contact histories and shedder status of participants are known. Contacts were documented and surfaces in the office space were targeted for sampling. Categories were set for target sampling that included different types of contact. Direct and indirect DNA transfer was detected in 55 % and 6 % of samples, respectively. Contactless DNA transfer was detected in 0.5 % of samples. The owner was observed as the sole/major/majority contributor in 77 % of the samples and as minor contributor in 10 % of samples. The intruder was observed as the sole/major/majority contributor in 14 % of samples and as the minor contributor in 16 %. An increased number of contacts increased the relative DNA contribution of the individual making the contact, however, not all observed direct contacts resulted in detectable DNA transfer. The outcome of this study will aid in better sample targeting strategies and contribute to the pool of data assisting in the development of activity level assessments.

Where did it go? A study of DNA transfer in a social setting.

The sensitivity of DNA analysis has progressed to the point that trace levels of DNA, originating from only a few cells, can generate informative profiles. This means that virtually any item or surface can be sampled with a reasonable chance of obtaining a DNA profile. As the presence of DNA does not suggest how it was deposited, questions are often raised as to how the DNA came to be at a particular location and the activity that led to its deposition. Therefore, understanding different modes of DNA deposition, reflective of realistic forensic casework situations, is critical for proper evaluation of DNA results in court. This study aimed to follow the movements of DNA to and from individuals and common household surfaces in a residential premises, while socially interacting. This took place over an hour and involved four participants, with known shedder status, designated as visitors (a male and a female) and hosts (a male and a female), who engaged in the activity of playing a board game while being served food. During the study, the participants were instructed to use the toilet on a single occasion to assess the transfer of DNA to new and unused underwear that was provided. All contacts made by the participants in the dining room and kitchen were video recorded to follow the movements of DNA. Samples were collected based on the history of contact, which included hands, fingernails and penile swabs. Direct contacts resulted in detectable transfer (LR > 1) in 87 % (87/100) of the non-intimate samples and clothing. For surfaces touched by multiple participants, DNA from the person who made the last contact was not always detectable. The duration and number of contacts did not significantly affect the detection of the person contacting the item. On the other hand, presence of background DNA and participant's shedder status appear to play an important role. Further, unknown contributors were detected in the majority of samples. Finally, indirect transfer was observed on a number of occasions including co-habiting partners of guests who were not present at the study location. The results of this study may assist with decision making for exhibit selection or targeting areas for sampling within the home environment. Our findings can also be used in conjunction with previous literature to develop activity-level evaluations in such situations where the source of the DNA is conceded, but the mode of deposition is disputed.

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.

Indirect DNA transfer without contact from dried biological materials on various surfaces.

DNA transfer is a well-recognised phenomenon impacting the probability of detecting the presence of a particular source of DNA and thus the likelihood of the evidence given considered events within forensic investigations. Comprehensive study is lacking on variables associated with indirect DNA transfer without physical contact. Additionally, the drying properties of forensically relevant biological materials are under researched despite the recognised potential for these properties to affect DNA transfer. This study investigated the drying properties and indirect DNA transfer of dried blood, saliva, semen, vaginal fluid and touch DNA without contact deposited on two different non-porous hard substrates (melamine and glass) and two different porous soft substrates (polyester and cotton) by tapping (all substrates) and stretching (only fabric substrates) agitations. Different apparent drying trends were observed between the volumes, substrates and biological materials tested with substrate type generally having a greater influence than biological material. The rate and percentage of indirect transfer appeared to be dependent on agitation, substrate type, biological material and its drying properties. The outcomes of this study may assist those evaluating the likelihood of the evidence given proposed events during activity level assessments.

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.

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.

DNA transfer: DNA acquired by gloves during casework examinations.

As loss and/or contamination of DNA evidence can have major negative implications, it is incumbent on forensic practitioners to minimise this risk. DNA transfer during examination of items, even when wearing gloves, is a potential risk. Here we observe a number of laboratory based biological evidence recovery personnel performing a range of examinations to help evaluate the risks. Assessments are made of; what is touched by each glove and in which sequence; the number, duration and types of contacts made by each glove with the item under examination, tools used and any other surfaces; when the gloves are replaced; as well as DNA profiles of samples taken of the worn gloves at time of replacement against those relating to the item under examination, the examiner and other staff members. Observations show that many different surface areas are touched by gloves during examinations. Differences were observed among examiners in what they touched and when they changed gloves. DNA was retrieved from the outer surfaces of the majority of gloves examined. In many instances the case associated person of interest was observed within the profile generated. So too were profiles of the examiner or other staff members, predominantly from the first and last gloves used during the examination, which were associated with removing the exhibit from its packaging and repackaging it. Several of the observed contacts made by the gloves were deemed high contamination risk events. This study is one of the first to investigate DNA transfer during actual casework examinations, increases our awareness of potential DNA contamination risks during the examination of exhibits, and highlights the need to consider improvements in procedures and/or training of those involved with forensic examinations.

DNA transfer in forensic science: A review.

Understanding the variables impacting DNA transfer, persistence, prevalence and recovery (DNA-TPPR) has become increasingly relevant in investigations of criminal activities to provide opinion on how the DNA of a person of interest became present within the sample collected. This review considers our current knowledge regarding DNA-TPPR to assist casework investigations of criminal activities. There is a growing amount of information available on DNA-TPPR to inform the relative probabilities of the evidence given alternative scenarios relating to the presence or absence of DNA from a specific person in a collected sample of interest. This information should be used where relevant. However, far more research is still required to better understand the variables impacting DNA-TPPR and to generate more accurate probability estimates of generating particular types of profiles in more casework relevant situations. This review explores means of achieving this. It also notes the need for all those interacting with an item of interest to have an awareness of DNA transfer possibilities post criminal activity, to limit the risk of contamination or loss of DNA. Appropriately trained forensic practitioners are best placed to provide opinion and guidance on the interpretation of profiles at the activity level. However, those requested to provide expert opinion on DNA-related activity level issues are often insufficiently trained to do so. We advocate recognition of DNA activity associated expertise to be distinct from expertise associated with the identification of individuals. This is to be supported by dedicated training, competency testing, authorisation, and regular fit for purpose proficiency testing. The possibilities for experts to report on activity-related issues will increase as our knowledge increases through further research, access to relevant data is enhanced, and tools to assist interpretations are better exploited. Improvement opportunities will be achieved sooner, if more laboratories and agencies accept the need to invest in these aspects as well as the training of practitioners.

The complexities of DNA transfer during a social setting.

When questions relating to how a touch DNA sample from a specific individual got to where it was sampled from, one has limited data available to provide an assessment on the likelihood of specific transfer events within a proposed scenario. This data is mainly related to the impact of some key variables affecting transfer that are derived from structured experiments. Here we consider the effects of unstructured social interactions on the transfer of touch DNA. Unscripted social exchanges of three individuals having a drink together while sitting at a table were video recorded and DNA samples were collected and profiled from all relevant items touched during each sitting. Attempts were made to analyze when and how DNA was transferred from one object to another. The analyses demonstrate that simple minor everyday interactions involving only a few items in some instances lead to detectable DNA being transferred among individuals and objects without them having contacted each other through secondary and further transfer. Transfer was also observed to be bi-directional. Furthermore, DNA of unknown source on hands or objects can be transferred and interfere with the interpretation of profiles generated from targeted touched surfaces. This study provides further insight into the transfer of DNA that may be useful when considering the likelihood of alternate scenarios of how a DNA sample got to where it was found.

Evaluation of multiple transfer of DNA using mock case scenarios.

DNA transfer and its possible role in explaining the presence of a biological sample at a crime scene is becoming more prevalent in criminal investigations and related court proceedings. To assist understanding of DNA transfer and assess the extent to which we can utilise already available information regarding transfer of DNA we compare transfer rates determined from mock multi-step transfer scenarios with transfer rates predicted by the application of currently available transfer rate data. The transfer results obtained from the scenarios tested were, in some instances, different (both lower and higher rates) from those predicted. These discrepancies are most likely the result of the impact of as yet untested variables. These may include the variations in substrate type, transfer area size and environmental factors such as temperature and humidity among others. Whilst detailed re-enactments of proposed transfer scenarios, that take into account the many possibly relevant aspects affecting transfer are desirable, to provide an accurate likelihood estimate, these are not always possible. The application of detailed transfer rate tables that include data on the many factors affecting transfer could provide a useful substitute for evaluating the likelihood of specific transfer events. The value and accuracy derived from applying such tables will improve as more research in this area is conducted and the tables expanded and refined.

DNA transfer within forensic exhibit packaging: potential for DNA loss and relocation.

Crime scene samples after their collection are packaged and transported to the laboratory for examination and DNA analysis. The amount and location of DNA-containing material retrieved from an exhibit can be critical in acquiring a profile for incrimination or exclusion purposes and for elucidating criminal events. This paper shows that significant quantities of DNA are frequently: (a) transferred from the exhibit to the inside of the packaging and (b) transferred from its area of initial deposit to other areas of the same exhibit and/or to other exhibits within the same package. There is a distinct possibility of failing to generate adequate profiles in instances where the DNA content may otherwise have been adequate, and for the misinterpretation of a result that could impact negatively on the criminal investigation and court outcome. These findings highlight the need for improvements in the collection and packaging of forensic casework exhibits for DNA analysis.

Investigation of secondary DNA transfer of skin cells under controlled test conditions.

There is a paucity of data on the relative transfer rates of deposited biological substances which could assist evaluation of the probability of given crime scene scenarios, especially for those relating to objects originally touched by hand. This investigation examines factors that may influence the secondary transfer of DNA from this source, including the freshness of the deposit, the nature of the primary and secondary substrate and the manner of contact between the surfaces. The transfer rates showed that both the primary and secondary type of substrate and the manner of contact are important factors influencing transfer of skin cells, but, unlike other biological fluids, such as blood and saliva, the freshness of the deposit in most instances is not. Skin cells deposited on a non-porous primary substrate transferred more readily to subsequent substrates than those deposited on a porous substrate. Porous secondary substrates, however, facilitated transfer more readily than non-porous secondary substrates, from both porous and non-porous surfaces. Friction as the manner of contact significantly increased the rate of transfer. The findings of this study improve our general understanding of the transfer of DNA material contained in fingerprints that is left on a surface, and assist in the evaluation of the probability of secondary and further DNA transfer under specific conditions.

Secondary DNA transfer of biological substances under varying test conditions.

This research investigates factors that may influence the secondary transfer of DNA. These include the type of biological substance deposited, the nature of the primary and secondary substrate, moisture content of the deposit and type of contact between the surfaces. Results showed that secondary transfer is significantly affected by both the type of primary substrate and the moisture (wetness) of the biological sample. Porous substrates and/or dry samples diminished transfer (with on average only 0.36% of biological material being transferred from one site to another), whereas non-porous substrates and/or wet samples facilitated transfer events (approximately 50-95% of biological material was transferred from one site to another). Further, the type of secondary substrate also influenced transfer rate, with porous surfaces, absorbing transferred biological substances more readily than non-porous ones. No significant differences were observed among the biological substances tested (pure DNA, blood and saliva). Friction contact between the two substrates significantly enhanced secondary transfer compared to either passive or pressure contact. These preliminary results will assist in developing general assumptions when estimating probability of a secondary DNA transfer event under simple conditions.