Investigative use of human environmental DNA in forensic genetics.

Individuals leave behind traces of their DNA wherever they go. DNA can be transferred to surfaces and items upon touch, can be released into the air, and may be deposited in indoor dust. The mere presence of individuals in a location is sufficient to facilitate either direct or indirect DNA transfer into the surrounding environment. In this study, we analyzed samples recovered from commonly touched surfaces such as light switches and door handles in an office environment. We evaluated two different methods to isolate DNA and co-extract DNA and RNA from the samples. DNA profiles were compared to the references of the inhabitants of the different locations and were analyzed taking into consideration the type of sampled surface, sampling location and information about the activities in a room during the sampling day. Results from DNA samples collected from surfaces were also compared to those from air and dust samples collected in parallel from the same areas. We characterized the amount and composition of DNA found on various surfaces and showed that surface DNA sampling can be used to detect occupants of a location. The results also indicate that combining information from environmental samples collected from different DNA sources can improve our understanding of DNA transfer events in an indoor setting. This study further demonstrates the potential of human environmental DNA as an investigative tool in forensic genetics.

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.

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 invisible witness: air and dust as DNA evidence of human occupancy in indoor premises.

Humans constantly shed deoxyribonucleic acid (DNA) into the surrounding environment. This DNA may either remain suspended in the air or it settles onto surfaces as indoor dust. In this study, we explored the potential use of human DNA recovered from air and dust to investigate crimes where there are no visible traces available-for example, from a recently vacated drugs factory where multiple workers had been present. Samples were collected from three indoor locations (offices, meeting rooms and laboratories) characterized by different occupancy types and cleaning regimes. The resultant DNA profiles were compared with the reference profiles of 55 occupants of the premises. Our findings showed that indoor dust samples are rich sources of DNA and provide an historical record of occupants within the specific locality of collection. Detectable levels of DNA were also observed in air and dust samples from ultra-clean forensic laboratories which can potentially contaminate casework samples. We provide a Bayesian statistical model to estimate the minimum number of dust samples needed to detect all inhabitants of a location. The results of this study suggest that air and dust could become novel sources of DNA evidence to identify current and past occupants of a crime scene.

Quantitative PCR analysis of bloodstains of different ages.

An accurate method to estimate the age of a stain or the time since deposition (TsD) would represent an important tool in police investigations for evaluating the true relevance of a stain. In this study, two laboratories reproduced an mRNA-based method for TsD estimation published by another group. The qPCR-based assay includes four transcripts (B2M, LGALS2, CLC, and S100A12) and showed preferential degradation of the 5' end over the 3' end. In this study, the blood-specific marker ALAS2 was added to examine whether it would show the same degradation pattern. Based on our qPCR data several elastic net models with different penalty combinations were created, using training data from the two laboratories separately and combined. Each model was then used to estimate the age of bloodstains from two independent test sets each laboratory had prepared. The elastic net model built on both datasets with training samples up to 320 days old displayed the best prediction performance across all test samples (MAD=18.9 days). There was a substantial difference in the prediction performance for the two laboratories: Restricting TsD to up to 100 days for test data, one laboratory obtained an MAD of 2.0 days when trained on its own data, whereas the other laboratory obtained an MAD of 15 days.

Body Fluid Identification in Samples Collected after Intimate and Social Contact: A Comparison of Two mRNA Profiling Methods and the Additional Information Gained by cSNP Genotypes.

The ability to associate a contributor with a specific body fluid in a crime stain can aid casework investigation. The detection of body fluids combined with DNA analyses may supply essential information, but as the two tests are independent, they may not be associated. Recently, the analysis of coding region SNPs (cSNPs) within the RNA transcript has been proven to be a promising method to face this challenge. In this study, we performed targeted RNA sequencing of 158 samples (boxershorts, fingernail swabs and penile swabs) collected from 12 couples at different time points post-intimate contact and after non-intimate contact, using the Ion S5™ System and BFID-cSNP-6F assay. The aim of the study was to compare the performance of the MPS and CE methods in the detection of mRNA markers, and to associate body fluids with contributors by their cSNP genotypes. The results of the study show a lower success rate in the detection of vaginal mucosa by the MPS compared to the CE method. However, the additional information obtained with the cSNP genotypes could successfully associate body fluids with contributors in most cases.

Limitations of qPCR to estimate DNA quantity: An RFU method to facilitate inter-laboratory comparisons for activity level, and general applicability.

The application of qPCR to estimate the quantity of DNA present is usually based upon a short amplicon (typically c.80bp) and a longer amplicon (typically c.200-300bp) where the latter is used to determine the amount of degradation present in a sample. The data are used to make decisions about a) whether there is sufficient template to amplify? b) how much of the elution volume to forward to PCR? A typical multiplex amplifies template in the region of 100-500bp. Consequently, the results from an 80bp amplicon will tend to overestimate the actual amplifiable quantity that is present in a degraded sample. To compensate, a method is presented that relates the quantity of amplifiable DNA to the average RFU of the amplified fragments. This provides greatly improved accuracy of the estimated quantity of DNA present, which may differ by more than an order of magnitude compared to qPCR. The relative DNA quantities can be apportioned per contributor once mixture proportions are ascertained with probabilistic genotyping software (EuroForMix). The motivation for this work was to provide an improved method to generate data to prepare distributions that are used to inform activity level propositions. However, other applications will benefit, particularly those where extraction and quantification are bypassed: For example direct PCR and Rapid DNA technology. The overall aim of this work was to provide a method of quantification that is standardised and can be used to compare results between different laboratories that use different multiplexes. A software solution "ShinyRFU" is provided to aid calculations.

Transfer, persistence and recovery of DNA and mRNA vaginal mucosa markers after intimate and social contact with Bayesian network analysis for activity level reporting.

In sexual assault cases, it can be challenging to identify the type of body fluids/ cell types present in a crime scene sample, especially the origin of epithelial cells. Therefore, more labs are applying mRNA body fluid analysis for saliva, skin and vaginal mucosa markers. To address activity level propositions, it is necessary to assign probabilities of transfer, persistence, prevalence and recovery of DNA and mRNA markers. In this study we analysed 158 samples (fingernail swabs, penile swabs and boxershorts) from 12 couples collected at different time points post intimate contact and after non-intimate contact in order to detect DNA from the person of interest (POI) and mRNA vaginal mucosa markers. Samples were DNA and RNA co-extracted and analysed with PowerPlex®Fusion 6C System and 19-plex mRNA primer mix respectively, using Endpoint PCR and the CE platform. Vaginal mucosa was detected up to 36 h post intimate contact, but also detected in one non-intimate contact sample. In 94% of intimate contact and 50 % of non-intimate contact samples the DNA results support the proposition that POI is the donor (LR ≥ 10,000). There was a strong association between the detection of vaginal mucosa and the average RFU value of the POI. The data were used to instantiate a comprehensive Bayesian network to evaluate the evidence at activity level, given alternate propositions conditioned upon indirect or direct transfer events. It is shown that the value of the evidence is mainly affected by the high DNA quantity (measured as mean RFU) that is recovered from the POI. The detection of vaginal mucosa had low impact upon the resultant likelihood ratio.

Non-self DNA on the neck: a 24 hours time-course study.

Non-self DNA is normally present on skin due to DNA transfer occurring during daily activities. The understanding of persistence and accumulation of foreign DNA on the neck can assist in the interpretation of DNA evidence collected from an assaulted victim. Establishing the composition and level of non-self DNA present is relevant, especially in cases where the victim cohabits with other individuals, such as partner and children. This study investigated the persistence and accumulation of non-self DNA on the neck, over the course of 24 h. DNA samples were collected from the neck of 20 adult volunteers at three time-points, on two days. The detection of a partner's DNA and DNA from unknown sources was studied in relation to the living arrangement and to the activities performed by each individual. An increased number of non-self alleles were detected over time. Partner's DNA was observed to accumulate during the day and to persist when an individual was absent from the shared home environment. DNA from unknown contributors was found on the neck of individuals that used public transport, attended public spaces and had social interactions. The data acquired from this study will help to increase knowledge on the composition of DNA present on an individual's neck in a daily situation.

RFU derived LRs for activity level assignments using Bayesian Networks.

A comparative study has been carried out, comparing two different methods to estimate activity level likelihood ratios (LRa) using Bayesian Networks. The first method uses the sub-source likelihood ratio (log10LRϕ) as a 'quality indicator'. However, this has been criticised as introducing potential bias from population differences in allelic proportions. An alternative method has been introduced that is based upon the total RFU of a DNA profile that is adjusted using the mixture proportion (Mx) which is calculated from quantitative probabilistic genotyping software (EuroForMix). Bayesian logistic regressions of direct transfer data showed that the two methods were comparable. Differences were attributed to sampling error, and small sample sizes of secondary transfer data. The Bayesian approach facilitates comparative studies by taking account of sampling error; it can easily be extended to compare different methods.

Evaluating the performance of five up-to-date DNA/RNA co-extraction methods for forensic application.

The importance of RNA evidence is growing with new developments in RNA profiling methods and purposes. As forensic samples often can be of small quantity, extraction methods with high yields of both DNA and RNA are desirable. In order to identify the optimal DNA/RNA co-extraction workflow for forensic samples, we evaluated the performance of three frequently-used methods, two new approaches for DNA/RNA co-extraction and a manual phenol/chloroform RNA-only extraction method on blood and saliva samples. Based on a comprehensive analysis of the RNA and DNA quantities, as well as the STR genotyping and mRNA profiling results, we conclude that the two frequently-used co-extraction methods, combining commercially available DNA and RNA extraction kits, achieved the best performance. However, not any combination of commercially available DNA and RNA extraction kits works well and extensive optimization is necessary, as seen in the poor results of the two new approaches.

Determination of shedder status: A comparison of two methods involving cell counting in fingerprints and the DNA analysis of handheld tubes.

The shedder status of an individual may be important to consider in the context of DNA transfer, persistence and recovery and in Bayesian networks where a person's shedder status may have an impact on the outcome. In this study we compared two methods to determine shedder status: the handheld tube (HH) method and a fluorescent cell count (CC) method. A poor association was observed between the numbers of detected cells in a fingerprint using the CC method and the strength of the DNA result with the HH method. The 20 participants were classified into low (25%), medium (50%) and high (25%) shedders based on the HH method. While the low and high shedders showed a good consistency between the replicates, the medium shedders varied more and have to be considered more carefully as they may act as either a high or a low shedder in an event of DNA transfer.

An LR framework incorporating sensitivity analysis to model multiple direct and secondary transfer events on skin surface.

Bayesian logistic regression is used to model the probability of DNA recovery following direct and secondary transfer and persistence over a 24 h period between deposition and sample collection. Sub-source level likelihood ratios provided the raw data for activity-level analysis. Probabilities of secondary transfer are typically low, and there are challenges with small data-sets with low numbers of positive observations. However, the persistence of DNA over time can be modelled by a single logistic regression for both direct and secondary transfer, except that the time since deposition must be compensated by an offset value for the latter. This simplifies the analysis. Probabilities are used to inform an activity-level Bayesian Network that takes account of alternative propositions e.g. time of assault and time of social activities. The model is extended in order to take account of multiple contacts between person of interest and 'victim'. Variables taken into account include probabilities of direct and secondary transfer, along with background DNA from unknown individuals. The logistic regression analysis is Bayesian - for each analysis, 4000 separate simulations were carried out. Quantile assignments enable calculation of a plausible range of probabilities and sensitivity analysis is used to describe the corresponding variation of LRs that occur when modelled by the Bayesian network. It is noted that there is need for consistent experimental design, and analysis, to facilitate inter-laboratory comparisons. Appropriate recommendations are made. The open-source program written in R-code ALTRaP (Activity Level, Transfer, Recovery and Persistence) enables analysis of complex multiple transfer propositions that are commonplace in cases-work e.g. between those who cohabit. A number of case examples are provided. ALTRaP can be used to replicate the results and can easily be modified to incorporate different sets of data and variables.

Who Packed the Drugs? Application of Bayesian Networks to Address Questions of DNA Transfer, Persistence, and Recovery from Plastic Bags and Tape.

When DNA from a suspect is detected in a sample collected at a crime scene, there can be alternative explanations about the activity that may have led to the transfer, persistence and recovery of his/her DNA. Previous studies have shown that DNA can be indirectly transferred via intermediate surfaces and that DNA on a previously used object can persist after subsequent use of another individual. In addition, it has been shown that a person's shedder status may influence transfer, persistence, prevalence, and recovery of DNA. In this study we have investigated transfer persistence and recovery on zip-lock bags and tape, which are commonly encountered in drug cases and how the shedder status of the participants influenced the results. A probabilistic framework was developed which was based on a previously described Bayesian network with case-specific modifications. Continuous modelling of data was used to inform the Bayesian networks and two case scenarios were investigated. In the specific scenarios only moderate to low support for Hp was obtained. Applying a continuous model based on the profile quality can change the LRs.

A retrospective study on the transfer, persistence and recovery of sperm and epithelial cells in samples collected in sexual assault casework.

Data from all sexual assault cases analysed at the Section of Forensic Biology at Oslo University Hospital in the period 2013-2015 were reviewed to study transfer and persistence of cells deposited on the body. Data were recorded on detection of both sperm and epithelial cells. The final dataset consist of 2141 samples from 765 cases. In this study "positive findings" refer to evidence to support the proposition that the DNA profile was contributed by the POI and do not only correspond to detection of cell type, e.g. sperm cells. Positive findings from analysis of sperm cells could be detected in samples collected up to 72 h after deposition, and was less frequently detected in oral swabs were the longest observed persistence time was 12 h. Positive findings from analysis of epithelial cells were observed up to 43 h after deposition. A high success rate was observed from penile swabs collected within 24 h of the incidence demonstrating the importance of collecting and analysing such samples in cases where no semen is detected.

The implications of shedder status and background DNA on direct and secondary transfer in an attack scenario.

In court questions are often raised related to how trace DNA was deposited, directly during the crime or innocently for instance by secondary transfer. It is therefore of interest to have knowledge of the probability of transfer or secondary transfer in different situations. Factors that could influence transfer probabilities are background DNA and the shedder status of the involved persons. In this study, we have classified participants as high or low DNA shedders. We observed DNA transfer in a simulated attack scenario, and demonstrated that shedder status has a significant influence of transfer rates. We have examined the background DNA in samples from T-shirts worn in an area with frequent human traffic and detected multiple contributors. We further demonstrated that DNA from co-workers of a T-shirt wearer can be secondarily transferred from the environment and detected in samples, and that the composition of background DNA is correlated with the shedder status of the wearer. Finally, we have illustrated the inference with the results of transfer probabilities and a fictive case with the use of a Bayesian network.

Contamination during criminal investigation: Detecting police contamination and secondary DNA transfer from evidence bags.

As the profiling systems used in forensic analyses have become more sensitive in recent years, the risk of detecting a contamination in a DNA sample has increased proportionally. This requires more stringent work protocols and awareness to minimize the chance of contamination. Although there is high consciousness on contamination and best practice procedures in forensic labs, the same requirements are not always applied by the police. In this study we have investigated the risk of contamination from police staff. Environmental DNA was monitored by performing wipe tests (sampling of hot spots) at two large police units (scenes of crime departments). Additionally, the DNA profiles of the scenes of crime officers were compared to casework samples that their own unit had investigated in the period of 2009-2015. Furthermore, a pilot study to assess whether DNA from the outside package of an exhibit could be transferred to a DNA sample was carried out. Environmental DNA was detected in various samples from hot spots. Furthermore, 16 incidences of previously undetected police-staff contamination were found in casework that had been submitted between 2009 and 2015. In 6 cases the police officers with a matching DNA profile reported that they had not been involved with the case. We have demonstrated that DNA from the outside package can be transferred to an exhibit during examination. This experience demonstrates that when implementing the new multiplex systems, it is important to ensure that 'best practice' procedures are upgraded, and appropriate training is provided in order to ensure that police are aware of the increased contamination risks.

Secondary and subsequent DNA transfer during criminal investigation.

With the introduction of new multiplex PCR kits and instrumentation such as the Applied Biosystems 3500xl, there has recently been a rapid change in technology that has greatly increased sensitivity of detection so that a DNA profile can routinely be obtained from only a few cells. Research to evaluate the risks of passive transfer has not kept pace with this development; hence the risk of innocent DNA transfer at the crime-scene is currently not properly understood. The purpose of this study was to investigate the possibility of investigator-mediated transfer of DNA traces with disposable nitrile-gloves used during crime-scene examinations. We investigated the primary transfer of freshly deposited DNA from touched plastic, wood or metal substrates and secondary and tertiary transfer by a person wearing disposable nitrile-gloves and onto a third object. We show that with use of the new highly sensitive technologies available in forensic DNA analysis there is an enhanced probability to obtain a DNA-profile which has not been directly deposited on the object but is an outcome of one or more transfer events. The nitrile-gloves used by investigators during exhibit examination can act as a vector for DNA transfer from one item to another. We have shown that the amount of DNA deposited on an object affects the probability of transfer. Secondly, the type of substrate material that DNA is deposited onto has an impact on transfer rates.

Regression models for DNA-mixtures.

This paper deals with the statistical interpretation of DNA mixture evidence. The conventional methods used in forensic casework today use something like 16 STR-markers. Power can be increased by rather using SNP-markers. New statistical methods are then needed, and we present a regression framework. The basic idea is that the traditional forensic hypotheses, commonly denoted HD and HP, are replaced by parametric versions: a person contributes to a mixture if and only if the fraction he contributes is greater than 0. This contributed fraction is a parameter of the regression model. The regression model uses the peak heights directly and there is no need to specify or estimate the number of contributors to the mixture. Also, drop-in and drop-out pose no principal problems. Data from 25 controlled blinded experiments were used to test the model. The number of contributors varied between 2 and 5, and the fractions contributed ranged from 0.01 to 0.99. The fractions were accurately estimated by the regression analyses. There were no false positives (i.e., in no cases were non-contributors declared to contributors). Some false negatives occurred for fractions of 0.1 or lower. Simulations were performed to test the model further. The analyses show that useful estimates can be obtained from a relatively small number of SNP-markers. Reasonable results are achieved using 300 markers which is close to the 313 SNPs in the controlled experiment. Increasing the number of SNPs, the analyses demonstrate that individuals contributing as little as 1% can reliably be detected, which suggests that cases beyond the reach of conventional forensic methods today can be reported.