Bas Kokshoorn

Response to Grisedale and Van Daal: comparison of STR profiling from low template DNA extracts with and without the consensus profiling method.

In a recent contribution to this journal Grisedale and Van Daal concluded that a single STR analysis of all available template DNA is to be preferred over replicate analyses and a consensus approach when analyzing low template DNA samples. A single STR analysis approach does not allow for an assessment of the validity of the resulting DNA profile. We argue that the use of replicate amplifications is the best way to objectively quantify the extent of the stochastic variation in the data. By applying consensus methodology and/or a probabilistic model, the interpretation of the data will therefore be more objective and reliable.Please see related article: http://www.investigativegenetics.com/content/3/1/14

Cell type determination and association with the DNA donor.

In forensic casework, evidence regarding the type of cell material contained in a stain can be crucial in determining what happened. For example, a DNA match in a sexual offense can become substantially more incriminating when there is evidence supporting that semen cells are present. Besides the question which cell types are present in a sample, also the question who donated what (association) is very relevant. This question is surprisingly difficult, even for stains with a single donor. The evidential value of a DNA profile needs to be combined with knowledge regarding the specificity and sensitivity of cell type tests. This, together with prior probabilities for the different donor-cell type combinations, determines the most likely combination. We present a Bayesian network that can assist in associating donors and cell types. A literature overview on the sensitivity and specificity of three cell type tests (PSA test for seminal fluid, RSID saliva and RSID semen) is helpful in assigning conditional probabilities. The Bayesian network is linked with a software package for interpreting mixed DNA profiles. This allows for a sensitivity analysis that shows to what extent the conclusion depends on the quantity of available research data. This can aid in making decisions regarding further research. It is shown that the common assumption that an individual (e.g. the victim) is one of the donors in a mixed DNA profile can have unwanted consequences for the association between donors and cell types.

Activity level DNA evidence evaluation: On propositions addressing the actor or the activity

More often than not, the source of DNA traces found at a crime scene is not disputed, but the activity or timing of events that resulted in their transfer is. As a consequence, practitioners are increasingly asked to assign a value to DNA evidence given propositions about activities provided by prosecution and defense counsel. Given that the dispute concerns the nature of the activity that took place or the identity of the actor that carried out the activity, several factors will determine how to formulate the propositions. Determining factors are (1) whether defense claims the crime never took place, (2) whether defense claims someone other than the accused (either an unknown individual or a known person) performed the criminal activity, and (3) whether it is claimed and disputed that the suspect performed an alternative, legitimate activity or has a relation to the victim, the object, or the scene of crime that implies a legitimate interaction. Addressing such propositions using Bayesian networks, we demonstrate the effects of the various proposition sets on the evaluation of the evidence.

An inter-laboratory comparison study on transfer, persistence and recovery of DNA from cable ties

To address questions on the activity that led to the deposition of biological traces in a particular case, general information on the probabilities of transfer, persistence and recovery of cellular material in relevant scenarios is necessary. These figures may be derived from experimental data described in forensic literature when conditions relevant to the case were included. The experimental methodology regarding sampling, DNA extraction, DNA typing and profile interpretation that were used to generate these published data may differ from those applied in the case and thus the applicability of the literature data may be questioned. To assess the level of variability that different laboratories obtain when similar exhibits are analysed, we performed an inter-laboratory study between four partner laboratories. Five sets of 20 cable ties bound by different volunteers were distributed to the participating laboratories and sampled and processed according to the in-house protocols. Differences were found for the amount of retrieved DNA, as well as for the reportability and composition of the DNA profiles. These differences also resulted in different probabilities of transfer, persistence and recovery for each laboratory. Nevertheless, when applied to a case example, these differences resulted in similar assignments of weight of evidence given activity-level propositions.

Results of an inter and intra laboratory exercise on the assessment of complex autosomal DNA profiles

The interpretation of complex DNA profiles may differ between laboratories and reporting officers, which can lead to discrepancies in the final reports. In this study, we assessed the intra and inter laboratory variation in DNA mixture interpretation for three European ISO17025-accredited laboratories. To this aim, 26 reporting officers analyzed five sets of DNA profiles. Three main aspects were considered: 1) whether the mixed DNA profiles met the criteria for comparison to a reference profile, 2) the actual result of the comparison between references and DNA profiling data and 3) whether the weight of the DNA evidence could be assessed. Similarity in answers depended mostly on the complexity of the tasks. This study showed less variation within laboratories than between laboratories which could be the result of differences between internal laboratory guidelines and methods and tools available. Results show the profile types for which the three laboratories report differently, which informs indirectly on the complexity threshold the laboratories employ. Largest differences between laboratories were caused by the methods available to assess the weight of the DNA evidence. This exercise aids in training forensic scientists, refining laboratory guidelines and explaining differences between laboratories in court. Undertaking more collaborative exercises in future may stimulate dialog and consensus regarding interpretation. For training purposes, DNA profiles of the mixed stains and questioned references are made available.

Cale CM, Earll ME, Latham KE, Bush GL. Could Secondary DNA Transfer Falsely Place Someone at the Scene of a Crime? J Forensic Sci 2016;61(1):196-203.

Sir, We would like to comment on the paper by Cale et al. (1). In this paper, the authors evaluated whether the increased sensitivity of current DNA profiling methods could lead to an increased detection of DNA, secondarily transferred to a knife handle following hand-to-hand contact. While the core results of their study add to our understanding of DNA transfer and persistence, in our opinion some of the claims made in the paper, and in subsequent commentaries in scientific and popular media (2–6) overstate the implications of the results obtained and do not fully acknowledge the many relevant studies that have been conducted since the original discovery of secondary transfer of DNA left by touch in 1997 (7). It is our concern that the methodology employed substantially overestimates the true rate of DNA transfer, and thus, the conclusions drawn and commentary made about the study are not an accurate reflection of such occurrences in casework situations. Cale et al. (1) conclude that “The demonstrated possibility of secondary DNA transfer could have major ramifications in a forensic investigation; secondary DNA transfer should not be regarded as an event that may only occur under optimal experimental conditions.” We completely agree with this statement, and hope that, given the large body of research within this area, no laboratory would regard DNA transfer as impossible in normal conditions, and positively infer activity on the basis of DNA presence alone. Beyond this however, particular concern was raised that the transferred profile may comprise the major contributor in a mixture (a phenomenon we term here “contributor inversion,” where the relative contributions do not match what is expected under those circumstances), or indeed the sole contributor. To assert this possibility renders DNA evidence unreliable, and transfer may cause strong genetic evidence to become insignificant (1,2), which is in our opinion misinterpreting the data and its implications and confusing source and activity-level assessment of evidence. Contributor inversion in DNA mixtures has been previously demonstrated, and is not unexpected, although is normally seen at a much lower rate than reported by the authors of this study. Such inversions, seen in 25% (5 of 25) of samples within Cale et al. (1), have only been observed at similarly high rates in one other study, that by Lowe et al. (8). As with the study by Cale et al. (1), the secondary transfer samples examined by Lowe et al. (8), from simulations where DNA was transferred from hand to hand to object, were also unrealistically maximized, in that the experimental design was biased toward transfer, and the subsequent detection of transferred DNA. Other studies examining the transfer of “touch” DNA in less controlled scenarios, considered more realistic examples of secondary transfer rates, have observed the transferred DNA as the major or only component to the profile on far fewer occasions, if at all: ~2.8% in studies where the source of the transferred DNA was known (9– 12); ~1.4% in studies where the source of the transferred DNA was unknown (13,14). Several other studies allude to the detection of secondarily transferred DNA from known and/or unknown sources within the samples obtained, but provide insufficient information with respect to their presence forming a minor or major component of the profile, and in the case of foreign alleles, whether they originated from a single or multiple sources (15–26). It is important that transfer probabilities are estimated using realistic scenarios if inferences are to be drawn for casework. Those maximize the probability of DNA on participant’s hands to be outside the normal range of variation under normal conditions, or use increased duration of contact, or include limited/unrealistic interval periods between actions, and/or use inappropriately pressured contact to maximize transfer (as was the case in this study) are of less value for estimation of transfer probabilities in casework than those that replicate real-life conditions. Likewise, any modifications to normal case working procedures, in terms of sample collection, processing, or interpretation, should be minimized and certainly noted as a limitation that may cause variations in the detection of transfer. Further, Cale et al. (1) posit that the transfer of DNA through an intermediary has not been systematically evaluated with current technology. Studies have provided empirical evidence of secondary (and further, including tertiary and quaternary) transfer (9,10,16,27), have investigated variables that may affect the transfer and subsequent detection of touch DNA (7,8,13,18,20,22–24,28,29), have provided casework relevant examples and simulations to model the possible effect of transfer on conclusions (9–11,14,16,17,21,30–32), and have provided statistical models to evaluate the possibility of transfer (33–35). Such studies have been conducted with various autosomal STR methods employed in forensic biology, including the current sensitive technologies, and, more recently, with alternate methods such as mRNA profiling (18,21). While the articles cited above refer only to the transfer of “touch” DNA, the authors would like to acknowledge that there are numerous other studies investigating the transfer of other biological fluids not cited here that also contribute to our general knowledge of DNA transfer within the forensic context. The community is well aware of the impact of more sensitive technologies on the detection of DNA transfer (36,37) and is actively developing models and generating casework-relevant data to ensure that the conclusions being reported are incorporating activity-level considerations, not simply source or subsource level (34,35,38,39). Likewise, the legal community is growing more aware of potential issues around DNA transfer. There have been many cases where the primary issue has surrounded the probability of transfer including Fitzgerald v The Queen (40,41), R v Reed and Reed, R v Garmson (42), and the infamous death of Meredith Kercher (43). Ultimately, DNA should not be used as the sole form of evidence, but should be presented as one of multiple pieces of evidence that may assist the trier of fact. Reliance upon DNA alone can result in erroneous conclusions, not just due to transfer, but also contamination and adventitious matching (16,36,43–45). In casework, it is the responsibility of an expert to offer as much guidance as possible to the court on the probability of the DNA evidence given various transfer mechanisms (46) and to ensure that limitations in the evidence or methodology, and potential sources of error in the opinions provided, are clear and understood by the trier of fact (47). Withholding such information prevents an accurate estimation of the probative value of the evidence and is inconsistent with legal, ethical, and scientific norms (48,49). If the expert has considered the probability of DNA

Transfer and persistence of non-self DNA on hands over time: Using empirical data to evaluate DNA evidence given activity level propositions

Questions relating to how DNA from an individual got to where it was recovered from and the activities associated with its pickup, retention and deposition are increasingly relevant to criminal investigations and judicial considerations. To address activity level propositions, investigators are typically required to assess the likelihood that DNA was transferred indirectly and not deposited through direct contact with an item or surface. By constructing a series of Bayesian networks, we demonstrate their use in assessing activity level propositions derived from a recent legal case involving the alleged secondary transfer of DNA to a surface following a handshaking event. In the absence of data required to perform the assessment, a set of handshaking simulations were performed to obtain probabilities on the persistence of non-self DNA on the hands following a 40min, 5h or 8h delay between the handshake and contact with the final surface (an axe handle). Variables such as time elapsed, and the activities performed and objects contacted between the handshake and contact with the axe handle, were also considered when assessing the DNA results. DNA from a known contributor was transferred to the right hand of an opposing hand-shaker (as a depositor), and could be subsequently transferred to, and detected on, a surface contacted by the depositor 40min to 5h post-handshake. No non-self DNA from the known contributor was detected in deposits made 8h post-handshake. DNA from the depositor was generally detected as the major or only contributor in the profiles generated. Contributions from the known contributor were minor, decreasing in presence and in the strength of support for inclusion as the time between the handshake and transfer event increased. The construction of a series of Bayesian networks based on the case circumstances provided empirical estimations of the likelihood of direct or indirect deposition. The analyses and conclusions presented demonstrate both the complexity of activity level assessments concerning DNA evidence, and the power of Bayesian networks to visualise and explore the issues of interest for a given case.

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.

Sharing data on DNA transfer, persistence, prevalence and recovery: Arguments for harmonization and standardization

Sharing data between forensic scientists on DNA transfer, persistence, prevalence and recovery (TPPR) is crucial to advance the understanding of these issues in the criminal justice community. We present the results of a collaborative exercise on reporting forensic genetics findings given activity level propositions. This exercise outlined differences in the methodology that was applied by the participating laboratories, as well as limitations to the use of published data on DNA TPPR. We demonstrate how publication of experimental results in scientific journals can be further improved to allow for an adequate use of these data. Steps that can be taken to share and use these data for research and casework purposes are outlined, and the prospects for future sharing of data through publicly accessible databases are discussed. This paper also explores potential avenues to proceed with implementation and is intended to fuel the discussion on sharing data pertaining to DNA TPPR issues. It is further suggested that international standardization and harmonization on these topics will benefit the forensic DNA community as it has been achieved in the past with the harmonization of STR typing systems.