Law enforcement use of genetic genealogy databases in criminal investigations: Nomenclature, definition and scope.

Although law enforcement use of commercial genetic genealogy databases has gained prominence since the arrest of the Golden State Killer in 2018, and it has been used in hundreds of cases in the United States and more recently in Europe and Australia, it does not have a standard nomenclature and scope. We analyzed the more common terms currently being used and propose a common nomenclature: investigative forensic genetic genealogy (iFGG). We define iFGG as the use by law enforcement of genetic genealogy combined with traditional genealogy to generate suspect investigational leads from forensic samples in criminal investigations. We describe iFGG as a proper subset of forensic genetic genealogy, that is, FGG as applied by law enforcement to criminal investigations; hence, investigative FGG or iFGG. We delineate its steps, compare and contrast it with other investigative techniques involving genetic evidence, and contextualize its use within criminal investigations. This characterization is a critical input to future studies regarding the legal status of iFGG and its implications on the right to genetic privacy.

Assessing the consistency of shedder status under various experimental conditions.

Shedder status is defined as the propensity of an individual to leave DNA behind on touched items or surfaces and has been suggested as one of the major factors influencing DNA transfer. However, little is known about whether shedder status is a constant property of an individual across multiple measurements or when the environmental conditions are changed. We have assessed DNA depositions of six males on 20 occasions to acquire a reference data set and to classify the participants into high, intermediate, or low shedders. This data set was also used to investigate how the probability of a correct shedder status classification changed when the number of DNA deposition measurements increased. Individual sweat rates were measured with a VapoMeter and data regarding hygiene routines were collected through a questionnaire on each sampling occasion. Next, we investigated how changes in the experimental conditions such as seasonal variation, hygiene routines, the temperature of the touched object, and repeated handling of an object influenced the DNA shedding. Additionally, we assessed DNA collected from the face and from T-shirts worn by the six participants to explore whether shedder status may be associated with the relative amount of DNA obtained from other body parts. Our results indicate that shedder status is a stable property across different seasons and different temperatures of handled objects. The relative DNA amounts obtained from repeatedly handled tubes, worn T-shirts, and from faces reflected the shedder status of the participants. We suggest that an individual's shedder status is highly influenced by the DNA levels on other body parts than hands, accumulating on the palms by frequently touching e.g., the face or previously handled items harboring self-DNA. Assessing physiological differences between the participants revealed that there were no associations between DNA shedding and individual sweat rates.

Individual shedder status and the origin of touch DNA.

Due to improved laboratory techniques, touched surfaces and items are increasingly employed as sources of forensic DNA evidence. This has urged a need to better understand the mechanisms of DNA transfer between individuals. Shedder status (i.e. the propensity to leave DNA behind) has been identified as one major factor regulating DNA transfer. It is known that some individuals tend to shed more DNA than others, but the mechanisms behind shedder status are largely unknown. By comparing the amounts of DNA deposited from active hands (i.e. used "as usual") and inactive hands (i.e. not allowed to touch anything), we show that some of the self-DNA deposited from hands is likely to have accumulated on hands from other parts of the body or previously handled items (active hands: 2.1 ± 2.7 ng, inactive hands: 0.83 ± 1.1 ng, paired t-test: p = 0.014, n = 27 pairs of hands). Further investigation showed that individual levels of deposited DNA are highly associated with the level of DNA accumulation on the skin of the face (Pearson's correlation: r = 0.90, p < 0.00001 and Spearman's ranked correlation: rs = 0.56, p = 0.0016, n = 29). We hypothesized that individual differences in sebum secretion levels could influence the amount of DNA accumulation in facial areas, but no such correlation was seen (Pearson's correlation: r = - 0.13, p = 0.66, n = 14). Neither was there any correlation between DNA levels on hands or forehead and the time since hand or face wash. We propose that the amount of self-DNA deposited from hands is highly influenced by the individual levels of accumulated facial DNA, and that cells/DNA is often transferred to hands by touching or rubbing one's face.

Getting the conclusive lead with investigative genetic genealogy - A successful case study of a 16 year old double murder in Sweden.

On the morning of October 19, 2004, an eight-year-old boy and a 56-year-old woman were stabbed to death on an open street in the city of Linköping, Sweden. The perpetrator left his DNA at the crime scene, and after 15 years of various investigation efforts, including more than 9000 interrogations and mass DNA screening of more than 6000 men, there were still no clues about the identity of the unknown murderer. The successful application of investigative genetic genealogy (IGG) in the US raised the interest for this tool within the Swedish Police Authority. After legal consultations it was decided that IGG could be applied in this double murder case as a pilot case study. From extensive DNA analysis, including whole-genome sequencing and genotype imputation, DNA data sets were established and searched within both GEDmatch and FamilyTree DNA genealogy databases. A number of fairly distant relatives were found from which family trees were created. The genealogy work resulted in two candidates, two brothers, one of whom matched the crime scene samples by routine STR profiling. The suspect confessed the murders at the initial police hearing and was later convicted of the murders. In this paper we describe the successful application of an emerging technology. We disclose details of the DNA analyses which, due to the poor quality and low quantity of the DNA, required reiterative sequencing and genotype imputation efforts. The successful application of IGG in this double murder case exemplifies its applicability not only in the US but also in Europe. The pressure is now high on the involved authorities to establish IGG as a tool for cold case criminal investigations and for missing person identifications. There is, however, a continuous need to accommodate legal, social and ethical aspects as well.

Enhanced forensic DNA recovery with appropriate swabs and optimized swabbing technique.

Efficient sampling with swabs is crucial for optimal forensic DNA analysis. The DNA recovery is determined by the skill of the practitioner and the compatibility between the applied swab and the surface. Here we investigate the impact of swabbing technique and swab type on the DNA yield. Thirteen different swabs from four categories (cotton, flocked nylon, small foam and large foam) provided equal DNA yields for smooth/non-absorbing surfaces. Large foam swabs gave higher DNA recovery for an absorbing wood surface. Factorial design of experiments and ANOVA was applied to study swabbing techniques for cotton swabs. Two key factors for efficient sampling were found to be 1) holding the swab with an approximate 60° angle against the surface and 2) to rotate the swab during sampling. For absorbing wood, it was beneficial to wet the swab heavily. The results of the factorial experiments were used to develop swabbing protocols for different surfaces. When ten experienced practitioners sampled according to these protocols, the DNA yield was increased for ridged plastic (around 1.25 times more DNA) and absorbing wood (2.2-6.2 times more DNA). For window glass, representing a smooth/non-absorbing surface, sampling according to the protocol gave DNA yields equivalent to applying individual sampling techniques. The protocol lowered person-to-person variation for ridged plastic. In conclusion, we have developed instructive protocols for cotton swab sampling on three types of surfaces: smooth/non-absorbing, ridged/non-absorbing and smooth/absorbing. We believe that such swabbing protocols will streamline and simplify the training of new practitioners and improve sampling efficiency for invisible DNA residues in casework.

Factors affecting DNA recovery from cartridge cases.

Cartridge cases are often the sole items left behind after a shooting incident and DNA traces from these can identify persons connected to the shooting. However, the chance of retrieving usable DNA profiles from cartridge cases is limited, due to the low amounts of deposited DNA and subsequent DNA loss associated with the firing process. In the current study, we set out to increase the DNA recovery from cartridge cases and cartridges by evaluating different swab types and detergents used for trace collection. A protocol applying nylon-flocked swabs instead of cotton swabs was implemented in casework at the Swedish National Forensic Centre (NFC), increasing DNA yield. The number of samples providing a DNA concentration ≥ 0.001 ng/µL (the in-house cut-off for processing low-template samples) increased from 11.1 to 28.6 % for cartridge cases and from 16.0 to 43.3 % for cartridges. There was also a substantial increase in mixed STR profiles, too complex to use for comparisons. Thus, it was not possible to take the full advantage of the elevated DNA yield provided by nylon-flocked swabs. The number of usable STR profiles increased from 5.0 to 8.0 % for cartridge cases and remained unchanged for cartridges. Controlled studies were performed to assess the impact on the DNA recovery from different persons handling the ammunition, different material and size of the cartridge cases, and the type of firearm used. These studies reflected an ideal situation, where all cartridges were extensively handled and loaded without gloves, thus providing a higher expected DNA yield compared to most casework samples. The total peak height differed by up to a factor of ∼50 when 20 different persons handled cartridges prior to shooting. By evaluating eleven combinations of different firearms and ammunition, it was found that the casing material and type of firearm also have a substantial impact on DNA yield.

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

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

The double-swab technique versus single swabs for human DNA recovery from various surfaces.

Most crime scene DNA evidence is retrieved using cotton swabs. Since the late 90's, the double-swab technique has been favoured by many practitioners throughout the world. However, the superiority of double-swabbing over applying single wet swabs has not been broadly verified. Here we set out to evaluate the need for the second dry swab for various surfaces, aiming at mimicking the range of surfaces encountered at crime scenes: flat and ridged, absorbing and non-absorbing. For the tested non-absorbing surfaces, i.e., window glass, steel, brass, synthetic leather and ridged plastic, the first wet swabs gave at least 16 times higher DNA yields compared to the second dry swabs. In addition, second wet swabs gave more DNA than second dry ones, opposing the common notion that the purpose of the second swab is to absorb excess liquid. When ten experienced staff members sampled saliva stains on a window glass surface the variation between persons was considerable, with mean DNA yields for the first wet swabs ranging from 0.045 ± 0.022 to 0.13 ± 0.024 ng/µL. The first wet swabs gave 4-162 times more DNA than the second dry swabs, with higher DNA amounts on second swabs coinciding with lower amounts for first swabs. We show that for non-absorbing surfaces, the first wet swab takes up most of the cells in dried stains, making it less valuable to apply a second dry swab. The differences in DNA recovery between first and second swabs were notable also for absorbing surfaces. Double-swabbing may be preferable for some complex surfaces, but focusing on efficient sampling technique with single wet swabs is likely a better general approach.

Assessment of the transfer, persistence, prevalence and recovery of DNA traces from clothing: An inter-laboratory study on worn upper garments.

Among the various items recovered from crime scenes or persons involved in a crime event, clothing items are commonly encountered and submitted for forensic DNA sampling. Depending on the case circumstances and the activity-of-interest, sampling of the garment may concentrate on collecting DNA from the wearer, or from one or more offenders who have allegedly contacted the item and/or wearer. Relative to the targeted DNA, background DNA already residing on the item from previous contacts, or transferred during or after the crime event, may also be collected during sampling and observed in the resultant DNA profile. Given our limited understanding of how, and from where, background DNA is derived on clothing, research on the transfer, persistence, prevalence, and recovery (TPPR) of DNA traces from upper garments was conducted by four laboratories. Samples were collected from several areas of two garments, each worn on separate working or non-working days and individually owned by four individuals from each of the four laboratories, and processed from DNA extraction through to profiling. Questionnaires documented activities relating to the garment prior to and during wearing, and reference profiles were obtained from the wearer and their close associates identified in the questionnaire. Among the 448 profiles generated, variation in the DNA quantity, composition of the profiles, and inclusion/exclusion of the wearer and their close associates was observed among the collaborating laboratories, participants, garments worn on different occasions, and garment areas sampled.

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.

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.

Accurate Digital Polymerase Chain Reaction Quantification of Challenging Samples Applying Inhibitor-Tolerant DNA Polymerases.

Digital PCR (dPCR) enables absolute quantification of nucleic acids by partitioning of the sample into hundreds or thousands of minute reactions. By assuming a Poisson distribution for the number of DNA fragments present in each chamber, the DNA concentration is determined without the need for a standard curve. However, when analyzing nucleic acids from complex matrixes such as soil and blood, the dPCR quantification can be biased due to the presence of inhibitory compounds. In this study, we evaluated the impact of varying the DNA polymerase in chamber-based dPCR for both pure and impure samples using the common PCR inhibitor humic acid (HA) as a model. We compared the TaqMan Universal PCR Master Mix with two alternative DNA polymerases: ExTaq HS and Immolase. By using Bayesian modeling, we show that there is no difference among the tested DNA polymerases in terms of accuracy of absolute quantification for pure template samples, i.e., without HA present. For samples containing HA, there were great differences in performance: the TaqMan Universal PCR Master Mix failed to correctly quantify DNA with more than 13 pg/nL HA, whereas Immolase (1 U) could handle up to 375 pg/nL HA. Furthermore, we found that BSA had a moderate positive effect for the TaqMan Universal PCR Master Mix, enabling accurate quantification for 25 pg/nL HA. Increasing the amount of DNA polymerase from 1 to 5 U had a strong effect for ExTaq HS, elevating HA-tolerance four times. We also show that the average Cq values of positive reactions may be used as a measure of inhibition effects, e.g., to determine whether or not a dPCR quantification result is reliable. The statistical models developed to objectively analyze the data may also be applied in quality control. We conclude that the choice of DNA polymerase in dPCR is crucial for the accuracy of quantification when analyzing challenging samples.

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.

High-throughput DNA extraction of forensic adhesive tapes.

Tape-lifting has since its introduction in the early 2000's become a well-established sampling method in forensic DNA analysis. Sampling is quick and straightforward while the following DNA extraction is more challenging due to the "stickiness", rigidity and size of the tape. We have developed, validated and implemented a simple and efficient direct lysis DNA extraction protocol for adhesive tapes that requires limited manual labour. The method uses Chelex beads and is applied with SceneSafe FAST tape. This direct lysis protocol provided higher mean DNA yields than PrepFiler Express BTA on Automate Express, although the differences were not significant when using clothes worn in a controlled fashion as reference material (p=0.13 and p=0.34 for T-shirts and button-down shirts, respectively). Through in-house validation we show that the method is fit-for-purpose for application in casework, as it provides high DNA yields and amplifiability, as well as good reproducibility and DNA extract stability. After implementation in casework, the proportion of extracts with DNA concentrations above 0.01ng/µL increased from 71% to 76%. Apart from providing higher DNA yields compared with the previous method, the introduction of the developed direct lysis protocol also reduced the amount of manual labour by half and doubled the potential throughput for tapes at the laboratory. Generally, simplified manual protocols can serve as a cost-effective alternative to sophisticated automation solutions when the aim is to enable high-throughput DNA extraction of complex crime scene samples.

Enhanced low-template DNA analysis conditions and investigation of allele dropout patterns.

Forensic DNA analysis applying PCR enables profiling of minute biological samples. Enhanced analysis conditions can be applied to further push the limit of detection, coming with the risk of visualising artefacts and allele imbalances. We have evaluated the consecutive increase of PCR cycles from 30 to 35 to investigate the limitations of low-template (LT) DNA analysis, applying the short tandem repeat (STR) analysis kit PowerPlex ESX 16. Mock crime scene DNA extracts of four different quantities (from around 8-84 pg) were tested. All PCR products were analysed using 5, 10 and 20 capillary electrophoresis (CE) injection seconds. Bayesian models describing allele dropout patterns, allele peak heights and heterozygote balance were developed to assess the overall improvements in EPG quality with altered PCR/CE settings. The models were also used to evaluate the impact of amplicon length, STR marker and fluorescent label on the risk for allele dropout. The allele dropout probability decreased for each PCR cycle increment from 30 to 33 PCR cycles. Irrespective of DNA amount, the dropout probability was not affected by further increasing the number of PCR cycles. For the 42 and 84 pg samples, mainly complete DNA profiles were generated applying 32 PCR cycles. For the 8 and 17 pg samples, the allele dropouts decreased from 100% using 30 cycles to about 75% and 20%, respectively. The results for 33, 34 and 35 PCR cycles indicated that heterozygote balance and stutter ratio were mainly affected by DNA amount, and not directly by PCR cycle number and CE injection settings. We found 32 and 33 PCR cycles with 10 CE injection seconds to be optimal, as 34 and 35 PCR cycles did not improve allele detection and also included CE saturation problems. We find allele dropout probability differences between several STR markers. Markers labelled with the fluorescent dyes CXR-ET (red in electropherogram) and TMR-ET (shown as black) generally have higher dropout risks compared with those labelled with JOE (green) and fluorescein (blue). Overall, the marker D10S1248 has the lowest allele dropout probability and D8S1179 the highest. The marker effect is mainly pronounced for 30-32 PCR cycles. Such effects would not be expected if the amplification efficiencies were identical for all markers. Understanding allele dropout risks and the variability in peak heights and balances is important for correct interpretation of forensic DNA profiles.

Collaborative EDNAP exercise on the IrisPlex system for DNA-based prediction of human eye colour.

The IrisPlex system is a DNA-based test system for the prediction of human eye colour from biological samples and consists of a single forensically validated multiplex genotyping assay together with a statistical prediction model that is based on genotypes and phenotypes from thousands of individuals. IrisPlex predicts blue and brown human eye colour with, on average, >94% precision accuracy using six of the currently most eye colour informative single nucleotide polymorphisms (HERC2 rs12913832, OCA2 rs1800407, SLC24A4 rs12896399, SLC45A2 (MATP) rs16891982, TYR rs1393350, and IRF4 rs12203592) according to a previous study, while the accuracy in predicting non-blue and non-brown eye colours is considerably lower. In an effort to vigorously assess the IrisPlex system at the international level, testing was performed by 21 laboratories in the context of a collaborative exercise divided into three tasks and organised by the European DNA Profiling (EDNAP) Group of the International Society of Forensic Genetics (ISFG). Task 1 involved the assessment of 10 blood and saliva samples provided on FTA cards by the organising laboratory together with eye colour phenotypes; 99.4% of the genotypes were correctly reported and 99% of the eye colour phenotypes were correctly predicted. Task 2 involved the assessment of 5 DNA samples extracted by the host laboratory from simulated casework samples, artificially degraded, and provided to the participants in varying DNA concentrations. For this task, 98.7% of the genotypes were correctly determined and 96.2% of eye colour phenotypes were correctly inferred. For Tasks 1 and 2 together, 99.2% (1875) of the 1890 genotypes were correctly generated and of the 15 (0.8%) incorrect genotype calls, only 2 (0.1%) resulted in incorrect eye colour phenotypes. The voluntary Task 3 involved participants choosing their own test subjects for IrisPlex genotyping and eye colour phenotype inference, while eye photographs were provided to the organising laboratory and judged; 96% of the eye colour phenotypes were inferred correctly across 100 samples and 19 laboratories. The high success rates in genotyping and eye colour phenotyping clearly demonstrate the reproducibility and the robustness of the IrisPlex assay as well as the accuracy of the IrisPlex model to predict blue and brown eye colour from DNA. Additionally, this study demonstrates the ease with which the IrisPlex system is implementable and applicable across forensic laboratories around the world with varying pre-existing experiences.

Pre-PCR processing in bioterrorism preparedness: improved diagnostic capabilities for laboratory response networks.

Diagnostic DNA analysis using polymerase chain reaction (PCR) has become a valuable tool for rapid detection of biothreat agents. However, analysis is often challenging because of the limited size, quality, and purity of the biological target. Pre-PCR processing is an integrated concept in which the issues of analytical limit of detection and simplicity for automation are addressed in all steps leading up to PCR amplification--that is, sampling, sample treatment, and the chemical composition of PCR. The sampling method should maximize target uptake and minimize uptake of extraneous substances that could impair the analysis--so-called PCR inhibitors. In sample treatment, there is a trade-off between yield and purity, as extensive purification leads to DNA loss. A cornerstone of pre-PCR processing is to apply DNA polymerase-buffer systems that are tolerant to specific sample impurities, thereby lowering the need for expensive purification steps and maximizing DNA recovery. Improved awareness among Laboratory Response Networks (LRNs) regarding pre-PCR processing is important, as ineffective sample processing leads to increased cost and possibly false-negative or ambiguous results, hindering the decision-making process in a bioterrorism crisis. This article covers the nature and mechanisms of PCR-inhibitory substances relevant for agroterrorism and bioterrorism preparedness, methods for quality control of PCR reactions, and applications of pre-PCR processing to optimize and simplify the analysis of various biothreat agents. Knowledge about pre-PCR processing will improve diagnostic capabilities of LRNs involved in the response to bioterrorism incidents.

Purification of crime scene DNA extracts using centrifugal filter devices.

BACKGROUND: The success of forensic DNA analysis is limited by the size, quality and purity of biological evidence found at crime scenes. Sample impurities can inhibit PCR, resulting in partial or negative DNA profiles. Various DNA purification methods are applied to remove impurities, for example, employing centrifugal filter devices. However, irrespective of method, DNA purification leads to DNA loss. Here we evaluate the filter devices Amicon Ultra 30 K and Microsep 30 K with respect to recovery rate and general performance for various types of PCR-inhibitory crime scene samples. METHODS: Recovery rates for DNA purification using Amicon Ultra 30 K and Microsep 30 K were gathered using quantitative PCR. Mock crime scene DNA extracts were analyzed using quantitative PCR and short tandem repeat (STR) profiling to test the general performance and inhibitor-removal properties of the two filter devices. Additionally, the outcome of long-term routine casework DNA analysis applying each of the devices was evaluated. RESULTS: Applying Microsep 30 K, 14 to 32% of the input DNA was recovered, whereas Amicon Ultra 30 K retained 62 to 70% of the DNA. The improved purity following filter purification counteracted some of this DNA loss, leading to slightly increased electropherogram peak heights for blood on denim (Amicon Ultra 30 K and Microsep 30 K) and saliva on envelope (Amicon Ultra 30 K). Comparing Amicon Ultra 30 K and Microsep 30 K for purification of DNA extracts from mock crime scene samples, the former generated significantly higher peak heights for rape case samples (P-values <0.01) and for hairs (P-values <0.036). In long-term routine use of the two filter devices, DNA extracts purified with Amicon Ultra 30 K were considerably less PCR-inhibitory in Quantifiler Human qPCR analysis compared to Microsep 30 K. CONCLUSIONS: Amicon Ultra 30 K performed better than Microsep 30 K due to higher DNA recovery and more efficient removal of PCR-inhibitory substances. The different performances of the filter devices are likely caused by the quality of the filters and plastic wares, for example, their DNA binding properties. DNA purification using centrifugal filter devices can be necessary for successful DNA profiling of impure crime scene samples and for consistency between different PCR-based analysis systems, such as quantification and STR analysis. In order to maximize the possibility to obtain complete STR DNA profiles and to create an efficient workflow, the level of DNA purification applied should be correlated to the inhibitor-tolerance of the STR analysis system used.

Assessment of forensic findings when alternative explanations have different likelihoods-"Blame-the-brother"-syndrome.

Assessment of forensic findings with likelihood ratios is for several cases straightforward, but there are a number of situations where contemplation of the alternative explanation to the evidence needs consideration, in particular when it comes to the reporting of the evidentiary strength. The likelihood ratio approach cannot be directly applied to cases where the proposition alternative to the forwarded one is a set of multiple propositions with different likelihoods and different prior probabilities. Here we present a general framework based on the Bayes' factor as the quantitative measure of evidentiary strength from which it can be deduced whether the direct application of a likelihood ratio is reasonable or not. The framework is applied on DNA evidence in forms of an extension to previously published work. With the help of a scale of conclusions we provide a solution to the problem of communicating to the court the evidentiary strength of a DNA match when a close relative to the suspect has a non-negligible prior probability of being the source of the DNA.