Choice of population database for forensic DNA profile analysis

When evaluating the weight of evidence (WoE) for an individual to be a contributor to a DNA sample, an allele frequency database is required. The allele frequencies are needed to inform about genotype probabilities for unknown contributors of DNA to the sample. Typically databases are available from several populations, and a common practice is to evaluate the WoE using each available database for each unknown contributor. Often the most conservative WoE (most favourable to the defence) is the one reported to the court. However the number of human populations that could be considered is essentially unlimited and the number of contributors to a sample can be large, making it impractical to perform every possible WoE calculation, particularly for complex crime scene profiles. We propose instead the use of only the database that best matches the ancestry of the queried contributor, together with a substantial F_ST adjustment. To investigate the degree of conservativeness of this approach, we performed extensive simulations of one- and two-contributor crime scene profiles, in the latter case with, and without, the profile of the second contributor available for the analysis. The genotypes were simulated using five population databases, which were also available for the analysis, and evaluations of WoE using our heuristic rule were compared with several alternative calculations using different databases. Using F_ST = 0.03, we found that our heuristic gave WoE more favourable to the defence than alternative calculations in well over 99% of the comparisons we considered; on average the difference in WoE was just under 0.2 bans (orders of magnitude) per locus. The degree of conservativeness of the heuristic rule can be adjusted through the F_ST value. We propose the use of this heuristic for DNA profile WoE calculations, due to its ease of implementation, and efficient use of the evidence while allowing a flexible degree of conservativeness.     

Enhancing accurate data collection in mass fatality kinship identifications: Lessons learned from Hurricane Katrina

A mass fatality DNA identification effort is a complex process in which direct matching and kinship analysis is used for identifying human remains. Kinship DNA identification is an important tool in the identification process in which victim's DNA profiles are compared to the profiles of “known” biologically related reference samples. Experience from the 9/11 World Trade Center DNA identification efforts showed that forms used to record biological relationships are important and that inaccurately documented information may hamper the kinship analysis and DNA identification process. In the identification efforts following Hurricane Katrina, a Family and/or Donor Reference Collection (FDRC) form was used as a means to document the reported relationship between the reference DNA donor and the purported missing individual. This FDRC form was developed based upon lessons learned from 9/11 and the Tsunami identification efforts. This paper analyses the effectiveness of the FDRC form used in the Hurricane Katrina kinship DNA identification efforts and proposes an improved sample collection form for kinship and other donor reference samples. The data presented can be used to enhance the accuracy of the data collection process through an improved sample collection form, streamlining the DNA kinship identification process and decreasing the burden on valuable resources.     

Street politics in Damascus: Kinship and other social categories as bases of political action, 1830–1841

The study examines popular politics in Damascus during the 1830s with a focus on kinship and other social categories that served as bases for political action. It is based on a close reading of one text, an anonymous Arabic chronicle known as ‘Historical Memoirs’ (Mudhakkirāt tārīkhiyya), which is analysed as a repertoire of contemporary social and political concepts. This analysis reveals an ideology of ‘localist’ resistance against the centralising state and its ‘loyalist’ allies in Damascus. Kinship played an important role in this struggle on the social plane (e.g. by using family networks for political ends) as well as on the cultural plane (e.g. through the use of kinship metaphors or through criticism of kinship ties in politics). It is concluded that the function of kinship in Damascene politics can only be understood in the context of other social categories such as factionalism, religious affiliation, class and ethnicity.     

Choosing relatives for DNA identification of missing persons

DNA-based analysis is integral to missing person identification cases. When direct references are not available, indirect relative references can be used to identify missing persons by kinship analysis. Generally, more reference relatives render greater accuracy of identification. However, it is costly to type multiple references. Thus, at times, decisions may need to be made on which relatives to type. In this study, pedigrees for 37 common reference scenarios with 13 CODIS STRs were simulated to rank the information content of different combinations of relatives. The results confirm that first-order relatives (parents and fullsibs) are the most preferred relatives to identify missing persons; fullsibs are also informative. Less genetic dependence between references provides a higher on average likelihood ratio. Distant relatives may not be helpful solely by autosomal markers. But lineage-based Y chromosome and mitochondrial DNA markers can increase the likelihood ratio or serve as filters to exclude putative relationships.     

Evaluating probabilistic genotyping for low-pass DNA sequencing

Most genomic methods consider the sample genotype. Data are evaluated at some location, and if the signal strength is sufficient, a genotype call is made. Conversely, sites that lack sufficient signal are treated as missing data. Such methods for genotype calling are binary, and this dichotomy limits genomic analyses to relatively high-coverage (and high-cost) massively parallel sequencing (MPS) data. It follows that bioinformatic methods that rely on genotypes may not be ideal for trace DNA samples, such as those sometimes encountered in forensic investigations, but even when applicable such analyses can be expensive. However, there are some genomic analyses where having many uncertain genotypes (with measured uncertainty) assayed over the entirety of the genome may be more powerful than current multi-locus approaches that consider a limited number of well-characterized markers. Methods for such problems may rely on genotype likelihood, which expresses the likelihood of alternative genotype calls in addition to the most likely call. One application that can benefit from genotype likelihoods is kinship analysis. NgsRelate is a bioinformatic tool that infers pairwise relatedness using a probabilistic genotyping framework, which accommodates the uncertainty associated with genotype calls for low-pass MPS data. Here, NgsRelate was used to infer kinship coefficients from low-pass whole genome sequencing data from a known pedigree. Multiple samples in a titration series (ranging from 50 ng to 0.5 ng) on a single MPS S4 flow cell were assessed. A reproducible scientific bioinformatic workflow was developed to evaluate kinship coefficients considering up to 3rd degree relatives. NgsRelate was found to provide robust assessments of kinship. Further, the use of low-pass MPS data provides a more cost-effective way to conduct forensic investigations.     

DNAc: A clustering method for identifying kinship relations between DNA profiles using a novel similarity measure

After decades of refinement, DNA testing methods have become essential tools in forensic sciences. They are essentially based on likelihood ratio test principle, which is utilized specifically, by using as prior knowledge the allele frequencies in the population, to confirm or refute a given kinship hypothesis made on two genotypes. This makes these methods ill suited when allele frequencies or kinship hypotheses are unavailable. In this paper, we introduce DNAc, a new clustering methodology for DNA testing based on a new similarity measure that allows an accurate retrieval of the degree of relatedness among two or more genotypes, without relying on kinship hypotheses or allele frequencies in the population. We used DNAc in analyzing microsatellite DNA sequences distributed among 12 genotypes from normal individuals from two distinct families. The results show that DNAc accurately determines kinship among genotypes and further gathers them in the appropriate kinship groups.     

The contribution of DNA to the disaster victim identification (DVI) effort

As part of the disaster victim identification (DVI) response to the 2009 Victorian bushfires disaster, a number of scientific disciplines contributed to the human identification process--forensic pathology, anthropology and odontology, as well as fingerprinting and DNA profiling. The DNA laboratory received 182 post-mortem (PM) samples from 120 DVI cases and 236 reference samples corresponding to 163 missing persons (and two non-DVI cases). DNA analysis yielded full DNA profiles for 102 DVI cases and 190 ante-mortem (AM) samples (relating to all 163 missing persons), respectively. Subsequent comparison of DNA profiles, through direct and kinship matching, resulted in the submission of 76 DNA reports to the DVI Reconciliation Centre which assisted in the identification of 67 deceased. This paper describes the contribution of DNA analysis towards the DVI response to the 2009 Victorian bushfires disaster.     

Molecular identification of the potentially forensically relevant cluster flies Pollenia rudis (Fabricius) and Pollenia vagabunda (Meigen) (Diptera: Polleniidae) — non-recorded species in Algeria

Cluster flies are represented by the genus Pollenia Robineau-Desvoidy, 1830 of the family Polleniidae Brauer and Bergenstamm, 1889. Their larvae are known to be internal parasites or predators of earthworms. Herein, we report for the first time the occurrence of the cluster flies Pollenia rudis Fabricius, 1794 and Pollenia vagabunda (Meigen, 1826) (Diptera: Polleniidae) on carcasses in Algeria and identify them through DNA barcoding. A region of the mitochondrial cytochrome c oxidase I gene (COI) was amplified and sequenced. Genetic distances were determined. A phylogenetic tree was constructed with the maximum parsimony method using 10 000 bootstrap replicates. A total number of 157 adults of P. rudis were collected together with 325 adults of Pollenia vagabunda. The occurrence of Pollenia on animal carcasses does not seem to be correlated with a particular stage of decomposition. All the sequences were correctly identified using the BLASTn tool from the GenBank database and the BOLD identification engine. Intra- and interspecific sequence divergence values were less than 1% and greater than 3%, respectively. COI barcodes obtained from this study were robust enough to identify and distinguish unambiguously between P. rudis and P. vagabunda. In the tree-based analysis, the cluster flies were all assigned to their respective species separately from each other confirming the morphological identification. These results provide DNA barcodes that contribute to the growth of reference databases and allow fast and accurate identification.     

Utility validation of extraction of genomic DNA from hard tissues, bone and nail, using PrepFiler™ Forensic DNA Extraction Kit

STR profiling using hard tissues obtained from a severely decomposed body is sometimes a laborious work. There is now on a market a new DNA extraction kit, PrepFiler™ Forensic DNA Extraction Kit (AppliedBiosystems), and we tested it for missing persons. Postmortem intervals ranged from weeks to several years. Fifteen bone fragments and eleven nails were used in this report. Genomic DNA was quantified by QuantiFiler^® DUO Quantification Kit (AppliedBiosystems), and STRs were analyzed using AmpFlSTR^® Identifiler^® PCR Amplification Kit (AppliedBiosystems). The profiling of 16 STR loci was successful in all nail samples. However, STR profiling was successful in only 6 of 15 bone materials. Nine cases failed to analyze STR polymorphisms using another DNA extraction kit, the QIAamp DNA Mini Kit (QIAGEN). For bone samples, it seems that STR profiling depends on the quality of samples.     

Kinship assignment with the ForenSeq™ DNA Signature Prep Kit: Sources of error in simulated and real cases

Kinship recognition between anonymous DNA samples is becoming a relevant issue in forensics, more so with the increasing number of DNA profiles in databanks. Also, NGS-based genotyping is being increasingly used in routine personal identification, to simultaneously type large numbers of markers of different kind. In the present work, we explored computationally and experimentally the performance of the ForenSeq™ DNA Signature Prep Kit in identifying the true relationship between two anonymous samples, distinguishing it from other possible relationships. We analyzed with Familias R series of 10,000 pairs with 9 different simulated relationships, corresponding to different degrees of autosomal sharing. For each pair we obtained likelihood ratios for five kinship hypotheses vs. unrelatedness, and used their ranking to identify the preferred relationship. We also typed 21 subjects from two pedigrees, representing from parent-child to 4th cousins relationships. As expected, the power for identifying the true relationship decays in the order of autosomal sharing. Parent-child and full siblings can be robustly identified against other relationships. For half-siblings the chance of reaching a significant conclusion is already small. For more distant relationships the proportion of cases correctly and significantly identified is 10% or less. Bidirectional errors in kinship attribution include the suggestion of relatedness when this does not exist (10–50%), and the suggestion of independence in pairs of individuals more than 4 generations apart (25–60%). The real cases revealed a relevant effect of genotype miscalling at some loci, which could only be partly avoided by modulating the analysis parameters. In conclusion, with the exception of first degree relatives, the kit can be useful to inform additional investigations, but does not usually provide probatory results.     

Kinship,ritual kinship and political milieus in an alpine Valley in 19th century

In my paper I will present some results about ritual kinship and political mobilization of popular groups in an alpine Valley: the Val de Bagnes, in the Swiss canton of Valais. There are two major reasons to choose the Val de Bagnes for our inquiry about social networks: the existence of sharp political and social conflicts during the 18th and the 19th century and the availability of almost systematic genealogical data between 1700 and 1900.

The starting point of my research focuses on this question: what role did kinship and ritual kinship play in the political mobilization of popular groups and in the organization of competing factions? This question allows us to shed light on some other uses and meanings of ritual kinship in the local society. Was ritual kinship a significant instrument for economic cooperation? Or was it a channel for patronage or for privileged social contacts? The analysis highlights the importance of kinship and godparentage for the building of homogeneous social and political networks.

If we consider transactions between individuals, the analysis of 19th century Val de Bagnes gives the impression of quite open networks. Men and women tried to diversify their relations in order to avoid strong dependency from powerful patrons. Nevertheless, when we consider the family networks, we can notice that most relations took place in a structured social space or a specific “milieu”, were intense contacts enhanced trust, although political allegiances and social choices were not fully predictable on the basis of such preferential patterns.

In a politically conflictual society, like 19th century Bagnes, ritual kinship interacted with kinship solidarities and ideological factors shaping dense social networks mostly based on a common political orientation. Such milieus sustained the building of political factions, which show surprising stability over time. In this sense, milieus are important factors to understand political and religious polarization in 19th century Switzerland.     

Bhaṭṭa Rāmakaṇṭha’s Elaboration of Self-Awareness (svasaṃvedana), and How it Differs from Dharmakīrti’s Exposition of the Concept

The article considers what happened to the Buddhist concept of self-awareness (svasa?vedana) when it was appropriated by ?aiva Siddhānta. The first section observes how it was turned against Buddhism by being used to attack the momentariness of consciousenss and to establish its permanence. The second section examines how self-awareness differs from I-cognition (ahampratyaya). The third section examines the difference between the kind of self-awareness elaborated by Rāmaka??ha (‘reflexive awareness’) and a kind elaborated by Dharmakīrti (‘intentional self-awareness’). It is then pointed out that Dharmakīrti avails himself not only of intentional self-awareness but also of reflexive awareness. Some remarks on the relationship between these two strands of Dharmakīrtian Buddhism are offered. The conclusion points out that although self-awareness occurs in Buddhism as inextricably linked with anātmavāda, the doctrine of no-self, and sākāravāda, the view that the forms we perceive belong not to external objects but to consciousness, it is used by Rāmaka??ha to refute both of these views. An appendix addresses the problem of how precisely to interpret Dharmakīrti’s contention that conceptual cognition is non-conceptual in its reflexive awareness of itself.     

Analysis of genetic polymorphisms associated with the presence of freckles for phenotypic prediction

The prediction of externally visible characteristics (EVCs) is a commonly used practice by the forensic sciences as an important resource in the investigation of criminal cases in which the identity of perpetrators or victims is unknown or even to recognize decomposed cadavers. With this purpose, genetic markers associated with pigmentation traits have been widely studied by forensic scientists and, nowadays, it is possible to predict phenotypic characteristics such as hair, eyes and skin colour, as well as the presence of skin freckles by analysing single nucleotide polymorphisms (SNPs). In this study, we analysed the association of six SNPs located in pigmentation genes to the presence of freckles in individuals from the Brazilian population for forensic DNA phenotyping. The study was based within the context of a larger project on a population sample of 534 adult Brazilians of both sexes and different skin colours. DNA was extracted from peripheral blood and genotyped using the TaqMan® OpenArray® Real-Time PCR System (ThermoFischer Scientific) technique. Statistical analyses were carried out with the R software (version 4.0.2). As for the results obtained, three SNPs were shown to be statistically associated to the freckling, rs12203592, rs1800404 and rs222847, with CT, AG and AA genotypes being the main contributors, respectively. Variables such as sex of the individuals and skin colour were found to also contribute to the manifestation of this pigmentation trait. Further statistical analyses will be carried out to evaluate the possibility of using the SNPs in this study for phenotyping prediction of the Brazilian population, improving existing DNA phenotyping models in forensic sciences.     

DNA identification of skeletal remains by investigator’s intuition

In June 2006 a decapitated woman was found in a parking area of the motorway in the area of Prato (Florence). Since the body was beheaded and no victim’s documents or objects were present at the crime scene, identification at that time was impossible. However, DNA profile from woman’s bones were collected. In the same year (2003), a mother had reported her daughter's disappearance but the two events were not related at that time. About ten years later the mother’s DNA profile was finally acquired for a genetic identification of another girl’s body found in the Ferrara area. These genetic profiles were completely discordant. All these genetic comparisons were carried out on behalf of the prosecutors of the cities involved in the findings of the bodies and in the disappearance complaints, but due to the lack of a database the events remained disconnected. In January 2017, the head of the scientific police of Prato who had followed the investigation and questioned the mother of the missing girl found about ten years later, suggested to the magistrate to order the comparison of the mother's DNA with the genetic profile of the bones found in 2006. This comparison finally allowed the identification of the missing daughter.This story highlights the importance of having forensic DNA database to search for missing persons and how the investigator's intuition can play a key role in resolving criminal cases. In fact, databases of unknown bodies and relatives of missing persons were created in Italy as a part of national DNA database just at the beginning of 2018.     

Robust detection of cow and female buffalo DNA through Real-Time PCR assay

Cow, Bos taurus, and female buffalo, Bubalus bubalis, are considered sacred animals that are a part of rural livelihood in India. The purity of products from these bovine species has significant sentimental implications in the dairy and meat industry. Therefore, the mitochondrial DNA and the sex origin, targeting the X and Y chromosomes from these bovine species, were selected to design three multiplex real-time probe PCR assays: Hi-PCR® Cow Detection Kit (MBPCR184), Hi-PCR® Buffalo Detection Kit (MBPCR185) and Hi-PCR® Cattle Sex Determination Kit (MBPCR186). Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines were followed to perform different studies using reference control DNAs. An Internal Reagent Control (IRC) was part of every assay, thus ensuring a successful reaction. The assays were 100% specific, with no cross-amplification of the two bovine species. The amplification of the X chromosomal target was observed for male and female DNAs, whereas Y chromosome amplification was observed only for the male DNA. The assays were 100% specific to the target genes in these organisms with no non-specificity towards any other targets or organisms. The limit of detection for sex determination was 0.01 ng/µl, whereas the differential capability of the assay was 3 copies/µl and 30 copies/µl for Bos taurus and Bubalus bubalis, respectively. The assays were reproducible at 1 ng/µl genomic DNA with 95% CI. The assays are open and compatible with other brands of Real-Time PCR systems used in forensic labs. The experiments presented here verify that the developed real-time PCR assays are robust, produce reliable and reproducible results for detection and differentiation of Bos taurus and Bubalus bubalis and their sex even at low DNA concentrations.     

Missing Data Problems in Criminological Research: Two Case Studies

This paper considers the problem of missing data in two circumstances commonly confronted by criminologists. In the first circumstance, there is missing data due to subject attrition—some cases drop out of a study. In this context, analysts are frequently interested in examining the association between an independent variable measured at time t(x _t ) and an outcome variable that is measured at time t + 1(y _t + 1); the problem is that the outcome variable is only observed for those cases which do not drop out of the study. In the second circumstance there is missing data on an independent variable of interest for typical reasons (i.e., the respondent did not wish to answer a question or could not be located). In this case, researchers are interested in estimating the association between the independent variable with missing data and an outcome variable that is fully observed. Criminologists often handle these two missing data problems by conducting analyses on the subsample of observations with complete data. In this paper, we explore this problem with two case studies and we then illustrate the use of methods that directly address the uncertainty produced by missing data.     

Examples of kinship analysis where Profiler Plus™ was not discriminatory enough for the identification of victims using DNA identification

The identification of the victims of the 2009 Victorian bushfires disaster, as in other mass disasters, relied on a number of scientific disciplines - including DNA analysis. As part of the DVI response, DNA analysis was performed to assist in the identification of victims through kinship (familial matching to relatives) or direct (self source of sample) matching of DNA profiles. The majority of the DNA identifications made (82%) were achieved through kinship matching of familial reference samples to post mortem (PM) samples obtained from the victims. Although each location affected by the bushfires could be treated as a mini-disaster (having a small closed-set of victims), with many such sites spread over vast areas, DNA analysis requires that the short tandem repeat (STR) system used be able to afford enough discrimination between all the DVI cases to assign a match. This publication highlights that although a 9-loci multiplex was sufficient for a DVI of this nature, there were instances that brought to light the short comings of using a 9-loci multiplex for kinship matching--particularly where multiple family members are victims. Moreso it serves to reinforce the recommendation that a minimum of 12 autosomal STR markers (plus Amelogenin) be used for DNA identification of victims which relies heavily on kinship matching.     

Start-up of the criminal genetic database in Mendoza,Argentina

The usefulness of DNA databases is widely known and demonstrated. After the successful experiences of the UK and the USA the creation of databases increased rapidly around the world. In Latin America the implementation was slower and more problematic, with Chile and Uruguay being the first to implement them. In Argentina the problems were greater and more persistent.Although the lack of legislation or applicable laws is a generalized problem, the most difficult one to overcome was the lack of decision, interest and resources by those responsible at an institutional level.In 2016, Mendoza province modified its database law by creating the “Registro Provincial de Huellas Genéticas Digitalizadas” which allowed the process of construction and consolidation to begin. From January 2017 all prisoners, convicted and imputed of all types of crimes began to be sampled. This made the database to grow rapidly, reaching 13.821 samples in that year.During 2018, in addition to the daily imputed individuals, we began with the sampling from all the Mendoza Police Department, including the Scientific Police that deals with the crime scene. At present the database has a total of 40.652 individuals.In August 2018, the FBI's CODIS system was installed, and later the data loading process began. In 12 months we have reached 87 match or hits of which 46 correspond to sexual assault, 17 to robbery, 16 to homicides and the rest to other cases. Given that within the sexual abuse cases we were able to identify several serial sexual offenders, the 87 hits allowed clarifying 174 criminal cases.These results reaffirm the potential of the databases and gives a light of hope for victims of crime.In this work we present the advances and challenges that we faced in a chronological order.     

Automated evaluation of approximate matching algorithms on real data

Bytewise approximate matching is a relatively new area within digital forensics, but its importance is growing quickly as practitioners are looking for fast methods to screen and analyze the increasing amounts of data in forensic investigations. The essential idea is to complement the use of cryptographic hash functions to detect data objects with bytewise identical representation with the capability to find objects with bytewise similar representations.Unlike cryptographic hash functions, which have been studied and tested for a long time, approximate matching ones are still in their early development stages and evaluation methodology is still evolving. Broadly, prior approaches have used either a human in the loop to manually evaluate the goodness of similarity matches on real world data, or controlled (pseudo-random) data to perform automated evaluation.This work's contribution is to introduce automated approximate matching evaluation on real data by relating approximate matching results to the longest common substring (LCS). Specifically, we introduce a computationally efficient LCS approximation and use it to obtain ground truth on the t5 set. Using the results, we evaluate three existing approximate matching schemes relative to LCS and analyze their performance.