<div>Forensic entomology is a subdiscipline of entomology that involves the use of insect behavior and developmental data to aid in criminal investigations. Genetic data has become increasingly important to the field as there has been a push for DNA-based species identification methods of forensically relevant insects. Genetic data can also elucidate population structure and relatedness of these insects, and such knowledge can contribute to the development of more specific datasets for insects in different regions. The first study presented here investigated the phylogenetics of sister species <i>Lucilia cuprina</i> and <i>Lucilia sericata</i> to identify possible subspecies divisions and issues with DNA-based identifications in the United States. The initial aim of this study was to identify genetic differences between specimens of <i>L. cuprina</i> that preferred live versus carrion flesh. Flies collected from Indiana, USA and South Africa were sequenced and analyzed. Upon sequencing of the genes <i>COI, Period,</i> and <i>28s,</i> our results indicated that <i>L. cuprina</i> from Indiana possess a unique combination of nuclear and mitochondrial haplotypes that suggest a unique lineage, possibly indicating modern hybridization with <i>L. sericata. </i>The inability of both nuclear and mitochondrial genes to distinguish between <i>L. cuprina</i> and <i>L. sericata</i> raises questions about the capabilities of DNA-based species identifications within this genus. Additionally, the inability of these genes to distinguish between specimens that preferred live versus carrion flesh highlights a need for continued research of these behavioral differences. The second study presented here investigated the population structure and relatedness of house flies in the American southwest in relation to a civil lawsuit where neighbors of a poultry farm alleged that flies were emanating from the farm to their homes. <i>Musca domestica</i> (house fly) specimens were collected from the chicken farm and from locations in varying directions and distances from the farm. Amplified fragment length polymorphism (AFLP) analysis was performed and the data were used in a number of analyses. Population reallocation simulations generally indicated that samples from different locations were not genetically different enough from other locations to allocate to their true origin population over others. Kinship analysis showed differences in samples collected in a later season that indicate a genetic bottleneck over time. Population structure analysis indicated the presence of two intermixing genetic populations in the dataset. AMOVA revealed that the majority of genetic variation laid within, rather than among, populations. A Mantel test revealed no significant correlation between genetic and geographic distances. These results indicate that the <i>M. domestica</i> population in this region of southwestern America is large and intermixing, with no clear genetic distinctions between specimens collected at the poultry farm versus the surrounding locations. In regard to the civil lawsuit, it was not possible to conclude that the flies did not emanate from the poultry farm. In a broader perspective, these data can be utilized to develop pest management strategies in this region. Overall, the data from both studies presented here will be useful to forensic investigations, development of more specific and detailed data and identification techniques, and pest control measures.</div>

In forensic casework analysis it is necessary to obtain genetic profiles from increasingly smaller amounts of biological material left behind by perpetrators of crime. The ability to obtain profiles from trace biological evidence is demonstrated with so-called ‘touch DNA evidence’ which is perceived to be the result of DNA obtained from shed skin cells transferred from donor to an object or person during physical contact. However, the current method of recovery of trace DNA involves cotton swabs or adhesive tape to sample an area of interest. This "blindswabbing" approach may result in the recovery of biological material from different individuals resulting in admixed DNA profiles which are often difficult to interpret. Profiles recovered from these samples are reported to be from shed skin cells with no biological basis for that determination. A specialized approach for the isolation of single or few cells from ‘touch DNA evidence’ is necessary to improve the analysis and interpretation of recovered profiles. Here we describe the development of optimized and robust micro volume PCR reactions (1-5 μL) to improve the sensitivity and efficiency of ‘touch DNA’ analysis. These methods will permit not only the recovery of the genetic profile of the donor of the biological material, but permit an identification of the tissue source of origin using mRNA profiling. Results showed that the 3.5 uL amplification volume, a fraction of the standard 25 uL amplification volume, was the most ideal volume for the DNA assay, as it had very minimal evaporation with a 50% profile recovery rate at a single cell equivalent input (~5 pg) with reducing amplification volume alone. Findings for RNA showed that by reducing both amplification steps, reverse transcriptase PCR (20 uL) and body fluid multiplex PCR (25 uL), to iv 5 uL, ideal results were obtained with an increase in sensitivity and detection of six different body fluids down to 50 pg. Once optimized at the trace level, the assays were applied to the collection of single and few cells. DNA findings showed that about 40% of a full profile could be recovered from a single buccal cell, with nearly 80% of a full profile recovered from only two cells. RNA findings from collected skin particles of "touched" surfaces showed accurate skin detection down to 25 particles and detection in one clump of particles. The profiles recovered were of high quality and similar results were able to be replicated through subsequent experiments. More studies are currently underway to optimize these developed assays to increase profile recovery at the single cell level. Methods of doing so include comparing different locations on touched surfaces for highest bio-particle recovery and the development of physical characteristics of bio-particles that would provide the most ideal results

<p>Ancestry is a complex and layered concept, but it must be operationalized for its objective use in genetic studies. Critical decisions in research analyses, clinical practice, and forensic investigations are based on genetic ancestry inference. For example, in genetic association studies for clinical and applied research, investigators may need to isolate one population of interest from a worldwide dataset to avoid false positive results, or in human identification, ancestry inferences can help reveal the identity of unknown DNA evidence by narrowing down a suspect list. Many studies seek to improve ancestry inference for these reasons. The research presented here offers valuable resources for exploring and improving genetic ancestry inference and intelligence toward identity. </p> <p>First, analyses with ‘big data’ in genomics is a resource-intensive task that requires optimization. Therefore, this research introduces a suite of automated Snakemake workflows, <em>Iliad</em>, that was developed to give the research community an easy-to-learn, hands-off computational tool for genomic data processing of multiple data formats. <em>Iliad</em> can be installed and run on a Google Cloud Platform remote server instance in less than 20 minutes when using the provided installation code in the ReadTheDocs documentation. The workflows support raw data processing from various genetic data types including microarray, sequence, and compressed alignment data, as well as performing micro-workflows on variant call format (VCF) files to merge data or lift over variant positions. When compared to a similar workflow, <em>Iliad </em>completed processing one sample’s raw paired-end sequence reads to a human-legible VCF file in 7.6 hours which was three-times faster than the other workflow. This suite of workflows is paramount towards building reference population panels from human whole-genome sequence (WGS) data which is useful in many research studies including imputation, ancestry estimation, and ancestry informative marker (AIM) discovery.</p> <p>Second, there are persistent challenges in ancestry inference for individuals of the Middle East, especially with the use of AIMs. This research demonstrates a population genomics study pertaining to the Middle East, novel population data from Lebanon (n=190), and an unsupervised genetic clustering approach with WGS data from the 1000 Genomes Project and Human Genome Diversity Project. These efforts for AIM discovery identified two single nucleotide polymorphisms (SNPs) based on their high allelic frequency differences between the Middle East and populations in Eurasia, namely Europe and South/Central Asia. These candidate AIMs were evaluated with the most current and comprehensive AIM panel to date, the VISAGE Enhanced Tool (ET), using an external validation set of Middle Eastern WGS data (n=137). Instead of relying on pre-defined biogeographic ancestry labels to confirm the accuracy of validation sample ancestry inference, this research produced a deep, unsupervised ADMIXTURE analysis on 3,469 worldwide WGS samples with nearly 2 million independent SNPs (r2 < 0.1) which provided a genetic “ground truth”. This resulted in 136/137 validation samples as Middle East and provided valuable insights toward reference samples with varying co-ancestries that ultimately affects the classification of admixed individuals. Novel deep learning methods, specifically variational autoencoders, were introduced for visualizing one hundred percent of the genetic variance found using these AIMS in an alternative method to PCA and presents distinct population clusters in a robust ancestry space that remains static for the projection of unknown samples to aid in ancestry inference and human identification. </p> <p>Third, this research delves into a craniofacial study that makes improvements toward key intelligence information about physical identity by exploring the relationship between dentition and facial morphology with an advanced phenotyping approach paired with robust dental parameters used in clinical practice. Cone-beam computed tomography (CBCT) imagery was used to analyze the hard and soft tissue of the face at the same time. Low-to-moderate partial correlations were observed in several comparisons of dentition and soft tissue segments. These results included partial correlations of: i) inter-molar width and soft tissue segments nearest the nasal aperture, the lower maxillary sinuses, and a portion of the upper cheek, and ii) of lower incisor inclination and soft tissue segments overlapping the mentolabial fold. These results indicate that helpful intelligence information, potentially leading towards identity in forensic investigations, may be present where hard tissue structures are manifested in an observable way as a soft tissue phenotype. This research was a valuable preliminary study that paves the way towards the addition of facial hard tissue structures in combination with external soft tissue phenotypes to advance fundamental facial genetic research. Thus, CBCT scans greatly add to the current facial imagery landscape available for craniofacial research and provide hard and soft tissue data, each with measurable morphological variation among individuals. When paired with genetic association studies and functional biological experiments, this will ultimately lead to a greater understanding of the intricate coordination that takes place in facial morphogenesis, and in turn, guide clinical orthodontists to better treatment modalities with an emphasis on personalized medicine. Lastly, it aids intelligence methodologies when applied within the field of forensic anthropology.</p>

Criminology is a science dealing with the protection of citizens and state from infringement. Criminology uses mostly biological or genetic methods for crime detection. Forensic traces which are collected by forensic experts on the scene are the key items of those methods. Forensic genetics is among the most important forensic subdisciplines. Forensic genetics uses DNA analysis for identification. The main aims of this study are description and importance of biological, anthropological and genetic methods in criminology, different ways of forensic identification, division and collection of forensic traces, characterization and course of forensic DNA analysis and DNA profiling. Key words Criminology, forensic methods, forensic identification, forensic trace, forensic biology, anthropology and genetics, information systems, forensic DNA analysis, DNA profile.

Des recherches en sciences judiciaires ont montré récemment une possible corrélation entre le temps d’entreposage d’échantillons de fluides corporels et la dégradation de l’ARN dans ceux-ci. Le moment où une tache a été déposée sur une scène de crime peut être important pour déterminer la pertinence d’un échantillon dans une enquête. Dans ce mémoire, nous rapportons les profils de dégradation de quatre ARN différents mesurés par RT-qPCR, soit l’ARN ribosomique 18S et les ARNm de la β-actine, de la glyceraldehyde-3-phosphate déhydrogénase et de la cyclophiline A, obtenus de taches de sang, de salive et de sperme, entreposés à la température de la pièce ou au congélateur à -80°C sur une période de 6 mois. Nos résultats montrent une faible variation interindividuelle pour le sang et le sperme, mais une différence importante entre les donneurs pour la salive. De plus, le profil de dégradation est semblable pour tous les transcrits, mais diffère entre les fluides. La congélation des échantillons stabilise les ARN avant leur analyse. Finalement, la quantité d’ARN détecté est en relation avec le temps d’entreposage et pourrait être utilisée afin d’estimer l’âge des échantillons lorsque l’impact des conditions d’entreposage sur la dégradation de l’ARN sera mieux connu. / Recent studies in forensic science have shown a possible correlation between the degradation rate of some RNA transcripts and the age of bloodstains. The time of deposition of a stain can be of major importance to determine the relevance of a sample in a forensic investigation. In this thesis, we describe the degradation profiles of the 18S ribosomal RNA and the β-actin, glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A mRNAs, measured by RT-qPCR and obtained from dried blood, semen and saliva stains stored at room temperature or frozen at -80°C up to 6 months. Our results showed low inter-individual variation for blood and semen stains, but a high variation was observed between donors for saliva. Moreover, degradation profile of each transcripts was similar, but differed between fluids. Freezing samples prevented RNA degradation over time. Finally, RNA quantity was in relation with the time of storage and could be used to estimate the time since deposition of a stain when the effects of various storage conditions on RNA degradation profiles will be better documented. / Projet de recherche réalisé en collaboration avec la section Biologie/ADN du Laboratoire de sciences judiciaires et de médecine légale (LSJML) de Montréal.

Des recherches en sciences judiciaires ont montré récemment une possible corrélation entre le temps d’entreposage d’échantillons de fluides corporels et la dégradation de l’ARN dans ceux-ci. Le moment où une tache a été déposée sur une scène de crime peut être important pour déterminer la pertinence d’un échantillon dans une enquête. Dans ce mémoire, nous rapportons les profils de dégradation de quatre ARN différents mesurés par RT-qPCR, soit l’ARN ribosomique 18S et les ARNm de la β-actine, de la glyceraldehyde-3-phosphate déhydrogénase et de la cyclophiline A, obtenus de taches de sang, de salive et de sperme, entreposés à la température de la pièce ou au congélateur à -80°C sur une période de 6 mois. Nos résultats montrent une faible variation interindividuelle pour le sang et le sperme, mais une différence importante entre les donneurs pour la salive. De plus, le profil de dégradation est semblable pour tous les transcrits, mais diffère entre les fluides. La congélation des échantillons stabilise les ARN avant leur analyse. Finalement, la quantité d’ARN détecté est en relation avec le temps d’entreposage et pourrait être utilisée afin d’estimer l’âge des échantillons lorsque l’impact des conditions d’entreposage sur la dégradation de l’ARN sera mieux connu. / Recent studies in forensic science have shown a possible correlation between the degradation rate of some RNA transcripts and the age of bloodstains. The time of deposition of a stain can be of major importance to determine the relevance of a sample in a forensic investigation. In this thesis, we describe the degradation profiles of the 18S ribosomal RNA and the β-actin, glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A mRNAs, measured by RT-qPCR and obtained from dried blood, semen and saliva stains stored at room temperature or frozen at -80°C up to 6 months. Our results showed low inter-individual variation for blood and semen stains, but a high variation was observed between donors for saliva. Moreover, degradation profile of each transcripts was similar, but differed between fluids. Freezing samples prevented RNA degradation over time. Finally, RNA quantity was in relation with the time of storage and could be used to estimate the time since deposition of a stain when the effects of various storage conditions on RNA degradation profiles will be better documented.

<div><div>In the forensic science community, there is a vast need for tools to help assist investigations when standard DNA profiling methods are uninformative. Methods such as Forensic DNA Phenotyping (FDP) and proteomics aims to help this problem and provide aid in investigations when other methods have been exhausted. FDP is useful by providing physical appearance information, while proteomics allows for the examination of difficult samples, such as hair, to infer human identity and ancestry. To create a “biological eye witness” or develop informative probability of identity match statistics through proteomically inferred genetic profiles, it is necessary to constantly strive to improve these methods. </div><div><br></div><div>Currently, two developmentally validated FDP prediction assays, ‘HIrisPlex’ and ‘HIrisplex-S’, are used on the capillary electrophoresis to develop a phenotypic prediction for eye, hair, and skin color based on 41 variants. Although highly useful, these assays are limited in their ability when used on the CE due to a 25 variant per assay cap. To overcome these limitations and expand the capacities of FDP, we successfully designed and validated a massive parallel sequencing (MPS) assay for use on both the ThermoFisher Scientific Ion Torrent and Illumina MiSeq systems that incorporates all HIrisPlex-S variants into one sensitive assay. With the migration of this assay to an MPS platform, we were able to create a semi-automated pipeline to extract SNP-specific sequencing data that can then be easily uploaded to the freely accessible online phenotypic prediction tool (found at https://hirisplex.erasmusmc.nl) and a mixture deconvolution tool with built-in read count thresholds. Based on sequencing reads counts, this tool can be used to assist in the separation of difficult two-person mixture samples and outline the confidence in each genotype call.<br></div><div><br></div><div>In addition to FDP, proteomic methods, specifically in hair protein analysis, opens doors and possibilities for forensic investigations when standard DNA profiling methods come up short. Here, we analyzed 233 genetically variant peptides (GVPs) within hair-associated proteins and genes for 66 individuals. We assessed the proteomic methods ability to accurately infer and detect genotypes at each of the 233 SNPs and generated statistics for the probability of identity (PID). Of these markers, 32 passed all quality control and population genetics criteria and displayed an average PID of 3.58 x 10-4. A population genetics assessment was also conducted to identify any SNP that could be used to infer ancestry and/or identity. Providing this information is valuable for the future use of this set of markers for human identification in forensic science settings. </div></div><div><br></div>

Indiana University-Purdue University Indianapolis (IUPUI) / Short tandem repeat (STR) analysis is currently the most common method for processing biological forensic evidence. STRs are highly polymorphic and allow for a strong statistical power of discrimination when comparing deoxyribonucleic acid (DNA) samples. Since sample testing and court proceedings occur months, if not years apart, samples must be stored appropriately in the event additional testing is needed. There are generally accepted methods to store DNA extracts long-term; however, one universally recognized method does not exist. The goal of this project was to examine various methods of storage and make recommendations for a universal storage method that maintained DNA integrity over time. Four variables were evaluated: storage buffer, storage temperature, initial storage concentration and the effects of repeated freeze-thaw cycles. DNA quantity was assessed using real-time polymerase chain reaction and DNA quality was evaluated using STR genotyping. Overall, the Tris-EDTA (TE) buffer outperformed nuclease free water as a long-term storage buffer for DNA extracts. Stock tubes stabilized concentration better than single use aliquots when eluted with TE while tube type was not significant when water was the buffer. For samples stored in TE, temperature had no effect on DNA integrity over time, but samples stored in water were largely affected at room temperature. Additionally, the greater the initial DNA concentration, the less likely it was to degrade in water. As a result of this research, DNA extracts from forensic samples should be stored long-term in TE buffer with a minimum concentration of 0.1 ng/μL. When water is the buffer, frozen storage is recommended.

Democratic and accountable policing is one of the hallmarks of democracy. In a healthy democracy, a police service exists to protect and support the rights of its community by successfully listening to those who are laying complaints and resolving to assist them by bringing the perpetrators to the grinding wheels of justice. Encouraging and ensuring that police officials utilise the most modern means of investigation such as the DNA technology, provides the necessary balance to the exercise of professional discretion and heightened conviction rate by the police officials. The utilisation of biological evidence in investigation of rape cases is such a modern intervention – a way of providing insulation against internal and external interference with the proper and successful investigation of rape cases. / Forensic Investigation / M. Tech. (Forensic Investigation)

Democratic and accountable policing is one of the hallmarks of democracy. In a healthy democracy, a police service exists to protect and support the rights of its community by successfully listening to those who are laying complaints and resolving to assist them by bringing the perpetrators to the grinding wheels of justice. Encouraging and ensuring that police officials utilise the most modern means of investigation such as the DNA technology, provides the necessary balance to the exercise of professional discretion and heightened conviction rate by the police officials. The utilisation of biological evidence in investigation of rape cases is such a modern intervention – a way of providing insulation against internal and external interference with the proper and successful investigation of rape cases. / Forensic Investigation / M. Tech. (Forensic Investigation)