<p> When an evidential DNA profile does not match identified suspects or profiles from available databases, further DNA analyses targeted at inferring the possible ancestral origin and phenotypic characteristics of the perpetrator could yield valuable information. Single Nucleotide Polymorphisms (SNPs), the most common form of genetic polymorphisms, have alleles associated with specific populations and/or correlated to physical characteristics. With this research, single base primer extension (SBE) technology was used to develop a 50 SNP assay designed to predict ancestry among the primary U.S. populations (African American, East Asian, European, and Hispanic/Native American), as well as pigmentation phenotype. The assay has been optimized to a sensitivity level comparable to current forensic DNA analyses, and has shown robust performance on forensic-type samples. In addition, three prediction models were developed and evaluated for ancestry in the U.S. population, and two models were compared for eye color prediction, with the best models and interpretation guidelines yielding correct information for 98% and 100% of samples, respectively. Also, because data from additional DNA markers (STR, mitochondrial and/or Y chromosome DNA) may be available for a forensic evidence sample, the possibility of including this data in the ancestry prediction was evaluated, resulting in an improved prediction with the inclusion of STR data and decreased performance when including mitochondrial or Y chromosome data. Lastly, the possibility of using next-generation sequencing (NGS) to genotype forensic STRs (and thus, the possibility of a multimarker multiplex incorporating all forensic markers) was evaluated on a new platform, with results showing the technology incapable of meeting the needs of the forensic community at this time.</p>

Thesis (M.S.)--Michigan State University. Forensic Science, 2008. / Title from PDF t.p. (Proquest, viewed on Aug. 11, 2009) Includes bibliographical references (p. 77-84).

<p>Forensic science has gained popularity in the last few decades. Many new techniques are being studied and implemented. It includes a wide range of scientific disciplines, such as entomology, physics and biology. An important improvement to forensic science is the development of different DNA techniques which are implemented during an investigation, making DNA a gold standard for forensics. Most of the time DNA is mentioned it is in terms of human DNA, but there are microscopic organisms with useful DNA. In the last decade, with the development of next-generation sequencing (NGS), studies focusing on bacterial communities have been published, but fungal communities have not been extensively studied.</p> For this project, the potential of fungi in forensic science was investigated through three different studies. Human flora was looked at by NGS from thirty-seven human bodies with differing post-mortem intervals (PMIs). The communities were analyzed statistically and quantitatively, resulting in unique operational taxonomic units (OTUs) and genera which were only present in certain PMIs, and in some which were present through the entire PMI time scale. These attributes can help, not only to give a better view on human mycoflora during decomposition, but they can also help in determining fungal signatures during decomposition. These signatures can help in a PMI determination. Moreover, swine carcasses – the model animals for human forensic studies – were investigated as well to create a checklist of fungal flora after five months of winter decomposition in the West Lafayette, Indiana area. Furthermore, due to the increased importance of wildlife forensics, a wildlife study was also conducted using four wildlife species (mute swan, red tailed hawk, river otter, bobcat). The fungal flora from these species were compared within species at the beginning of the study and at skeletonization stage to look at any indicator fungal species and to create a general checklist for wildlife studies in the West Lafayette, Indiana area for future studies. Additionally, the fungal communities were compared across species as well.

<p>In terms of forensic entomology, one area that is scrutinized most is the estimation of a minimum post mortem interval (mPMI) based on insects that are present at a crime scene. The identification of the insects found at the scene if the first step to calculate a mPMI. However, currently there are no methods that can present the courts with accurate statistical error rates in identification, because the current methods are reliant on an expert’s use of a morphological key to identify the specimen, and this identification method does not produce a confidence value. This project aimed to test a method of identification using geometric morphometrics that can produce confidence intervals to provide to the courtrooms. </p><p> Before any identification could start, a standard preservation protocol was developed to ensure that all diagnostic features are preserved, and specimens can be identified in the same way. A clearing method was designed to clear specimens within 24hrs using potassium hydroxide, so they can be dissected and mounted the next day. The dissection of the specimens was a simple six-step procedure to split the mouth hooks, the cuticle and the posterior spiracle. This procedure ensures that all diagnostic features are preserved on a microscope slide.</p><p> With all of the features preserved, the microscope slide is photographed for storage and an investigator can perform geometric morphometrics to identify the insect. This study tested the application of geometric morphometrics to distinguish between three genera of Calliphoridae (<i>Calliphora</i>,<i>Lucilia</i>, <i>Phormia</i>), from three locations in the US (Delaware, Indiana, California). Results showed significant (p-value: <0.05) variation in shape among all genera. When genera were tested for shape differences based on location, these variations were also significant (p-value: <0.05). The implication of these results is that enough shape difference exists to distinguish between these genera and to distinguish between populations.</p>

<p>Obesity is a medical condition whose rates have seen a rise in both the United States and worldwide in recent decades. Numerous studies have been done to understand obesity, and through the use of GWAS researchers have been able to find multiple genetic factors that can contribute to obesity in mammals. One proposed cause of obesity are genetic impacts on cilia formation in the CNS, which causes downstream effects on food intake and energy expenditure, causing obesity via overeating and decreased activity.</p> <p>In the first half of this thesis, I describe a study, in collaboration with the Berbari Lab at IUPUI, that explored the human chromosome 16:53801550-53808600 (GRCh37/hg19), an intron of the FTO alpha-ketoglutarate Dependent Dioxygenase (<i>FTO</i>) gene for transcriptional regulators that impact BMI and obesity. First, using control DNA, PCR, and gel-electrophoresis, we created an assay for 44 primer sets (forward and reverse) covering the genomic region. After optimizing the assay, we then selected 111 human DNA samples across three weight groups (underweight, normal weight, and obese) to sequence using the assay. The samples were selected from subjects enrolled in the Walsh Lab FDP study. Sequencing was completed using the Illumina MiSeq System, and sequenced results were viewed using the Integrative Genomics Viewer (IGV) program. Variants that showed in the results were analyzed across and within the weight groups, and their locations were researched for previously known BMI or enhancer activity using online genome browsers Ensembl and UCSC Genome Browser.</p> <p>The results of this study revealed two SNPs, rs8055197 and rs11642015, that provided the best correlation with the weight categories among the samples. These results were consistent with literature that previously linked these single-nucleotide polymorphisms (SNPs) to obesity, particularly in relation to genes that are regulated by <i>FTO</i> (<i>CUX1</i>, <i>POMC</i>, and <i>IRX3/5</i>). Both SNPs lie within areas that show high enhancer activity in neural crest cells, important cells for cilia formation. Although there were SNPs in high LD within both regions, these two SNPs were chosen due to their homologous variant locations within the mouse genome (rs8055197 - GRCm38/mm10 8:91376305; rs11642015 - GRCm38/mm10 8:91375651), which provides a means of testing this obesity correlation, with a proposed enhancer relationship through <i>FTO</i>, in mouse models. </p> <p>In the second half of this thesis, I explored new methods for quantitatively defining natural hair color categories, and attempted to find novel SNPs impacting hair color in a GWAS using the quantitative values as phenotypes. In previous publications, the development and validation of the HIrisPlex-S Prediction Tool for hair prediction was made using categorical hair colors, which were defined and classified by individual researchers or lab personnel. Using spectrophotometer measurements and HSV color values, we used a machine-learning tool to objectively classify sample hair photos into natural hair color classes. We then used this quantitative data as the input phenotype for a GWAS, using both linear regression and linear mixed model regression, to search for new genetic associations with these objectively defined hair color classes. Lastly, we also measured correlations between these hair color phenotypes and a SNP array consisting of all currently known pigment SNPs cited in recent literature.</p> <p>The results of this study showed that quantitative values can be used as a means of classifying human hair colors. Both models used in the GWAS highlighted previously known SNPs that contributed to quantitative hair color. By utilizing the linear mixed model approach which has the ability to generate more power due to the normalization of hidden population structure, there was one near genome-wide significant SNP found that is currently not linked with hair color, rs2037697 (<i>IQUB</i>), which showed strong associations with light brown hair (p-value = 1.83192E-07), however this would need to be confirmed with increased numbers to validate its association. </p> <p>The results of the correlation analysis showed that SNPs cited as having impacts on pigmentation (eye, skin, and hair) also show strong associations with these objectively defined quantitative hair color classes and these rankings may prove very useful as the field moves towards quantitative hair color prediction.</p>

Indiana University-Purdue University Indianapolis (IUPUI) / Modern DNA profiling techniques have increased in sensitivity allowing for higher success in producing a DNA profile from limited evidence sources. However, this can lead to the amplification of more DNA profiles that do not get a hit on a suspect or DNA database and more mixture profiles. The work here aims to address or improve these consequences of current DNA profiling techniques. Based on allele-specific PCR and quantitative color measurements, a 24-SNP forensic phenotypic profile (FPP) assay was designed to simultaneously predict eye color, hair color, skin color, and ancestry, with the potential for age marker incorporation. Bayesian Networks (BNs) were built for model predictions based on a U.S sample population of 200 individuals. For discrete pigmentation traits using an ancestry influenced pigmentation prediction model, AUC values were greater than 0.65 for the eye, hair, and skin color categories considered. For ancestry using an all SNPs prediction model, AUC values were greater than 0.88 for the 5 continental ancestry categories considered. Quantitative pigmentation models were also built with prediction output as RGB values; the average amount of error was approximately 7% for eye color, 12% for hair color, and 8% for skin color. A novel sequencing method, methyl-RADseq, was developed to aid in the discovery of candidate age-informative CpG sites to incorporate into the FPP assay. There were 491 candidate CpG sites found that either increased or decreased with age in three forensically relevant xii fluids with greater than 70% correlation: blood, semen, and saliva. The effects of exogenous microbial DNA on human DNA profiles were analyzed by spiking human DNA with differing amounts of microbial DNA using the Promega PowerPlex® 16 HS kit. Although there were no significant effects to human DNA quantitation, two microbial species, B. subtilis and M. smegmatis, amplified an allelic artifact that mimics a true allele (‘5’) at the TPOX locus in all samples tested, interfering with the interpretation of the human profile. Lastly, the number of contributors of theoretically generated 2-, 3-, 4-, 5-, and 6-person mixtures were evaluated via allele counting with the Promega PowerPlex® Fusion 6C system, an amplification kit with the newly expanded core STR loci. Maximum allele count in the number of contributors for 2- and 3-person mixtures was correct in 99.99% of mixtures. It was less accurate in the 4-, 5-, and 6-person mixtures at approximately 90%, 57%, and 8%, respectively. This work provides guidance in addressing some of the limitations of current DNA technologies.

Indiana University-Purdue University Indianapolis (IUPUI) / Criminal investigation and the science behind evidence analysis is an ever- growing niche, and forensic DNA phenotyping (FDP) is no exception. For years the only information given to authorities regarding DNA found at a crime scene was STR analysis and matching to a comparative DNA sample from a known source. However, what happens when there is no suspect to compare DNA profiles, or the case involves a missing person where the only available piece of evidence is a biological sample found at the scene? Before FDP, not much could be done with the DNA sample and the investigation would be stalled. Now it is becoming possible to statistically predict an individual’s visual characteristics using FDP. Currently, with the use of Irisplex, HIrisplex, and HIrisplex-S, statistical analyses and predictions can be done for categorical eye, hair, and skin color by looking at specific genes and their associative SNPs, such as HERC2 and OCA2. The more that is understood about trait-determining genes and their functional significance with regards to our physical traits, the more phenotypes can be added to these prediction tools. In an effort to discover additional genes associated with human phenotypes, this study looked at thirty-two pigmentation-associated candidate genes, and eleven hair structure and morphology associated genes in owl monkey pelage samples. Although the samples were not of human origin, it is important to point out the high conservation between humans and their non-human primate relatives. The owl monkeys used in this study were helpful for tracking expression levels of genes controlling differentpigmentation and hair structure types, because each monkey had intra-individual variation in thickness and in coat color which allowed the generation of potential candidate genes for human investigation. Of the 43 total candidate genes analyzed, 36 had successful amplification, and 28 showed a significant difference in expression when comparing the different samples. The second part of this study was to compare quantitative characteristics of human hair in physical samples and two-dimensional (2D) photos. A test set of 45 individuals had 3-5 hairs from the vertex of their head plucked and analyzed, and a 2D photograph was taken of their scalp hair. The idea was to be able to make quantitative phenotypes in hair (such as hair width, amount of curl) from 2D imagery, when physical samples are not available for analysis. This is due to the fact that the majority of genotype-phenotype databases consist solely of photographic imagery, and seldom have hairs that can be microscopically prepared for analysis. Defining measurable phenotypes from 2D photos that strongly correlate with their physical counterparts allow for the generation of a more accurate phenotype for future genome wide association studies (GWAS) within and outside this laboratory that study hair thickness and hair curl. Three different quantitative phenotypes were compared between the microscopic and 2D photo- analysis.

Indiana University-Purdue University Indianapolis (IUPUI) / In the forensic science community, there is an immense need for tools to help assist investigations where conventional DNA profiling methods have been non-informative. Forensic DNA Phenotyping (FDP) aims to bridge that gap and aid investigations by providing physical appearance information when other investigative methods have been exhausted. To create a “biological eye witness”, it becomes necessary to constantly improve these methods in order to develop a complete and accurate image of the individual who left the sample. To add to our previous prediction systems IrisPlex and HIrisPlex, we have developed the HIrisPlex-S system for the all-in-one combined prediction of eye, hair, and skin color from DNA. The skin color prediction model uses 36 variants that were recently proposed for the accurate prediction of categorical skin color on a global scale, and the system is completed by the developmental validation of a 17-plex capillary electrophoresis (CE) genotyping assay that is run in conjunction with the HIrisPlex assay to generate these genotypes. The predicted skin color output includes Very Pale, Pale, Intermediate, Dark and Dark-to-Black categories in addition to categorical eye (Blue, Intermediate, and Brown) and hair (Black, Brown, Blond, and Red) color predictions. We demonstrate that the HIrisPlex-S assay performs in full agreement with guidelines from the Scientific Working Group on DNA Analysis Methods (SWGDAM), achieving high sensitivity levels with a minimum 63pg DNA input. In addition to adding skin color to complete the pigmentation prediction system termed HIrisPlex-S, we successfully designed a Massively Parallel Sequencing (MPS) assay to complement the system and bring Next Generation Sequencing (NGS) to the forefront of forensic DNA analyses methods. Using Illumina’s MiSeq system enables the generation of HIrisPlex-S’s 41 variants using sequencing data that has the capacity to xiii better deconvolute mixtures and perform with even more sensitivity and accuracy. This transition opens the door for a plethora of new ways in which this physical appearance assay can grow as sequencing technology is not limited by variant number; therefore, in essence many more traits have the potential to be included in this one assay design. For now, the HIrisPlex-S design of 41 variants using MPS is being fully assessed according to SWGDAM validated guidelines; therefore, this design paves the way for Forensic DNA Phenotyping to be used in any forensic laboratory. This new and improved HIrisPlex-S system will have a profound impact on casework, missing persons cases, and anthropological cases, as it is relatively inexpensive to run, HIrisPlex-S is easy to use, developmentally validated and one of the largest systems freely available online for physical appearance prediction from DNA using the freely available online web tool found at https://hirisplex.erasmusmc.nl/. Lastly, moving forward in our aim to include additional traits for prediction from DNA, we contributed to a large-scale research collaboration to unearth variants associated with hair morphology. 1026 samples were successfully sequenced using an inhouse MPS design at 91 proposed hair morphological loci. From this reaction, we were able to contribute to the identification of significant correlations between the SNPs rs2219783, rs310642 and rs80293268 with categorical hair morphology: straight, wavy or curly.

Indiana University-Purdue University Indianapolis (IUPUI) / 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 Lucilia cuprina and Lucilia sericata 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 L. cuprina that preferred live versus carrion flesh. Flies collected from Indiana, USA and South Africa were sequenced and analyzed. Upon sequencing of the genes COI, Period, and 28s, our results indicated that L. cuprina from Indiana possess a unique combination of nuclear and mitochondrial haplotypes that suggest a unique lineage, possibly indicating modern hybridization with L. sericata. The inability of both nuclear and mitochondrial genes to distinguish between L. cuprina and L. sericata 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. Musca domestica (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 M. domestica 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.

<p> Though it is well established that insects are attracted to decomposing vertebrates, little is known about the mechanism of attraction. Olfaction is thought to be the primary sense involved in resource location by necrophilous insects, and several studies have attempted to identify cadaveric-produced volatile organic compounds (VOCs) that contribute to insect attraction. This previous research has been completed almost exclusively in lab settings, with no field studies to verify these findings. </p><p> The goal of my thesis was to test necrophilous insect attraction to nine cadaveric VOCs (1-butanol, 1-pentanol, <i>p­</i>-xylene, <i>o</i>-xylene, ethanol, cyclohexanone, acetamide, dimethyl disulfide, and dimethyl trisulfide) in a field setting. Pitfall traps at six research locations were baited with the chemicals for one week. Specimens collected from the traps were identified and species assemblages were compared to those of a pig carcass. Replicates were conducted in fall 2018 and spring 2019. </p><p> Two chemicals were identified as important attractants for a variety of necrophilous insects: dimethyl disulfide and dimethyl trisulfide. These chemicals were especially attractive to <i>Phormia regina</i>, a blow fly (Diptera: Calliphoridae) found worldwide. VOC insect communities did not include all of the insects found on carcasses, possibly indicating the necessity of chemical blends for effective attraction. This study is only the second study to test necrophilous insect attraction in a field setting. </p>