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.

In traditional forensic DNA casework, the inclusion or exclusion of individuals who may have contributed to an item of evidence may be dependent upon the assumption on the number of individuals from which the evidence arose. Typically, the determination of the minimum number of contributors (NOC) to a mixture is achieved by counting the number of alleles observed above a given analytical threshold (AT); this technique is known as maximum allele count (MAC). However, advances in polymerase chain reaction (PCR) chemistries and improvements in analytical sensitivities have led to an increase in the detection of complex, low template DNA (LtDNA) mixtures for which MAC is an inadequate means of determining the actual NOC. Despite the addition of highly polymorphic loci to multiplexed PCR kits and the advent of interpretation softwares which deconvolve DNA mixtures, a gap remains in the DNA analysis pipeline, where an effective method of determining the NOC needs to be established. The emergence of NOCIt -- a computational tool which provides the probability distribution on the NOC, may serve as a promising alternative to traditional, threshold- based methods. Utilizing user-provided calibration data consisting of single source samples of known genotype, NOCIt calculates the a posteriori probability (APP) that an evidentiary sample arose from 0 to 5 contributors. The software models baseline noise, reverse and forward stutter proportions, stutter and allele dropout rates, and allele heights. This information is then utilized to determine whether the evidentiary profile originated from one or many contributors. In short, NOCIt provides information not only on the likely NOC, but whether more than one value may be deemed probable. In the latter case, it may be necessary to modify downstream interpretation steps such that multiple values for the NOC are considered or the conclusion that most favors the defense is adopted. Phase I of this study focused on establishing the minimum number of single source samples needed to calibrate NOCIt. Once determined, the performance of NOCIt was evaluated and compared to that of two other methods: the maximum likelihood estimator (MLE) -- accessed via the forensim R package, and MAC. Fifty (50) single source samples proved to be sufficient to calibrate NOCIt, and results indicate NOCIt was the most accurate method of the three. Phase II of this study explored the effects of template mass and sample complexity on the accuracy of NOCIt. Data showed that the accuracy decreased as the NOC increased: for 1- and 5-contributor samples, the accuracy was 100% and 20%, respectively. The minimum template mass from any one contributor required to consistently estimate the true NOC was 0.07 ng -- the equivalent of approximately 10 cells' worth of DNA. Phase III further explored NOCIt and was designed to assess its robustness. Because the efficacy of determining the NOC may be affected by the PCR kit utilized, the results obtained from NOCIt analysis of 1-, 2-, 3-, 4-, and 5-contributor mixtures amplified with AmpFlstr® Identifiler® Plus and PowerPlex® 16 HS were compared. A positive correlation was observed for all NOCIt outputs between kits. Additionally, NOCIt was found to result in increased accuracies when analyzed with 1-, 3-, and 4-contributor samples amplified with Identifiler® Plus and with 5-contributor samples amplified with PowerPlex® 16 HS. The accuracy rates obtained for 2-contributor samples were equivalent between kits; therefore, the effect of amplification kit type on the ability to determine the NOC was not substantive. Cumulatively, the data indicate that NOCIt is an improvement to traditional methods of determining the NOC and results in high accuracy rates with samples containing sufficient quantities of DNA. Further, the results of investigations into the effect of template mass on the ability to determine the NOC may serve as a caution that forensic DNA samples containing low-target quantities may need to be interpreted using multiple or different assumptions on the number of contributors, as the assumption on the number of contributors is known to affect the conclusion in certain casework scenarios. As a significant degree of inaccuracy was observed for all methods of determining the NOC at severe low template amounts, the data presented also challenge the notion that any DNA sample can be utilized for comparison purposes. This suggests that the ability to detect extremely complex, LtDNA mixtures may not be commensurate with the ability to accurately interpret such mixtures, despite critical advances in software-based analysis. In addition to the availability of advanced comparison algorithms, limitations on the interpretability of complex, LtDNA mixtures may also be dependent on the amount of biological material present on an evidentiary substrate.

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Produzidos pela inserção ou deleção de um ou mais nucleotídeos em um ou ambos os cromossomos homólogos, os marcadores InDel são os polimorfismos mais abundantes depois dos SNPs. Apresentam baixa taxa de mutação quando comparados a outros polimorfismos, ampla distribuição no genoma, simples e rápida genotipagem e frequências alélicas diferentes entre populações distintas. Além disso, uma aplicação relevante dos InDel é a possibilidade de se direcionar a amplificação de sequências alvo específicas, através do desenho de primers que se ligam com regiões de interesse e amplifiquem alelos específicos em um lócus, capacidade esta que melhora a detecção e quantificação do DNA, inclusive do DNA fetal na circulação materna. Apesar de marcadores autossômicos serem os mais utilizados para estudos de mistura em populações, o cromossomo X tem ganhado significativa importância em estudos populacionais e de genética forense, graças ao seu padrão especial de transmissão, a uma deriva genética menor e à sua menor taxa de recombinação. Dessa forma, marcadores genéticos do cromossomo X têm potencial de apresentar parâmetros forenses mais eficientes do que autossômicos em casos de investigação complexa de parentesco, complementar informações fornecidas pelos autossomos e permitir identificar haplótipos com mais facilidade. Neste trabalho, propomos a seleção e descrição formal de um número adequado de lócus do tipo InDel bialélicos ligados ao cromossomo X que possam ser aplicados em estudos de análises populacionais, a fim de complementar os trabalhos de padronização de conjuntos de InDel autossômicos anteriormente desenvolvidos neste laboratório. Foram selecionados sete lócus do cromossomo X que formam dois blocos de haplótipos. Primers flanqueadores e inserção-específicos desenhados para estes lócus tiveram suas condições de PCR convencional padronizadas e foram aplicados na análise fenotípica de uma amostra da população urbana brasileira, composta por 80 trios (Mãe-Filha(o)-Pai), para estimar parâmetros populacionais e de interesse forense. A estimativa das frequências alélicas dos lócus revelou que o alelo inserção é mais frequente para todos os lócus estudados, com exceção do lócus MIDX550. Foram encontrados 60 haplótipos, com o mais frequente correspondendo a 7,5%. Os parâmetros forenses gerados com base nestes lócus estudados mostraram-se eficazes, todos com heterozigose acima de 0,2. Não é possível fornecer afirmações definitivas sobre a paternidade com estes lócus, uma vez que não foi encontrado nenhuma Probabilidade Média de Paternidade W > 99,99%. Entretanto, se combinados com outras informações que aumentem seu poder de discriminação, integrados em painéis e combinados com marcadores autossômicos, os lócus analisados neste trabalho são capazes de fornecer alta informatividade na identificação humana, ancestralidade e quantificação e dosagem de misturas desbalanceadas de DNA. / Originated by the insertion or deletion of one or more nucleotides in one or both homologous chromosomes, InDel markers are the polymorphisms more abundant after SNPs. They have a low mutation rate when compared to other polymorphisms, wide spreading in the genome, simple and rapid genotyping and distinct allelic frequencies between different populations. In addition, a relevant application of the InDel is the possibility to target a specific sequence in PCR, by designing primers that anneal to the regions of interest and amplify specifics alleles in that locus, which improves the DNA detection and quantification in DNA mixtures situations, including fetal DNA in the maternal circulation. Despite being the autosomic markers the most used for DNA mixtures in populations studies, the X chromosome has significant importance in population and forensic genetics studies, thanks to its special transmission pattern, to a lesser genetic drift and to the lower recombination rate. Thus, the genetic markers of the X chromosome have the potential to generate more efficient parameters than the autosomal ones in cases of complex kinship invetigation, adding information in that provided by the autosomes and allow haplotypes with more easily identification. In this work, we propose the selection and formal description of a set of biallelic X-InDel loci that can be applied in population analysis, to complement the standardization work of autosomes InDel sets previously developed in this laboratory. Seven loci of the X chromosome forming two haplotypes blocks were selected. We standardized flanking and insertion-specific primers designed for these loci in conventional PCR conditions. Then, these primers were applied in the phenotypic analysis of a brazilian urban population sample, composed of 80 trios (MotherChild-Father), to estimate the population and forensic interest parameters. The estimation of allele frequencies of loci showed that the insertion allele is more frequent for all the loci studied, with the exception of the MIDX550 locus. Sixty haplotypes were found, most frequently corresponding to 7.5%. The forensic parameters generated for these loci were effective, all with heterozygosis above 0.2. It was not possible to provide statements about paternity with these loci, since no Average Paternity Probability W> 99.99% was found. However, combined with other information that increase its power of discrimination, integrated in panels and combined with the autosomal markers, the analyzed loci in this work are able to provide high informativity for human identification, ancestrality and quantification and dosage of unbalanced DNA mixtures.