Browsing by Subject "massively parallel sequencing"
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Item A Continuous Statistical Phasing Framework for the Analysis of Forensic Mitochondrial DNA Mixtures(MDPI, 2021-01-20) Smart, Utpal; Cihlar, Jennifer Churchill; Mandape, Sammed N.; Muenzler, Melissa; King, Jonathan L.; Budowle, Bruce; Woerner, August E.Despite the benefits of quantitative data generated by massively parallel sequencing, resolving mitotypes from mixtures occurring in certain ratios remains challenging. In this study, a bioinformatic mixture deconvolution method centered on population-based phasing was developed and validated. The method was first tested on 270 in silico two-person mixtures varying in mixture proportions. An assortment of external reference panels containing information on haplotypic variation (from similar and different haplogroups) was leveraged to assess the effect of panel composition on phasing accuracy. Building on these simulations, mitochondrial genomes from the Human Mitochondrial DataBase were sourced to populate the panels and key parameter values were identified by deconvolving an additional 7290 in silico two-person mixtures. Finally, employing an optimized reference panel and phasing parameters, the approach was validated with in vitro two-person mixtures with differing proportions. Deconvolution was most accurate when the haplotypes in the mixture were similar to haplotypes present in the reference panel and when the mixture ratios were neither highly imbalanced nor subequal (e.g., 4:1). Overall, errors in haplotype estimation were largely bounded by the accuracy of the mixture's genotype results. The proposed framework is the first available approach that automates the reconstruction of complete individual mitotypes from mixtures, even in ratios that have traditionally been considered problematic.Item Development of a Comprehensive Massively Parallel Sequencing Panel of Single Nucleotide Polymorphism and Short Tandem Repeat Markers for Human Identification(2015-08-01) Warshauer, David H.; Bruce Budowle; Ranajit Chakraborty; Bobby L. LaRueMassively parallel sequencing (MPS) technologies allow for the detection of an unparalleled amount of genetic information with unprecedented speed and relative ease. These qualities make the technology desirable for generating DNA profiles that may be uploaded into forensic offender, arrestee, and family reference database files. This doctoral dissertation research was conducted under the hypothesis that MPS, with its exquisitely high throughput, can provide a system whereby reference samples can be typed for a large battery of markers, providing more discrimination power for forensic DNA typing and offering increased opportunities to develop investigative leads. The design and implementation of large marker panels for the typing of reference samples will reduce debates on the best core markers for forensic utility, generate innovation because focus will not be solely on a core set of autosomal STRs, promote the development of better systems that can analyze more challenging samples, and enable sharing of data across laboratories worldwide. The primary goal of this project was to develop the capability of typing reference samples for a large battery of markers: 84 autosomal, Y-chromosome, and X-chromosome short tandem repeats (STRs), Amelogenin, and 275 human identity single nucleotide polymorphisms (SNPs), in a single multiplex analysis. To that end, a bioinformatic software package, STRait Razor, was developed to detect STR alleles in raw MPS data. A proof-of-concept study was performed to evaluate the efficacy of using MPS to type forensically relevant markers, using a PCR multiplex-based SNP assay. The proposed comprehensive capture-based MPS panel then was designed and extensively tested. Finally, the benefits of the additional genetic data afforded by MPS, as opposed to traditional methods, were illustrated through the characterization of intra-repeat nucleotide variation within Y-chromosome STR alleles. The results of this dissertation research indicate that MPS is capable of providing robust genetic data from a wide variety of forensically-relevant STR and SNP loci in a single analysis. To date, the comprehensive MPS panel developed during the course of these studies is the most potentially informative assay for reference sample testing for human identification.Item Developmental Validation of a MPS Workflow with a PCR-Based Short Amplicon Whole Mitochondrial Genome Panel(MDPI, 2020-11-13) Cihlar, Jennifer Churchill; Amory, Christina; Lagace, Robert; Roth, Chantal; Parson, Walther; Budowle, BruceFor the adoption of massively parallel sequencing (MPS) systems by forensic laboratories, validation studies on specific workflows are needed to support the feasibility of implementation and the reliability of the data they produce. As such, the whole mitochondrial genome sequencing methodology-Precision ID mtDNA Whole Genome Panel, Ion Chef, Ion S5, and Converge-has been subjected to a variety of developmental validation studies. These validation studies were completed in accordance with the Scientific Working Group on DNA Analysis Methods (SWGDAM) validation guidelines and assessed reproducibility, repeatability, accuracy, sensitivity, specificity to human DNA, and ability to analyze challenging (e.g., mixed, degraded, or low quantity) samples. Intra- and inter-run replicates produced an average maximum pairwise difference in variant frequency of 1.2%. Concordance with data generated with traditional Sanger sequencing and an orthogonal MPS platform methodology was used to assess accuracy, and generation of complete and concordant haplotypes at DNA input levels as low as 37.5 pg of nuclear DNA or 187.5 mitochondrial genome copies illustrated the sensitivity of the system. Overall, data presented herein demonstrate that highly accurate and reproducible results were generated for a variety of sample qualities and quantities, supporting the reliability of this specific whole genome mitochondrial DNA MPS system for analysis of forensic biological evidence.Item Highly Informative Short Tandem Repeat Markers for Enhanced DNA Mixture Deconvolution(2018-08-01) Novroski, Nicole M. M.; Bruce Budowle; Robert C. Barber; Bobby L. LaRueDNA typing in forensic genetics relies on amplification of short tandem repeat (STR) markers using the polymerase chain reaction (PCR), subsequently allele sizes are determined for each locus, using capillary electrophoresis (CE) and fluorescent detection. The resulting profiles are compared to reference sample profiles or to query existing profiles, such as those stored in the FBI Combined DNA Index System, to develop investigative leads to help solve crimes. The success of commercial STR kits to facilitate analysis of challenging samples has led to a demand to analyze increasingly complex DNA mixtures. Low quantity/low quality DNA samples have become commonplace in casework, but the interpretation of the resultant DNA profiles continues to remain challenging. Massively parallel sequencing (MPS) for typing forensically-relevant STR loci has dramatically enhanced the ability to identify allele diversity due to sequence variation within STR repeat and flanking regions. Sequence variation within the currently utilized STR loci for forensic genetic analysis is quite large. However, recent studies have demonstrated that some of the current core CODIS loci are devoid of repeat and/or flanking region sequence variation, minimizing the relative information via MPS for these STRs. Thus, novel STRs with increased sequence variation should be sought to facilitate mixture deconvolution. The primary goal of this research was to identify and characterize STR genetic variation, which in turn would allow for the development of a novel panel of highly polymorphic STR markers (referred to as the STR DECoDE panel; STR DNA EnhanCed DEconvolution panel) that is capable of deconvolving simple to complex DNA mixture samples better than current systems. A list of candidate STRs was generated by mining the 1000 Genomes Project using the criteria of 1) a repeat size of at least 4 nucleotides; 2) a minimum of 80% locus heterozygosity; and 3) generally an allele length spread of 10 nominal alleles or less. A preliminary panel of 248 candidate markers was designed, and a bioinformatics pipeline for MPS was created and implemented to assess the analytical performance and biological properties of each STR. The STR DECoDE panel is comprised of 73 of the 248 STRs that displayed the highest heterozygosity. This panel was compared to the current core CODIS loci regarding an ability to resolve in silico two-person mixtures from 443 population samples comprising three US populations. Additionally, each of the 73 loci was extensively characterized for its underlying genetic variation, and population genetic analyses were performed. The results of this dissertation research indicate that the STR DECoDE panel improves upon current mixture deconvolution efforts by employing markers that allow for better allele resolution of component contributors in a mixed DNA sample. The DECoDE panel loci offer a substantial degree of diversity compared with the current core CODIS STR loci used for forensic identity typing. In turn, use of this panel could facilitate complex downstream statistical modeling (probabilistic genotyping) and subjective interpretation that are currently utilized for analysis of DNA mixture samples in forensic laboratories. Finally, integration of DECoDE STR loci into current multiplexes will allow the field of forensic genetic investigation to increase the number of resolved genotypes in mixed samples being compared to reference and suspect profiles, and expand the DNA database by increasing the number of samples uploaded. The benefit to society from this revolutionary application will be an increase in the number of investigative leads and the overall resolution of more crimes.Item Pharmacogenetics of Select Genes in the Opiate Metabolism and Response Pathways(2018-08) Wendt, Frank R.; Budowle, Bruce; Phillips, Nicole R.; LaRue, Bobby L.; Luedtke, Robert R.; Clark, Abbot F.Pharmacogenetics and pharmacogenomics aim to elucidate the underlying genetic variation contributing to adverse drug reactions, differential enzyme activity, and resulting appropriate drug dosage on the individual and population levels. Studies with this goal in mind typically rely on targeted genotyping of select single nucleotide polymorphisms (SNPs) and/or insertion/deletion (INDEL) polymorphisms within a gene that have demonstrated significant association with the rate of drug absorption, distribution, excretion, and/or metabolism. This approach may enable association and characterization of clinically relevant polymorphisms with a phenotype of interest and may provide guidance regarding appropriate prescription medication practices for medical professionals. Additionally, these data, namely those of the cytochrome p450, family 2, subfamily D, polypeptide 6 gene (CYP2D6), have contributed to identifying cause and/or manner of death in some death investigations which initially were negative medico-legal autopsies. Though invaluable to medical genetics, the chemistry of targeted genotyping approaches, including genome-wide association studies and SNP-targeted massively parallel sequencing, inherently lack the capability to discover novel or rare polymorphisms that may be enriched in pharmacogenetically-valuable cohorts (i.e., individuals who have experienced idiosyncratic responses to codeine/morphine). Relatively recently, the pharmacogenetics community has utilized comprehensive (i.e., full-gene) and/or combinatorial (i.e., multi-gene) genetic studies using multiple genes whose protein products are involved in a drug metabolism/response pathway. The multi-gene approach is demonstrably more successful in predicting phenotypic expressions and more efficacious for patient outcomes compared to as single-gene approach. While mainly elucidating multigenic profiles of psychiatric drugs and disorders, to date, it is reasonable to consider that more efficacious patient outcomes can be achieved using the pathways responsible for other pathologies or drug metabolism and response pathways. The goal of this dissertation was to develop a comprehensive genetic profiling system using the full gene region of five genes that have demonstrated associations between specific SNPs and opiate metabolism/response. The in silico phases of this dissertation aimed to characterize the genes encoding CYP2D6, uridine diphosphate glucuronosyltransferase family 1 polypeptide B7 (UGT2B7), adenosine triphosphate (ATP) binding cassette subfamily B number 1 (ABCB1; p-glycoprotein; multidrug resistance protein 1), opioid receptor mu 1 (OPRM1; MOR1), and catechol-O-methyltransferase (COMT) on the individual SNP and full-gene haplotype levels. Subsequent empirical evaluation of these genes was performed on a cohort of deceased tramadol-exposed Finns using targeted genotyping and exome-wide analyses. This dissertation research has 1) described previously uncharacterized individual SNPs that are associated with the metabolism of tramadol to its primary metabolite, O-desmethyltramadol; 2) evaluated the utility of full-gene information for predicting metabolizer phenotype; 3) produced a massively parallel sequencing panel to genotype opiate-metabolism genes in a more comprehensive and combinatorial manner than previously attempted; 4) demonstrated the increased predictive capabilities of a multigenic opiate metabolizer phenotyping system; and 5) identified additional genetic targets that may have predictive phenotypic value.