Browsing by Author "Zascavage, Roxanne R."
Now showing 1 - 13 of 13
- Results Per Page
- Sort Options
Item Admixture Effects on Coevolved Metabolic Systems(2016-05-01) Zascavage, Roxanne R.; Planz, John V.; Barber, Robert C.; Clark, Abbot F.Oxidative phosphorylation (OXPHOS) is the primary energy generating system in eukaryotic organisms. Consequently, any malfunctions or disruptions in the pathway significantly impact fitness and health. Errors in energy production have been linked to cancer, Alzheimer’s disease, Parkinson’s disease, various neuropathies, and general aging and health degeneration over time. However, there is a fundamental gap in the understanding of the genetic causes of deficiencies in energy production. The complexes within the OXPHOS pathway are of mixed origin; while most subunit-coding genes are located within the nuclear genome, thirteen are coded for in the mitochondrial genome. There is strong evidence to support coadaptation between the two genomes in these OXPHOS gene regions in order to create tight protein interactions necessary for a functional energetic system. While the effect of separating coevolved alleles is not fully understood, hybrid studies have indicated decreased energy production when combining different ancestral nuclear and mitochondrial backgrounds in various species. This suggests the common human practice of interpopulation matings between ancestrally distinct groupings influences health and relative fitness. The primary hypothesis is that admixture creates maladaptive combinations of nuclear and mitochondrial alleles in the OXPHOS-coding genes that have adverse effects on the efficiency of energy production, leading to a decrease in relative fitness. This dissertation project has: 1) identified the effects of admixture on OXPHOS activity in Mus musculus populations, showing that high admixture leads to significantly lower basal respiration rates; and 2) assessed the genetic composition of the strains of Mus musculus evaluated to identify to cause of the loss of respiration in highly admixed mice. It was determined that there were no genetic anomalies present that could explain the observations, meaning the cause is likely not due to a mutation, but instead an undetected difference, such as cyto-nuclear incompatibility. It is recommended that further energetic and genetic studies be performed to identify the source of the deficiency. Mice obtained from Jackson Laboratories and a previously published genotype dataset [82] were used for experiments. Laboratory experiments included: Liver and heart extraction, tissue preparation and bioenergetics analysis, statistical analysis, and genetic analysis.Item Assessment of Oxford Nanopore Technologies as a Sequencing Platform for Increased Taxonomic Resolution of Microbial Communities(2019-05) Valenti, Kiana L.; Planz, John V.; Allen, Michael S.; Roane, Brandy M.; Zascavage, Roxanne R.Microbiome profiling for forensic identification is an emerging area of research. Profiling the microbiome of trace evidence for forensic cases will become more prevalent as more reliable, accurate, and efficient methodology is developed. Sequencing the V4 region of the 16S rRNA gene using second-generation sequencing methods has traditionally been the means of characterizing the microbiome of microbial samples, however this method often does not garner species-level resolution. In this study, the MinION[TM] device from Oxford Nanopore Technologies, an instrument capable of sequencing the entire length of the 16S rRNA gene was used. The main goal of this was to determine whether using the MinION[TM] device is a more viable means of obtaining deeper yet still accurate taxonomic resolution of a mixed microbial standard that contains species of bacteria that might be found in a casework sample.Item Cell-Free mtDNA Quantification in Alzheimer's Patients from the Mexican American Population(2020-05) House, Sara R.; Phillips, Nicole R.; Hodge, Lisa M.; Zascavage, Roxanne R.Abstract Background AD is a continuous problem in the 65+ population but it is especially challenging in the Hispanic population where not only is it more prevalent but more severe than Caucasian populations. This study explores the efficacy of using peripheral blood plasma as an alternative tissue for testing as well as the usefulness for future research assisting in identifying the population structure most at risk for developing AD based upon CF-mtDNA quantity results. Materials and Methods Samples tested included a total cohort (Mexican American and Caucasian) of 177 individuals (AD=45, MCI=74, NC=58). The Mexican American subset contained 92 individuals (AD=21, MCI=53, and NC=18). Peripheral blood plasma was collected from the TARCC biobank and quantified. CF-mtDNA was then tested for significance using correlation analyses, logistic and linear regression models. Results CF-mtDNA was significantly negatively correlated with education, age, sex, and hypertensive samples in the total and Mexican American populations. The greatest difference was expected to be in CF-mtDNA quantity from NC to AD samples. Instead, the most significant difference was between MCI and NC samples. As CF-mtDNA quantity increased, the MMSE and CDRSOB scores were less impaired. Conclusion In conclusion, CF-mtDNA is an easily accessible and easily tested molecular marker of diseases that are relevant to studies for cognitive decline. Although our findings were inconsistent with current literature, they bring to light the weight of confounding factors within limited sample studies. With the completion of the full sample set associated with this study, more power is needed to overcome these issues.Item Examining the skin and workplace microbiome following the return to the University of North Texas Health Science Center after self-isolation(2021-08) Khichi, Ophelia J.; Allen, Michael S.; Zhang, Yan; Zascavage, Roxanne R.; Rosales, ArmandoThe human skin microbiome contains trillions of microbiomes that colonize the human body. It is unknown how social distancing and an increase in handwashing due to the COVID-19 pandemic affect the bacterial communities that reside on the human hands & fomites. In this research, bacterial swabs from individual's dominant hand, forearm of their dominant hand, and a fomite from the workplace environment were taken, and the resident microflora, i.e., the skin microbiome, was investigated. The DNA from the samples were extracted and amplified by PCR. Samples were then pooled for sequencing of the V4 region of the 16S ribosomal RNA gene using Illumina's MiSeq platform and subsequently analyzed for community composition. The results were compared against each other to determine how an individual and a fomite's microbiome shifted following their return to work. Furthermore, the results were used to determine if individual's microbiome became more similar to each other as they returned to work in the same building.Item Optimization and Evaluation of qPCR Duplex Assay for mtDNA Copy Number Quantification(2020-05) Johnson, Gretchen A.; Planz, John V.; Phillips, Nicole R.; Zascavage, Roxanne R.Purpose: The mitochondrial genome (mtDNA) encodes thirteen essential proteins in oxidative phosphorylation, the cell's primary energy-generating process. Depending on the cell type and stage of development, each cell contains an average of 103 to 104 copies of mtDNA. Current methods of quantification of mtDNA copy number can be imprecise due to low efficiencies of assays and inherent imbalance of mtDNA copy number with nuclear DNA (nDNA) copy number. Accurate quantification of both mtDNA and nDNA is important when calculating the ratio of mtDNA to nDNA. The goal of this project is to optimize a duplex assay that will give precise and accurate estimates in human samples. Methods: Here we employ synthetic oligomer standards for an absolute real-time qPCR assay. The significance of using absolute qPCR is that the standard curve allows for the direct comparison of unknowns to obtain a copy number. The mitochondrial target is a site in the minor arc (MinArc), and the nuclear target is a single copy locus ([beta]2M). The accuracy of this assay was evaluated using a standard reference material (SRM2372a) and the precision was evaluated via replications. Results: This design resulted in high R2 values for the standards as well as sufficiently high efficiencies. The precision of the assay was analyzed over 6 replicated runs and was deemed effectively reproducible. The accuracy was assessed with the use of a standard reference material (SRM 2372a) and was found to be problematic [Romsos et al., 2018]. This could be from a possible dilution bias of the SRM, effectively changing the copy number ratios in a difficult to predict way [Malik et al., 2011]. An attempt to mathematically correct the data was made but did not provide any solution. Conclusion: The optimization of this assay is ongoing due to the error in accuracy. The assay has proven to be precise and reproducible with sufficient efficiency. Possible future directions include sonication of samples and SRMs to examine if dilution bias could be the cause of inaccurate SRM quantification. Other methods of possibly reducing dilution bias mentioned in Malik et al. [2011] include manual shearing and the use of DNA carriers such as tRNA. Another avenue of future research could include a different method of mathematically correcting the data post run to improve accuracy. This assay has the potential to provide data which can be used to indicate overall mitochondrial health and can be utilized in various research areas such as aging, cancer, forensics and neurodevelopment.Item Sequencing Long Amplicon Microsatellite Loci Using the Oxford Nanopore Technologies MinION Device(2019-03-05) Zascavage, Roxanne R.; Sedlazeck, Fritz; Planz, John V.; Hall, CourtneyPurpose: Forensic DNA typing exploits the high variability of short tandem repeat (STR) markers to differentiate individuals. Typical STR workflow consists of PCR followed by separation and detection via capillary electrophoresis (CE). Despite the power and reliability of current techniques, variations in nucleotide sequences are masked in size-based DNA profiles. Nanopore sequencing has the ability to provide long-read DNA sequence data that allows for accurate alignment and identification of single nucleotide polymorphisms (SNPs) both within and around STRs of interest. Detection of hidden sequence variation significantly expands the resolving power of STRs and aids in interpretation of more challenging samples. This project aimed to evaluate the applicability of nanopore sequencing to forensically-relevant autosomal and Y STR markers. Methods: Twenty unrelated individuals, two control DNAs, and three NIST-traceable standards were evaluated for 45 STR loci. Primer sets targeting 800 base pair amplicons containing the repeat and flanking regions were designed and multiplexed. Amplicons from each sample were barcoded and sequenced on the ONT MinION device using 1D read chemistry and SpotON flow cells (vR9.4.1). Raw reads were basecalled with MinKNOW’s real-time, local base caller. Sequence data were separated by barcode, merged by sample, and mapped to the human reference sequence (GRCh37/hg19) using NextGenMap-LR (NGMLR). Variations in motif composition and flanking SNPs were detected using Sniffles and visualized with Integrative Genomics Viewer (IGV). Consensus sequences and variant reports were compiled for each sample. Size-based allelic designations were predicted and compared to those generated via CE to evaluate concordance between the STR typing approaches. Results: High quality sequencing results were obtained for all STR loci interrogated. Concordance between size-based allelic designations revealed the reliability of nanopore sequencing data analyzed using this customized pipeline. Identification of flanking SNPs within the longer amplicons added variations that could differentiate alleles with the same motif structure, enriching discrimination potential. Conclusions: Complete nucleotide sequence data for repeat and flanking regions enhances the resolving power over that of current STR typing techniques. This project sets the foundation for future development of STRs for biomedically-relevant regions and potential forensic applications.Item Sequencing Long Amplicon Microsatellite Loci Using the Oxford Nanopore Technologies MinION[TM] Device(2019-05) Hall, Courtney L.; Planz, John V.; Zascavage, Roxanne R.; Phillips, Nicole R.; Menegaz, Rachel A.Forensic DNA typing utilizes highly variable short tandem repeat (STR) markers to differentiate individuals. Despite the power and reliability of current techniques, sequence-level variations are masked in the length-based profiles generated. Nanopore sequencing has the ability to provide long-read data, allowing for accurate alignment and identification of single nucleotide polymorphisms (SNPs) within and around microsatellite loci. To evaluate the applicability of nanopore sequencing to forensically-relevant autosomal and Y chromosome markers, selected STRs and their flanking regions (~800 bp) were amplified using custom primer sets, barcoded by sample, and sequenced on the MinION[TM] device. High quality sequencing data were obtained for all 24 samples at the 45 STRs interrogated using a customized data analysis pipeline. This project sets the foundation for future development of STRs for potential forensic applications as well as biomedically-relevant regions.Item SNP Genotyping of Native DNA using Oxford Nanopore MinION Sequencing(2018-05) Ludwick, Whitney N.; Planz, John V.; Zascavage, Roxanne R.; Phillips, Nicole R.; Yang, ShaohuaShort tandem repeats (STRs) are the primary system of genetic variation used for human identity testing in forensics; however, STR typing relies on the use of time- consuming polymerase chain reaction and expensive laboratory equipment. The use of single nucleotide polymorphisms (SNPs) in forensics have several advantages over STRs. In this study, a panel of Identity SNPs were interrogated and typed from native genomic DNA sequencing libraries using the Oxford Nanopore Technologies (ONT) MinION sequencer. We determined that SNPs could be effectively captured using existing software. Four different methods of alignment were investigated, and we found that aligning sequence data to the human genomic sequence (hg19) provided partial profiles, while aligning data to a merged reference profile resulted in more complete profiles. As ONT?s platform continues to improve, SNP genotyping using the MinION may be used to generate complete SNP profiles with the sufficient depth of coverage for reliable genotype determination.Item Taking the bait: A PCR-free enrichment strategy for nanopore sequencing applications(2020) Planz, John V.; Sedlazeck, Fritz; Zascavage, Roxanne R.; Hall, CourtneyPurpose: DNA methylation is a critical epigenetic modification involved in regulating gene expression. Although aberrant cytosine methylation has been implicated in diseases ranging from neurological disorders to cancer, current understanding is obscured by the inherent limitations of bisulfite conversion. Nanopore sequencing offers the ability to simultaneously ascertain genetic variation and base modifications without chemical treatment. While numerous copies of target regions may be present within a sample, this represents a small fraction of total genetic material competing for pore access. Here we evaluate RNA bait hybridization capture for enrichment of mitochondrial DNA (mtDNA) prior to nanopore sequencing. Methods: Heavy and light strands were individually captured using the Arbor Biosciences myBaits Mito panel. Elutant from the first capture served as rebaiting input with the opposite probe set. Double-stranded products subsequently generated were multiplexed and sequenced on the MinION device. Resultant data were separated by barcode and mapped to the human reference genome. Read counts were normalized for coverage comparisons and methylation was detected using Tombo. Results: Overall input and throughput were significantly lower than a typical whole genome sequencing run, however mtDNA read counts indicate successful enrichment. In addition to ensuring target regions outcompete background DNA, these techniques allowed for methylation detection within native strands. Conclusion: The significance of a PCR-free enrichment strategy for nanopore sequencing applications extends beyond mtDNA. These techniques have been used to capture challenging regions within the rat genome and could provide novel insights into the genetic and epigenetic landscape of other biomedically relevant regions.Item Taking the Bait: Utilization of Probe-Capture Enrichment in Human Remains Identification(2024-03-21) McBroom, Katherine; Kesharwani, Rupesh; Kapema, Bupe; Hall, Courtney; Phillips, Nicole; Sedlazeck, Fritz; Zascavage, Roxanne R.Purpose: Human remains are frequently encountered in forensic laboratories, coming from crime scenes, mass graves, historical samples, mass disasters, and military conflicts. Short tandem repeat (STR) markers evaluated via capillary electrophoresis (CE) are the gold standard for human remains identification (HRID) in forensic investigations due to their high variability and robust database of comparative samples. However, CE excludes valuable sequence-level information both within and around STRs and is often unsuccessful when used on challenged and degraded samples. The problem forensic laboratories face is choosing between depleting sample volumes to repeat individualizing STR analysis or perform costly, time-consuming, and less discriminatory mitochondrial DNA analysis. New DNA sequencing methodologies combined with novel enrichment techniques may provide a more effective platform that overcomes the most common challenges associated with HRID. Our goal for this portion of the project was to design a RNA-baiting assay to capture forensically relevant STRs. Methods: A custom myBaits panel targeting forensically relevant regions of interest was designed with Arbor Biosciences. DNA extract from whole blood samples was used. Samples were barcoded with Illumina-compatible barcode adapters, pooled, and then probe-captured, resulting in single stranded DNA covering our targeted regions. Fill-in PCR was performed on the captured single stranded DNA using Illumina primers to create DS DNA, which was then sequenced on the MiSeq. Our data was analyzed using our custom script, STRspy, which uses minimap 2 or BWA-mem to align the data to the designated reference database. STRspy then evaluates the reads aligning to each designated loci (forensically relevant STRs in this case), classifying the allele calls as well as the SNPs in the flanking region to create sequence-based genetic profiles. These calls also include the length-based allele designation for each sequence-based allele, therefore allowing the results to be compatible with current CODIS data. Results: We found that our RNA-baiting assay was successful in capturing our targeted regions of interest. The STR regions targeted for enrichment showed significantly enhanced coverage compared to the coverage across the entire genome. Conclusions: Our results indicate that probe-capture is a viable enrichment technique for DNA samples.Item The devil is in the details: Variable impacts of season, BMI, sampling site temperature, and presence of insects on the post-mortem microbiome(Frontiers Media S.A., 2022-12-27) Tarone, Aaron M.; Mann, Allison E.; Zhang, Yan; Zascavage, Roxanne R.; Mitchell, Elizabeth A.; Morales, Edgar; Rusch, Travis W.; Allen, Michael S.BACKGROUND: Post-mortem microbial communities are increasingly investigated as proxy evidence for a variety of factors of interest in forensic science. The reported predictive power of the microbial community to determine aspects of the individual's post-mortem history (e.g., the post-mortem interval) varies substantially among published research. This observed variation is partially driven by the local environment or the individual themselves. In the current study, we investigated the impact of BMI, sex, insect activity, season, repeat sampling, decomposition time, and temperature on the microbial community sampled from donated human remains in San Marcos, TX using a high-throughput gene-fragment metabarcoding approach. MATERIALS AND METHODS: In the current study, we investigated the impact of BMI, sex, insect activity, season, repeat sampling, decomposition time, and temperature on the microbial community sampled from donated human remains in San Marcos, TX using a high-throughput gene-fragment metabarcoding approach. RESULTS: We found that season, temperature at the sampling site, BMI, and sex had a significant effect on the post-mortem microbiome, the presence of insects has a homogenizing influence on the total bacterial community, and that community consistency from repeat sampling decreases as the decomposition process progresses. Moreover, we demonstrate the importance of temperature at the site of sampling on the abundance of important diagnostic taxa. CONCLUSION: The results of this study suggest that while the bacterial community or specific bacterial species may prove to be useful for forensic applications, a clearer understanding of the mechanisms underpinning microbial decomposition will greatly increase the utility of microbial evidence in forensic casework.Item The Future of Medicine: Expoloring the Acceleration of Drug Development with Decentralized Clinical Trials(2023-12) Ajape, Opemipo; Zascavage, Roxanne R.The present study explores the transformative potential of Decentralized Clinical Trials (DCT) within the field of medical research. The primary objective is to investigate how the adoption of DCT influences efficiency, patient engagement, and data quality in medical research. DCT offers the potential to streamline data collection, reduce administrative complexities, and overcome geographical constraints. This is achieved through real-time monitoring, electronic data capture, and remote data gathering techniques, making it particularly advantageous in complex scenarios like rare disease research. These efficiencies can expedite drug development, ensuring timely access to novel treatments; however, concerns about data security and privacy must be addressed to sustain these benefits. Patient engagement is a significant advantage of DCT. It allows patients to participate in research remotely, removing geographical barriers and promoting inclusion. The patient-centric approach in DCT design enhances diversity, recruitment, and retention rates. Nonetheless, maintaining patient engagement may be challenging due to limited face-to-face interactions with healthcare professionals. Data quality in DCT depends on methodologies that generate substantial data, but this complexity requires advanced analytics. DCT emphasizes a practical approach to clinical research, prioritizing patient care and valuable data collection. Ensuring the exclusion of ineligible or fraudulent participants, especially in cases involving financial incentives, is crucial. The implementation of Decentralized Clinical Trials holds the promise of fundamentally transforming medical research by enhancing efficiency, inclusivity, and data-centricity in trials. While challenges exist, current research suggests that the advantages of DCT outweigh the obstacles, paving the way for advancements in medical practices.Item Validation of the PowerPlex(R) Fusion 6C System and Alternate Robotic Methods on the Hamilton MICROLAB(R) STAR(2020-05) Smith, Briana N.; Planz, John V.; Barber, Robert C.; Zascavage, Roxanne R.Internal validations are done in forensic laboratories to ensure the methods and procedures are working as expected. For this internship practicum, I conducted an internal validation of Promega's PowerPlex(R) Fusion 6C System, as well as, a Quarter Plate Method for the Hamilton MICROLAB(R) STARlet Robotic System. This validation project was done for the Kansas Bureau of Investigation's DNA Databank Unit. This validation consisted of testing a PBS wash versus no wash, a sensitivity study, an injection time study, an analytical threshold study, a stochastic threshold study, a precision study a reproducibility study, and a contamination study. The results of these validation studies demonstrated that both the novel Quarter Plate Method that was created and Promega's PowerPlex(R) Fusion 6C System can be successfully used by the DNA Databank Unit for sample processing.