Browsing by Subject "High-Throughput Nucleotide Sequencing"
Now showing 1 - 4 of 4
- Results Per Page
- Sort Options
Item Clinical Diagnostic Potential and Characterization of Distinctly Hypermutated Antibodies in Multiple Sclerosis Patients(2016-08-12) Rounds, William Harold Alexander; Cowell, Lindsay G.; Monson, Nancy L.; Wakeland, Edward K.; Patrie, Steven M.; Ward, E. SallyMultiple sclerosis (MS) diagnosis primarily revolves around the use of brain lesion detection by MRI and the elimination of other possible neurological disorder diagnoses through clinical testing and history. For many patients first experiencing clinical symptoms that could be MS-related, this presents a challenge since diagnostic certainty based on clinical presentation and testing does not always reach a consensus among doctors who evaluate them. With a growing body of evidence for B cell involvement and dysregulation in MS, our group investigated and identified a potential biomarker in the cerebrospinal fluid of patients with MS based on B cell antibody sequencing. This work first identified a distinct mutation pattern in the antibody sequences of CSF-derived B cells, termed the antibody gene signature (AGS), that could be used to identify patients with MS or patients who would convert to MS subsequent to their first onset of clinically detectable symptoms. This thesis project outlines the transition from AGS testing in a laboratory setting to its use and implementation as an additional clinical diagnostic tool for MS (MSPrecise®) using next generation sequencing (NGS). One of its main goals is to thoroughly evaluate the performance of MSPrecise® using the far greater throughput which NGS allows for. Over the course of the project, NGS technology and accuracy optimization methods have advanced significantly. As our laboratory is the first to ever utilize NGS for somatic hypermutation evaluation, we focused strongly on the evaluation of challenges and features associated with NGS use for immune repertoire diversity and somatic hypermutation profiling of clinical samples. In this context, this project also highlights observations on sequence library preparation and post-sequencing data filtering that affect all immune repertoire research that uses these rapidly developing sequencing platforms.Item Illuminating Clonal Dynamics: Development and Use of a High-Throughput Cellular Barcoding System to Track Clonal Evolution(2012-12-06) Porter, Shaina N. 1983-; Scaglioni, Pier Paolo; Porteus, Matthew H.; Wu, Lani; Zhang, Chengcheng "Alec"It is increasingly recognized that tracking the clonal dynamics of large populations is important in understanding aspects of cancer and stem cell biology. Attempts to track the contributions of individual cells to the clonality of large homogenous populations, however, have been constrained by limitations in sensitivity and complexity. We have created an efficient and high throughput method to overcome these limitations by harnessing the power of viral marking and next generation sequencing technology, allowing us to track the clonal contributions of many thousands of cells with minimal perturbations to the population as a whole. We have applied this system to several of the most commonly used cell lines in biological research in order to validate this system and gain valuable biological insight into these ubiquitous research tools. Cell lines, often continuously passaged for years, are often assumed to be clonal, the most fit and stable clone having been selected during establishment and early passages. However, our results show that ongoing genomic and/or epigenomic instability within these cells leads to proliferative instability, resulting in the divergence of clones from one another and revealing unexpected clonal dynamics and rapid clonal evolution. These results have profound implications for the experimental use of cell lines, as well as broad applications in the fields of stem cells and cancer biology, among others.Item The Structural Distribution of Epistasis in a Pair of Essential Metabolic Enzymes(December 2021) Nguyen, Thuy Ngoc-Thi; Hibbs, Ryan E.; Reynolds, Kimberly A.; Rice, Luke M.; Yu, HongtaoInteractions between proteins provide the basis for cells to perform metabolism, grow, divide, move, and appropriately respond to external stimuli. Because proteins do not act as independent entities, the genetic background influences the effect of a mutation in unexpected ways. This context-dependence of mutational effects is epistasis. Extensive progress has been made in our ability to identify epistasis between proteins. However, how the epistasis between a pair of proteins is distributed across the amino acid sequence is less clear. Previous work characterized this sequence-level epistasis between proteins that bind to form a physical complex. Until now, the structural pattern and magnitude of epistasis between pairs of mutations spanning interacting metabolic enzymes remained uncharacterized. In my dissertation work, I deeply examined the context dependence of mutations for two essential enzymes in the bacterial folate metabolic pathway, Dihydrofolate Reductase (DHFR) and Thymidylate Synthase (TYMS). To achieve this goal, I used deep mutational scanning assays on DHFR in the context of varying activities of TYMS. The result is a rigorous dataset with epistasis measurements over the entire amino acid sequence of DHFR. I found that the positions with the greatest magnitude of epistasis within the structure of DHFR lied at the active site. However, the sign of epistasis at the DHFR active site was dependent on whether TYMS was active. Beyond the active site, the distribution of positive epistasis among the positions of DHFR was also context- dependent on the state of TYMS. Therefore, we can think of the active site as a non-physical "interface" between protein pairs that do not form a physical complex but share an intermediate. The potential consequences of this dataset on the epistasis between DHFR and TYMS are profound. This dataset is fundamental towards our understanding of how epistasis mechanistically emerges in nonlinearities between catalytic activity in enzymes, protein abundance, and cellular growth rate. This experimental dataset is also necessary to credibly validate predictions of epistasis from models of statistical co-evolution.Item Understanding RNA Regulation Through Analysis of CLIP-Seq Data(2015-11-18) Wang, Tao; Mendell, Joshua T.; Xie, Yang; Xiao, Guanghua; Mangelsdorf, David J.; Zhang, Michael Q.The past decades have witnessed a surge of discoveries revealing RNA regulation as a central player in cellular processes. The advent of cross-linking immunoprecipitation coupled with high-throughput sequencing (CLIP-Seq) technology has recently enabled the investigation of genome-wide RNA binding protein-RNA interactions, which is a very important component of RNA-regulation. However, proper and systematic bioinformatics analysis of CLIP-Seq data is still lacking and challenging. For the past few years, I have been devoting my research to methodological developments of CLIP-Seq data analysis, and developed MiClip and dCLIP for peak calling and differential analysis of CLLIP-Seq data, respectively. I have also applied my CLIP-Seq analysis pipelines in on-campus collaborating projects, in which I identified ORF57 and nuclear AGO2 binding sites. Finally, I conducted analysis of public CLIP-Seq datasets to systematically characterize RNA binding protein targeting sites on circular RNAs.