Manli Yang

Date of Award

Fall 2022

Document Type

Open Access Thesis

Department

College of Pharmacy

First Advisor

Michael D. Wyatt

Abstract

Specific genomic profiles associated with exposure to DNA damaging agents have been identified in cancer related genes, revealing that mutational patterns can be carcinogen- specific. However, there have been limited efforts to demonstrate similar clear relationships for endogenous mutational processes. One endogenous source of mutations is contamination of the deoxynucleotide pool with damaged bases that, if incorporated into DNA, cause mutations.

The Catalog of Somatic Mutations In Cancer (COSMIC) represents an international effort to characterize mutations in cancer. A mutational signature is the pattern of mutations generated by a mutational process. Each mutational process contains two parts: DNA damage and DNA repair. If DNA repair is successful, the DNA will be restored to its native sequence. However, if damaged bases are not faithfully removed and replaced, then damaged bases can be misinterpreted by DNA polymerases to cause mutations. A mutational signature refers to mutations that occur within a defined adjoining sequence 5’ and 3’ to the base of altered sequence.

Nudt15 and Nudt18 are part of a nucleotide hydrolase superfamily of enzymes that may play diverse and independent roles in modulation of nucleotide pools. The founding member of the family, Nudt1 (hMTH1) hydrolyzes the mutagenic deoxynucleotide, 8-oxo-deoxyguanosine-triphosphate, to a deoxy nucleoside monophosphate, thus preventing mutagenesis caused by this damaged dNTP precursor. However, little is known about the endogenous substrates of Nudt15 and Nudt18. This study was undertaken with two purposes. First, there is a need to develop long and short-read sequencing and mutational profile analysis workflows for COSMIC mutational signature and structural variation analysis. Second, the application of the workflow was applied to studying mutational signatures and structural variations in two genetic models, namely in Nudt15 and Nudt18 knockout ovarian cancer cells, and in Rad51d deficient murine embryonic fibroblasts. This study reports Illumina sequencing and mutational patterns analysis workflows to analyze mutational signatures and structural variations in ovarian cancer cells with reads shorter than 5000 base pairs. The results showed that the overall mutational patterns in ovarian cancer single-cell colonies include COSMIC signature 5. Structural variations (SVs) are large scale genomic changes, including insertions, deletions, duplications, inversions, and translocations. This study also reports Nanopore sequencing and structural variation analysis workflows to detect structural variations in Rad51d-deficient mouse embryonic fibroblasts (MEFs). Nanopore sequencing generated 652,000 base-called reads containing 4,404,372,528 bases by using 1 MinION flow cell sequencing. The results establish workflow pipelines for analysis of mutational signatures and for determining structural variations using long-read Nanopore sequencing.

Rights

© 2022, Manli Yang

Download

Share

COinS