Characterization Of Oxidative Stressinduced Genomic Structural Variations In Saccharomyces cerevisiae Through Optical Genome Mapping

Abstract

The genome of every known organism is composed of deoxyribonucleic acid (DNA). The DNA in organisms like humans, is organized into chromosomes and encodes the genetic instruction necessary for survival. When alterations occur in the genome, they can lead to disease. Structural variations (SVs) are large-scale genomic rearrangements, which can span several megabases, and are difficult to detect using traditional short-read sequencing methods. SVs have been observed in the widely used eukaryotic model organism Saccharomyces cerevisiae (S. cerevisiae). Particularly in strains with a compromised defense system against endogenous reactive oxygen species (ROS). In this project, optical genome mapping (OGM) was used to study SVs in a ROS-sensitive S. cerevisiae strain lacking the TSA1 gene. OGM is a technique that enables sequence-related information to be extracted across long DNA segments. OGM was performed using competitive binding of YOYO-1 and netropsin to fluorescently label DNA, followed by stretching od DNA via a nanofluidic approach. The DNA was then imaged using fluorescence microscopy. A cultivation scheme was developed for both wild-type and ROS-sensitive S. cerevisiae strains suitable for the purpose of this study. Additionally, chromosomal DNA extraction methods were evaluated to obtain long DNA fragments suitable for OGM. Fluorescence imaging data was processed to generate single molecule intensity profiles (barcodes). Barcodes where then compared to theoretical intensity profiles of the S. cerevisiae reference genome to assess coverage and detect structural changes. Amongst the samples, enough data was obtained to cover the entirety of the S. cerevisiae genome approximately 4-11 times. By focusing on chromosome II, comparisons between non-stressed and oxidatively stressed strains revealed intensity profile differences that may indicate SVs. However, further validation, preferably with higher coverage, is necessary to further characterize the genomic changes.