Exploring Repetitive Elements in Neurodegenerative Disease Using Targeted Long-Read Sequencing

Abstract

The repetitive nature of the human genome, with repeat sequences accounting for around 50% of its content, poses significant challenges to genomic analysis. With advances in long-read sequencing techniques, we are now better equipped to investigate variation in repeats and how they contribute to disease risk. My dissertation focuses on using nanopore targeted sequencing strategies to investigate two types of repetitive elements: short tandem repeats and transposable elements (TEs). Taking advantage of long-read technologies, we apply targeted sequencing to investigate neurodegenerative diseases. My work incorporates post-mortem brain tissue samples and cell lines to explore repeat variation in neurodegenerative diseases and control samples across diseases including late-onset ataxias and Alzheimer’s disease (AD). In addition, we apply these techniques to investigate variation among cells. Thus, this work provides insights into the role of repeat sequences in the human genome while simultaneously highlighting the potential of long-read sequencing approaches in unmasking the mechanisms driving neurodegenerative diseases. Throughout this dissertation, I explore the variation in the repetitive elements of the human genome, examining how they differ both within and between individuals. Despite their abundance and considerable contribution to our genetic makeup, repetitive sequences remain largely under-characterized, illustrating a notable gap in our understanding of genomics. In 2022, in a large-scale effort coordinated by the Telomere-to-Telomere consortium, a complete human genome was officially released. Prior to this, more than ninety percent of the human genome was assembled, but much of what remained unknown consisted of highly repetitive sequences. While the consortium's efforts have underscored the significance of a comprehensive depiction of the human genome, it is equally crucial to delve into the variation present within these repetitive elements, and to investigate their role in overall genome function. My dissertation investigates this repetitive landscape through the development and application of nanopore-based targeted sequencing approaches. First, I describe how the historical challenge of assembling an accurate reference genome (Chapter 1) spurred new advances in third-generation sequencing technologies, which now allow researchers to interrogate even the most difficult repetitive regions. Building on these innovations, I then detail a targeted sequencing panel, which uses CRISPR Cas9 to efficiently genotype disease-associated tandem repeats (Chapter 2). This strategy not only captures known pathogenic expansions, such as those implicated in late-onset ataxias, but also reveals unexpected or co-occurring expansions, illustrating the complexity and clinical relevance of repeat variation. I subsequently extend these techniques (Chapter 3) to characterize a broader array of repetitive elements, including human papillomavirus (HPV) integrations and TEs in Alzheimer’s disease, while also leveraging nanopore’s capacity for directly assessing CpG methylation. Finally, I address how these methods can be adapted to study somatic variation in human tissues (Chapter 4). By comparing whole-genome amplification strategies and applying nanopore sequencing to single cells, I explore the challenges of detecting low-frequency mutational events in post-mortem brain tissue, a setting where somatic mosaicism may contribute to neurodegeneration. Finally, in the conclusion (Chapter 5), I highlight how targeted long-read sequencing of repetitive elements is poised to accelerate our understanding of genomic structure and disease etiology. As large-scale consortium projects continue to refine both the technology and accompanying analytical tools, the comprehensive study of repeats, once considered inaccessible, has the potential to illuminate new mechanisms of pathology and advance both discovery and diagnostic based applications.