Application of Long-Read Sequencing to Modified Nucleotides for Detecting Chromatin Accessibility

Nucleosomes provide an additional layer of gene regulation by regulating chromatin ac-cessibility. We have found that factors involved in regulating nucleosome positioning, such as SMARCA4, are recurrently mutated in lung adenocarcinoma and can correlate with alternative splicing changes. As a result, it is crucial to understand nucleosome po- sitioning across an entire gene body to understand how they interact to cause changes in splicing. The key to being able to understand this is using long-read sequencing methods to understand the positioning of nucleosomes at once. My thesis focuses on developing methods and computational tools to understand nucleosome positioning with long reads. I developed a sequencing approach called Add-seq that uses a small molecule called angelicin to label accessible regions and determine those positions using nanopore sequencing. Based on the analyses for Add-seq, I also developed a toolkit called cawlr. This computational pipeline can automate calling nucleosomes on single molecules from any long-read sequencing technology. This work provides new ways of looking at nucle- osomes and understanding their positioning in the context of other biological processes