Advancement: Add-Seq: a novel method for genome-wide chromatin accessibility mapping with nanopore sequencing

Speaker Name: 
Brandon Saint-John
Speaker Title: 
PhD Student
Speaker Organization: 
Biomolecular Engineering & Bioinformatics
Start Time: 
Thursday, June 28, 2018 - 11:00am
End Time: 
Thursday, June 28, 2018 - 1:00pm
Physical Sciences, Room 305
Angela Brooks

Abstract:  Nucleosomes are comprised of 147 bp of DNA wrapped around a histone octamer, and are the building blocks for chromosomal structure. Nucleosomes play a role in gene regulation based on their position across the body of a gene. However, current next-generation sequencing methods rely on digesting DNA into short fragments that do not give information on multiple nucleosome positions across a gene locus. We propose a new method, Add-seq, which will be able to detect nucleosome positions across entire genomic loci. The method utilizes psoralen crosslinking of DNA using 4,5’,8-trimethylpsoralen (psoralen) not bound by nucleosomes. Crosslink reversal by alkali treatment leaves an adduct on the DNA. We propose that the DNA modified by the adduct can be detect by using nanopore sequencing. Nanopore sequencing involves threading single-stranded DNA through a biological pore with current running across it. As current drives the DNA through the por! e, each base will uniquely change current and can be used to identify the base identity as well as modified bases. Nanopore sequencing has already been successfully used to identify methylation and its length also allows for resolving structural variants. The psoralen modified bases will then to be used to footprint nucleosome positions with single-molecule resolution.I will use this method to look at chromatin accessibility in yeast and benchmark using the PHO5 promoter region. Furthermore, I will sequence lung cancer cell lines with loss-of-function mutations in the SWI/SNF chromatin remodeler subunit SMARCA4 to determine changes chromatin accessibility. This represents a novel method for determining chromatin accessibility across long stretches of DNA and will provide a deeper understanding of how they contribute to regulating genes. The ultimate goal for this method is to become a one-shot assay for measuring methylation, structural variants, and chromatin accessibility.