Chloe Ziemelis
Research Mentor(s): Brian Athey
Mentor Department: Computational Medicine & Bioinformatics
Authors: Chloe Ziemelis, Gregory Farnum, Brian Athey, Monica Holmes
Session: Session 1 (9:00am – 9:50am)
Presentation Type: Poster 100
Abstract
Nanopore ligation adapters are specialized molecules that facilitate the attachment of DNA fragments to nanopore sequencing flow cells, enabling the generation of high-fidelity long-read sequencing data and real-time nucleotide analysis. In this project, we utilize a targeted sequencing strategy using in vitro Cas9 cleavage to investigate structural variations and haplotypes in the CYP2D6 gene, a critical determinant of tamoxifen metabolism in breast cancer patients. Variations in CYP2D6 influence the conversion of tamoxifen into its active form, endoxifen, thereby impacting therapeutic efficacy and patient outcomes. By capturing high-fidelity sequencing data for CYP2D6, this project aims to overcome the limitations of current pharmacogenomic tests, which often fail to detect complex structural variations in genes. Foundational to this effort is the optimization of adapter ligation efficiency, ensuring consistent and precise attachment of nanopore sequencing adapters to Cas9-cleaved DNA fragments. Quantifying the ligation efficiency metric enables fine-tuning of adapter-to-sample DNA ratios. ONT performance is maximal at ~75 fmol of adapter-ligated DNA per ~600 ng total library DNA. Loading less than 75 fmol of target DNA can result in a low pore occupancy and can lead to inadequate reads, however, loading a larger amount of DNA can damage the pores in the technology. In consequence, this decreases the accuracy of detecting genetic variations. Understanding ligation efficiency is essential to maximizing target molecule concentration when sequencing with ONT. Beyond its direct application to CYP2D6, refining ligation protocols has broader implications for advancing long-read sequencing workflows across diverse genomic targets. This approach not only addresses gaps in current pharmacogenomic technologies but also sets the stage for improved genetic research and clinical diagnostics, driving progress in personalized medicine.
