(1184-A) Optimization of Tagmentation Chemistries and Workflows for Fully Automated Next-Generation Sequencing Library Preparation

Abstract: Advancements in Illumina sequencing methods have necessitated novel approaches for Next Generation Sequencing (NGS) library preparation. In recent years, the library preparation process has undergone biochemical optimizations as well as mechanical approaches for automation. Several library preparation protocols employ tagmentation chemistries, which promise workflow simplification and reduced bias in DNA cleavage sites compared to sonication and enzymatic fragmentation, making it an attractive option for NGS researchers. Our group’s previous work has adapted enzymatic fragmentation and sonication-based library preparation assays to a custom platform, integrating in-capillary thermal cycling, fluid transport, and biochemical reactions. In this work we expand on previous strategies by automating a tagmentation-based protocol, addressing several concerns that distinguish tagmentation assay automation from enzymatic fragmentation library preparation assays. As a bead-based chemistry, tagmentation requires specialized liquid handling techniques that ensure optimal reaction conditions. Bead-linked transposomes (BLTs) differ from SPRI purification beads in magnetic behavior, size, surface morphology, and biomechanical reactivity. We have developed custom mixing and liquid handling protocols, as well as a method of manipulating the magnetic field throughout the assay to achieve more homogenous reaction mixtures. The protocol is fully automated at less than six hours of on-platform run time, with both final library concentration and electropherograms comparable to manual positive controls.