P211 A roadmap for successful robotic automation of next generation sequencing based HLA typing

Abstract

Aim DNA library preparation for next generation sequencing (NGS) consists of several hundred material transfer and wash steps, which are time consuming, labor intensive, and increase the risk of repetitive strain injury. Robotic liquid handlers can automate and standardize library preparation, but few HLA laboratories have experience with high-complexity robotic platforms. We sought to evaluate the use of robotics for library preparation in conjunction with a NGS based HLA typing assay. Methods A Beckman Coulter Biomek NXp Span-8 robotic liquid handler was acquired along with the vendor supplied robotics method for the Illumina TruSight HLA v2 sequencing panel. The robotics method was first optimized with a series of runs using water, 50% glycerol, 85% ethanol, and AMPure beads. Next, DNA and reagents were run to identify sample viscosity and reagent issues. After each run, issues were communicated to the vendor for method improvement. The finalized method was run on 78 DNA samples over seven runs to assess the quality of normalization, pooling, sequencing, and reproducibility of results. Results Robotic issues were encountered with pipette calibration, labware definitions, proper labware selection, and efficiency. To add complexity, these issues had subtle compounding effects on the overall success of the method, which required that each be addressed individually and in detail. Eleven versions of the method were subsequently produced and tested to ensure robustness and consistency. The finalized method produced consistent high quality sequencing metrics on the NGS typing platform which agreed with the known HLA typing results. Conclusions Detailed documentation of issues by lab staff and clear communication with the vendor allowed for a rapid and iterative improvement of the robotics method. The lessons learned during the implementation process formed a roadmap that allowed for successful implementation of a robotics platform for NGS based HLA typing.