(1228-A) Automated Single-Cell Sequencing Using Evercode Split-Pool Combinatorial Barcoding on a Hamilton Vantage Liquid Handler

Abstract: Demand for single-cell sequencing and the resulting data continues to grow, both in cell count and sample number. To meet this demand, Parse Bioscience’s Evercode combinatorial barcoding technology was developed to allow for sequencing of up to 1 million cells in as many as 96 samples. We have developed a system to automate >95% of the pipetting steps in the Evercode workflow using a Hamilton Vantage liquid handler equipped with 8-span channels, a 96 head, and plate gripper. Steps that were automated include combinatorial barcoding, amplification of barcoded cDNA, and library preparation for sequencing. Verification of the system was performed by processing identical samples of human peripheral blood mononuclear cells (PBMCs) in parallel both manually and using our automated workflow. After sequencing, analysis of the resulting data showed >99.7% correlation between the genes detected by the two workflows, indicating that the automated system performs comparably to manual. In the future, leveraging the 96 head of the liquid handler would allow for scaling the amplification and library prep portions of the workflow up to 6-fold without significant changes to system hardware or increases in hands-on time.