Automation of Amplicon-Based Library Preparation for Next-Generation Sequencing by Centrifugal Microfluidics.

Next-generation sequencing (NGS) has become a mainstream method in bioanalysis. Improvements in sequencing and bioinformatics turned the complex and cumbersome library preparation to the bottleneck in terms of reproducibility and costs in the complete NGS workflow. Here, we introduce an automated library preparation approach based on a generic centrifugal microfluidic cartridge. Multiplex polymerase chain reaction amplification and subsequent cleanup were performed with all reagents prestored on the disk, including cell-line-based DNA as quality control. Exchange of prestored reagents allows applying the cartridge to different target genes. Sequencing of automatically prepared libraries from T-cell receptor and immunoglobulin gene rearrangements in context of lymphoproliferative disorders demonstrated excellent cleanup performance between 91.9 and 99.9% of target DNA reads and successful amplification of all target regions by up to 15 forward primers combined with 4 reverse primers. The fully automated library preparation by centrifugal microfluidics thus offers attractive automation options in diagnostic settings.