Automation of the amplification and library preparation steps for HLA typing using the NGS-based Holotype HLA kit and the Hamilton STARlet system

Automate amplification and library preparation using the NGS-based Holotype HLA kit for 7 loci (A, B, C, DRB1, DPB1, DQA1 and DQB1). Amplification and library preparation as dictated by the Omixon Holotype kit were automated on 2 Hamilton STARlet systems. PCR setup is performed on a dedicated pre-PCR STARlet with no peripheral equipment. A user interface allows input of sample ID, DNA concentration, and loci to be amplified from a .csv file. Pre-PCR programs include master mix preparation, DNA dilution, and distribution of DNA and master mix to 2 PCR plates (Class I+ Class II). Plates are sealed by a technologist and placed on thermal cyclers. After amplification, plates are placed on the post-PCR STARlet equipped with an Agilent plate sealer, temperature controlled carrier, and Trobot (thermal cycler). Post-PCR setup includes amplicon clean up with ExoSAP-IT, followed by Quantiflour reaction preparation (read on a SpectroMax fluorometer), and amplicon normalization with water. In the Holotype X4 configuration, libraries are created for samples as individual loci and as pools of seven loci in parallel. For library preparation on the STARlet, diluted amplicons are fragmented, end repaired and ligated to indexed adaptors. Indexed libraries are pooled, concentrated with AMPure beads, then size selected on a Blue Pippin. Library is quantified by qPCR and loaded on an Illunima Miseq for sequencing. Methods automated on STARlet: Pre-PCR: master mixture preparation X4 PCR preparation Post-PCR: ExoSAP-IT quantitation with promega quantifluor normalization and pooling of amplicons fragmentation end repair adaptor ligation pooling of final library qPCR preparation manual. Methods: Bead concentration of final poolsize selection The pre-PCR methods have been validated using 145 samples, each amplified for 7 HLA loci and implemented in clinical practice. Post-PCR library preparation methods have been generated and are in the validation stage. The Holotype HLA typing protocol is relatively simple and easy to perform manually. However, automation has been a key factor in reducing overall cost, hands-on time and opportunities for errors. The final result of this work is an efficient robotic method central to an optimized and high-throughput workflow for HLA typing. D. Ferriola: Other (Identify); Company/Organization; Royalty. D. Monos: Other (Identify); Company/Organization; Royalty.