P253 Automating holotype HLA™ on the Agilent Bravo NGS workstation liquid handler and validating for routine HLA genotyping

Abstract

Aim HLA typing by Next Generation Sequencing (NGS) is increasingly being adopted for high-resolution HLA genotyping due to the technological advancements that allow complete characterization of the HLA genes. For almost all combinations of alleles, the genotyping results present with no ambiguity, eliminating reflexive testing. To ensure reproducibility and repeatability across technical staff and to reduce the possibility of human error, pre-PCR and post-PCR steps of the protocol can be automated on liquid handling robots to provide the most reliable results within a 3-day turnaround time from sample to answer. The Agilent Bravo NGS Workstation provides high flexibility due to its 96-tip pipetting head, that is capable of significantly reducing the hands-on time for technologists both pre- and post-PCR in the Holotype HLA workflow, providing an end-to-end liquid handling solution for any HLA lab. Methods The automated workflow includes: input DNA normalization, Long-Range PCR setup, amplicon quantitation and normalization, per-sample pooling of HLA loci, library preparation and final pooling. Holotype HLA 24/11 kits were run on both pre- and post-PCR steps on the Agilent Bravo as part of the liquid handler validation. Results Here we present results from the validation study that was performed during the automation of Holotype HLA 24/11 kits on the Agilent Bravo NGS Workstation. Without automation, the total hands-on time required for completing the entire protocol is about 4 h for experienced users, which is reduced to less than 40 min with the automated methods for both pre- and post-PCR steps. Conclusions All data was automatically analyzed using Omixon HLA Twin demonstrating that the results are of high quality, accurate and reproducible making the Agilent Bravo NGS Workstation a very suitable partner in efficient HLA typing by NGS using Holotype HLA.