Authors:
T. Vágó (Budapest, HU)
F. Felegyhazi (Budapest, HU)
T. Yu (New Taipei City, TW)
W. Hsu (New Taipei City, TW)
E. Melista (Budapest, US)
P. Meintjes (Budapest, US)
T. Hague (Budapest, HU)
Next Generation Sequencing (NGS) is a powerful new method for HLA genotyping. Currently, the rate of NGS adoption by HLA laboratories is rapidly increasing driven by the accuracy, resolution, reproducibility and high-throughput nature of the technology. The elimination of human and random error is a desirable attribute of any diagnostic test and the implementation of a liquid handling system can significantly reduce both sources of error in an NGS workflow. Holotype HLA is a commercially available HLA genotyping product developed in a clinical lab with clinical routine in mind. With Holotype HLA one can easily scale up to 96 samples at 11 loci on a single MiSeq run. Due to its flexibility, it is compatible with multiple liquid handling systems, such as the TBG DX-A, which is a small footprint robot capable of reducing the hands-on time for technicians for both pre- and post-PCR steps in the Holotype workflow. Together, Holotype HLA and DX-A provides an ideal solution for labs who wish to fully automate their HLA genotyping workflow, reducing variation and human error, thus increasing the specificity, sensitivity and accuracy of the genotyping. The DX-A reduces the total hands-on time of the Holotype workflow to 40 minutes. An Illumina’s MiniSeq system was used to test a set of 192 samples. In this study, the PCR setup, amplicon quantitation and normalisation, and library preparation were automated on the DX-A for HLA-A, -B, -C (class I) and HLA-DRB1, -DQA1, -DQB1, DPB1 (class II) loci for each sample. In each setup, 24 samples were prepared on the robot, then four runs of 24 samples were collected together for each sequencing run on the MiniSeq. Two MiniSeq runs have been performed to sequence 96 samples for 7 loci per run. The MiniSeq results were compared to known genotyping calls previously determined from Sanger-based sequencing data. All MiniSeq data was analysed using Omixon HLA Twin. Here we present the results of our validation of Holotype HLA automated on DX-A and sequenced on the MiniSeq system - an accurate, reproducible and high-throughput system for HLA typing in less than 48 hours from gDNA to genotype.