Aim: NGS-Turbo® is a fast and reliable high resolution HLA-typing technique that is used during time-sensitive solid organ transplantation. Organ transplantations regularly occur outside of regular working hours, when less laboratory staff is available and available staff may have to divide their attention. This makes it crucial that HLA-typing is performed without error and with minimal hands-on time. To further simplify the workflow and reduce the hands-on time of NGS-Turbo®, the workflow was automated on Integra’s Miro Canvas microfluidics system. Ultimately providing highly reliable HLA-typing for solid organ transplantation in less than three and a half hours.
Methods: 24 DNA samples from the Genetic Testing Reference Material Coordination Program (GeT-RM) HLA58 panel were analyzed by NGS-Turbo®. NGS-Turbo® consists of a two-stage workflow of amplification (NGS-TurboAmp®) and library preparation (NGS-TurboPrep®) followed by Oxford Nanopore Technologies (ONT) sequencing. For each sample, a single NGS-TurboAmp® was performed, which was used for both a manual and automated NGS-TurboPrep® library preparation. The prepared libraries were sequenced using MinION R10.4.1 flow cells on a GridION. Data was processed using super accuracy basecalling and finally analyzed using NGSengine®.
Results: The performance of the manual and automated NGS-Turbo® workflows was compared. Library yields were similar between the workflows and ONT pore occupancy remained consistent. 3-field typing results between the workflows were 100% concordant. Furthermore, there were no significant differences in sample mappability and noise levels. The automated workflow from blood to sequencing was completed in 3 and a half hours, compared to the manual workflow that required just under 3 hours. Even though the total time to completion for the automated workflow was longer, the hands-on time was only 25 minutes compared to 50 minutes for the manual workflow.
Conclusion: Here the automation of NGS-Turbo® on the Integra Miro Canvas system is shown. Time and accuracy is crucial during solid organ transplantation and without losing library yield, data quality or typing resolution, a 50% reduction in hands-on time for NGS-Turbo® HLA-typing is achieved.
