Revolutionising High Resolution HLA Genotyping for Transplant Assessment: Validation, Implementation and Challenges of Oxford Nanopore Technologies' Q20+ Sequencing

ABSTRACT The advent of third‐generation sequencing (TGS) represents a significant shift in the field of genetic sequencing, enabling single‐molecule sequencing to overcome limitations of short‐read NGS platforms. Several studies have assessed the utilisation of TGS in HLA genotyping, though many of these studies have described the high error rate as an obstacle to achieving a robust and highly accurate HLA typing assay. In 2021, Oxford Nanopore Technologies (ONT) released the high‐accuracy sequencing Kit 14 and the MinION flow cell model R10.4.1, which were reported to achieve sequencing accuracies up to 99%. The aim of this study was to validate this novel high‐accuracy sequencing kit for HLA genotyping coupled with a full‐gene HLA PCR assay. Comparison with historical data obtained using legacy flow cell models such as R9.4, R10.3 and R10.4 was also done to assess progressive improvement in sequencing performance with each sequential release. The workflow was validated based on data throughput, sequence quality and accuracy, and HLA genotyping resolution. An initial validation was performed using an internal reference panel of 42 samples representing common HLA allele groups, followed by an analysis of data obtained from 111 sequencing batch runs since the implementation, to assess assay performance and define quality control metrics to assess inter‐run variability and monitor quality. Furthermore, challenges arising from MinION flow cell stability and use, and assessment of barcode contamination are discussed. The findings of this study highlight advantages of ONT sequencing kit 14/R10.4.1 for HLA genotyping and the implementation considerations for the routine diagnostic HLA laboratory.