Sequenzierung mit Nanoporen - die Zukunft der hochauflösenden HLA-Genotypisierung Erfahrungen aus einer multizentrischen Studie
Stéphane Buhler (Geneva / CH), Maja Nørgaard (Aarhus / DK), Rudi Steffensen (Aalborg / DK), Kirstine Kløve-Mogensen (Aalborg / DK), Bjarne Kuno Møller (Aarhus / DK), Rebecca Grossmann (Leipzig / DE), Sylvie Ferrari-Lacraz (Geneva / CH), Claudia Lehmann (Leipzig / DE)
Molecular techniques for human leukocyte antigen typing have become an integral part of everyday laboratory work. While real-time polymerase chain reaction delivers fast results in low-resolution, high-resolution HLA typing is generate using next-generation-sequencing, essential for stem cell transplantations. High-resolution typing will become increasingly important in organ transplantation in the future, however the minimal time for deceased donor HLA typing is challenging.
HLA typing using nanopores has undergone enormous development in recent years and shows the potential for high resolution HLA typing of 11 loci within a few hours. The aim of this multicentre study was to investigate the nanopore sequencing in terms of concordance with NGS HLA typing results and practicability for diagnostic laboratories. A total of 381 samples were sequenced in four European laboratories with NanoTYPE™ (Omixon Biocomputing Ltd.) on a MinION (Oxford Nanopore Technologies).
The results show a concordance of 99.6% for the HLA loci, HLA-A, -B, -C, -DRB1, -DRB3, -DRB4, -DRB5, -DQA1, -DQB1, -DPA1 and -DPB1. The fastq files analyzed with NanoTyperTM software (Omixon Biocomputing Ltd.), whereby quality data generated for each sample. A total of 34926 (97.15%) quality control values were issued as "passed". Only 2.4% of the QC values were assessed by the software as "inspect" and 0.45% as "failed". Our results show, that correct HLA typing results achieved despite software warnings ("inspect","failed").
The advantages of nanopore sequencing are shown in simple implementation in the laboratory, fewer ambiguities and cost efficiency, which is demonstrated by the reusability of the flow cells. The current limitation with regard to exon coverage will be resolved surely in the next few years, so that this technology can use in diagnostics for high resolution HLA typing for deceased donor HLA typing. This could offer opportunities for routine epitope determinations in the future.
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