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通过MinION牛津纳米孔测序进行快速高分辨率HLA基因分型用于 deceased 供体器官分配

Rapid high-resolution HLA genotyping by MinION Oxford nanopore sequencing for deceased donor organ allocation.

作者信息

De Santis Dianne, Truong Linh, Martinez Patricia, D'Orsogna Lloyd

机构信息

Department of Clinical Immunology, PathWest, Fiona Stanley Hospital, Perth, Western Australia, Australia.

出版信息

HLA. 2020 Aug;96(2):141-162. doi: 10.1111/tan.13901. Epub 2020 Apr 26.

Abstract

Recently, HLA epitopes on donor HLA molecules have been shown to be important in the success of solid organ transplantation. However, these epitopes can only be defined using high-resolution typing results of which are often not available prior to deceased donor allocation. The ability to perform high-resolution typing at all HLA loci for deceased organ donor allocation prior to transplantation would have major clinical benefits, in particular for highly sensitised recipients. We, therefore, developed a rapid high-resolution next generation sequencing (NGS) HLA typing (ONT-Rapid HR HLA) method for on-call deceased donor allocation using the AllType 11 loci single-tube assay (OneLambda Inc), modified in-house to reduce polymerase chain reaction amplification time, and the Oxford Nanopore single-molecule sequencing platform on the Flongle flow cell. The ONT-Rapid HR HLA method was validated on 42 samples previously typed by current on-call sequence-specific oligonucleotide (HistoSpot) and NGS methods (AllType/Ion Torrent). High-resolution typing obtained using the ONT-Rapid HR HLA typing method was 100% concordant with both the current SSO and NGS methods, and in some cases, obtained higher resolution than either of the current methods. The rapid ONT-Rapid HR HLA typing method was able to obtain these typing results at all loci in 4 to 4.5 hours. The novel ONT-Rapid HR HLA typing method is the first reported NGS HLA typing method utilised for deceased donor allocation. The ability to provide high-resolution HLA typing on deceased donors before implantation will in the future allow improvements in matching, which will ultimately provide clinical benefits to patients.

摘要

最近研究表明,供体HLA分子上的HLA表位在实体器官移植的成功中起着重要作用。然而,这些表位只能通过高分辨率分型结果来确定,而在脑死亡供体分配前往往无法获得高分辨率分型结果。在移植前对脑死亡器官供体的所有HLA位点进行高分辨率分型,将具有重大的临床益处,特别是对于高敏受者。因此,我们开发了一种快速高分辨率下一代测序(NGS)HLA分型方法(ONT-Rapid HR HLA),用于脑死亡供体的紧急分配,该方法使用AllType 11位点单管检测法(OneLambda公司),并在内部进行了改进以缩短聚合酶链反应扩增时间,同时结合Flongle流动槽上的牛津纳米孔单分子测序平台。ONT-Rapid HR HLA方法在42个先前通过当前紧急序列特异性寡核苷酸(HistoSpot)和NGS方法(AllType/Ion Torrent)分型的样本上进行了验证。使用ONT-Rapid HR HLA分型方法获得的高分辨率分型结果与当前的SSO和NGS方法100%一致,在某些情况下,获得的分辨率高于当前任何一种方法。快速的ONT-Rapid HR HLA分型方法能够在4至4.5小时内获得所有位点的分型结果。这种新型的ONT-Rapid HR HLA分型方法是首次报道用于脑死亡供体分配的NGS HLA分型方法。在植入前为脑死亡供体提供高分辨率HLA分型的能力,未来将有助于改善配型,最终为患者带来临床益处。

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