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

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.

Utilizing nanopore sequencing technology for the rapid and comprehensive characterization of eleven HLA loci; addressing the need for deceased donor expedited HLA typing

“…Earlier reported efforts to utilize the nanopore technology for HLA genotyping fell short of providing a comprehensive solution of characterizing all loci within a time frame necessary for all of our clinical needs [ 28 ]. Most recently, however, De Santis et al using a combination of a multiplex PCR and ONT reported successful characterization of all 11 HLA loci [ 29 ]. Our report introduces nanopore technology as a viable alternative to existing methods for reliable clinical HLA typing of all eleven loci within a significantly shorter time.…”

Growing evidence that 2-field high-resolution HLA typing is important for kidney transplantation

“…This limitation may soon be resolved by third-generation sequencing, which has been reported to achieve 2F-HR typing for 11 HLA loci in 4 to 4.5 hours. 5 Time and cost have been barriers to routine use of 2F-HR typing for kidney transplantation. Commercial kits for HLA typing using next-generation sequencing (NGS) offer options for completing 2F-HR typing in 1 to 5 days.…”

“…However, LR HLA typing can cause false‐positive virtual crossmatches, which preclude compatible transplants. This limitation may soon be resolved by third‐generation sequencing, which has been reported to achieve 2F‐HR typing for 11 HLA loci in 4 to 4.5 hours 5 …”

The Future of Livestock Management: A Review of Real-Time Portable Sequencing Applied to Livestock

“…Initially, single-pass accuracies of only 68.4% (Phred score of 5) were reported with the MinION in 2015 [13]. Using the R9.4.1 flow cell chemistry and Guppy base caller, raw read accuracies of 85-90% have been reported [14]. This is considerably less than the 99.999% accuracy in sanger sequencing [15] and 99.92% in Illumina [16].…”