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

Abstract

The comprehensive characterization of human leukocyte antigen (HLA) genomic sequences remains a challenging problem. Despite the significant advantages of next-generation sequencing (NGS) in the field of Immunogenetics, there has yet to be a single solution for unambiguous, accurate, simple, cost-effective, and timely genotyping necessary for all clinical applications. This report demonstrates the benefits of nanopore sequencing introduced by Oxford Nanopore Technologies (ONT) for HLA genotyping. Samples (n = 120) previously characterized at high-resolution three-field (HR-3F) for 11 loci were assessed using ONT sequencing paired to a single-plex PCR protocol (Holotype) and to two multiplex protocols OmniType (Omixon) and NGSgo®-MX6-1 (GenDx). The results demonstrate the potential of nanopore sequencing for delivering accurate HR-3F typing with a simple, rapid, and cost-effective protocol. The protocol is applicable to time-sensitive applications, such as deceased donor typings, enabling better assessments of compatibility and epitope analysis. The technology also allows significantly shorter turnaround time for multiple samples at a lower cost. Overall, the nanopore technology appears to offer a significant advancement over current next-generation sequencing platforms as a single solution for all HLA genotyping needs.

Keywords: HLA genotyping; Human Leukocyte Antigens; NGS; Oxford Nanopore sequencing; Transplantation.

Fig. 1.

Distribution of reads per sample for the five MinION experiments. The dashed line represents the expected representation of a sample if all 24 samples in an experiment are equally represented.

Fig. 2.

Distribution of reads among the 11 HLA loci for the MinION and Flongle experiments. A) Percent Usable Reads – MinION; B) Count of Usable Reads – MinION; C) Percent usable reads – OmniType; D) Count of usable Reads – OmniType; E) Percent usable reads – NGSgo^®-MX6-1; F) Count of usable reads – NGSgo^®-MX6-1. The y-axis represents the percentage of the reads assigned that were used for analysis for each locus out of the total reads that were used for the sample (A, C and E) or the count of reads used for analysis per locus (B, D, and F).

Fig. 3.

Percentage of the minor allele for all heterozygous loci sequenced for the MinION and Flongle experiments. A) Holotype PCR on MinION; B) OmniType multiplex PCR on Flongle. All samples with DRB3 and DRB4 were hemizygous. C) NGSgo^®-MX6-1 multiplex PCR on Flongle.

Fig. 4.

Timing Diagram for Flongle Experiments. * Amplification time varies based on method. Shown here is 2 h for the OmniType protocol, whereas the NGSgo^®-MX6-1 multiplex takes 3 h and 15 min.

Fig. 5.

Percent error of sequencing on the two different ONT platforms: MinION and Flongle. The error rate is broken down and colored by locus and includes substitutions, insertions and deletions. For the MinION experiments, the error rate is combined for the five sequencing experiments (n = 120 samples). For the Flongle, the error rate is grouped by the PCR method (n = 9 samples each).