Aim: With the growing need for unambiguous HLA typing to support precision medicine, achieving high accuracy in HLA typing is paramount. The long-range PCR products still require fragmentation prior to short-read sequencing on routine next-generation sequencing (NGS). HLA class II genes, particularly HLA-DPB1, present a unique challenge because of its large intron 1 and intron 2. This study aimed to evaluate improvements achievable through long-read sequencing using Oxford Nanopore Technology (ONT) without fragmenting long-range PCR products, and to determine the coverage required for unambiguous DPB1 typing.
Methods: DNA was extracted from 67 whole blood (n=52) and buccal swab samples (n=15). Challenging samples previously typed using One Lambda’s AllType NGS HLA typing platform on the IonS5 system were selected. The samples were sequenced using GenDx NGS-Pronto, and the data were analyzed with NGSengine-Turbo software (v1.3.0) with IMGT v3.58.0. In addition, 179 specimens without previous high-resolution typing were tested to further evaluate the platform.
Results: The mean length of routine NGS (IonS5) is with 293 bp compared to ONT mean length of 4,268 bp (Figure 1A). We investigated the read yield for each sample, with 65 samples (97.0%) achieving more than 2,000 reads and 59 samples (88%) achieving more than 10,000 reads per sample (Figure 1B). The Nanopore platform provided unambiguous 3-field DPB1 typing for 100% (67/67) samples due to complete phasing with as few as 2000 reads per sample, indicating that ONT data can potentially reduce sequencing time, increase throughput, and shorten overall analysis time (Figure 1C). We further investigated cis/trans ambiguity between the IonS5 and ONT platforms (Figure 2). While the IonS5 platform provides high-resolution HLA typing, it exhibited ambiguity in 31.3% of cases across both Class I and Class II loci. In contrast, the ONT platform demonstrated significantly reduced ambiguity, with only DRB4*01:01:01G affected, resulting in an unambiguous call rate exceeding 99.1%. Additionally, 100% of an independent set of 179 specimens yielded unambiguous DPB1 typing results using as few as 2000 reads per sample.
Conclusion: Overall, 246 samples were tested, and Nanopore sequencing addresses this limitation by enabling unambiguous HLA typing, particularly for DPB1 assignment. Furthermore, our coverage analysis offers a distinct advantage for phasing and resolving allele-level ambiguity.
