Long-Read Nanopore Sequencing Validated for Human Leukocyte Antigen Class I Typing in Routine Diagnostics

Abstract

Matching of human leukocyte antigen (HLA) gene polymorphisms by high-resolution DNA sequence analysis is the gold standard for determining compatibility between patient and donor for hematopoietic stem cell transplantation. Single-molecule sequencing (PacBio or MinION) is a newest (third) generation sequencing approach. MinION is a nanopore sequencing platform, which provides long targeted DNA sequences. The long reads provide unambiguous phasing, but the initial high error profile prevented its use in high-impact applications, such as HLA typing for HLA matching of donor and recipient in the transplantation setting. Ongoing developments on instrumentation and basecalling software have improved the per-base accuracy of 1D2 nanopore reads tremendously. In the current study, two validation panels of samples covering 70 of the 71 known HLA class I allele groups were used to compare third field sequences obtained by MinION, with Sanger sequence-based typing showing a 100% concordance between both data sets. In addition, the first validation panel was used to set the acceptance criteria for the use of MinION in a routine setting. The acceptance criteria were subsequently confirmed with the second validation panel. In summary, the present study describes validation and implementation of nanopore sequencing HLA class I typing method and illustrates that nanopore sequencing technology has advanced to a point where it can be used in routine diagnostics with high accuracy.