A novel approach to simultaneous genotyping of human platelet antigen systems and human leucocyte antigen class I loci using PacBio long-read sequencing.

Abstract

Background and objectives: Accurate human leucocyte antigen (HLA) and human platelet antigen (HPA) typing is essential for establishing a blood platelet donor bank to deal with refractoriness in patients undergoing multiple platelet transfusions. Current methods, such as Sanger and next-generation sequencing, encounter difficulties in haplotyping. Herein, the aim of this study was to establish a method for HLA and HPA typing based on the long read sequencing.

Study design and methods: The HPA and HLA class I genotypes of 268 platelet donors from the Taiyuan Blood Center, China were identified using long-read sequencing on the PacBio platform. Allele frequencies for HPA systems and HLA class I genes were calculated, and genetic variability within HPA system genes was analysed.

Results: Polymorphisms were identified in 8 of the 35 HPA systems (HPA-1 to HPA-6w, HPA-15 and HPA-21w), with the frequencies of the 'b' allele at 0.0187, 0.0709, 0.4086, 0.0075, 0.0149, 0.0317, 0.4310 and 0.0019, respectively. The alleles with the highest frequencies at the HLA-A, HLA-B and HLA-C loci are HLA-A02:01, B51:01, B46:01 and C06:02, respectively. Additionally, several genetic patterns in HPA systems were identified, including the c.166-1029C>T variant, which was found exclusively in samples carrying the HPA-1b allele.

Conclusion: This study developed a targeted long-read sequencing method characterized by high throughput and simultaneity, capable of resolving allele ambiguities for effective HLA class I genotyping in establishing a platelet donor bank.