Resolving genotype-phenotype discrepancies of the Kidd blood group system using long-read nanopore sequencing

Abstract

Due to substantial improvement in read accuracy, third-generation long-read sequencing holds great potential in blood group diagnostics, particularly in cases where traditional genotyping or sequencing techniques, primarily targeting exons, are unable to explain serologic phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype-phenotype discrepancies in the Kidd blood group system (JK,SLC14A1) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry based platform at Blood Transfusion Service Zurich. Discrepant results of standard serological typing and donor genotyping were confirmed by commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region ofSLC14A1(∼24 kb, exons 3 to 10) in two overlapping long-range PCRs in all samples. Amplicons were barcoded and sequenced on a MinION flow cell. Sanger sequencing and bridge-PCRs were used to confirm findings. Among 11,972 donors who had both serology and genotypic data available for the Kidd system, we identified 10 cases with unexplained conflicting results. Five were linked to known weak and null alleles caused by variants not included in the routine donor genotyping. In two cases, we identified novel null alleles on theJK*01(Gly40Asp; c.119G>A) andJK*02(Gly242Glu; c.725G>A) haplotype, respectively. Remarkably, the remaining three cases were linked to a yet unknown deletion of ∼5 kb spanning over exon 9-10 of theJK*01allele, which other molecular methods had failed to detect. Overall, nanopore sequencing demonstrated reliable and accurate performance for detecting both single nucleotide and structural variants. It possesses the potential to become a robust tool in the molecular diagnostic portfolio, particularly for addressing challenging structural variation such as hybrid genes, deletions and duplications.