Resolving Genotype-Phenotype Discrepancies of the Kidd Blood Group System Using Long-Read Nanopore Sequencing

Abstract

Due to substantial improvements 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, fail to explain serological phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype-phenotype discrepancies in the Kidd blood group system (JK, encoded by SLC14A1) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry-based platform at the Blood Transfusion Service, Zurich. Discrepant results from standard serological typing and donor genotyping were confirmed using commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region of SLC14A1 (~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 with both serological and genotype 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 the JK*01 (Gly40Asp; c.119G>A) and JK*02 (Gly242Glu; c.725G>A) haplotypes, respectively. Remarkably, the remaining three cases were associated with a yet unknown deletion of ~5 kb spanning exons 9-10 of the JK*01 allele, 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 variants such as hybrid genes, deletions and duplications.

Keywords: Kidd blood group system; Oxford nanopore sequencing; haplotype; high-throughput genotyping; long-read sequencing; structural variant.

Figure 1

Flowchart depicting identification and processing of genotype–phenotype discrepancies. Main bioinformatics tools are provided in green. MALDI-TOF MS stands for Matrix-Assisted Laser Desorption Ionization–Time-of-Flight Mass Spectrometry; PCR-SSP for Sequence-Specific-Priming PCR; LR-PCR for Long-Range PCR; SV for Structural Variant; and ISBT for International Society of Blood Transfusion. Erytra is an automated gel cards system used to serologically phenotype donors.

Figure 2

Genetic structure of the SLC14A1 gene encoding the JK blood group system, and details of the identified large novel deletion. (a) SLC14A1 gene showing exons, coding DNA sequence (CDS), newly identified ~5 kb deletion (red), positions of long-range PCRs (LR1/LR2), as well as overlap between both amplicons. (b) Region of ~5 kb deletion with breakpoints and primer positions used for bridge-PCR. (c) Gel electrophoresis of long-range PCR products (LR1 and LR2) of all samples. (d) Gel electrophoresis of bridge-PCR products for the three samples with novel deletion and wild-type control reactions.

Figure 3

Nanopore sequencing coverage by nucleotide position for each sample. Coverages were computed by mapping length-filtered reads to SLC14A1 reference sequence (NG_011775.4) using minimap2. For readability, samples showing a drop in coverage in LR2 were grouped together. Graphical representation of mapping location on SLC14A1 gene is given underneath the coverage plots. Coverage scales are not fixed.