Abstract
Introduction: In immunohematology case studies the knowledge about variants of the blood group gene of interest can facilitate antibody diagnosis. Known gene variants can be rapidly genotyped by specific methods, but the identification of unknown variants requires sequencing of the gene. High throughput next-generation sequencing (NGS) technologies represent important tools for DNA sequencing of many targets in larger numbers of samples but are less suitable for the analysis of one or few genes in single samples. Nanopore sequencing (Oxford Nanopore Technologies, ONT) is a fast sequencing technology that could fulfill the requirements for targeted gene sequencing in case studies. Here, we describe an optimized protocol for long-read nanopore sequencing of blood group genes that enables analysis of whole genes within less than 7 h from DNA extraction to genotype determination. Methods: Primers for long-range PCR (LR-PCR) were designed for the blood group genes ACKR1, CD151, BCAM, KEL, SLC14A1, GYPA, GYPB, GYPE, RHD, and RHCE with amplicon sizes in the range of 2.4-15.8 kilo base pairs (kbp). For evaluation of the sequencing data, 22 samples with 25 known gene variants were selected. The optimized sequencing workflow included DNA extraction from EDTA blood, LR-PCR amplification, library preparation, nanopore sequencing on the MinION Mk1D sequencing device with FLO-MIN114 (MinION) flow cells and data analysis including variant detection and genotyping. In addition, the workflow was tested on the MinION sequencing device with FLO-FLG114 (Flongle) flow cells and the PromethION 2 Solo sequencing device with FLO-PRO114 (PromethION) flow cells. Results: Using the outlined long-read nanopore sequencing protocol, sequencing data for reliable variant calling were obtained. All alleles were identified and the zygosity could be determined based on the read counts, except for GYPB in one sample. Besides sequencing on the MinION Mk1D sequencing device MinION flow cells, successful application of the sequencing protocol to the Flongle flow cells and the PromethION sequencing device using PromethION flow cells was demonstrated. As expected, mean coverage and mean Q scores varied between the flow cells and devices. Conclusion: The optimized nanopore sequencing protocol enabled the generation of long-read sequence data and identification of blood group gene variants within a working day. This approach is suitable for molecular analyses of different blood group genes in immunohematology case studies under the same LR-PCR and sequencing conditions.