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The Rh blood group system (ISBT004) is the second most important blood group after ABO and the most polymorphic one, with 55 antigens encoded by two genes, RHD and RHCE. The two genes are homologous and located on chromosome 1 with 10 coding exons. This research uses next generation sequencing (NGS) on Ion Torrent Personal Genome Machine™ (Ion PGM™) to sequence the Rh genes (RHD, RHCE, RHAG) using overlapping Long Range-PCR (LR-PCR) amplicons. The aim was to study different RHD and RHCE alleles present in the population to establish reference RHD and RHCE allele sequences by utilising the analysis of intronic single nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. We also aimed to use single molecule sequencing (SMS), MinION™, to test the feasibility of applying SMS in blood group genotyping (BGG). Genomic DNA (gDNA) samples (n=218) were sequenced for the RHD gene and 100 samples were sequenced for the RHCE gene using Ion PGM™. The RHAG gene was also sequenced from samples where no mutation was detected in the RHD gene that would explain weak D reactivity in serological testing. The RHD gene was sequenced from samples (n=13) using MinION™. Data generated was mapped to the human genome reference sequence hg38 and variants were called. Variants detected by the MinION™ were compared to the ones detected from the Ion PGM™. The RHD gene data analysis lead to the detection of different exonic SNPs that correlate to known variants and the identification of 10 novel RHD variant alleles. Multiple RHD intronic SNPs were found in all samples: 21 intronic SNPs were present in all samples indicating their specificity to the RHD*DAU0 (RHD*10.00) allele which the hg38 reference sequence encodes. Twenty-three intronic SNPs were found to be R2 haplotype specific, and 15 were linked to R1,R0,RZ haplotypes which lead to the establishment of two RHD reference sequences one for R2 and the other for R1,R0,Rz. Intronic SNPs were also detected in the RHCE gene in which 89 SNPs were specific to samples with C+/c- and 22 SNPs were found in C-/c+ samples which lead to the conclusion that these SNPs are Rhc specific. This work resulted in the establishment of three RHCE allele specific reference sequences in which one is C+ specific, one is c+ specific and the other is for e+ allele. MinION™ was successful for determining the RHD allele in all 13 samples sequenced. NGS is a high throughput technique that enables the sequencing of polymorphic blood group genes and identification of novel variants present in the population which cannot be detected using the available BGG molecular platforms. Intronic SNPs may represent a novel diagnostic approach to investigate known and novel variants of the RHD and RHCE genes, whilst being a useful approach to establish reference RHD and RHCE allele sequences. MinION™ is a powerful tool and was successful here in BGG. However, challenges remain with developing a straight forward user-friendly data analysis pipeline for translating this work into transfusion facilities.