The Rh blood group system (ISBT004) is the second most important blood group after ABO and is the most polymorphic one, with 55 antigens encoded by 2 genes, RHD and RHCE. This research uses next-generation sequencing (NGS) to sequence the complete RHD gene by amplifying the whole gene using overlapping long-range polymerase chain reaction (LR-PCR) amplicons. The aim was to study different RHD alleles present in the population to establish reference RHD allele sequences by using the analysis of intronic single-nucleotide polymorphisms (SNPs) and their correlation to a specific Rh haplotype. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including R₁R₁ (DCe/DCe), R₂R₂ (DcE/DcE), R₁R₂ (DCe/DcE), R₂R_Z (DcE/DCE), R₁r (DCe/dce), R₂r (DcE/dce), and R₀r (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38. We focused on the analysis of hemizygous samples, as these by definition will only have a single copy of RHD. For the 69 samples sequenced, different exonic SNPs were detected that correlate with known variants. Multiple 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) haplotype which the hg38 reference sequence encodes. Twenty-three intronic SNPs were found to be R₂ haplotype specific, and 15 were linked to R₁, R₀, and R_Z haplotypes. In conclusion, intronic SNPs may represent a novel diagnostic approach to investigate known and novel variants of the RHD and RHCE genes, while being a useful approach to establish reference RHD allele sequences. Key Points We have used LR-PCR and NGS to completely sequence RHD genes in a variety of blood donors. The results show correlation between intronic SNPs and common Rh haplotypes, thus establishing reference alleles. Visual Abstract

Background: Paternal zygosity testing is used for determining homo- or hemizygosity of RHD in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the Rhesus box PCR, which can be difficult to interpret and unreliable, particularly for black African populations. Methods: DNA samples extracted from 58 blood donors were analyzed using 2 multiplex reactions for RHD-specific targets against a reference (AGO1)² to determine gene dosage by digital PCR. Results were compared with serological data, and the correct genotype for 2 discordant results was determined by long-range PCR, next-generation sequencing, and conventional Sanger sequencing. Results: The results showed clear and reliable determination of RHD zygosity using digital PCR and revealed that 4 samples did not match the serologically predicted genotype. Sanger sequencing and long-range PCR (LR-PCR) followed by next-generation sequencing revealed that the correct genotypes for samples 729M and 351D, which were serologically typed as R₁R₂ (DCe/DcE), were R₂r9 (DcE/dCe) for 729M and R₁r” (DCe/dcE), R₀r^y (Dce/dCE), or R_Zr (DCE/dce) for 351D, in concordance with the digital PCR data. Conclusions: Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal RH zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous RHD-positive individuals.

Background: Nanopore sequencing is direct sequencing of a single-stranded DNA molecule using biological pores. A portable nanopore-based sequencing device from Oxford Nanopore Technologies (MinION) depends on driving a DNA molecule through nanopores embedded in a membrane using a voltage. Changes in current are then measured by a sensor, thousands of times per second and translated to nucleobases.Methods: Genomic DNA (gDNA) samples (n = 13) were tested for Rh blood group D antigen (RHD) gene zygosity using droplet digital PCR. The RHD gene was amplified in 6 overlapping amplicons using long-range PCR. Amplicons were purified, and the sequencing library was prepared following the 1D Native barcoding gDNA protocol. Sequencing was carried out with 1D flow cells R9 version. Data analysis included basecalling, aligning to the RHD reference sequence, and calling variants. Variants detected were compared to the results acquired previously by the Ion Personal Genome Machine (Ion PGM).Results: Up to 500× sequence coverage across the RHD gene allowed accurate variant calling. Exonic changes in the RHD gene allowed RHD allele determination for all samples sequenced except 1 RHD homozygous sample, where 2 heterozygous RHD variant alleles are suspected. There were 3 known variant RHD alleles (RHD*01W.02, RHD*11, and RHD*15) and 6 novel RHD variant alleles, as previously seen in Ion PGM sequencing data for these samples.Conclusions: MinION was effective in blood group genotyping, provided enough sequencing data to achieve high coverage of the RHD gene, and enabled confident calling of variants and RHD allele determination.

Fetal RHD screening for targeted routine antenatal anti-D prophylaxis has been implemented in many countries, including Finland, since the 2010s. Comprehensive knowledge of the RHD polymorphism in the population is essential for the performance and safety of the anti-D prophylaxis program. During the first 3 years of the national screening program in Finland, over 16 000 samples from RhD− women were screened for fetal RHD; among them, 79 samples (0.5%) containing a maternal variant allele were detected. Of the detected maternal variants, 35 cases remained inconclusive using the traditional genotyping methods and required further analysis by next-generation sequencing (NGS) of the whole RHD gene to uncover the variant allele. In addition to the 13 RHD variants that have been previously reported in different populations, 8 novel variants were also detected, indicating that there is more variation of RHD in the RhD− Finnish population than has been previously known. Three of the novel alleles were identified in multiple samples; thus, they are likely specific to the original Finnish population. National screening has thus provided new information about the diversity of RHD variants in the Finnish population. The results show that NGS is a powerful method for genotyping the highly polymorphic RHD gene compared with traditional methods that rely on the detection of specific nucleotides by polymerase chain reaction amplification.

Background: The Rh blood group system (ISBT004) is encoded by two homologous genes, RHD and RHCE. Polymorphism in these two genes gives rise to 56 antigens, which are highly immunogenic and clinically significant. This study extended previous work on the establishment of RHD allele specific reference sequences using next generation sequencing (NGS) with the Ion Personal Genome Machine (Ion PGM) to sequence the complete RHCE gene.Study Design and Methods: Genomic DNA (gDNA) samples (n = 87) from blood donors of different serologically predicted genotypes including R₁R₁ (DCe/DCe), R₂R₂ (DcE/DcE), R₁R₂ (DCe/DcE), R₂R_Z (DcE/DCE), R₁r (DCe/dce), R₂r (DcE/dce), R₀r (Dce/dce), rr (dce/dce), r'r (dCe/dce), and r″r (dcE/dce) were used in this study. The RHCE gene was amplified through overlapping long range‐polymerase chain reaction (LR‐PCR) amplicons and then sequenced with the Ion PGM. Data were analyzed against the human genome reference sequence build hg38 and variants were called.Results: Referen variant allel VS. In addition to the RHCE reference alleles, different exonic single nucleotide variants (SNVs) were detected that encode known RHCE variant alleles including RHCE*Ce.09, RHCE*ceAR, and RHCE*ceVS.03. Numerous intronic SNVs were detected and compared from samples with different Rh genotypes, to determine their link to a specific Rh haplotype. Based on the exonic and intronic changes detected in different RHCE alleles, three RHCE reference sequences were established and submitted to Genbank (one for the RHCE*Ce allele, one for the RHCE*cE allele, and one for the RHCE*ce allele).Conclusion: Intronic SNVs may represent a novel alternative diagnostic approach to investigate known and novel variants of the RH genes and the prediction of Rh haplotype.