Samples (n = 123 Table 3) with different Rh genotypes presumed from serology results were first tested using dPCR to determine RHD zygosity. Library construction, NGS, and data analysis To validate PCR amplification, PCR products were run on a 0.7% wt/vol agarose gel in 1× Tris-acetate-EDTA buffer next to a Quick-Load 1-kb Extend DNA Ladder (New England Biolabs). The last extension was at 65☌ for 10 minutes finally, samples were held at 4☌. Annealing temperature varied for each primer set ( Tables 1 and 2). The Veriti Thermal Cycler (Applied Biosystems) program was set as follows: denaturation at 95☌ for 5 minutes, 30 cycles of 95☌ for 30 seconds, annealing for 30 seconds, and extension at 65☌ for 10 minutes. In a 50-μL reaction, 1× master mix of LongAmp Hot Start Taq 2× Master Mix (New England Biolabs) was used with 200 ng of gDNA template 1 μM of the forward and reverse primers was used for all amplicons except for RHD amplicon 3, where 0.2 μM of the forward and reverse primers was used. To optimize PCR conditions, different annealing temperatures and primer concentrations were tested to ensure specific amplification from the target gene. The number of RHD copies per microliter present in a sample was compared with the reference gene AGO1 copies per microliter.
12,39 The droplet reader in combination with QuantaSoft software v1.7.4 analyzed the droplet signals and differentiated between negative and positive ones, creating an absolute concentration of DNA. 12,39 Samples were tested for RHD exon 5 ( RHD5) and RHD exon 7 ( RHD7) against the reference gene AGO1 on chromosome 1. The RHD zygosity was determined for all samples using dPCR to determine whether a sample was hemizygous (Dd) or homozygous (DD). Samples were tested for zygosity with the aim of knowing the number of RHD alleles present for subsequent sequence analysis. gDNA concentration was determined on the Qubit 2.0 Fluorometer (Life Technologies) using the Qubit double-stranded DNA High Sensitivity assay kit (Life Technologies). Genomic DNA (gDNA) was extracted from buffy coat using the QIAamp DNA Blood Mini kit (Qiagen Ltd) following the manufacturer’s guidelines. 13,20 Complete DNA sequencing could be the most effective technique to thoroughly study blood group variations and overcome limitations in other assays. They are designed to target predefined nucleotides or DNA regions through polymerase chain reaction (PCR), whereas novel variants remain unknown. 14-19 Although these assays are very accurate, they have limitations. 13 Different DNA microarray-based tests were introduced that enable genotyping of variant blood groups by targeting specific single-nucleotide polymorphisms (SNPs). Extensive efforts have been made to alternatively use molecular genotyping ranging from low to high throughput. 1 Complete blood group genotyping (BGG) could be widely used in transfusion practice where serology fails to clarify issues or resolve discrepancies. Unlike serological testing, genotyping provides the freedom to analyze a wider range of blood antigens including low-frequency antigens, for instance: Go a, BARC, and Tar which can cause HDFN and alloimmunization. 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. Twenty-three intronic SNPs were found to be R 2 haplotype specific, and 15 were linked to R 1, R 0, and R Z haplotypes. 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. For the 69 samples sequenced, different exonic SNPs were detected that correlate with known variants. We focused on the analysis of hemizygous samples, as these by definition will only have a single copy of RHD. Genomic DNA samples (n = 69) from blood donors of different serologically predicted genotypes including R 1R 1 (DCe/DCe), R 2R 2 (DcE/DcE), R 1R 2 (DCe/DcE), R 2R Z (DcE/DCE), R 1r (DCe/dce), R 2r (DcE/dce), and R 0r (Dce/dce) were sequenced and data were then mapped to the human genome reference sequence hg38. 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. 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 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.