Overcoming methodical limits of standard RHD genotyping by next-generation sequencing

Background and objectives Molecular variations of the RHD gene may result in the reduced expression of the D antigen and altered Rh phenotypes. In many occasions, they cannot be typed reliably by standard serological methods. Sequence‐based typing is the gold standard to determine rare and unknown RHD genotypes. For this pilot study, sequence‐based typing by standard Sanger sequencing was compared to a newly established next‐generation sequencing approach based on pyrosequencing. Materials and methods Twenty‐six DNA samples were selected after primary serological testing exhibiting a weak reaction in Rh phenotype. Parallel sequence analysis of the complete coding sequence including adjacent intronic sequences allowed a comparison of the methodical potency in mutation detection of Sanger with next‐generation sequencing. Results Sanger sequencing revealed 39 RHD polymorphisms in 21 of 26 samples in the RHD coding region, while pyrosequencing detected all but two alterations resulting in a concordance rate of 94·9% and clearly revealed a heterozygous compound mutation in one sample with RHDψ and Weak D type 4 alleles. The resolution of cis/trans linkage of polymorphisms and exact characterization of a 37 bp duplication was achieved by next‐generation sequencing. Conclusion Our data suggest that next‐generation sequencing offers a new development for high‐throughput and clonal sequencing for molecular RHD genotyping. However, further attempts in the methodical set‐up have to be undertaken prior to validation and introduction as a routine service.