Overcoming the challenges of interpreting complex and uncommon RH alleles from whole genomes

Background and objectives: Rh is one of the most diverse and complex blood group systems. Recently, next generation sequencing (NGS) has proven to be a viable option for RH genotyping. We have developed automated software (bloodTyper) for determining alleles encoding RBC antigens from NGS-based whole genome sequencing (WGS). The bloodTyper algorithm has not yet been optimized and evaluated for complex and uncommon RH alleles. Materials and methods: Twenty-two samples with previous polymerase chain reaction (PCR) and Sanger sequencing-based RH genotyping underwent WGS. bloodTyper was used to detect RH alleles including those defined by structural variation (SV) using a combination of three independent strategies: sequence read depth of coverage, split reads and paired reads. Results: bloodTyper was programmed to identify D negative and positive phenotypes as well as the presence of alleles encoding weak D, partial D and variant RHCE. Sequence read depth of coverage calculation accurately determined RHD zygosity and detected the presence of RHD/RHCE hybrids. RHCE*C was determined by sequence read depth of coverage and by split read methods. RHD hybrid alleles and RHCE*C were confirmed by using a paired read approach. Small SVs present in RHCE*CeRN and RHCE*ceHAR were detected by a combined read depth of coverage and paired read approach. Conclusions: The combination of several different interpretive approaches allowed for automated software based-RH genotyping of WGS data including RHD zygosity and complex compound RHD and RHCE heterozygotes. The scalable nature of this automated analysis will enable RH genotyping in large genomic sequencing projects.