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

Justin B.L. Halls, Sunitha Vege, Daimon P. Simmons, Judith Aeschlimann, Baderha Bujiriri, Helen H. Mah, Matthew S. Lebo, Prathik K. Vijay Kumar, Connie M. Westhoff, William J. Lane

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Pages (from-to)790-801
Number of pages12
JournalVox Sanguinis
Issue number8
StatePublished - Nov 2020
Externally publishedYes


  • RHCE
  • RHD
  • blood group
  • genomics
  • whole genome sequencing

ASJC Scopus subject areas

  • Hematology


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