Genomic characterisation of clinically significant blood group variants in Aboriginal Australians.

BACKGROUND: Hematological disorders are often treated with blood transfusions. Many blood group antigens and variants are population-specific, and for patients with rare blood types, extensive donor screening is required to find suitable matches for transfusion. There is a scarcity of knowledge regarding blood group variants in Aboriginal Australian populations, despite a higher need for transfusion due to the higher prevalence of renal diseases and anaemia. MATERIALS AND METHODS: In this study, we applied next-generation sequencing and analysis to 245 samples obtained from Aboriginal Australians from South-East Queensland, to predict antigen phenotypes for 36 blood group systems. RESULTS: We report potential weak antigens in blood group systems RH, FY and JR that have potential clinical implications in transfusion and pregnancy settings. These include partial DIII type 4, weak D type 33, and Del RHD (IVS2-2delA). The rare Rh phenotypes D+ C+ E+ c- e+ and D+ C+ E+ c+ e- were also detected. DISCUSSION: The comprehensive analyses of blood group genetic variant profiles identified in this study will provide insight and an opportunity to improve Aboriginal health by aiding in the identification of appropriate blood products for population-specific transfusion needs.

A cold case of hemolytic disease of the fetus and newborn resolved by genomic sequencing and population studies to define a new antigen in the Rh system.

BACKGROUND: We report an obstetric case involving an RhD-positive woman who had developed a red blood cell (RBC) antibody that was not detected until after delivery of a newborn, who presented with a positive direct antiglobulin test result. Immunohematology studies suggested that the maternal antibody was directed against a low-prevalence antigen on the paternal and newborn RBCs. RESULTS: Comprehensive blood group profiling by targeted exome sequencing revealed a novel nonsynonymous single nucleotide variant (SNV) RHCE c.486C>G (GenBank MZ326705) on the RHCE*Ce allele, for both the father and newborn. A subsequent genomic-based study to profile blood groups in an Indigenous Australian population revealed the same SNV in 2 of 247 individuals. Serology testing showed that the maternal antibody reacted specifically with RBCs from these two individuals. DISCUSSION: The maternal antibody was directed against a novel antigen in the Rh blood group system arising from an RHCE c.486C>G variant on the RHCE*Ce allele linked to RHD*01. The variant predicts a p.Asn162Lys change on the RhCE protein and has been registered as the 56th antigen in the Rh system, ISBT RH 004063. CONCLUSION: This antibody was of clinical significance, resulting in a mild to moderate hemolytic disease of the fetus and newborn (HDFN). In the past, the cause of such HDFN cases may have remained unresolved. Genomic sequencing combined with population studies now assists in resolving such cases. Further population studies have potential to inform the need to design population-specific red cell antibody typing panels for antibody screening in the Australian population.

Genetic Characterization of Blood Group Antigens for Polynesian Heritage Norfolk Island Residents.

Improvements in blood group genotyping methods have allowed large scale population-based blood group genetics studies, facilitating the discovery of rare blood group antigens. Norfolk Island, an external and isolated territory of Australia, is one example of an underrepresented segment of the broader Australian population. Our study utilized whole genome sequencing data to characterize 43 blood group systems in 108 Norfolk Island residents. Blood group genotypes and phenotypes across the 43 systems were predicted using RBCeq. Predicted frequencies were compared to data available from the 1000G project. Additional copy number variation analysis was performed, investigating deletions outside of RHCE, RHD, and MNS systems. Examination of the ABO blood group system predicted a higher distribution of group A1 (45.37%) compared to group O (35.19%) in residents of the Norfolk Island group, similar to the distribution within European populations (42.94% and 38.97%, respectively). Examination of the Kidd blood group system demonstrated an increased prevalence of variants encoding the weakened Kidd phenotype at a combined prevalence of 12.04%, which is higher than that of the European population (5.96%) but lower than other populations in 1000G. Copy number variation analysis showed deletions within the Chido/Rodgers and ABO blood group systems. This study is the first step towards understanding blood group genotype and antigen distribution on Norfolk Island.

Next generation sequencing to identify iron status and individualise blood donors' experience.

BACKGROUND: Young adults form the majority of first-time blood donors to Australian Red Cross Lifeblood. However, these donors pose unique challenges for donor safety. Young blood donors, who are still undergoing neurological and physical development, have been found to have lower iron stores, and have higher risks of iron deficiency anaemia when compared to older adults and non-donors. Identifying young donors with higher iron stores may improve donor health and experience, increase donor retention, and reduce the burden on product donation. In addition, these measures could be used to individualise donation frequency. MATERIALS AND METHODS: Stored DNA samples from young male donors (18-25 years; No.=47) were sequenced using a custom panel of genes identified in the literature to be associated with iron homeostasis. The custom sequencing panel used in this study identified and reported variants to human genome version 19 (Hg19). RESULTS: 82 gene variants were analysed. Only one of which, rs8177181, was found to have a statistically significant (p<0.05) association with plasma ferritin level. Heterozygous alleles of this Transferrin gene variant, rs8177181T>A, significantly predicted a positive effect on ferritin levels (p=0.03). DISCUSSION: This study identified gene variants involved in iron homeostasis using a custom sequencing panel and analysed their association with ferritin levels in a young male blood donor population. Additional studies of factors associated with iron deficiency in blood donors are required if a goal of personalised blood donation protocols is to be achieved.

Inactivation of SARS-CoV-2 infectivity in platelet concentrates or plasma following treatment with ultraviolet C light or with methylene blue combined with visible light.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unlikely to be a major transfusion-transmitted pathogen; however, convalescent plasma is a treatment option used in some regions. The risk of transfusion-transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB-plasma and THERAFLEX UV-Platelets systems. Here we examined the capability of these PI systems to inactivate SARS-CoV-2. STUDY DESIGN AND METHODS: SARS-CoV-2 spiked plasma units were treated using the THERAFLEX MB-Plasma system in the presence of methylene blue (~0.8 µmol/L; visible light doses: 20, 40, 60, and 120 [standard] J/cm2 ). SARS-CoV-2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV-platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 [standard] J/cm2 ). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay. RESULTS: Treatment of spiked plasma with the THERAFLEX MB-Plasma system resulted in an average ≥5.03 log10 reduction in SARS-CoV-2 infectivity at one third (40 J/cm2 ) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV-Platelets system resulted in an average ≥5.18 log10 reduction in SARS-CoV-2 infectivity at the standard UVC dose (0.2 J/cm2 ). CONCLUSIONS: SARS-CoV-2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB-Plasma and THERAFLEX UV-Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion-transmitted emerging pathogens.

Using whole-genome sequencing to characterize clinically significant blood groups among healthy older Australians.

There have been no comprehensive studies of a full range of blood group polymorphisms within the Australian population. This problem is compounded by the absence of any databases carrying genomic information on chronically transfused patients and low frequency blood group antigens in Australia. Here, we use RBCeq, a web server-based blood group genotyping software, to identify unique blood group variants among Australians and compare the variation detected vs global data. Whole-genome sequencing data were analyzed for 2796 healthy older Australians from the Medical Genome Reference Bank and compared with data from 1000 Genomes phase 3 (1KGP3) databases comprising 661 African, 347 American, 503 European, 504 East Asian, and 489 South Asian participants. There were 661 rare variants detected in this Australian sample population, including 9 variants that had clinical associations. Notably, we identified 80 variants that were computationally predicted to be novel and deleterious. No clinically significant rare or novel variants were found associated with the genetically complex ABO blood group system. For the Rh blood group system, 2 novel and 15 rare variants were found. Our detailed blood group profiling results provide a starting point for the creation of an Australian blood group variant database.

RBCeq: A robust and scalable algorithm for accurate genetic blood typing.

BACKGROUND: While blood transfusion is an essential cornerstone of hematological care, patients requiring repetitive transfusion remain at persistent risk of alloimmunization due to the diversity of human blood group polymorphisms. Despite the promise, user friendly methods to accurately identify blood types from next-generation sequencing data are currently lacking. To address this unmet need, we have developed RBCeq, a novel genetic blood typing algorithm to accurately identify 36 blood group systems. METHODS: RBCeq can predict complex blood groups such as RH, and ABO that require identification of small indels and copy number variants. RBCeq also reports clinically significant, rare, and novel variants with potential clinical relevance that may lead to the identification of novel blood group alleles. FINDINGS: The RBCeq algorithm demonstrated 99·07% concordance when validated on 402 samples which included 29 antigens with serology and 9 antigens with SNP-array validation in 14 blood group systems and 59 antigens validation on manual predicted phenotype from variant call files. We have also developed a user-friendly web server that generates detailed blood typing reports with advanced visualization (https://www.rbceq.org/). INTERPRETATION: RBCeq will assist blood banks and immunohematology laboratories by overcoming existing methodological limitations like scalability, reproducibility, and accuracy when genotyping and phenotyping in multi-ethnic populations. This Amazon Web Services (AWS) cloud based platform has the potential to reduce pre-transfusion testing time and to increase sample processing throughput, ultimately improving quality of patient care. FUNDING: This work was supported in part by Advance Queensland Research Fellowship, MRFF Genomics Health Futures Mission (76,757), and the Australian Red Cross LifeBlood. The Australian governments fund the Australian Red Cross Lifeblood for the provision of blood, blood products and services to the Australian community.

Prevalence of Neutralising Antibodies to HCoV-NL63 in Healthy Adults in Australia.

The COVID-19 pandemic has highlighted the importance of understanding the immune response to seasonal human coronavirus (HCoV) infections such as HCoV-NL63, how existing neutralising antibodies to HCoV may modulate responses to SARS-CoV-2 infection, and the utility of seasonal HCoV as human challenge models. Therefore, in this study we quantified HCoV-NL63 neutralising antibody titres in a healthy adult population using plasma from 100 blood donors in Australia. A microneutralisation assay was performed with plasma diluted from 1:10 to 1:160 and tested with the HCoV-NL63 Amsterdam-1 strain. Neutralising antibodies were detected in 71% of the plasma samples, with a median geometric mean titre of 14. This titre was similar to those reported in convalescent sera taken from individuals 3-7 months following asymptomatic SARS-CoV-2 infection, and 2-3 years post-infection from symptomatic SARS-CoV-1 patients. HCoV-NL63 neutralising antibody titres decreased with increasing age (R2 = 0.042, p = 0.038), but did not differ by sex. Overall, this study demonstrates that neutralising antibody to HCoV-NL63 is detectable in approximately 71% of the healthy adult population of Australia. Similar titres did not impede the use of another seasonal human coronavirus (HCoV-229E) in a human challenge model, thus, HCoV-NL63 may be useful as a human challenge model for more pathogenic coronaviruses.

Low Genetic Diversity of Hepatitis B Virus Surface Gene amongst Australian Blood Donors.

Variants in the small surface gene of hepatitis B virus (HBV), which codes for viral surface antigen (HBsAg), can affect the efficacy of HBsAg screening assays and can be associated with occult HBV infection (OBI). This study aimed to characterise the molecular diversity of the HBV small surface gene from HBV-reactive Australian blood donors. HBV isolates from 16 HBsAg-positive Australian blood donors' plasma were sequenced and genotyped by phylogenies of viral coding genes and/or whole genomes. An analysis of the genetic diversity of eight HBV small surface genes from our 16 samples was conducted and compared with HBV sequences from NCBI of 164 international (non-Australian) blood donors. Genotypes A-D were identified in our samples. The region of HBV small surface gene that contained the sequence encoding the 'a' determinant had a greater genetic diversity than the remaining part of the gene. No escape mutants or OBI-related variants were observed in our samples. Variant call analysis revealed two samples with a nucleotide deletion leading to truncation of polymerase and/or large/middle surface amino acid sequences. Overall, we found that HBV small surface gene sequences from Australian donors demonstrated a lower level of genetic diversity than those from non-Australian donor population included in the study.

Targeted exome sequencing designed for blood group, platelet, and neutrophil antigen investigations: Proof-of-principle study for a customized single-test system.

BACKGROUND: Immunohematology reference laboratories provide red blood cell (RBC), platelet (PLT), and neutrophil typing to resolve complex cases, using serology and commercial DNA tests that define clinically important antigens. Broad-range exome sequencing panels that include blood group targets provide accurate blood group antigen predictions beyond those defined by serology and commercial typing systems and identify rare and novel variants. The aim of this study was to design and assess a panel for targeted exome sequencing of RBC, PLT, and neutrophil antigen-associated genes to provide a comprehensive profile in a single test, excluding unrelated gene targets. STUDY DESIGN AND METHODS: An overlapping probe panel was designed for the coding regions of 64 genes and loci involved in gene expression. Sequencing was performed on 34 RBC and 17 PLT/neutrophil reference samples. Variant call outputs were analyzed using software to predict star allele diplotypes. Results were compared with serology and previous sequence genotyping data. RESULTS: Average coverage exceeded 250×, with more than 94% of targets at Q30 quality or greater. Increased coverage revealed a variant in the Scianna system that was previously undetected. The software correctly predicted allele diplotypes for 99.5% of RBC blood groups tested and 100% of PLT and HNA antigens excepting HNA-2. Optimal throughput was 12 to 14 samples per run. CONCLUSION: This single-test system demonstrates high coverage and quality, allowing for the detection of previously overlooked variants and increased sample throughput. This system has the potential to integrate genomic testing across laboratories within hematologic reference settings.

In silico Analysis of Genetic Diversity of Human Hepatitis B Virus in Southeast Asia, Australia and New Zealand.

The extent of whole genome diversity amongst hepatitis B virus (HBV) genotypes is not well described. This study aimed to update the current distribution of HBV types and to investigate mutation rates and nucleotide diversity between genotypes in Southeast Asia, Australia and New Zealand. We retrieved 930 human HBV complete genomes from these regions from the NCBI nucleotide database for genotyping, detection of potential recombination, serotype prediction, mutation identification and comparative genome analyses. Overall, HBV genotypes B (44.1%) and C (46.2%) together with predicted serotypes adr (36%), adw2 (29%) and ayw1 (19.9%) were the most commonly circulating HBV types in the studied region. The three HBV variants identified most frequently were p.V5L, c.1896G>A and double mutation c.1762A>T/c.1764G>A, while genotypes B and C had the widest range of mutation types. The study also highlighted the distinct nucleotide diversity of HBV genotypes for whole genome and along the genome length. Therefore, this study provided a robust update to HBV currently circulating in Southeast Asia, Australia and New Zealand as well as an insight into the association of HBV genetic hypervariability and prevalence of well reported mutations.

Developments beyond blood group serology in the genomics era.

Blood group serology and single nucleotide polymorphism-based genotyping platforms are accurate but do not provide a comprehensive cover for all 36 blood group systems and do not cover the antigen diversity observed among population groups. This review examines the extent to which genomics is shaping blood group serology. Resources for genomics include the Human Reference Genome Sequence assembly; curated blood group tables listing variants; public databases providing information on genetic variants from world-wide studies; and massively parallel sequencing technologies. Blood group genomic studies span the spectrum, from bioinformatic data mining of huge data sets containing whole genome and whole exome information to laboratory investigations utilising targeted sequencing approaches. Blood group predictions based on genome sequencing and genomic studies are proving accurate, and have shown utility in both research and reference settings. Overall, studies confirm the potential for blood group genomics to reshape donor and patient transfusion management strategies to provide more compatible blood transfusions.

Comprehensive blood group antigen profile predictions for Western Desert Indigenous Australians from whole exome sequence data.

BACKGROUND: The distribution of RBC antigens, which define blood group types, differs among populations. In contrast to many world populations, blood group profiles for Indigenous Australians have not been well studied. As it is now possible to predict comprehensive blood group antigen profiles from genomic data sets, we aimed to apply this for Indigenous Australians and to provide a comparison to other major world populations. STUDY DESIGN AND METHODS: Whole exome sequence data for 72 Western Desert Indigenous Australians was provided by the Telethon Kids Institute. Variants (against hg19) were annotated using computer software (ANNOVAR, Qiagen Bioinformatics) and filtered to include only variants in genes for 36 blood group systems, and the transcription factors KLF1 and GATA1. The RHCE*C allele and RHD zygosity were identified by copy number variant analysis of sequence alignments. The impact of missense variants was investigated in silico using a meta-predictor of disease-causing variants (Meta-SNP). RESULTS: For 21 blood group systems the predicted blood group antigen frequencies were comparable to those for other major world populations. For 13 systems, interesting points of contrast were identified. Furthermore, we identified 12 novel variants, one novel D allele, and four rare variants with potential clinical significance. CONCLUSION: This is the first systematic assessment of genomic data to elucidate blood group antigen profiles for Indigenous Australians who are linguistically and culturally diverse. Our study paves the way to understanding the geographic distribution of blood group variants in different Indigenous groups and the associated RBC phenotypes. This in turn is expected to guide transfusion practice for Indigenous individuals.

Modeling the parvovirus B19 blood safety risk in Australia.

BACKGROUND: Three probable cases of transfusion-transmitted (TT) parvovirus B19 (B19V) occurred in Australia between 2014 and 2017. This study aimed to determine the B19V DNA prevalence among blood donors, to model the risk to recipients of fresh components, and to assess risk management options. STUDY DESIGN AND METHODS: Plasma samples from 4232 donors were tested for B19V DNA by polymerase chain reaction. Reactive samples were confirmed and viral load determined. A transmission-risk model was used to estimate recipient risk, and the risk from community exposure was estimated using seroprevalence data. RESULTS: Two samples (0.0473%, 95% confidence interval [CI] 0.0130-0.172) confirmed positive for B19V DNA had a potentially infectious viral load of 105 IU/mL or higher. The estimated risk of a TT-B19V-associated significant complication was low overall at approximately 1 in 300,000 (95% CI, 1 in 82,000 to 1 in 1 million) fresh components transfused, with 3.1 (95% CI, 0.85-11.3) complications modeled per year. Among vulnerable recipient groups, the risk was higher than 1 in 15,000 patients, but the risk from community exposure far exceeded the transfusion risk for all patient and age groups. CONCLUSION: In the context of the small contribution of transfusion to the burden of B19V disease, the significant costs that would be incurred by any strategy to reduce the risk, and given the significant uncertainties and likely overestimation of the risk, we conclude TT-B19V is a tolerable risk to blood safety, despite being high for some vulnerable recipient groups.

Molecular Basis and Clinical Overview of McLeod Syndrome Compared With Other Neuroacanthocytosis Syndromes: A Review.

Importance: McLeod syndrome, encoded by the gene XK, is a rare and progressive disease that shares important similarities with Huntington disease but has widely varied neurologic, neuromuscular, and cardiologic manifestations. Patients with McLeod syndrome have a distinct hematologic presentation with specific transfusion requirements. Because of its X-linked location, loss of the XK gene or pathogenic variants in this gene are principally associated with the McLeod blood group phenotype in male patients. The clinical manifestation of McLeod syndrome results from allelic variants of the XK gene or as part of a contiguous gene deletion syndrome involving XK and adjacent genes, including those for chronic granulomatous disease, Duchenne muscular dystrophy, and retinitis pigmentosa. McLeod syndrome typically manifests as neurologic and cardiologic symptoms that evolve in individuals beginning at approximately 40 years of age. Observations: Diagnosis of McLeod syndrome encompasses a number of specialties, including neurology and transfusion medicine. However, information regarding the molecular basis of the syndrome is incomplete, and clinical information is difficult to find. The International Society of Blood Transfusion has recently compiled and curated a listing of XK alleles associated with the McLeod phenotype. Of note, McLeod syndrome caused by structural variants as well as those cases diagnosed as part of a contiguous gene deletion syndrome were previously classified under a singular allele designation. Conclusions and Relevance: This review discusses the clinical manifestations and molecular basis of McLeod syndrome and provides a comprehensive listing of alleles with involvement in the syndrome published to date. This review highlights the clinical diversity of McLeod syndrome and discusses the development of molecular tools to elucidate genetic causes of disease. A more precise and systematic genetic classification is the first step toward correlating and understanding the diverse phenotypic manifestations of McLeod syndrome and may guide clinical treatment of patients and support for affected and carrier family members. This review provides a knowledge base for neurologists, hematologists, and clinical geneticists on this rare and debilitating disease.

A DEL phenotype attributed to RHD Exon 9 sequence deletion: slipped-strand mispairing and blood group polymorphisms.

BACKGROUND: The RhD blood group antigen is extremely polymorphic and the DEL phenotype represents one such class of polymorphisms. The DEL phenotype prevalent in East Asian populations arises from a synonymous substitution defined as RHD*1227A. However, initially, based on genomic and cDNA studies, the genetic basis for a DEL phenotype in Taiwan was attributed to a deletion of RHD Exon 9 that was never verified at the genomic level by any other independent group. Here we investigate the genetic basis for a Caucasian donor with a DEL partial D phenotype and compare the genomic findings to those initial molecular studies. STUDY DESIGN AND METHODS: The 3'-region of the RHD gene was amplified by long-range polymerase chain reaction (PCR) for massively parallel sequencing. Primers were designed to encompass a deletion, flanking Exon 9, by standard PCR for Sanger sequencing. Targeted sequencing of exons and flanking introns was also performed. RESULTS: Genomic DNA exhibited a 1012-bp deletion spanning from Intron 8, across Exon 9 into Intron 9. The deletion breakpoints occurred between two 25-bp repeat motifs flanking Exon 9 such that one repeat sequence remained. CONCLUSION: Deletion mutations bordered by repeat sequences are a hallmark of slipped-strand mispairing (SSM) event. We propose this genetic mechanism generated the germline deletion in the Caucasian donor. Extensive studies show that the RHD*1227A is the most prevalent DEL allele in East Asian populations and may have confounded the initial molecular studies. Review of the literature revealed that the SSM model explains some of the extreme polymorphisms observed in the clinically significant RhD blood group antigen.

Targeted exome sequencing defines novel and rare variants in complex blood group serology cases for a red blood cell reference laboratory setting.

BACKGROUND: We previously demonstrated that targeted exome sequencing accurately defined blood group genotypes for reference panel samples characterized by serology and single-nucleotide polymorphism (SNP) genotyping. Here we investigate the application of this approach to resolve problematic serology and SNP-typing cases. STUDY DESIGN AND METHODS: The TruSight One sequencing panel and MiSeq platform was used for sequencing. CLC Genomics Workbench software was used for data analysis of the blood group genes implicated in the serology and SNP-typing problem. Sequence variants were compared to public databases listing blood group alleles. The effect of predicted amino acid changes on protein function for novel alleles was assessed using SIFT and PolyPhen-2. RESULTS: Among 29 unresolved samples, sequencing defined SNPs in blood group genes consistent with serologic observation: 22 samples exhibited SNPs associated with varied but known blood group alleles and one sample exhibited a chimeric RH genotype. Three samples showed novel variants in the CROM, LAN, and RH systems, respectively, predicting respective amino acid changes with possible deleterious impact. Two samples harbored rare variants in the RH and FY systems, respectively, not previously associated with a blood group allele or phenotype. A final sample comprised a rare variant within the KLF1 transcription factor gene that may modulate DNA-binding activity. CONCLUSION: Targeted exome sequencing resolved complex serology problems and defined both novel blood group alleles (CD55:c.203G>A, ABCB6:c.1118_1124delCGGATCG, ABCB6:c.1656-1G>A, and RHD:c.452G>A) and rare variants on blood group alleles associated with altered phenotypes. This study illustrates the utility of exome sequencing, in conjunction with serology, as an alternative approach to resolve complex cases.

Evaluation of targeted exome sequencing for 28 protein-based blood group systems, including the homologous gene systems, for blood group genotyping.

BACKGROUND: Blood group single nucleotide polymorphism genotyping probes for a limited range of polymorphisms. This study investigated whether massively parallel sequencing (also known as next-generation sequencing), with a targeted exome strategy, provides an extended blood group genotype and the extent to which massively parallel sequencing correctly genotypes in homologous gene systems, such as RH and MNS. STUDY DESIGN AND METHODS: Donor samples (n = 28) that were extensively phenotyped and genotyped using single nucleotide polymorphism typing, were analyzed using the TruSight One Sequencing Panel and MiSeq platform. Genes for 28 protein-based blood group systems, GATA1, and KLF1 were analyzed. Copy number variation analysis was used to characterize complex structural variants in the GYPC and RH systems. RESULTS: The average sequencing depth per target region was 66.2 ± 39.8. Each sample harbored on average 43 ± 9 variants, of which 10 ± 3 were used for genotyping. For the 28 samples, massively parallel sequencing variant sequences correctly matched expected sequences based on single nucleotide polymorphism genotyping data. Copy number variation analysis defined the Rh C/c alleles and complex RHD hybrids. Hybrid RHD*D-CE-D variants were correctly identified, but copy number variation analysis did not confidently distinguish between D and CE exon deletion versus rearrangement. CONCLUSION: The targeted exome sequencing strategy employed extended the range of blood group genotypes detected compared with single nucleotide polymorphism typing. This single-test format included detection of complex MNS hybrid cases and, with copy number variation analysis, defined RH hybrid genes along with the RHCE*C allele hitherto difficult to resolve by variant detection. The approach is economical compared with whole-genome sequencing and is suitable for a red blood cell reference laboratory setting.