Complete RHD next-generation sequencing: establishment of reference RHD alleles

Tounsi, Wajnat A; Madgett, Tracey E.; Avent, Neil D.

doi:10.1182/bloodadvances.2018017871

Cited by 27 publications

(23 citation statements)

References 45 publications

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“…RBC genotyping has also increased the availability of RBC units for routine extended antigen matching in chronically transfused patients and has enabled selection of antigen‐matched units when compatibility cannot be demonstrated due to warm autoantibodies or drug interference in pretransfusion testing. Although single‐nucleotide polymorphism (SNP)‐based assays currently dominate the RBC and PLT genotyping market, next generation sequencing (NGS) approaches including targeted NGS, whole exome sequencing (WES), and whole genome sequencing (WGS) are being actively pursued by several groups …”

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confidence: 99%

Automated typing of red blood cell and platelet antigens from whole exome sequences

et al. 2019

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for the MilSeq Project BACKGROUND: Genotyping has expanded the number red blood cell (RBC) and platelet (PLT) antigens that can readily be typed, but often represents an additional testing cost. The analysis of existing genomic data offers a cost-effective approach. We recently developed automated software (bloodTyper) for determination of RBC and PLT antigens from whole genome sequencing. Here we extend the algorithm to whole exome sequencing (WES). STUDY DESIGN AND METHODS: Whole exomesequencing was performed on samples from 75 individuals. WES-based bloodTyper RBC and PLT typing was compared to conventional polymerase chain reaction (PCR) RHD zygosity testing and serologic and single-nucleotide polymorphism (SNP) typing for 38 RBC antigens in 12 systems (17 serologic and 35 SNPs) and 22 PLT antigens (22 SNPs). Samples from the first 20 individuals were used to modify bloodTyper to interpret WES followed by blinded typing of 55 samples. RESULTS:Over the first 20 samples, discordances were noted for C, M, and N antigens, which were due to WES-specific biases. After modification, bloodTyper was 100% accurate on blinded evaluation of the last 55 samples and outperformed both serologic (99.67% accurate) and SNP typing (99.97% accurate) reflected by two Fy b and one N serologic typing errors and one undetected SNP encoding a Jk null phenotype. RHD zygosity testing by bloodTyper was 100% concordant with a combination of hybrid Rhesus box PCR and PCRrestriction fragment length polymorphism for all samples. CONCLUSION:The automated bloodTyper software was modified for WES biases to allow for accurate RBC and PLT antigen typing. Such analysis could become a routing part of future WES efforts.

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confidence: 99%

Automated typing of red blood cell and platelet antigens from whole exome sequences

et al. 2019

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“…Others have used NGS to determine RH genotypes including targeted NGS focused on blood group genes, WES in which mostly the coding exons of the whole genome are sequenced, and WGS including the coding and non‐coding regions. [4–6, 8–17] Many relied on subject‐matter expert interpretation [4, 5, 10–13, 15, 16] and sometimes utilized additional DNA approaches to aid the interpretation [5]. We and others have used read depth methods to determine RHD zygosity, the presence of alleles encoding D and C antigens and to detect large SVs such as those present in DIIIa‐CE(4‐7)‐D [8–10, 12, 14, 15, 17].…”

Section: Discussionmentioning

confidence: 99%

“…In addition to accurate detection of SNVs, NGS data—especially that derived from whole genome sequencing (WGS)—can be analysed for SVs [3]. NGS, including WGS, whole exome sequencing (WES) and targeted NGS, has been applied to the Rh blood group system [4–17], but the volume and complexity of the raw data are difficult to interpret manually. To address this, we developed automated interpretive software (bloodTyper) and previously validated it against commonly typed RBC antigens including the Rh antigens: D, C, c, E, e, V and VS [14, 17].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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 (blood-Typer) 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.

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“…Meanwhile, long‐range PCR applications for RHD have been described and studies for the FY (Duffy) alleles were described above (Halawani et al , ; Lopez et al , ; Yin et al , ). A recent study describes an important advance whereby the complete RHD sequence was provided using MPS technology (Tounsi et al , ). Interestingly, testing of donor samples revealed characteristic intronic variants delineating the DcE (or R 2 ) haplotype from other Rh haplotypes.…”

Section: Current Challenges With Mps “Read Lengths” and Defining Refementioning

confidence: 99%

Developments beyond blood group serology in the genomics era

2019

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Summary 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.

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Complete RHD next-generation sequencing: establishment of reference RHD alleles

Cited by 27 publications

References 45 publications

Automated typing of red blood cell and platelet antigens from whole exome sequences

Automated typing of red blood cell and platelet antigens from whole exome sequences

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

Developments beyond blood group serology in the genomics era

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