Background: Conventional sequencing uses gene-specific primers to determine the location of RH variants and permits a qualitative assessment of zygosity. Whole-genome and whole-exome sequencing determine the genetic location of variants and enable a quantitative assessment of zygosity. Nonspecific sequencing uses RH-consensus primers to detect variants and sequencing-read ratios to quantify their copy number. Study Design and Methods: Two hundred seventy eight samples with diverse genotypes were analyzed by next-generation sequencing with RHconsensus primers. Custom-developed data analysis software was used to detect individual variants and infer the RH genotype. The method was evaluated for its quantitative nature, its ability to discriminate similar genotypes, its accuracy to detect variants, and its accuracy to assign them to RHD or RHCE. Results: As a measure of balanced amplification of RHD and RHCE sequences, observed ratio medians deviate from expected ratios by 3% or less of the ratio range. As a measure of discriminatory power, contiguous RHCE / [RHD + RHCE] ratio averages are separated by 4 or more standard deviations of the mean. Variants are detected with a sensitivity and specificity greater than 99%, and variants at consensus positions are correctly assigned to RHD vs RHCE with a sensitivity greater than 72% and a specificity greater than 99%. The method is successful in the identification of genotypes with large conversion events and in the detection of copy number variation. Conclusion: Nonspecific sequencing of homologous gene sets combines detection and quantification of genetic variation in a single assay. Evidence is provided for the quantitative nature of the method, its sensitivity and specificity, and its ability to identify complex RH genotypes. Conventional sequencing of homologous blood group genes such as RHD and RHCE makes use of gene-specific primers, with data analysis limited to a qualitative determination of homozygosity for the common sequence,
ID RHD XT genotype predictions for high-prevalence RhD negative and weak D types 1, 2 and 3 as well as for HPA-1a/HPA-1b antigens were accurate, which is of clinical significance in guiding transfusion needs.
Background: The most common large-deletion RHD allele (RHD*01N.01) includes the entire coding sequence, intervening regions and untranslated regions. The rest of large-deletion RHD alleles reported to-date consist of single-exon deletions, such as RHD*01N.67 which includes exon 1. Materials and Methods: Samples from two donors with RhD-negative serology yielded unclear or inconclusive results when subject to confirmatory testing on RHD genotyping arrays. To determine their RHD genotypes, genomic DNA was analyzed with a combination of allele-specific PCR, long-range PCR, Sanger sequencing, and next-generation sequencing assays. Results: Allele-specific PCR failed to detect products for RHD exons 1 to 3 in one sample and RHD exons 1 to 5 in the other. A quantitative next-generation sequencing assay confirmed deletion of exons 1 to 3 and 1 to 5 respectively, and detected the absence of an RHD gene in trans in both samples. Long-range PCR and Sanger sequencing enabled identification of the breakpoints for both alleles. Both deletions start within the 5 0 Rhesus box (upstream of the identity region for the 1-to-3 deletion, downstream of it for the 1-to-5 deletion), and end within introns. Conclusions: Resolution of unclear or inconclusive results from targeted genotyping arrays often leads to the discovery of new alleles. The 5 0 Rhesus box may be a hot spot for genetic recombination events, such as the large deletions described in this report.
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