Schoeman Elizna M, Lopez Genghis H, McGowan Eunike C, Millard Glenda M, O'Brien Helen, Roulis Eileen V, Liew Yew-Wah, Martin Jacqueline R, McGrath Kelli A, Powley Tanya, Flower Robert L, Hyland Catherine A
Research and Development.
Red Cell Reference Laboratory, Australian Red Cross Blood Service, Brisbane, Queensland, Australia.
Transfusion. 2017 Apr;57(4):1078-1088. doi: 10.1111/trf.14054. Epub 2017 Mar 24.
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.
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.
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.
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.
血型单核苷酸多态性基因分型探针针对的多态性范围有限。本研究调查了采用靶向外显子组策略的大规模平行测序（也称为新一代测序）是否能提供扩展的血型基因型，以及大规模平行测序在同源基因系统（如RH和MNS）中正确进行基因分型的程度。
使用TruSight One测序试剂盒和MiSeq平台对28份供体样本进行分析，这些样本已通过单核苷酸多态性分型进行了广泛的表型和基因型分析。分析了28个基于蛋白质的血型系统、GATA1和KLF1的基因。使用拷贝数变异分析来表征GYPC和RH系统中的复杂结构变异。
每个目标区域的平均测序深度为66.2±39.8。每个样本平均含有43±9个变异，其中10±3个用于基因分型。对于这28个样本，大规模平行测序变异序列与基于单核苷酸多态性基因分型数据的预期序列正确匹配。拷贝数变异分析确定了Rh C/c等位基因和复杂的RHD杂合子。正确识别了杂合RHD*D-CE-D变异，但拷贝数变异分析无法可靠地区分D和CE外显子缺失与重排。
与单核苷酸多态性分型相比，所采用的靶向外显子组测序策略扩展了检测到的血型基因型范围。这种单一检测形式包括检测复杂的MNS杂合病例，并且通过拷贝数变异分析，确定了RH杂合基因以及迄今难以通过变异检测解析的RHCE*C等位基因。与全基因组测序相比，该方法经济实惠，适用于红细胞参考实验室环境。
