(P-IG-21) Evaluation of multiplex blood group system genotype based on high-throughput exome capture NGS technology

Background/Case Studies: Currently, 44 blood group systems have been confirmed by the International Society for Blood Transfusion (ISBT). Genotyping is helpful to identify the blood group type of individuals, which is of great significance in clinical blood transfusion. Researchers have been committed to develope more efficient, accurate and high-throughput typing technologies, which can be widely used in blood group confirmation or large-scale screening of donors with rare blood groups. Here, we designed and validated a high-throughput extended genotyping setup based on exome capture NGS technology for targeting 36 different blood group systems.

Study

Design/Methods: The NGS technology base on whole exome capture was established for 36 different blood group systems(NO.1-No.36), including 41 genes. 200 blood donors were collected. 36 blood groups genotypes of the donors were performed using the established NGS method. Meanwhile, a multiple PCR-SBT technique for 26 blood group antigens was established and performed with blood group genotyping synchronously, in order to verify the accuracy of the exome capture based NGS technology. The raw sequence data were analyzed by CLC Gennomics workbench software.

Results/Findings: A high-throughput genotyping of the 36 blood group systems by next generation sequencing base on exon capture technology was established. 871 capture probes targeting the whole exome of 36 blood group system genes were designed. The typing platform could fully obtain the exon sequences of the corresponding genes of each blood group system, with wide coverage and high sequencing depth. The average sequencing depth per target region was more than 500. Software analysis could successfully obtain gene sequence characteristics and determine the genotype of blood groups, except RHCE antigen, Chido/Rodgers system and MNS system because of the high degree of homology between the different genes in the same system. Compared with PCR-SBT results, The genotyping results of Gill, Kidd, FORS, Knops, Raph, John Milton NGS scores of Hagen, P1PK, Duffy, Landsteiner-Wiener, Kell, Colton, Lutheran, Diego, Indian, Ok, Scianna, Cromer, Yt, I, Vel, CD59 and Augustine blood group system were 100% consistent with the PCR-SBT genotype results except only one sample in the Dombrock blood group system, indicating the high accuracy of this platform.

Conclusions: The platform could synchronize genotyping of multiple blood group systems of red blood cells with high throughput and high accuracy, which provides a new screening and confirmation technology for identification of blood groups and could improve the detection ability of difficult specimens.
This work was sponsored by Zhejiang Provincial Program for the Cultivation of High-Level Innovative Health Talents, the National Natural Foundation of China (82070195) and the Medical Science Research Foundation of Zhejiang Province(2022KY140).

Importance of research: Currently, the reported genetic analysis methods of blood group are limited to several ones, which cannot effectively target all exons and difficult to realize large-scale screen. In addition, some variants in the blood group system, whose molecular mechanism often involves the changes of the whole exon sequences. A new technology is urgently needed to analysis of all exon sequences of multiple blood group systems, to achieve accurate identification of blood group and improve blood safety.