P090 Development of a next generation sequencing based ABO blood group assay and typing software

Abstract

Aim ABO typing of recipients and donors is important for optimal transplant outcomes. ABO typing by serology requires a fresh blood sample, whereas living donor evaluations as well as collection of samples for the donor registry often involve a buccal swab for DNA isolation. ASHI and CAP have recently released guidelines that allow DNA-based genetic ABO determination as a screening tool for initial donor evaluation or for listing donors on the registry. We sought to develop a targeted ABO next generation sequencing (NGS) assay, along with companion interpretive software, for automated data processing and ABO prediction. Methods To develop a target approach, a series of PCR primer combinations spanning the ABO gene were evaluated on blood and buccal swab isolated DNA. The DNA products were run on agarose gel and Agilent bioanalyzer to determine product size and quality. PCR products underwent Illumina TruSight NGS library preparation and sequencing using Illumina MiSeq. A curated ABO allele table was created, and customize software developed to detect and phase ABO alleles from NGS data. Results PCR primer evaluation showed reproducible PCR products spanning exons 6–7 of the ABO gene, which yielded good quality NGS data. These exons encode the major ABO specificities and variations. To verify the ABO NGS assay, a combination of 50 blood and buccal swab samples were tested and found to be 100% concordant with serologic ABO typing which included 19 Group A, 18 Group O, 12 Group B, and 1 Group AB. Conclusions A targeted ABO NGS based assay was developed and optimized for blood and buccal swab isolated DNA. Companion interpretive software, which automates NGS ABO analysis, was developed and correctly determined the ABO type when compared with serology. This assay and software interpretation provides a screening tool for prediction of ABO blood type when no blood sample is available.