The ABO blood group must be determined for blood donors before use of their blood for transfusion and for patients requiring blood transfusion. In our work, due to the discrepant serological results of ABO donor blood group determination, the relatives (mother, aunt, brother and father) were tested as well. Blood group was determined with different serological and molecular methods, as PCR–SSP and NGS. Due to the suspicion of subgroup B, the donor, her relatives and 12 reference samples from the Registry of typed donors were sequenced using a new commercial kit for ABO sequencing with NGS method (Omixon) on the Illumina platform. Most of the determined references samples genotypes were incorrect comparing to the expected genotypes obtained by the PCR-SSP due to the incorrect use of allowed ISBT nomenclature by Omixon software. The alignment of obtained samples sequences with references shows the correct sequencing and matching. Subgroup B was not confirmed at blood donor, mother and aunt. In all three, a mutation A>C at position 533 in intron 5 of the B allele, which can not be found in any database. With some improvements, the used commercial set for determining blood group ABO by NGS could be used.
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