Abstract
Third-generation sequencing (TGS), also known as long-read sequencing, has become a promising tool in clinical and research laboratories because it delivers high-resolution results with unmatched throughput. Specialised immunohematology laboratories currently employ sequencing-based methods to characterise rare ABO blood group phenotypes that cannot be identified through serology and genotyping methods. However, routine clinical application of these methods remains elusive due to the absence of validated laboratory protocols and bioinformatics tools. In this study, we have developed and validated a TGS-based workflow for comprehensive determination of the clinically relevant ABO phenotypes from DNA isolated from buccal swabs or whole blood. The region spanning exons 2 to 7 of the ABO gene were amplified and sequenced on MinION 10.4.1 flow cells. Predicted ABO phenotypes were initially determined based on single-nucleotide variants at gDNA261 (rs8176719), gDNA796 (rs8176746), and gDNA803 (rs8176747). However, certain O subtypes lacked the distinguishing deletion (rs8176719) and instead exhibited variations in exon 7 at gDNA802 (rs41302905) and gDNA805, caused by gDNA804 (rs782782485), which differentiate them from A alleles sharing the same nucleotides at gDNA261, gDNA796, and gDNA803. These additional variants were added to the analysis pipeline to identify the additional subtypes. DNA sequence data were sufficient to distinguish between the four clinically relevant ABO blood group phenotypes based on five polymorphic positions. While high sequencing coverage allowed for higher resolution genetic analysis, as few as 20 reads are sufficient for determining the ABO genotype and predicted phenotype of an individual. Typing results generated by this pipeline showed remarkable concordance with both serological results and molecular typing results by an independent laboratory, indicating its accuracy and reliability. This study demonstrates a comprehensive characterisation of clinically relevant ABO blood genotypes and predicted phenotypes using TGS methods. The approach provided a scalable and precise method for routine ABO blood group screening and aided in the development of pioneering bioinformatics tools suitable for clinical and research application.