Mobegi Fredrick M, Bruce Samuel, El-Lagta Naser, Ayora Felipe, Matern Benedict M, Groeneweg Mathijs, D'Orsogna Lloyd J, De Santis Dianne
Department of Clinical Immunology, PathWest Laboratory Medicine WA, Fiona Stanley Hospital Network, Murdoch, WA 6150, Australia.
School of Biomedical Sciences, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands, WA 6009, Australia.
Int J Mol Sci. 2025 Jun 6;26(12):5443. doi: 10.3390/ijms26125443.
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 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.
第三代测序（TGS），也称为长读长测序，已成为临床和研究实验室中一种很有前景的工具，因为它能提供高分辨率结果且通量无与伦比。专业的免疫血液学实验室目前采用基于测序的方法来鉴定通过血清学和基因分型方法无法识别的罕见ABO血型表型。然而，由于缺乏经过验证的实验室方案和生物信息学工具，这些方法的常规临床应用仍然难以实现。在本研究中，我们开发并验证了一种基于TGS的工作流程，用于从口腔拭子或全血中分离的DNA全面确定临床相关的ABO表型。对该基因外显子2至7的区域进行扩增，并在MinION 10.4.1流动槽上进行测序。最初根据gDNA261（rs8176719）、gDNA796（rs8176746）和gDNA803（rs8176747）处的单核苷酸变异来确定预测的ABO表型。然而，某些O亚型缺乏特征性缺失（rs8176719），而是在gDNA802（rs41302905）和gDNA805的外显子7处表现出变异，这是由gDNA804（rs782782485）引起的，这使它们与在gDNA261、gDNA796和gDNA803处具有相同核苷酸的A等位基因区分开来。这些额外的变异被添加到分析流程中以识别额外的亚型。DNA序列数据足以根据五个多态性位点区分四种临床相关的ABO血型表型。虽然高测序覆盖度允许进行更高分辨率的遗传分析，但低至20条读数就足以确定个体的ABO基因型和预测表型。该流程产生的分型结果与血清学结果以及独立实验室的分子分型结果显示出显著的一致性，表明其准确性和可靠性。本研究展示了使用TGS方法对临床相关ABO血型基因型和预测表型进行的全面表征。该方法为常规ABO血型筛查提供了一种可扩展且精确的方法，并有助于开发适用于临床和研究应用的开创性生物信息学工具。
