PDF(Pubmed)
Abstract：
Due to substantial improvements in read accuracy, third-generation long-read sequencing holds great potential in blood group diagnostics, particularly in cases where traditional genotyping or sequencing techniques, primarily targeting exons, fail to explain serological phenotypes. In this study, we employed Oxford Nanopore sequencing to resolve all genotype-phenotype discrepancies in the Kidd blood group system (JK, encoded by SLC14A1) observed over seven years of routine high-throughput donor genotyping using a mass spectrometry-based platform at the Blood Transfusion Service, Zurich. Discrepant results from standard serological typing and donor genotyping were confirmed using commercial PCR-SSP kits. To resolve discrepancies, we amplified the entire coding region of SLC14A1 (~24 kb, exons 3 to 10) in two overlapping long-range PCRs in all samples. Amplicons were barcoded and sequenced on a MinION flow cell. Sanger sequencing and bridge-PCRs were used to confirm findings. Among 11,972 donors with both serological and genotype data available for the Kidd system, we identified 10 cases with unexplained conflicting results. Five were linked to known weak and null alleles caused by variants not included in the routine donor genotyping. In two cases, we identified novel null alleles on the JK*01 (Gly40Asp; c.119G>A) and JK*02 (Gly242Glu; c.725G>A) haplotypes, respectively. Remarkably, the remaining three cases were associated with a yet unknown deletion of ~5 kb spanning exons 9-10 of the JK*01 allele, which other molecular methods had failed to detect. Overall, nanopore sequencing demonstrated reliable and accurate performance for detecting both single-nucleotide and structural variants. It possesses the potential to become a robust tool in the molecular diagnostic portfolio, particularly for addressing challenging structural variants such as hybrid genes, deletions and duplications.
摘要：
由于读取精度的显著提高，第三代长读数测序在血型诊断中具有巨大潜力，特别是在传统的基因分型或测序技术的情况下，主要针对外显子，无法解释血清学表型。在这项研究中,我们采用牛津纳米孔测序来解决Kidd血型系统中所有基因型-表型差异(JK，由SLC14A1编码）使用基于质谱的平台在输血服务中观察到超过七年的常规高通量供体基因分型，苏黎世.使用商业PCR-SSP试剂盒证实标准血清学分型和供体基因分型的不同结果。为了解决差异，我们扩增了SLC14A1的整个编码区(~24kb，外显子3至10）在所有样品中的两个重叠的长程PCR中。将扩增子条形码化并在MinION流动池上测序。使用Sanger测序和桥-PCR来确认发现。在11,972名具有Kidd系统血清学和基因型数据的捐赠者中，我们确定了10例原因不明的结果相互矛盾.五个与由常规供体基因分型中未包括的变体引起的已知弱和无效等位基因相关。在两种情况下，我们在JK*01（Gly40Asp;c.119G>A）和JK*02（Gly242Glu;c.725G>A）单倍型上鉴定了新的无效等位基因，分别。值得注意的是,其余3例与未知的约5kb缺失相关，跨越JK*01等位基因的外显子9-10，其他分子方法未能检测到。总的来说,纳米孔测序证明了检测单核苷酸和结构变体的可靠和准确的性能。它具有成为分子诊断组合中强大工具的潜力，特别是解决具有挑战性的结构变异，如杂种基因，删除和重复。
