Table_2_Analysis of the Genomic Sequence of ABO Allele Using Next-Generation Sequencing Method.xlsx

BackgroundAlthough many molecular diagnostic methods have been used for ABO genotyping, there are few reports on the full-length genomic sequence analysis of the ABO gene. Recently, next-generation sequencing (NGS) has been shown to provide fast and high-throughput results and is widely used in the clinical laboratory. Here, we established an NGS method for analyzing the sequence of the start codon to the stop codon in the ABO gene.
Study Design and MethodsTwo pairs of primers covering the partial 5’-untranslated region (UTR) to 3’-UTR of the ABO gene were designed. The sequences covering from the start codon to the stop codon of the ABO gene were amplified using these primers, and an NGS method based on the overlap amplicon was developed. A total of 110 individuals, including 88 blood donors with normal phenotypes and 22 ABO subtypes, were recruited and analyzed. All these specimens were first detected by serological tests and then determined by polymerase chain reaction sequence-based typing (PCR-SBT) and NGS. The sequences, including all the intron regions for the specimens, were analyzed by bioinformatics software.
ResultsAmong the 88 blood donors with a normal phenotype, 48 homozygous individuals, 39 heterozygous individuals, and one individual with a novel O allele were found according to the results of the PCR-SBT method. Some single-nucleotide variants (SNV) in intronic regions were found to be specific for different ABO alleles from 48 homozygous individuals using the NGS method. Sequences in the coding region of all specimens using the NGS method were the same as those of the PCR-SBT method. Three intronic SNVs were found to be associated with the ABO subtypes, including one novel intronic SNV (c.28+5956T>A). Moreover, six specimens were found to exhibit DNA recombination.
ConclusionAn NGS method was established to analyze the sequence from the start codon to the stop codon of the ABO gene. Two novel ABO alleles were identified, and DNA recombination was found to exist in the ABO alleles.

背景：尽管诸多分子诊断方法已应用于ABO基因分型，但针对ABO基因全长基因组序列分析的相关报道仍较为匮乏。近年来，下一代测序（next-generation sequencing, NGS）因其可实现快速、高通量的检测结果，已在临床实验室中得到广泛应用。本研究建立了一种可分析ABO基因从起始密码子至终止密码子序列的NGS检测方法。

研究设计与方法：设计了两对可覆盖ABO基因部分5'-非翻译区（5'-untranslated region, UTR）至3'-UTR的引物。利用该引物扩增ABO基因从起始密码子至终止密码子的目标序列，并建立了基于重叠扩增子的NGS检测方法。本研究共招募110名受试者，其中包括88名表型正常的献血者与22名ABO亚型携带者，均纳入分析。所有标本首先通过血清学检测进行初筛，随后分别采用基于聚合酶链反应的序列分型（polymerase chain reaction sequence-based typing, PCR-SBT）与NGS进行检测。借助生物信息学软件对所有标本的序列（包含全部内含子区域）进行分析。

结果：基于PCR-SBT的检测结果，在88名表型正常的献血者中，共检出48名纯合子、39名杂合子，以及1名携带新型O等位基因的受试者。通过NGS方法对48名纯合子样本进行分析，发现内含子区域的部分单核苷酸变异（single-nucleotide variants, SNV）可作为不同ABO等位基因的特异性标志物。所有标本的NGS编码区序列与PCR-SBT检测结果完全一致。共发现3个与ABO亚型相关的内含子SNV，其中包括1个新型内含子SNV（c.28+5956T>A）。此外，6份标本被检出存在DNA重组现象。

结论：本研究成功建立了可分析ABO基因从起始密码子至终止密码子序列的NGS检测方法。共鉴定出2个新型ABO等位基因，并证实ABO等位基因中存在DNA重组现象。