Bone Marrow Transplantation Center, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China.
Institute of Transfusion medicine, Blood Center of Zhejiang Province, Hangzhou, China.
Front Immunol. 2022 Jul 6;13:814263. doi: 10.3389/fimmu.2022.814263. eCollection 2022.
Although many molecular diagnostic methods have been used for genotyping, there are few reports on the full-length genomic sequence analysis of the 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 gene.
Two pairs of primers covering the partial 5'-untranslated region (UTR) to 3'-UTR of the gene were designed. The sequences covering from the start codon to the stop codon of the 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.
Among the 88 blood donors with a normal phenotype, 48 homozygous individuals, 39 heterozygous individuals, and one individual with a novel 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 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.
An NGS method was established to analyze the sequence from the start codon to the stop codon of the gene. Two novel alleles were identified, and DNA recombination was found to exist in the alleles.
尽管已经有许多分子诊断方法用于基因分型，但关于基因全长基因组序列分析的报道很少。最近，下一代测序（NGS）已被证明可提供快速和高通量的结果，并广泛应用于临床实验室。在这里，我们建立了一种用于分析基因起始密码子到终止密码子序列的 NGS 方法。
设计了两对引物，涵盖基因的部分 5'-非翻译区（UTR）到 3'-UTR。使用这些引物扩增涵盖基因起始密码子到终止密码子的序列，并开发了一种基于重叠扩增子的 NGS 方法。共招募并分析了 110 名个体，包括 88 名表型正常的献血者和 22 名 ABO 亚型。所有这些标本首先通过血清学检测，然后通过聚合酶链反应序列基序分型（PCR-SBT）和 NGS 确定。通过生物信息学软件分析包括所有内含子区域的序列。
根据 PCR-SBT 方法的结果，在 88 名表型正常的献血者中发现了 48 名纯合子个体、39 名杂合子个体和 1 名具有新等位基因的个体。使用 NGS 方法，从 48 名纯合子个体中发现了一些内含子区域的单核苷酸变异（SNV）是不同等位基因特有的。使用 NGS 方法，所有标本的编码区序列与 PCR-SBT 方法相同。在 3 个内含子 SNV 与 ABO 亚型相关，包括一个新的内含子 SNV（c.28+5956T>A）。此外，发现 6 个标本存在 DNA 重组。
建立了一种用于分析基因起始密码子到终止密码子序列的 NGS 方法。鉴定了两个新的等位基因，并发现了等位基因中的 DNA 重组。
