Wei Xingyu, Yang Xu, Han Wanqing, Zhang Li, Ouyang Guojun, Qu Shoufang, Yang Fang, Yang Xuexi
Department of Fetal Medicine and Prenatal Diagnosis, Zhujiang Hospital, Southern Medical University, Guangzhou, People's Republic of China.
Institute of Antibody Engineering, School of Laboratory Medicine and Biotechnology, Southern Medical University, Guangzhou, People's Republic of China.
J Clin Lab Anal. 2025 Jun;39(11):e70044. doi: 10.1002/jcla.70044. Epub 2025 May 20.
Nanopore sequencing shows advantages in detecting single nucleotide variations (SNVs), deletions, and complex structural variants as a single test in thalassemia. However, the performance evaluation or verification of this method remains unestablished, which is essential before clinical utility and panel registration. Here, we developed a classification method for thalassemia mutations, enabling automated interpretation, visual representation, and identification of diverse mutation types.
We used a total of 36 samples, comprising 32 reference materials and four clinical samples to assess the performance of nanopore sequencing in identifying variants in terms of concordance, precision, and the lower limits of detection.
Our analysis successfully identified 19 SNVs, six deletions, and two triplications using nanopore sequencing across all samples. Notably, these variants showed complete concordance of 100% with the genotypes of the reference materials and known results. The precision of nanopore sequencing for detecting thalassemia variants was consistently high, with neither false positive nor false negative observed. Furthermore, the lower limits of detection achieved in our study were 3 ng/μL.
Overall, our study proved that the reference materials can be used to evaluate the performance of nanopore sequencing in identifying thalassemia mutations, and it is necessary to incorporate triplications when utilizing reference materials for performance evaluation of long-read sequencing. The consistent and robust performance of nanopore sequencing in this study demonstrates its potential as a reliable method for comprehensive variant detection in thalassemia and other genetic diseases diagnosis.
纳米孔测序在作为地中海贫血的单一检测方法来检测单核苷酸变异（SNV）、缺失和复杂结构变异方面显示出优势。然而，该方法的性能评估或验证尚未确立，而这在临床应用和检测组合注册之前至关重要。在此，我们开发了一种地中海贫血突变分类方法，能够实现自动解读、直观呈现并识别多种突变类型。
我们总共使用了36个样本，包括32个参考物质和4个临床样本，以评估纳米孔测序在一致性、精密度和检测下限方面识别变异的性能。
我们的分析通过纳米孔测序在所有样本中成功鉴定出19个SNV、6个缺失和2个三倍体。值得注意的是，这些变异与参考物质的基因型和已知结果显示出100%的完全一致性。纳米孔测序检测地中海贫血变异的精密度一直很高，未观察到假阳性或假阴性。此外，我们研究中实现的检测下限为3 ng/μL。
总体而言，我们的研究证明参考物质可用于评估纳米孔测序识别地中海贫血突变的性能，并且在利用参考物质进行长读长测序性能评估时纳入三倍体是必要的。本研究中纳米孔测序一致且稳健的性能证明了其作为地中海贫血和其他遗传疾病诊断中全面变异检测的可靠方法的潜力。
