Background Tandem repeats (TRs) are abundant in the human genome and associated with repeat expansion disorders. Our study aimed to develop a tandem repeat panel utilizing targeted long-read sequencing to evaluate known TRs associated with these disorders and assess its clinical utility. Methods We developed a targeted long-read sequencing panel for 70 TR loci, termed dynamic mutation third-generation sequencing (dmTGS), using the PacBio Sequel II platform. We tested 108 samples with suspected repeat expansion disorders and compared the results with conventional molecular methods. Results For 108 samples, dmTGS achieved an average of 8000 high-fidelity reads per sample, with a mean read length of 4.7 kb and read quality of 99.9%. dmTGS outperformed repeat-primed-PCR and fluorescence amplicon length analysis-PCR in distinguishing expanded from normal alleles and accurately quantifying repeat counts. The method demonstrated high concordance with confirmatory methods (rlinear = 0.991, P < 0.01), and detected mosaicism with sensitivities of 1% for FMR1 CGG premutation and 5% for full mutations. dmTGS successfully identified interruptive motifs in genes that conventional methods had missed. For variable number TRs in the PLIN4 gene, dmTGS identified precise repeat counts and sequence motifs. Screening 57 patients with suspected genetic muscular diseases, dmTGS confirmed repeat expansions in genes such as GIPC1, NOTCH2NLC, NUTM2B-AS1/LOC642361, and DMPK. Additionally, dmTGS detected CCG interruptions in CTG repeats in 8 myotonic dystrophy type 1 patients with detailed characterization. Conclusions dmTGS accurately detects repeat sizes and interruption motifs associated with repeat expansion disorders and demonstrates superior performance compared to conventional molecular methods.

中文翻译：

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dmTGS：用于串联重复检测的精确靶向富集长读长测序组合


背景 串联重复序列 （TR） 在人类基因组中含量丰富，并与重复扩增障碍有关。我们的研究旨在开发一个串联重复面板，利用靶向长读长测序来评估与这些疾病相关的已知 TR 并评估其临床效用。方法 我们使用 PacBio Sequel II 平台开发了一种针对 70 个 TR 位点的靶向长读长测序面板，称为动态突变第三代测序 （dmTGS）。我们测试了 108 个疑似重复扩增障碍的样本，并将结果与传统分子方法进行了比较。结果 对于 108 个样品，dmTGS 平均每个样品实现了 8000 个高保真读数，平均读数长度为 4.7 kb，读取质量为 99.9%。dmTGS 在区分扩增等位基因与正常等位基因和准确定量重复计数方面优于重复引物 PCR 和荧光扩增子长度分析-PCR。该方法与验证方法高度一致 （rlinear = 0.991，P < 0.01），并检测到嵌合体，FMR1 CGG 前突变的敏感性为 1%，完全突变的敏感性为 5%。dmTGS 成功地在基因中鉴定了传统方法遗漏的中断基序。对于 PLIN4 基因中数量可变的 TR，dmTGS 鉴定了精确的重复计数和序列基序。筛选 57 例疑似遗传性肌肉疾病患者，dmTGS 证实 GIPC1 、 NOTCH2NLC 、 NUTM2B-AS1/LOC642361 和 DMPK 等基因重复扩增。此外，dmTGS 在 8 例强直性肌营养不良症 1 型患者的 CTG 重复中检测到 CCG 中断，并具有详细的特征。 结论 dmTGS 准确检测与重复扩增障碍相关的重复大小和中断基序，与传统分子方法相比，表现出优异的性能。