Department of Neurology, St. Marianna University School of Medicine, Kawasaki, 2168511, Japan.
Artificial Intelligence Research Center, AIST, Tokyo, Japan.
Sci Rep. 2024 Oct 24;14(1):25161. doi: 10.1038/s41598-024-75749-8.
Variants in mitochondrial genomes (mtDNA) can cause various neurological and mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Given the 16 kb length of mtDNA, continuous sequencing is feasible using long-read sequencing (LRS). Herein, we aimed to show a simple and accessible method for comprehensive mtDNA sequencing with potential diagnostic applications for mitochondrial diseases using the compact and affordable LRS flow cell "Flongle." Whole mtDNA amplification (WMA) was performed using genomic DNA samples derived from four patients with mitochondrial diseases, followed by LRS using Flongle. We compared these results to those obtained using Cas9 enrichment. Additionally, the accuracy of heteroplasmy rates was assessed by incorporating mtDNA variants at equimolar levels. Finally, mtDNA from 19 patients with Parkinson's disease (PD) was sequenced using Flongle to identify disease risk-associated variants. mtDNA variants were detected in all four patients with mitochondrial diseases, with results comparable to those obtained from Cas9 enrichment. Heteroplasmy levels were accurately detected (r > 0.99) via WMA using Flongle. A reported variant was identified in three patients with PD. In conclusion, Flongle can simplify the traditionally cumbersome and expensive mtDNA sequencing process, offering a streamlined and accessible approach to diagnosing mitochondrial diseases.
线粒体基因组（mtDNA）的变异可导致各种神经和线粒体疾病，如线粒体肌病、脑病、乳酸酸中毒、卒中样发作（MELAS）。鉴于 mtDNA 的 16kb 长度，使用长读测序（LRS）可以连续测序。在此，我们旨在展示一种使用紧凑型且价格合理的 LRS 流片“Flongle”进行全面 mtDNA 测序的简单且易于获取的方法，该方法具有线粒体疾病的潜在诊断应用。使用来自 4 名线粒体疾病患者的基因组 DNA 样本进行全 mtDNA 扩增（WMA），然后使用 Flongle 进行 LRS。我们将这些结果与使用 Cas9 富集获得的结果进行了比较。此外，通过将 mtDNA 变体以等摩尔水平掺入来评估异质性率的准确性。最后，使用 Flongle 对 19 名帕金森病（PD）患者的 mtDNA 进行测序，以鉴定与疾病风险相关的变体。在所有 4 名线粒体疾病患者中均检测到 mtDNA 变体，结果与 Cas9 富集获得的结果相当。通过 Flongle 进行的 WMA 可以准确检测异质性水平（r＞0.99）。在 3 名 PD 患者中鉴定出了一个报道的变体。总之，Flongle 可以简化传统上繁琐且昂贵的 mtDNA 测序过程，为诊断线粒体疾病提供一种简化且易于获取的方法。
