Lyu Xue-Ying, Tsui Yu-Man, Tam Ivan Ka-Kit, Li Po-Man, Cheung Gary Cheuk-Hang, Lee Joyce Man-Fong, Ng Irene Oi-Lin, Ho Daniel Wai-Hung
State Key Laboratory of Liver Research, The University of Hong Kong, Hong Kong, China.
Department of Pathology, School of Clinical Medicine, The University of Hong Kong, Hong Kong, China.
Cancers (Basel). 2024 Aug 29;16(17):3012. doi: 10.3390/cancers16173012.
Mitochondria generate energy to support cells. They are important organelles that engage in key biological pathways. The dysfunction of mitochondria can be linked to hepatocarcinogenesis, which has been actively explored in recent years. To investigate the mitochondrial dysfunction caused by genetic variations, target-panel sequencing is a flexible and promising strategy. However, the copy number of mitochondria generally exceeds nuclear DNA, which raises a concern that uneven target enrichment of mitochondrial DNA (mtDNA) and nuclear DNA (ncDNA) in target-panel sequencing would lead to an undesirably biased representation of them. To resolve this issue, we evaluated the optimal pooling of mtDNA probes and ncDNA probes by a series of dilutions of mtDNA probes in both genomic DNA (gDNA) and cell-free DNA (cfDNA) samples. The evaluation was based on read count, average sequencing depth and coverage of targeted regions. We determined that an mtDNA:ncDNA probe ratio of around 1:10 would offer a good balance of sequencing performance and cost effectiveness. Moreover, we estimated the median physiological mtDNA:ncDNA copy ratio as 38.1 and 2.9 in cfDNA and gDNA samples of non-liver cancer subjects, respectively, whereas they were 20.0 and 2.1 in the liver cancer patients. Taken together, this study revealed the appropriate pooling strategy of mtDNA probes and ncDNA probes in target-panel sequencing and suggested the normal range of physiological variation of the mtDNA:ncDNA copy ratio in non-liver cancer individuals. This can serve as a useful reference for future target-panel sequencing investigations of the mitochondrial genome in liver cancer.
线粒体产生能量以支持细胞。它们是参与关键生物途径的重要细胞器。线粒体功能障碍可能与肝癌发生有关，近年来对此进行了积极探索。为了研究由基因变异引起的线粒体功能障碍，靶向测序是一种灵活且有前景的策略。然而，线粒体的拷贝数通常超过核DNA，这引发了一个担忧，即靶向测序中线粒体DNA（mtDNA）和核DNA（ncDNA）的目标富集不均会导致它们出现不理想的偏差。为了解决这个问题，我们通过在基因组DNA（gDNA）和游离DNA（cfDNA）样本中对mtDNA探针进行一系列稀释，评估了mtDNA探针和ncDNA探针的最佳混合比例。评估基于读数计数、平均测序深度和靶向区域的覆盖度。我们确定，mtDNA:ncDNA探针比例约为1:10能在测序性能和成本效益之间实现良好平衡。此外，我们估计非肝癌受试者的cfDNA和gDNA样本中生理mtDNA:ncDNA拷贝比的中位数分别为38.1和2.9，而肝癌患者的这一比例分别为20.0和2.1。综上所述，本研究揭示了靶向测序中mtDNA探针和ncDNA探针的合适混合策略，并提出了非肝癌个体中mtDNA:ncDNA拷贝比的生理变异正常范围。这可为未来肝癌线粒体基因组的靶向测序研究提供有用参考。
