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.
线粒体是产生能量以维持细胞功能的重要细胞器，参与多种关键生物学通路。近年研究表明，线粒体功能障碍与肝癌发生发展密切相关。为探究基因变异引起的线粒体功能异常，靶向测序是一种灵活且具有前景的研究策略。然而，线粒体DNA拷贝数通常远超核DNA，这可能导致靶向测序中线粒体DNA与核DNA捕获效率不均，从而造成检测结果的系统性偏差。为解决此问题，本研究通过梯度稀释线粒体DNA探针浓度，在基因组DNA和游离DNA样本中系统评估了线粒体DNA探针与核DNA探针的最佳混合比例。评估指标包括测序读数、平均测序深度及目标区域覆盖度。实验结果表明，当线粒体DNA探针与核DNA探针比例约为1:10时，可在测序性能与成本效益间取得最佳平衡。此外，研究测得非肝癌人群游离DNA与基因组DNA中线粒体DNA与核DNA拷贝数中位比值分别为38.1和2.9，而肝癌患者对应比值分别为20.0和2.1。本研究不仅明确了靶向测序中线粒体DNA与核DNA探针的最佳混合策略，同时揭示了非肝癌人群中线粒体DNA与核DNA拷贝数比值的生理变异范围，为未来肝癌线粒体基因组靶向测序研究提供了重要参考依据。
肿瘤(癌症)患者之家