NGS-based accurate and efficient detection of circulating cell-free mitochondrial DNA in cancer patients
Yang Liu, Kaixiang Zhou, Shanshan Guo, Yang Wang, Xiaoying Ji, Qing Yuan, Liping Su, Xu Guo, Xiwen Gu, Jinliang Xing
Abstract
Mitochondrial DNA (mtDNA) mutations are closely implicated in the pathogenesis of multiple cancers, making circulating cell-free mtDNA (ccf-mtDNA) as a potential non-invasive tumor biomarker. However, an effective approach to comprehensively profile ccf-mtDNA mutations is still lacking. In this study, we first characterized ccf-mtDNA by low-depth whole-genome sequencing (WGS) and found that plasma DNA samples exhibited a dramatic decrease in mtDNA copy number when compared with fresh tumor tissues. Further analysis revealed that plasma ccf-mtDNA had a biased distribution of fragment size with a peak around 90 bp. Based on these insights, we developed a robust captured-based mtDNA deep-sequencing approach that enables accurate and efficient detection of plasma ccf-mtDNA mutations by systematic optimization of probe quantity and length, hybridization temperature, and PCR amplification cycles. Moreover, we found that placement of isolated plasma for 6 h at both 4°C and room temperature (RT) led to a dramatic decrease of ccf-mtDNA stability, highlighting the importance of proper plasma sample processing. We further showed that the optimized approach can successfully detect a substantial fraction of tumor-specific mtDNA mutations in plasma ccf-mtDNA specifically from hepatocellular carcinoma (HCC) patients but not from colorectal cancer (CRC) patients, suggesting the presence of a potential cancer-specific difference in the abundance of tumor-derived mtDNA in plasma.