Rare stochastic mutations may accumulate during dormancy of stem-like cells, but technical limitations in DNA sequencing have limited exploring this possibility. In this study, we employed a recently established deep sequencing method termed Duplex Sequencing to conduct a genome-wide analysis of mitochondrial (mt) DNA mutations in a human breast stem cell model that recapitulates the sequential stages of breast carcinogenesis. Using this method, we found significant differences in mtDNA amongst normal stem cells, immortal/preneoplastic cells, and tumorigenic cells. Putative cancer stem-like cell (CSC) populations and mtDNA copy numbers increased as normal stem cells become tumorigenic cells. Transformed cells exhibited lower rare mutation frequencies of whole mtDNA than did normal stem cells. The predicted mtDNA rare mutation pathogenicity was significantly lower in tumorigenic cells than normal stem cells. Major rare mutation types in normal stem cells are C>T/G>A and T>C/A>G transitions, while only C>T/G>A are major types in transformed cells. We detected a total of 1220 rare point mutations, 678 of which were unreported previously. With only one possible exception (m10342T>C), we did not find specific mutations characterizing mtDNA in human breast CSC; rather, the mitochondrial genome of CSC displayed a decrease in rare mutations overall. Based on our work, we suggest that this decrease (in particular T>C/A>G transitions), rather than the presence of specific mitochondrial mutations, may constitute an early biomarker for breast cancer detection. Our findings support the hypothesis that the mitochondrial genome is altered greatly as a result of the transformation of normal stem cells to CSC, and that mtDNA mutation signatures may aid in delineating normal stem cells from CSC.

<p>Supplementary methods</p>

<p>Figure legends for Supplementary figures 1, 2, and 3</p>

<p>Seven supplementary tables. Table S1. Data yield and Duplex Sequencing statistics. Table S2. Mutation contexts significantly differ between normal stem cells and transformed cells. Table S3. Homoplasmic variants in the whole mtDNA identified using Duplex Sequencing. Table S4. Rare variants in the whole mtDNA of HBEC identified using Duplex Sequencing. Table S5. Mutations that are clonally expanded or negatively selected toward tumorigenesis. Table S6. Distribution of nonsynonymous mutations of rare variants in the whole mtDNA and within mtDNA coding regions. Table S7. The number of non-synonymous mutations of rare variants in mitochondrial protein-coding regions.</p>

<p>g Score sum graph</p>

<p>Mutation signature correlation graph</p>

<p>Rare and low heteroplasmic mutations frequency and mutation (%) fraction graphs</p>

<p>Mutation signature correlation graph</p>