3.3. Genomic Mutation Analyses of Single Cells
To compare the mutation rate in cells cultured for different lengths of time, we performed mitochondrial genomic sequence analyses. Recently, many single-cell genomics technologies have been developed. However, it is difficult to obtain genomic/epigenomic information from limited numbers of cells because of sample loss during chromatin preparation and inefficient immunoprecipitation (Harada et al., 2018). Each cell has only a single nuclear genome but several mitochondrial genomes. Using our single-cell transcriptome sequence data, we obtained sufficient coverage of the mitochondrial genome to call SNPs and short indels, with 498 genomic loci identified with variation at single-cell resolution. We annotated variations as “heteroplasmy,” “homo alternative,” “homo reference,” or “low coverage” (Figure 2). Homo alternative and homo reference variations were shared between most cells. However, heteroplasmic SNPs were unique to individual cells. Stochastically, recently acquired mutations do not show a high frequency in the gene pool. The mutations that arose during our experiment should have appeared as new hetero SNPs; indeed, we could not reject the null hypothesis that the number of novel mutations did not increase during culture, based on a correlation test between the culture time and the hetero SNP/homo SNP ratio in each cell (p = 0.14; Figure 3). This indicated that there was a low genomic mutation rate during cultivation and that the increasing heterogeneity observed in genome-wide gene expression was independent of genomic mutations.
We would like to emphasize the usefulness of the identified heteroplasmic mitochondrial mutations in the context of single-cell cloning. Regulatory authorities require that cell lines used in the commercial production of recombinant proteins must be derived from a single cell progenitor or clone (Evans et al., 2015). To provide evidence of monoclonality, multiple rounds of the limiting dilution method or cell sorting coupled with high-resolution cell imaging are necessary. However, our study indicates that the unique heteroplasmic mitochondrial mutations of individual cultured cells could potentially provide evidence of monoclonality. In other words, evidence of monoclonality could be obtained by single-cell sequencing of the cell strain in question.