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.