Direct, genome-wide assessment of DNA mutations in single cells

Abstract

DNA mutations are the inevitable consequences of errors that arise during replication and repair of DNA damage. Because of their random and infrequent occurrence, quantification and characterization of DNA mutations in the genome of somatic cells has been difficult. Random, low-abundance mutations are currently inaccessible by standard high-throughput sequencing approaches because they cannot be distinguished from sequencing errors. One way to circumvent this problem and simultaneously account for the mutational heterogeneity within tissues is whole genome sequencing of a representative number of single cells. Here, we show elevated mutation levels in single cells from Drosophila melanogaster S2 and mouse embryonic fibroblast populations after treatment with the powerful mutagen N-ethyl-N-nitrosourea. This method can be applied as a direct measure of exposure to mutagenic agents and for assessing genotypic heterogeneity within tissues or cell populations.

Figure 1.

Somatic mutation detection using single cell sequencing. (A) Somatic mutations in tissues are rare and therefore found only in single sequencing reads from which they are routinely filtered out as sequencing errors during post-alignment processing. Adopting a single cell approach overcomes this limitation by transforming each somatic event into a consensus variant call. (B) Schematic depiction of the single cell sequencing protocol used for Drosophila S2 cells.

Figure 2.

Observed somatic point mutations. (A) Genome-wide sequence coverage and mutation localization in an untreated cell and an ENU-treated cell. The outer track represents binned coverage with an upper cutoff of 50×. The inner track shows the location of detected point mutations (represented as dark points). (B) Histograms of mutant read frequencies for point mutations on chr2L and chrX. The dotted lines indicate a normal distribution with mean of 25 for chr2L and 50 for chrX with standard deviations of 22.

Figure 3.

Somatic point mutation spectra and localization. (A) Mutation spectra for the control and ENU-treated S2 and MEF single cells. Due to a limited number of point mutations, the control cells were combined for the analysis. (B) Strand of origin for ENU-induced mutations found in genic regions of MEFs. (C) Strand of origin for ENU-induced mutations found in genic regions of S2 cells.