Detection of "hidden" mitotic crossovers by long-read DNA sequencing

Mitotic crossovers in diploid organisms are usually detected by looking for loss of heterozygosity (LOH) for a marker. Assuming the crossover occurs between two duplicated chromatids, a single reciprocal crossover will result in two recombinant chromatids and two nonrecombinant chromatids. LOH will only be observed when a recombinant chromatid cosegregates with a nonrecombinant chromatid. Cells containing both recombinant chromosomes will not result in LOH. Below, we use long-range DNA sequencing to detect crossovers that do not produce LOH, the "hidden" crossovers. In wild-type diploids of Saccharomyces cerevisiae, these crossovers occur at a rate of about 4 × 10^-4/cell division. Long-range sequencing also allows the detection of gene conversions associated with crossovers in samples derived from a single cell. Last, we showed that hydrogen peroxide treatment of G1-arrested cells not only induces frequent recombination, but also a high proportion of two-strand mutation events in which both DNA strands are mutated at the same position prior to replication.