Recombination hotspots measured at ultra-high resolution with error-corrected sequencing
Sprache des Vortragstitels:
Englisch
Original Tagungtitel:
EMBO Meiosis
Sprache des Tagungstitel:
Englisch
Original Kurzfassung:
It is well known that DNA sequences quickly evolve at recombination hotspots by the accumulation of de novo mutations (DNMs) and by GC-biased gene conversion introduced during double strand break (DSB) repair. These events can be examined by collecting a large number of single recombination products. This has been achieved by analyzing a focused hotspot region or a large number of family trios or by whole genome single sperm sequencing. Here, we examined these processes using budding yeast as a model system. Applying CRISPR/Cas, we artificially introduced polymorphisms in close proximity to highly active DSB hotspots in S. cerevisiae. To assess the outcomes of DSB repair at the single molecule DNA-level, we established a highly accurate deep-sequencing approach based on information of the forward and reverse strand (duplex sequencing). With this approach, we obtained data on hundreds of thousands of single molecules of a ~300 bp hotspot region in a single experiment. We achieved an average coverage of ~70k per hotspot with substitution frequencies of 1-3 x 10-7. At this accuracy and resolution, we can quantify the accumulation of DNMs and assess the repair of polymorphic sites during meiosis, also in the genetic background of repair deficient yeast strains. Further, this approach can be easily modified for hotspot analyses in other species and is a powerful approach to examine mutagenesis and repair outcomes at the molecular level driven by meiosis.
Grants:
SFB F-88