An Optimized Genotyping Workflow for Identifying Highly SCRaMbLEd Synthetic Yeasts.

Synthetic Sc2.0 yeast strains contain hundreds to thousands of loxPsym recombination sites that allow restructuring of the Saccharomyces cerevisiae genome by SCRaMbLE. Thus, a highly diverse yeast population can arise from a single genotype. The selection of genetically diverse candidates with rearranged synthetic chromosomes for downstream analysis requires an efficient and straightforward workflow. Here we present loxTags, a set of qPCR primers for genotyping across loxPsym sites to detect not only deletions but also inversions and translocations after SCRaMbLE. To cope with the large number of amplicons, we generated qTagGer, a qPCR genotyping primer prediction tool. Using loxTag-based genotyping and long-read sequencing, we show that light-inducible Cre recombinase L-SCRaMbLE can efficiently generate diverse recombination events when applied to Sc2.0 strains containing a linear or a circular version of synthetic chromosome III.