iSeq 2.0: A Modular and Interchangeable Toolkit for Interaction Screening in Yeast.

We developed a flexible toolkit for combinatorial screening in Saccharomyces cerevisiae, which generates large libraries of cells, each uniquely barcoded to mark a combination of DNA elements. This interaction sequencing platform (iSeq 2.0) includes genomic landing pads that assemble combinations through sequential integration of plasmids or yeast mating, 15 barcoded plasmid libraries containing split selectable markers (URA3AI, KanMXAI, HphMXAI, and NatMXAI), and an array of ∼24,000 "double-barcoder" strains that can make existing yeast libraries iSeq compatible. Various DNA elements are compatible with iSeq: DNA introduced on integrating plasmids, engineered genomic modifications, or entire genetic backgrounds. DNA element libraries are modular and interchangeable, and any two libraries can be combined, making iSeq capable of performing many new combinatorial screens by short-read sequencing.

The dynamics of adaptive genetic diversity during the early stages of clonal evolution.

The dynamics of genetic diversity in large clonally evolving cell populations are poorly understood, despite having implications for the treatment of cancer and microbial infections. Here, we combine barcode lineage tracking, sequencing of adaptive clones and mathematical modelling of mutational dynamics to understand adaptive diversity changes during experimental evolution of Saccharomyces cerevisiae under nitrogen and carbon limitation. We find that, despite differences in beneficial mutational mechanisms and fitness effects, early adaptive genetic diversity increases predictably, driven by the expansion of many single-mutant lineages. However, a crash in adaptive diversity follows, caused by highly fit double-mutant 'jackpot' clones that are fed from exponentially growing single mutants, a process closely related to the classic Luria-Delbrück experiment. The diversity crash is likely to be a general feature of asexual evolution with clonal interference; however, both its timing and magnitude are stochastic and depend on the population size, the distribution of beneficial fitness effects and patterns of epistasis.