Barcode sequencing and a high-throughput assay for chronological lifespan uncover ageing-associated genes in fission yeast

Abstract

Ageing-related processes are largely conserved, with simple or-ganisms remaining the main platform to discover and dissect new ageing-associated genes. Yeasts provide potent model systems to study cellular age-ing owing their amenability to systematic functional assays under controlled conditions. Even with yeast cells, however, ageing assays can be laborious and resource-intensive. Here we present improved experimental and computa-tional methods to study chronological lifespan in Schizosaccharomyces pom-be. We decoded the barcodes for 3206 mutants of the latest gene-deletion library, enabling the parallel profiling of ∼700 additional mutants compared to previous screens. We then applied a refined method of barcode sequencing (Bar-seq), addressing technical and statistical issues raised by persisting DNA in dead cells and sampling bottlenecks in aged cultures, to screen for mutants showing altered lifespan during stationary phase. This screen identified 341 long-lived mutants and 1246 short-lived mutants which point to many previ-ously unknown ageing-associated genes, including 46 conserved but entirely uncharacterized genes. The ageing-associated genes showed coherent en-richments in processes also associated with human ageing, particularly with respect to ageing in non-proliferative brain cells. We also developed an auto-mated colony-forming unit assay to facilitate medium- to high-throughput chronological-lifespan studies by saving time and resources compared to the traditional assay. Results from the Bar-seq screen showed good agreement with this new assay. This study provides an effective methodological platform and identifies many new ageing-associated genes as a framework for analys-ing cellular ageing in yeast and beyond.