Abstract
BackgroundBarcodes are unique DNA sequence tags that can be used to specifically label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as easily studied. To further support genome-scale research in S. pombe, we generated a barcode-tagged fission yeast insertion mutant library that has the potential of generating viable mutations in both essential and non-essential genes and can be easily analyzed using standard molecular biological techniques.ResultsAn insertion vector containing a selectable ura4+ marker and a random barcode was used to generate a collection of 10,000 fission yeast insertion mutants stored individually in 384-well plates and as six pools of mixed mutants. Individual barcodes are flanked by Sfi I recognition sites and can be oligomerized in a unique orientation to facilitate barcode sequencing. Independent genetic screens on a subset of mutants suggest that this library contains a diverse collection of single insertion mutations. We present several approaches to determine insertion sites.ConclusionsThis collection of S. pombe barcode-tagged insertion mutants is well-suited for genome-wide studies. Because insertion mutations may eliminate, reduce or alter the function of essential and non-essential genes, this library will contain strains with a wide range of phenotypes that can be assayed by their associated barcodes. The design of the barcodes in this library allows for barcode sequencing using next generation or standard benchtop cloning approaches.
Highlights
Barcodes are unique DNA sequence tags that can be used to label individual mutants
The availability of the open reading frame (ORF) deletion mutant collections in the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe has proven to be advantageous in this approach
A linear DNA vector loses sequences from the ends during non-homologous recombination-mediated insertion in S. pombe As a first test of using insertion mutagenesis to generate barcode-tagged insertion mutations in S. pombe, an initial barcoded insertion vector was made by PCR amplification of the ura4+ gene using ura4+-specific primers to produce a ura4+ gene with a random barcode sequence at its 3’ end (Figure 1A)
Summary
Barcodes are unique DNA sequence tags that can be used to label individual mutants. The barcode-tagged open reading frame (ORF) haploid deletion mutant collections in the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe allow for high-throughput mutant phenotyping because the relative growth of mutants in a population can be determined by monitoring the proportions of their associated barcodes. While these mutant collections have greatly facilitated genome-wide studies, mutations in essential genes are not present, and the roles of these genes are not as studied. Following barcode frequencies can identify mutants with a desired growth phenotype from a population of diverse mutants, an approach referred to as parallel analysis [3,4]
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