Generation and analysis of a barcode-tagged insertion mutant library in the fission yeast Schizosaccharomyces pombe

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.

Identification of a Lifespan Extending Mutation in the Schizosaccharomyces pombe Cyclin Gene clg1+ by Direct Selection of Long-Lived Mutants

“…To allow the isolation of long-lived mutants using this approach, we generated a collection of bar code-tagged insertion mutants where each mutant contains a stably integrated insertion vector bearing a random bar code in an unknown genomic location (Figure 1A) [27]. While this library has the advantage of insertions in ORFs, 5’ and 3’ gene regulatory regions and non-coding RNAs, a disadvantage is that the insertions are random and complex which precludes their identification by high-throughput sequencing [27].…”

“…While this library has the advantage of insertions in ORFs, 5’ and 3’ gene regulatory regions and non-coding RNAs, a disadvantage is that the insertions are random and complex which precludes their identification by high-throughput sequencing [27]. This disadvantage was overcome by including a 27 nt random bar code in each insertion that could be used to track the proportions of individual mutants without predetermining the bar code sequences, and to provide a unique primer to identify the insertion site.…”

“…We have generated an S. pombe DNA insertion mutant library in which each mutant bears a stable insertion tagged with a unique bar code [27]. A commercially available library of bar code-tagged S.…”

“…pombe gene deletion mutants has also been constructed [28]. An advantage of our insertion mutant library is that it is designed to include viable haploid mutants in essential genes as well as mutations in non-coding RNAs, which are usually excluded from ORF deletion libraries [27]. Work in C.…”

“…aging) without prior knowledge of the bar code sequences and without a large investment in the production of microarrays. In contrast, these bar codes can be analyzed using standard cloning and sequencing approaches to identify those mutants with a growth advantage under a given condition [27]. …”