Global fitness profiling of fission yeast deletion strains by barcode sequencing

Han et al. Genome Biology 2010, 11:R60 http://genomebiology.com/2010/11/6/R60 Open Access METHOD Global fitness profiling of fission yeast deletion strains by barcode sequencing Method Tian Xu Han†, Xing-Ya Xu†, Mei-Jun Zhang, Xu Peng and Li-Lin Du* Abstract A genome-wide deletion library is a powerful tool for probing gene functions and one has recently become available for the fission yeast Schizosaccharomyces pombe. Here we use deep sequencing to accurately characterize the barcode sequences in the deletion library, thus enabling the quantitative measurement of the fitness of fission yeast deletion strains by barcode sequencing. Background Over the past decade, the availability of whole genome sequences for several major model organisms has spurred the development of many powerful reverse genetics approaches and, as a consequence, brought about dramatic changes to the way gene functions are analyzed. The ultimate reverse genetics tool, whole-genome deletion mutant libraries, were first created for the budding yeast Saccharomyces cerevisiae [1,2]. This resource allows all predicted open reading frames in the budding yeast genome to be studied by analyzing the phenotypes of their deletion mutants. Numerous screens have been conducted with the budding yeast deletion libraries to uncover new genes involved in various biological pathways [3]. In addition, new approaches based on the deletion libraries, such as synthetic genetic array analysis, have been developed to map global genetic interaction networks [4]. The utility of the deletion libraries goes even beyond studying gene functions, as profiling drugsensitive yeast mutants has allowed the targets of therapeutic compounds to be defined [5-8]. The construction of the budding yeast deletion libraries incorporated the ingenious idea of molecular barcodes, which are a pair of 20-nucleotide-long unique DNA sequences flanking each deletion cassette [9]. The two barcodes for each gene are called uptag (barcode upstream of the KanMX marker gene) and dntag (barcode downstream of the KanMX marker gene), respec* Correspondence: National Institute of Biological Sciences, 7 Science Park Road, Zhongguancun Life Science Park, Beijing, 102206, PR China † Contributed equally Full list of author information is available at the end of the article tively. These barcodes revolutionized the way yeast mutants are phenotyped by allowing thousands of mutant strains to be pooled and analyzed together in a highly parallel fashion. The barcodes can be easily amplified by PCR from genomic DNA extracted from the yeast cells in the mutant pool. The amounts of barcode PCR products serve as a quantitative measure of the cell number of each deletion strain in the mutant pool. Traditionally, oligonucleotide microarrays have been used to deconvolute the identity of the strains in the mutant pool and quantify the amount of each barcode PCR product. Recently, deep sequencing was found to perform equally well [10]. Compared to one-by-one screen of individual deletion mutants, barcode-based analyses of pooled mutants significantly improve the throughput of screens, reduce the amount of reagents used, and avoid the problems associated with strain cross-contamination. The most frequently analyzed phenotype of pooled mutants is the growth rates, or fitness, of the mutant strains. Fitness profiling of mutants under hundreds of growth conditions has led to the conclusion that 97% of the genes in the budding yeast genome are required for optimal growth under at least one condition [11]. In addition to phenotyping single-gene mutants, barcode-based analysis has also been used to study gene-gene interactions [12,13]. Besides budding yeast, the only other major eukaryotic model organism in which gene deletion can be carried out with ease is the fission yeast Schizosaccharomyces pombe. With its facile genetics, fission yeast has long been a favorite for biologists studying cell cycle control and chromosome dynamics [14,15]. The fission yeast genome contains about 5,000 protein-coding genes, the © 2010 Han et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Han et al. Genome Biology 2010, 11:R60 http://genomebiology.com/2010/11/6/R60 smallest number among the commonly used eukaryotic model organisms [16]. Comparative genomic analysis showed that around 500 fission yeast genes have no homologs in the budding yeast, but are conserved in other eukaryotic species, including human, apparently due to lineage-specific gene losses that happened during the evolution of S. cerevisiae [17]. The recent availability of genome-wide fission yeast deletion libraries has paved the way for global analysis of fission yeast genes, allowing researchers to take full advantage of the differences between the two yeast models [18]. Importantly, the fission yeast deletion libraries have built-in DNA barcodes, similar to the ones used in the budding yeast deletion libraries. The barcode sequences in each strain need to be experimentally characterized as up to 30% of the barcodes in the budding yeast deletion libraries are known to deviate from the original design [10,19]. Here we report a deep sequencing-based characterization of the barcode sequences in the deletion library and describe a fitnessprofiling pipeline that allows the analysis of a fission yeast haploid deletion library in pooled cultures by deep sequencing of the DNA barcodes. Results We used two independent deep sequencing approaches to sequence and deduce the 20-mer barcodes in the haploid Bioneer version 1.0 deletion library (Additional files 1 and 2). We obtained at least one unique barcode sequence for 2,560 strains, which represent about 90% of the strains in the library; and for 2,235 strains, both unique uptag and unique dntag sequences were obtained (Additional file 3). A byproduct of our characterization of the barcodes is the identification of certain defects of the deletion library, including duplicated barcodes, misplaced strains, and contaminated wells (Additional files 4, 5, 6, and 7). The Illumina Genome Analyzer II sequencing platform can generate over 10 million sequence reads in one sequencing lane. On average, one million reads are sufficient to allow each barcode in a library of 3,000 mutants to be sequenced more than 100 times. To take advantage of the sequencing depth and to reduce the cost of barcode sequencing per screen, we adopted a multiplexing strategy to sequence multiple samples in a single lane. A 4nucleotide sequence called the multiplex index was incorporated into the PCR primers that harbor the Illumina sequencing primer sequence (Figure 1) [20,21]. Thus, all sequencing reads begin with the index sequences, which allow reads fr (...truncated)