Yeast Yeast 2006; 23: 1097–1106. Published online in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/yea.1423 Research Article Systematic hybrid LOH: a new method to reduce false positives and negatives during screening of yeast gene deletion libraries David Alvaro 1 , Ivana Sunjevaric 1 , Robert J. D. Reid 1 , Michael Lisby 2 , David J. Stillman 3 and Rodney Rothstein 1 * 1 Department of Genetics and Development, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA 2 Department of Genetics, Institute of Molecular Biology and Physiology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark 3 Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA *Correspondence to: Rodney Rothstein, Department of Genetics and Development, Columbia University Medical Center, 701 West 168th Street, New York, NY 10032, USA. E-mail: roth- stein@cancercenter.columbia.edu Received: 4 August 2006 Accepted: 9 September 2006 Abstract We have developed a new method, systematic hybrid loss of heterozygosity, to facilitate genomic screens utilizing the yeast gene deletion library. Screening is performed using hybrid diploid strains produced through mating the library haploids with strains from a different genetic background, to minimize the contribution of unpredicted recessive genetic factors present in the individual library strains. We utilize a set of strains where each contains a conditional centromere construct on one of the 16 yeast chromosomes that allows the destabilization and selectable loss of that chromosome. After mating a library gene deletion haploid to such a conditional centromere strain, which corresponds to the chromosome carrying the gene deletion, loss of heterozygosity (LOH) at the gene deletion locus can be generated in these otherwise hybrid diploids. The use of hybrid diploid strains permits complementation of any spurious recessive mutations in the library strain, facilitating attribution of the observed phenotype to the documented gene deletion and dramatically reducing false positive results commonly obtained in library screens. The systematic hybrid LOH method can be applied to virtually any screen utilizing the yeast non-essential gene deletion library and is particularly useful for screens requiring the introduction of a genetic assay into the library strains. Copyright  2006 John Wiley & Sons, Ltd. Keywords: deletion library; loss of heterozygosity; homologous recombination; hybrid Introduction The creation of sets of isogenic haploid strains containing deletions of all non-essential genes by the Yeast Genome Deletion project has provided a critical tool for genomic studies in Saccha- romyces cerevisiae (Winzeler et al., 1999). The gene deletion library permits simultaneous cov- erage of the genome in identical experiments and alleviates the need for cloning to deter- mine the mutation causing a given phenotype. Further, the KanMX4 –marked gene deletions in the library strains can be easily amplified by PCR and cloned or transferred into other genetic backgrounds by genetic recombination, facilitat- ing phenotype confirmation and subsequent experi- ments (Hudson et al., 1997). High-throughput stud- ies analysing drug sensitivity (Chang et al., 2002; Giaever et al., 2004; Lum et al., 2004; Parsons et al., 2004), synthetic lethality (Tong et al., 2001, 2004), and other assays have yielded many results not found in traditional mutagenesis screens. The Copyright  2006 John Wiley & Sons, Ltd.