Genotyping 1000 yeast strains by next-generation sequencing

BMC Genomics

Methodology Article

David W Lazinski¹  Andrew Camilli¹  Julien Gagneur²  Stefan Wilkening³  Wu Wei³  Lars M Steinmetz³  Manu M Tekkedil³  Emilie S Fritsch³  Gen Lin³ 

[1] Department of Molecular Biology & Microbiology and Howard Hughes Medical Institute, Tufts University, 136 Harrison Avenue, 02111-1817, Boston, MA, USA;Gene Center Munich, Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Str. 25, 81377, Munich, Germany;Genome Biology Unit, European Molecular Biology Laboratory, Meyerhofstr. 1, 69117, Heidelberg, Germany;

关键词: Next-generation sequencing;    High throughput;    DNA isolation;    Yeast;    DNA fragmentation;    Heat inactivation;    Recombination;    Aneuploidy;   

DOI  :  10.1186/1471-2164-14-90

 received in 2012-12-07, accepted in 2013-02-06,  发布年份 2013

来源: Springer

【 摘 要 】

BackgroundThe throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, comprising a 96-well based method for DNA isolation for yeast cells, a low-cost DNA shearing alternative, and adapter ligation using heat inactivation of enzymes instead of bead cleanups.ResultsUp to 384 whole-genome libraries can be prepared from yeast cells in one week using this method, for less than 15 euros per sample. We demonstrate the robustness of this protocol by sequencing over 1000 yeast genomes at ~30x coverage. The sequence information from 768 yeast segregants derived from two divergent S. cerevisiae strains was used to generate a meiotic recombination map at unprecedented resolution. Comparisons to other datasets indicate a high conservation of recombination at a chromosome-wide scale, but differences at the local scale. Additionally, we detected a high degree of aneuploidy (3.6%) by examining the sequencing coverage in these segregants. Differences in allele frequency allowed us to attribute instances of aneuploidy to gains of chromosomes during meiosis or mitosis, both of which showed a strong tendency to missegregate specific chromosomes.ConclusionsHere we present a high throughput workflow to sequence genomes of large number of yeast strains at a low price. We have used this workflow to obtain recombination and aneuploidy data from hundreds of segregants, which can serve as a foundation for future studies of linkage, recombination, and chromosomal aberrations in yeast and higher eukaryotes.

【 授权许可】

Unknown   
© Wilkening et al.; licensee BioMed Central Ltd. 2013. This article is published under license to 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.