Department of Genetics, Case Western Reserve University School of Medicine, 10900 Euclid Avenue, Cleveland, OH 44106, USA.
BMC Genomics. 2012 May 3;13:161. doi: 10.1186/1471-2164-13-161.
Barcodes 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.
An 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.
This 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.
条码是唯一的 DNA 序列标签，可以用于特异性标记个体突变体。在芽殖酵母酿酒酵母和裂殖酵母 Schizosaccharomyces pombe 中，带有条码标签的开放阅读框（ORF）单倍体缺失突变体库允许高通量突变体表型分析，因为通过监测其相关条码的比例，可以确定群体中突变体的相对生长情况。虽然这些突变体库极大地促进了全基因组研究，但不存在必需基因中的突变，并且这些基因的作用也不容易研究。为了进一步支持 S. pombe 的全基因组研究，我们生成了一个带有条码标签的裂殖酵母插入突变体库，该文库有可能在必需基因和非必需基因中产生可行的突变，并且可以使用标准分子生物学技术轻松分析。
含有可选择的 ura4+ 标记和随机条码的插入载体被用于生成一个包含 10000 个裂殖酵母插入突变体的文库，这些突变体分别储存在 384 孔板中，并作为六个混合突变体池存储。每个条码都被 Sfi I 识别位点包围，可以以独特的取向进行寡聚化，以方便条码测序。对部分突变体的独立遗传筛选表明，该文库包含了多样化的单插入突变体集合。我们提出了几种确定插入位点的方法。
这个带有条码标签的 S. pombe 插入突变体文库非常适合全基因组研究。由于插入突变可能消除、减少或改变必需和非必需基因的功能，因此该文库将包含具有广泛表型的菌株，可以通过其相关条码进行检测。该文库中条码的设计允许使用下一代或标准台式克隆方法进行条码测序。
