Department of Molecular Genetics, Lerner Research Institute, Cleveland Clinic Lerner College of Medicine at Case Western Reserve University, Cleveland, OH 44195, USA.
Department of Genetics and Genomic Sciences, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA.
Nucleic Acids Res. 2022 Sep 23;50(17):e102. doi: 10.1093/nar/gkac546.
Arrayed libraries of defined mutants have been used to elucidate gene function in the post-genomic era. Yeast haploid gene deletion libraries have pioneered this effort, but are costly to construct, do not reveal phenotypes that may occur with partial gene function and lack essential genes required for growth. We therefore devised an efficient method to construct a library of barcoded insertion mutants with a wider range of phenotypes that can be generalized to other organisms or collections of DNA samples. We developed a novel but simple three-dimensional pooling and multiplexed sequencing approach that leveraged sequence information to reduce the number of required sequencing reactions by orders of magnitude, and were able to identify the barcode sequences and DNA insertion sites of 4391 Schizosaccharomyces pombe insertion mutations with only 40 sequencing preparations. The insertion mutations are in the genes and untranslated regions of nonessential, essential and noncoding RNA genes, and produced a wider range of phenotypes compared to the cognate deletion mutants, including novel phenotypes. This mutant library represents both a proof of principle for an efficient method to produce novel mutant libraries and a valuable resource for the S. pombe research community.
在基因组时代，已经使用定义明确的突变体排列文库来阐明基因功能。酵母单倍体基因缺失文库率先开展了这方面的工作，但构建成本高，不能揭示部分基因功能时可能出现的表型，并且缺乏生长所需的必需基因。因此，我们设计了一种高效的方法来构建具有更广泛表型的条码插入突变体文库，该方法可以推广到其他生物体或 DNA 样本集合。我们开发了一种新颖但简单的三维池化和多路复用测序方法，利用序列信息将所需测序反应的数量减少了几个数量级，并能够仅通过 40 个测序准备来识别 4391 个裂殖酵母插入突变的条码序列和 DNA 插入位点。插入突变发生在非必需、必需和非编码 RNA 基因的基因和非翻译区，与同源缺失突变体相比，产生了更广泛的表型，包括新的表型。这个突变体文库不仅证明了一种高效产生新型突变体文库的方法的原理，而且为裂殖酵母研究社区提供了一个有价值的资源。
