Invasive fungal infections caused by Candida species are life threatening with high mortality, posing a severe public health threat. New technologies for rapid, genome-wide identification of virulence genes and therapeutic targets are urgently needed. Our recent engineering of a piggyBac (PB) transposon-mediated mutagenesis system in haploid Candida albicans provides a powerful discovery tool, which we anticipate should be adaptable to other haploid Candida species. In this protocol, we use haploid C. albicans as an example to present an improved version of the mutagenesis system and provide a detailed description of the protocol for constructing high-quality mutant libraries. We also describe a method for quantitative PB insertion site sequencing, PBISeq. The PBISeq library preparation procedure exploits tagmentation to quickly and efficiently construct sequencing libraries. Finally, we present a pipeline to analyze PB insertion sites in a de novo assembled genome of our engineered haploid C. albicans strain. The entire protocol takes ~7 d from transposition induction to having a final library ready for sequencing. This protocol is highly efficient and less labor intensive than alternative approaches and significantly accelerates genetic studies of Candida.

由念珠菌引起的侵袭性真菌感染威胁生命，死亡率高，严重威胁公共健康。迫切需要用于在全基因组范围内快速鉴定毒力基因和治疗靶标的新技术。我们最近在白色单倍体念珠菌中对piggyBac（PB）转座子介导的诱变系统进行的工程设计提供了强大的发现工具，我们预计该工具应适用于其他单倍体念珠菌物种。在此协议中，我们使用单倍体白色念珠菌作为示例，介绍了诱变系统的改进版本，并提供了有关构建高质量突变体文库的协议的详细说明。我们还描述了定量PB插入位点测序PBISeq的方法。PBISeq文库制备程序利用标记技术来快速有效地构建测序文库。最后，我们提出了一个管道，用于分析我们的工程单倍体白色念珠菌菌株从头组装的基因组中的PB插入位点。从转座诱导到最终的测序文库，整个实验过程需约7 d。与替代方法相比，该协议高效且劳动强度低，并且显着加快了遗传学研究的速度。念珠菌。