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.
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Demo data derived from using a freshly induced transposon mutant library has been deposited in the NCBI Sequence Read Archive under accession numbers SRX6817047, SRX6817048 and SRX6817049 (https://www.ncbi.nlm.nih.gov/bioproject/PRJNA564479). The haploid C. albicans GZY892 assembly and annotation reported here are available as Supplementary Data 1. Raw sequence reads from the Illumina library and the PacBio library have been deposited in NCBI under accession numbers PRJNA605578 and PRJNA605577, respectively. This Whole Genome Shotgun project has also been deposited at DDBJ/ENA/GenBank under the accession number JAAGWN000000000. The version described in this paper is the first version, JAAGWN010000000.
The in-house scripts of PBISeq are publicly available in GitHub at https://github.com/xchromosome219/PBseq.pipeline under an MIT license.
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This work was supported by the Thousand Young Talents Program (J.W.), Ministry of Science and Technology of China (2016YFC0900103 to J.W.), National Natural Science Foundation of China (21675098 to J.W.), THU-PKU Center for Life Sciences (J.G. and J.W.) and the Agency for Sciences, Technology and Research of Singapore (NMRC/OFIRG/0072/2018 to Y.W.). J.G. thanks T. Ha for her continued support and company.
The authors declare no competing interests.
Peer review information Nature Protocols thanks Damian Krysan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key reference using this protocol
Gao, J. et al. Nat. Commun. 9, 4495 (2018): https://www.nature.com/articles/s41467-018-06944-1
Supplementary Results, Supplementary Fig. 1 and Supplementary Tables 1 and 2.
The haploid C. albicans GZY892 assembly sequence and its annotation file.
Output file contains the processed mapping results from Step 69.
Output file lists genes mapped by insertions from Step 69.
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Li, Z., Wang, H., Cai, C. et al. Genome-wide piggyBac transposon-based mutagenesis and quantitative insertion-site analysis in haploid Candida species. Nat Protoc 15, 2705–2727 (2020). https://doi.org/10.1038/s41596-020-0351-3