Genome-wide piggyBac transposon-based mutagenesis and quantitative insertion-site analysis in haploid Candida species

Nature Protocols
Zeyao LiYue Wang

Abstract

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 accelera...Continue Reading

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Citations

Jun 29, 2021·Frontiers in Oncology·Faroogh MarofiMostafa Jarahian

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Methods Mentioned

BETA
PCR
electrophoresis
chip
Illumina sequencing
genotyping

Software Mentioned

Perl
PBISeq
Qubit
R
FASTX
fastq
Conda
- toolkit
samtools
Bedtools

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