Insertion Pool Sequencing (iPool-Seq) for theHigh-Throughput Quantification of Ustilago maydis Mutant Fitness.

Insertion pool sequencing (iPool-Seq) is a robust functional genomic technique that combines insertion mutagenesis with next-generation sequencing to comprehensively analyze the genome-wide gene function in maize infecting fungus Ustilago maydis. This high-throughput method tracks the abundance of individual mutants in pooled populations under defined selective conditions. It relies on the construction of saturated mutant libraries, where each mutant carries a unique insertion, followed by tagmentation for efficient DNA fragmentation, tagging with unique molecular identifiers (UMIs), and affinity purification to enrich the pathogen DNA from host tissues. Sequencing of genome insertion junctions then allows determination of mutant representation in Ustilago maydis. Together, this strategy provides a powerful framework to uncover the genes essential for fungal development and virulence within the host.