Identification of Transposon Insertion Sites in Maize <i>Mu</i>-Tagged Mutants Using Mu-Seq

<i>Mutator</i> (<i>Mu</i>) transposons facilitate untargeted insertional mutagenesis in maize by moving within the genome and disrupting genes. Such an approach has been used to generate collections such as the <i>BonnMu</i> resource, a <i>Mu-</i>tagged maize population for functional genomics studies. Mutant-Seq (Mu-Seq) is a sequencing-based method for the high-throughput identification and mapping of <i>Mu</i> insertion sites. The approach involves the construction of multiplexed sequencing libraries (known as Mu-Seq libraries) from <i>Mu</i>-tagged populations, followed by high-throughput sequencing and data processing using the Mu-Seq Workflow Utility (MuWU) tool, to determine the location of <i>Mu</i> insertions. Here, we provide a detailed protocol for Mu-Seq, from the generation of the maize <i>Mu</i>-tagged mutant population to data analysis. Researchers can use this approach to develop mutant collections customized to specific genetic backgrounds of interest, which can aid in characterizing genotype-specific mutations and identifying candidate genes linked to visible mutant phenotypes.