Random insertional mutagenesis screens are important tools in microbial genetics studies. Investigators in fungal systems have used the plant pathogen @entity3261 to create tagged, random mutations for genetic screens in their fungal species of interest through a unique process of trans-kingdom cellular transconjugation. However, identifying the locations of insertion has traditionally required tedious PCR-based methods, limiting the effective throughput of this system. We have developed an efficient genomic sequencing and analysis method (AIM-Seq) to facilitate identification of randomly generated genomic insertions in microorganisms. AIM-Seq combines batch sampling, whole genome sequencing, and a novel bioinformatics pipeline, AIM-HII, to rapidly identify sites of genomic insertion. We have specifically applied this technique to @entity3261 -mediated transconjugation in the @entity1 fungal pathogen @entity1327 . With this approach, we have screened a library of @entity1327 cell wall mutants, selecting twenty-seven mutants of interest for analysis by AIM-Seq. We identified thirty-five putative genomic insertions in known and previously unknown regulators of cell wall processes in this pathogenic fungus. We confirmed the relevance of a subset of these by creating independent mutant strains and analyzing resulting cell wall phenotypes. Through our sequence-based analysis of these mutations, we observed "typical" insertions of the @entity3261 transfer DNA as well as atypical insertion events, including large deletions and chromosomal rearrangements. Initially applied to @entity1327 , this mutant analysis tool can be applied to a wide range of experimental systems and methods of mutagenesis, facilitating future microbial genetic screens.

Rapid mapping of insertional mutations to probe cell wall regulation in XXXX .

@entity1 :: ('9606', 'Species') :: ['human'] @entity1327 :: ('5207', 'Species') :: ['Cryptococcus neoformans', 'C. neoformans'] @entity3261 :: ('358', 'Species') :: ['Agrobacterium tumefaciens', 'Agrobacterium']
@entity1327 :: (5207,Species) :: ['Cryptococcus neoformans']
