Rapid curation of gene disruption collections using Knockout Sudoku

Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as 3 weeks of dedicated labor and at a cost of similar to$10,000 for a collection for a single organism. The method uses manual 4D combinatorial pooling, next-generation sequencing, and a Bayesian inference algorithm to rapidly process and then accurately annotate the extremely large progenitor transposon insertion mutant collections needed to achieve saturating coverage of complex microbial genomes. This method is similar to 100x faster and 30x lower in cost than the next comparable method (In-seq) for annotating transposon mutant collections by combinatorial pooling and next-generation sequencing. This method facilitates the rapid, algorithmically guided condensation and curation of the progenitor collection into a high-quality, nonredundant collection that is suitable for rapid genetic screening and gene discovery.