Knockout Sudoku is a method for the construction of whole-genome knockout collections for a wide range of microorganisms with as little as three weeks of dedicated labor and at a cost of approximately $10,000. The method uses manual four-dimensional 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 approximately 100× faster and 30× lower in cost than the next comparable method.
Figure. Combinatorial pooling step of the Knockout Sudoku workflow