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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 approximately $10,000. The method uses manual 4-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. Here we present a protocol for the generation, combinatorial pooling and annotation of highly oversampled progenitor collections and their subsequent algorithmically guided condensation and curation into high-quality collections suitable for rapid genetic screening and gene discovery.