Baym, MichaelShaket, LevAnzai, Isao A.Adesina, OluwakemiBarstow, Buz2017-02-182016Baym, Michael, Lev Shaket, Isao A. Anzai, Oluwakemi Adesina, and Buz Barstow. 2016. “Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku.” Nature Communications 7 (1): 13270. doi:10.1038/ncomms13270. http://dx.doi.org/10.1038/ncomms13270.2041-1723http://nrs.harvard.edu/urn-3:HUL.InstRepos:30371059Whole-genome knockout collections are invaluable for connecting gene sequence to function, yet traditionally, their construction has required an extraordinary technical effort. Here we report a method for the construction and purification of a curated whole-genome collection of single-gene transposon disruption mutants termed Knockout Sudoku. Using simple combinatorial pooling, a highly oversampled collection of mutants is condensed into a next-generation sequencing library in a single day, a 30- to 100-fold improvement over prior methods. The identities of the mutants in the collection are then solved by a probabilistic algorithm that uses internal self-consistency within the sequencing data set, followed by rapid algorithmically guided condensation to a minimal representative set of mutants, validation, and curation. Starting from a progenitor collection of 39,918 mutants, we compile a quality-controlled knockout collection of the electroactive microbe Shewanella oneidensis MR-1 containing representatives for 3,667 genes that is functionally validated by high-throughput kinetic measurements of quinone reduction.en-USJournal Article2017-02-1810.1038/ncomms13270