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Rapid curation of gene disruption collections using Knockout Sudoku

Nature Protocols volume 12, pages 21102137 (2017) | Download Citation


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 $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 100× faster and 30× 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.

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We thank N. Ando, K. Davis, A. Palmer, S. Meisburger, B. Chang, E. Adler, K. Malzbender, and C. Kyauk for experimental assistance; W. Metcalf for providing the E. coli strain WM3064; J. Gralnick for providing Shewanella oneidensis MR-1; L. Kovacs, J. Miller, L.R. Parsons, S. Silverman, W. Wang, and J. Wiggins for assistance with next-generation sequencing and media preparation; and N. Ando for critical reading of the manuscript. This work was supported by a Career Award at the Scientific Interface from the Burroughs Wellcome Fund and Princeton University startup funds (B.B.) and Fred Fox Class of 1939 funds (I.A.A.).

Author information

Author notes

    • Isao A Anzai
    • , Lev Shaket
    •  & Oluwakemi Adesina

    These authors contributed equally to this work.


  1. Department of Chemistry, Princeton University, Princeton, New Jersey, USA.

    • Isao A Anzai
    • , Lev Shaket
    • , Oluwakemi Adesina
    •  & Buz Barstow
  2. Department of Biomedical Informatics, Harvard Medical School, Boston, Massachusetts, USA.

    • Michael Baym


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M.B. and B.B. conceived the Knockout Sudoku method; L.S., M.B., and B.B. conceived and designed the experiments. I.A.A., L.S., O.A., and B.B performed the experiments; I.A.A., O.A., and B.B. wrote the manuscript; I.A.A., O.A., and B.B. prepared the figures and/or tables; B.B. analyzed the data and developed the KOSUDOKU software; all authors reviewed and revised the manuscript.

Competing interests

All authors of this study are currently seeking patent protection for this method.

Corresponding authors

Correspondence to Michael Baym or Buz Barstow.

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  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–5, Supplementary Tables 1–6, and Supplementary Data 1–13.


  1. 1.

    Combinatorial pooling process for Knockout Sudoku.

    Knockout Sudoku uses a 96-channel pipettor to pool a 96-well plate in four pipetting operations. (i) All wells are pooled into row pools (A–H) using a specialized row tray. (ii) All wells are pooled into column pools (1–12) using a specialized column tray. (iii) All wells are pooled by plate column using an OmniTray specific to the plate's plate column (PC1–PC4 in the example in Figure 1). (iv) All wells are pooled by plate row using an OmniTray specific to the plate's plate row (PR1–PR4 in the example in Figure 1).

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