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Rapid curation of gene disruption collections using Knockout Sudoku

Abstract

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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Figure 1: Combinatorial pooling scheme for Knockout Sudoku.
Figure 2: Workflow for creation and annotation of progenitor collection (Steps 1–49).
Figure 3: Software workflow and important input and output files for progenitor collection catalog solution (Steps 32–43).
Figure 4: Detailed schematic of pool amplicon library generation.
Figure 5: Construction of the condensed and quality-controlled collections from the progenitor collection (Steps 50–76).
Figure 6: Workflow for creation of condensed and quality-controlled collections (Steps 50–79).
Figure 7: Software workflow for Sanger verification of Knockout Sudoku predictions (Steps 47–49).

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Acknowledgements

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

Affiliations

Authors

Contributions

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.

Corresponding authors

Correspondence to Michael Baym or Buz Barstow.

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Competing interests

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

Integrated supplementary information

Supplementary Figure 1 Antibiotic titration of S. oneidensis progenitor collection plating.

To find the best choice of antibiotic concentration for preparing the progenitor collection we plated the S. oneidensis progenitor transposon insertion mutant library (left column) and wild-type S. oneidensis (right column) on LB agar Petri dishes with increasing concentrations of kanamycin. We chose a concentration (30 μg/mL) that was slightly higher than that necessary to eliminate the appearance of wild-type colonies and that eliminated the appearance of a lawn of transposon insertion mutants.

Supplementary Figure 2 Testing the freeze-thaw tolerance of the S. oneidensis progenitor collection.

Plate 4 from the S. oneidensis progenitor collection was repeatedly thawed and re-frozen and at each step a new copy was made and the number of surviving mutants on the plate was recorded. This indicates that under the conditions used to preserve the collection (LB with 30 μg/mL kanamycin mixed with an equal volume of 20% v/v glycerol), a plate can withstand at least three thaw-freeze cycles.

Supplementary Figure 3 Molecular weight distribution of progenitor collection pool amplicon libraries

The molecular weight distributions of the amplicon libraries generated from the pooled S. oneidensis progenitor collection were measured by gel electrophoresis on 1% w/v agarose gel in TAE buffer. The gel was run at 150 V for 30 minutes. Amplicon libraries were also generated from wild-type S. oneidensis (SoG); a single mutant with a disruption in the phosphate acetyltransferase gene (Δpta); and a blank sample (Blank).

Supplementary Figure 4 Molecular weight distribution of condensed collection pool amplicon libraries.

The molecular weight distributions of the amplicon libraries generated from the pooled S. oneidensis condensed collection were measured by gel electrophoresis on 1% w/v agarose-TAE gel. The gel was run at 150 V for 30 minutes. Amplicon libraries were also generated for a blank sample (Blank).

Supplementary Figure 5 Purification and size selection of combined pool amplicon libraries.

The amplicon libraries generated from the pooled amplicon libraries were purified by molecular weight prior to next generation sequencing by agarose gel electrophoresis. Approximately 3 μg of DNA from the combined amplicon generation reactions was loaded onto an 8.3 cm long 2% w/v agarose-TAE gel. The gel was run at 150 V for 30 minutes. Immediately after the voltage was removed, the portion of the gel containing amplicons with molecular weights between 500 and 1,000 bp was removed with a scalpel and purified by gel extraction.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Tables 1–6, and Supplementary Data 1–13. (PDF 23076 kb)

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). (MP4 26513 kb)

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Anzai, I., Shaket, L., Adesina, O. et al. Rapid curation of gene disruption collections using Knockout Sudoku. Nat Protoc 12, 2110–2137 (2017). https://doi.org/10.1038/nprot.2017.073

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