Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Rapid curation of gene disruption collections using Knockout Sudoku

Nature Protocols volume 12, pages 2110–2137 (2017)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

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).

Similar content being viewed by others

References

  1. van Opijnen, T. & Camilli, A. Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat. Rev. Microbiol. 11, 435–442 (2013).

    Article  CAS  Google Scholar 

  2. Hutchison, C.A. et al. Design and synthesis of a minimal bacterial genome. Science 351, aad6253 (2016).

    Article  Google Scholar 

  3. Baym, M., Shaket, L., Anzai, I.A., Adesina, O. & Barstow, B. Rapid construction of a whole-genome transposon insertion collection for Shewanella oneidensis by Knockout Sudoku. Nat. Commun. 7, 13270 (2016).

    Article  CAS  Google Scholar 

  4. Richie, D.L. et al. Identification and evaluation of novel acetolactate synthase inhibitors as antifungal agents. Antimicrob. Agents Chemother. 57, 2272–2280 (2013).

    Article  CAS  Google Scholar 

  5. Brotcke, A. & Monack, D.M. Identification of fevR, a novel regulator of virulence gene expression in Francisella novicida. Infect. Immun. 76, 3473–3480 (2008).

    Article  CAS  Google Scholar 

  6. Ahlund, M.K., Rydén, P., Sjöstedt, A. & Stöven, S. Directed screen of Francisella novicida virulence determinants using Drosophila melanogaster. Infect. Immun. 78, 3118–3128 (2010).

    Article  Google Scholar 

  7. Fey, P.D. et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. MBio 4, e00537-12 (2013).

    Article  CAS  Google Scholar 

  8. Zerikly, M., & Challis, G.L. Strategies for the discovery of new natural products by genome mining. Chembiochem 10, 625–633 (2009).

    Article  CAS  Google Scholar 

  9. Seyedsayamdost, M.R. High-throughput platform for the discovery of elicitors of silent bacterial gene clusters. Proc. Natl. Acad. Sci. USA 111, 7266–7271 (2014).

    Article  CAS  Google Scholar 

  10. Winzeler, E.A. et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science 285, 901–906 (1999).

    Article  CAS  Google Scholar 

  11. Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).

    Article  Google Scholar 

  12. Giaever, G. & Nislow, C. The yeast deletion collection: a decade of functional genomics. Genetics 197, 451–465 (2014).

    Article  CAS  Google Scholar 

  13. van Opijnen, T., Bodi, K.L. & Camilli, A. Tn-seq: high-throughput parallel sequencing for fitness and genetic interaction studies in microorganisms. Nat. Methods 6, 767–772 (2009).

    Article  CAS  Google Scholar 

  14. Erlich, Y. et al. DNA Sudoku--harnessing high-throughput sequencing for multiplexed specimen analysis. Genome Res. 19, 1243–1253 (2009).

    Article  CAS  Google Scholar 

  15. Goodman, A.L. et al. Identifying genetic determinants needed to establish a human gut symbiont in its habitat. Cell Host Microbe 6, 279–289 (2009).

    Article  CAS  Google Scholar 

  16. Goodman, A.L., Wu, M. & Gordon, J.I. Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat. Protoc. 6, 1969–1980 (2011).

    Article  CAS  Google Scholar 

  17. Gallagher, L.A. et al. Sequence-defined transposon mutant library of Burkholderia thailandensis. MBio 4, e00604–e00613 (2013).

  18. Porwollik, S. et al. Defined single-gene and multi-gene deletion mutant collections in Salmonella enterica sv Typhimurium. PLoS One 9, e99820 (2014).

    Article  Google Scholar 

  19. Gallagher, L.A. et al. Resources for genetic and genomic analysis of emerging pathogen Acinetobacter baumannii. J. Bacteriol. 197, 2027–2035 (2015).

    Article  CAS  Google Scholar 

  20. Vandewalle, K. et al. Characterization of genome-wide ordered sequence-tagged Mycobacterium mutant libraries by Cartesian pooling-coordinate sequencing. Nat. Commun. 6, 7106 (2015).

    Article  CAS  Google Scholar 

  21. Lane, J.M. & Rubin, E.J. Scaling down: a PCR-based method to efficiently screen for desired knockouts in a high density Mycobacterium tuberculosis picked mutant library. Tuberculosis (Edinb) 86, 310–313 (2006).

    Article  CAS  Google Scholar 

  22. Chun, K.T., Edenberg, H.J., Kelley, M.R. & Goebl, M.G. Rapid amplification of uncharacterized transposon-tagged DNA sequences from genomic DNA. Yeast 13, 233–240 (1997).

    Article  CAS  Google Scholar 

  23. Manoil, C. Tagging exported proteins using Escherichia coli alkaline phosphatase gene fusions. Methods Enzymol. 326, 35–47 (2000).

    Article  CAS  Google Scholar 

  24. Jacobs, M.A. et al. Comprehensive transposon mutant library of Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 100, 14339–14344 (2003).

    Article  CAS  Google Scholar 

  25. Spradling, A.C., Bellen, H.J. & Hoskins, R.A. Drosophila P elements preferentially transpose to replication origins. Proc. Natl. Acad. Sci. USA 108, 15948–15953 (2011).

    Article  CAS  Google Scholar 

  26. Barquist, L., Boinett, C.J. & Cain, A.K. Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biol. 10, 1161–1169 (2014).

    Article  Google Scholar 

  27. Picelli, S. et al. Tn5 transposase and tagmentation procedures for massively scaled sequencing projects. Genome Res. 24, 2033–2040 (2014).

    Article  CAS  Google Scholar 

  28. Deutschbauer, A. et al. Evidence-based annotation of gene function in Shewanella oneidensis MR-1 using genome-wide fitness profiling across 121 conditions. PLoS Genet. 7, e1002385 (2011).

    Article  CAS  Google Scholar 

  29. Bouhenni, R., Gehrke, A. & Saffarini, D. Identification of genes involved in cytochrome c biogenesis in Shewanella oneidensis, using a modified mariner transposon. Appl. Environ. Microbiol. 71, 4935–4937 (2005).

    Article  CAS  Google Scholar 

  30. Melnyk, R.A., Clark, I.C., Liao, A. & Coates, J.D. Transposon and deletion mutagenesis of genes involved in perchlorate reduction in Azospira suillum PS. MBio 5 e00769-13 (2013).

  31. Zou, L. et al. SlyA regulates Type III Secretion System (T3SS) genes in parallel with the T3SS master regulator HrpL in Dickeya dadantii 3937. Appl. Environ. Microbiol. 78, 2888–2895 (2012).

    Article  CAS  Google Scholar 

  32. Maier, T.M., Pechous, R., Casey, M., Zahrt, T.C. & Frank, D.W. In vivo Himar1-based transposon mutagenesis of Francisella tularensis. Appl. Environ. Microbiol. 72, 1878–1885 (2006).

    Article  CAS  Google Scholar 

  33. Rollefson, J.B., Levar, C.E. & Bond, D.R. Identification of genes involved in biofilm formation and respiration via mini-Himar transposon mutagenesis of Geobacter sulfurreducens. J. Bacteriol. 191, 4207–4217 (2009).

    Article  CAS  Google Scholar 

  34. Bonis, B.M. & Gralnick, J.A. Marinobacter subterrani, a genetically tractable neutrophilic Fe(II)-oxidizing strain isolated from the Soudan Iron Mine. Front. Microbiol. 6, 719 (2015).

    Article  Google Scholar 

  35. Yu, R. & Kaiser, D. Gliding motility and polarized slime secretion. Mol. Microbiol. 63, 454–467 (2007).

    Article  CAS  Google Scholar 

  36. Dey, A. & Wall, D. A genetic screen in Myxococcus xanthus identifies mutants that uncouple outer membrane exchange from a downstream cellular response. J. Bacteriol. 196, 4324–4332 (2014).

    Article  Google Scholar 

  37. Zhu, L.-P. et al. Allopatric integrations selectively change host transcriptomes, leading to varied expression efficiencies of exotic genes in Myxococcus xanthus. Microb. Cell Fact. 14, 105 (2015).

    Article  Google Scholar 

  38. Kwon, Y.M., Ricke, S.C. & Mandal, R.K. Transposon sequencing: methods and expanding applications. Appl. Microbiol. Biotechnol. 100, 31–43 (2015).

    Article  Google Scholar 

  39. Kumar, A. Using yeast transposon-insertion libraries for phenotypic screening and protein localization. Cold Spring Harb. Protoc. 2016, http://dx.doi.org/10.1101/pdb.prot085217 (2016).

  40. Alonso, J.M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).

    Article  Google Scholar 

  41. Newman, D. & Kolter, R. A role for excreted quinones in extracellular electron transfer. Nature 405, 94–97 (2000).

    Article  CAS  Google Scholar 

Download references

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

Author notes

  1. Isao A Anzai, Lev Shaket and Oluwakemi Adesina: These authors contributed equally to this work.

Authors and Affiliations

  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

Authors
  1. Isao A Anzai
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lev Shaket
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Oluwakemi Adesina
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Michael Baym
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Buz Barstow
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Ethics declarations

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)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2017.073

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing AI and Robotics

Sign up for the Nature Briefing: AI and Robotics newsletter — what matters in AI and robotics research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: AI and Robotics