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Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries

Nature Protocols volume 6, pages 1969–1980 (2011)Cite this article

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Abstract

Insertion sequencing (INSeq) is a method for determining the insertion site and relative abundance of large numbers of transposon mutants in a mixed population of isogenic mutants of a sequenced microbial species. INSeq is based on a modified mariner transposon containing MmeI sites at its ends, allowing cleavage at chromosomal sites 16–17 bp from the inserted transposon. Genomic regions adjacent to the transposons are amplified by linear PCR with a biotinylated primer. Products are bound to magnetic beads, digested with MmeI and barcoded with sample-specific linkers appended to each restriction fragment. After limited PCR amplification, fragments are sequenced using a high-throughput instrument. The sequence of each read can be used to map the location of a transposon in the genome. Read count measures the relative abundance of that mutant in the population. Solid-phase library preparation makes this protocol rapid (18 h), easy to scale up, amenable to automation and useful for a variety of samples. A protocol for characterizing libraries of transposon mutant strains clonally arrayed in a multiwell format is provided.

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Figure 1: Overview of insertion sequencing protocol.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (DK30292, DK70977, and DK064540 to J.I.G.; F32AI078628 and K01DK089121 to A.L.G.) and by the Crohn's and Colitis Foundation of America. We thank L. Kyro and J. Eberle for their assistance in making the movies that accompany this article.

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Author notes

  1. Andrew L Goodman

    Present address: Present address: Section of Microbial Pathogenesis and Microbial Diversity Institute, Yale School of Medicine, New Haven, Connecticut, USA (A.L.G.).,

Authors and Affiliations

  1. Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, Missouri, USA

    Andrew L Goodman, Meng Wu & Jeffrey I Gordon

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Contributions

A.L.G., J.I.G. and M.W. designed the experiments and software. A.L.G., M.W. and J.I.G. wrote the manuscript.

Corresponding authors

Correspondence to Andrew L Goodman or Jeffrey I Gordon.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Table 1

Primer sequences. (DOCX 137 kb)

Supplementary Dataset 1

INSeq_analysis.zip: INSeq data analysis pipeline and README.txt file. (ZIP 14615 kb)

Supplementary Movie 1

Pipetting technique for bead-based separation. This video demonstrates the process of removing the supernatant from beads with a multichannel pipettor as described in Step 34. (MOV 25545 kb)

Supplementary Movie 2

Mapping an arrayed INSeq library by combinatorial pooling. This video demonstrates the process of pooling strains using an EpMotion liquid handling robot as described in Box 1, Step 5. (MOV 25601 kb)

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Goodman, A., Wu, M. & Gordon, J. Identifying microbial fitness determinants by insertion sequencing using genome-wide transposon mutant libraries. Nat Protoc 6, 1969–1980 (2011). https://doi.org/10.1038/nprot.2011.417

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