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

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Nature Protocols
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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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Author information


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

    • Andrew L Goodman


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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Word documents

  1. Supplementary Table 1 (137K)

    Primer sequences.

Zip files

  1. Supplementary Dataset 1 (15M) INSeq data analysis pipeline and README.txt file.


  1. Supplementary Movie 1 (26M)

    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.

  2. Supplementary Movie 2 (26M)

    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.


  1. Report this comment #30618

    Christopher Surridge said:

    The videos associated with this protocol have been added to our youtube channel. They are embedded below:

    Supplementary Video 1

    Supplementary Video 2

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