Article series: New technologies: methods and applications

Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms

Journal name:
Nature Reviews Microbiology
Year published:
Published online


Our knowledge of gene function has increasingly lagged behind gene discovery, hindering our understanding of the genetic basis of microbial phenotypes. Recently, however, massively parallel sequencing has been combined with traditional transposon mutagenesis in techniques referred to as transposon sequencing (Tn-seq), high-throughput insertion tracking by deep sequencing (HITS), insertion sequencing (INSeq) and transposon-directed insertion site sequencing (TraDIS), making it possible to identify putative gene functions in a high-throughput manner. Here, we describe the similarities and differences of these related techniques and discuss their application to the probing of gene function and higher-order genome organization.

At a glance


  1. Four methods of massively parallel sequencing of transposon insertions.
    Figure 1: Four methods of massively parallel sequencing of transposon insertions.

    Each of four methods of transposon sequencing is illustrated, starting from pooled genomic DNA from the transposon insertion library and ending with sequencing of the left and right transposon junctions. The number of sequences (reads) for each junction can differ between the start of the experiment (t1) and the end (t2), after a selection has been carried out on the library of transposon insertion mutants. In both examples shown, the transposon insertion mutation decreases fitness during growth under the conditions tested, as indicated by there being fewer reads at the end of the experiment than at the start. a | The Tn-seq (named for transposon sequencing) and insertion sequencing (INSeq) methods are highly similar, but INSeq includes a PAGE gel purification step following adaptor ligation and PCR, whereas Tn-Seq includes an agarose gel purification at this point. A recent study66 introduced additional steps to the original INSeq protocol: a linear-PCR step using a biotinylated primer and subsequent purification of the product with magnetic streptavidin beads were added following adapter ligation. These steps reduce both the amount of sample and the amount of enzymes needed. Although they make the protocol more laborious, the results suggest that these modifications increase the sensitivity of the technique. b | The high-throughput insertion tracking by deep sequencing (HITS) and transposon-directed insertion site sequencing (TraDIS) methods are more similar to each other than to Tn-seq and INSeq: after shearing of the DNA, the DNA ends are repaired, and a poly(A) tail is added. However, the methods diverge after the PCR step; in HITS, the PCR products undergo size selection (on a gel) and affinity purification before sequencing, whereas in TraDIS, the PCR products are sequenced directly.

  2. Pathway analysis in vitro and in different host niches.
    Figure 2: Pathway analysis in vitro and in different host niches.

    a | Fitness (W) of individual Streptococcus pneumoniae transposon insertion mutants with insertions in three loci involved in pyrimidine biosynthesis. The genes and genome coordinates of the insertions are indicated. In vitro fitness was calculated during growth in a semi-defined minimal glucose medium42. The data show that SP2193, which encodes a response regulator (a type of transcription factor) and positive regulator of pyrimidine biosynthesis genes, is a suppressor of the fitness defects that result from insertion mutations in each gene involved in the pyrimidine biosynthesis pathway, as determined by genetic interaction analysis. b | Three metabolic pathways in S. pneumoniae lead to the production of inosine monophosphate (IMP) and uridine monophosphate (UMP), which are essential precursors for purine and pyrimidine synthesis, respectively. The effect of disrupting each gene in these three pathways, in terms of the change in bacterial fitness, is indicated for infections of the nasopharynx and of the lung. A question mark indicates that there is no data available. The pathway leading to the synthesis of IMP and purines is required for colonization of the nasopharynx and lung. For the synthesis of UMP and pyrimidines, the pathway that uses glutamine as a precursor is required only for colonization of the nasopharynx, whereas the alternative pathway, which uses uracil as a precursor, can be used for UMP synthesis in the lung.


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Author information


  1. Tim van Opijnen is at the Biology Department, Boston College, 140 Commonwealth Avenue, 420 Higgins Hall, Chestnut Hill, Massachusetts 02467, USA.

  2. Andrew Camilli is at the Howard Hughes Medical Institute and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, Massachusetts 02111, USA.

Competing interests statement

The authors declare no competing interests.

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Author details

  • Tim van Opijnen

    Tim van Opijnen is an assistant professor at Boston College, Chestnut Hill, Massachusetts, USA, where he uses high-throughput robotics, next-generation sequencing and computational biology to develop new antibiotics and engineer bacteria with novel properties that can aid in curing disease.

  • Andrew Camilli

    Andrew Camilli is a Howard Hughes Medical Institute investigator and Professor in the Department of Molecular Biology and Microbiology at Tufts University School of Medicine, Boston, Massachusetts, USA. His laboratory studies the life cycles of bacterial pathogens and is developing new vaccines against these pathogens.

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