Approaches to querying bacterial genomes with transposon-insertion sequencing

Abstract

In this review, we discuss transposon-insertion sequencing, variously known in the literature as TraDIS, Tn-seq, INSeq, and HITS. By monitoring a large library of single transposon-insertion mutants with high-throughput sequencing, these methods can rapidly identify genomic regions that contribute to organismal fitness under any condition assayable in the laboratory with exquisite resolution. We discuss the various protocols that have been developed and methods for analysis. We provide an overview of studies that have examined the reproducibility and accuracy of these methods, as well as studies showing the advantages offered by the high resolution and dynamic range of high-throughput sequencing over previous methods. We review a number of applications in the literature, from predicting genes essential for in vitro growth to directly assaying requirements for survival under infective conditions in vivo. We also highlight recent progress in assaying non-coding regions of the genome in addition to known coding sequences, including the combining of RNA-seq with high-throughput transposon mutagenesis.

Keywords: bacteria; essential genes; sRNA; sequencing; systems biology; transposon mutagenesis.

Figure 1. An illustration of the workflow typical of transposon-insertion sequencing protocols. Transposons are represented by pink lines, sequencing adaptors by blue, genomic DNA by black and PCR primers by green. Mutants are generated through either in vivo or in vitro transposition and subsequent selection for antibiotic resistance. These mutants are pooled, and optionally competed in test conditions, then genomic DNA is extracted and fragmented by restriction digest or physical shearing. Sequencing adaptors are ligated, some protocols then perform a step to purify fragments containing transposon insertions, and PCR with transposon- and adaptor-specific primers is used to specifically enrich for transposon-containing fragments. The fragments are then sequenced and mapped back to a reference genome to uniquely identify insertion sites with nucleotide-resolution. Dashed boxes indicate steps which differ between protocols.

Figure 2. Applications of transposon-insertion sequencing to non-coding RNAs. (A) Plots of genomic regions in Mycobacterium tuberculosis containing the required non-coding RNAs RNase P (top) and tmRNA (bottom). Tracks, from top to bottom, 1. Histogram of insertion counts, 2. Comprehensive heat-map of requirement of 500-bp windows, 3. Position of annotated genes, 4. Position of TA dinucleotide sites, 5. Position of non-coding RNA. Reproduced from reference . (B) 1 × 1 competition assays validate attenuating Streptococcus pneumoniae sRNA mutants identified by transposon-insertion sequencing. Mice were infected with defined deletions of sRNAs identified as attenuating by Tn-seq and wild-type S. pneumoniae TIGR4 at the body site indicated and bacterial densities were compared 24 h post-infection. These plots show the derived competitive index in blood (top) and the nasopharnyx (bottom). Each point represents the result of a competition experiment between an sRNA deletion mutant and wild-type TIGR4. A competitive index of 1 indicates equivalent numbers of mutants and wild-type were recovered. Modified from reference .