Abstract

Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. However, all TIS methods described to date use short nucleotide sequence reads which cannot uniquely determine the locations of transposon insertions within repeating genomic sequences where the repeat units are longer than the sequence read length. To overcome this limitation, we have developed a TIS method using Oxford Nanopore sequencing technology that generates and uses long nucleotide sequence reads; we have called this method LoRTIS (Long-Read Transposon Insertion-site Sequencing). LoRTIS enabled the unique localisation of transposon insertion sites within long repetitive genetic elements of E. coli, such as the transposase genes of insertion sequences and copies of the ~ 5 kb ribosomal RNA operon. We demonstrate that LoRTIS is reproducible, gives comparable results to short-read TIS methods for essential genes, and better resolution around repeat elements. The Oxford Nanopore sequencing device that we used is cost-effective, small and easily portable. Thus, LoRTIS is an efficient means of uniquely identifying transposon insertion sites within long repetitive genetic elements and can be easily transported to, and used in, laboratories that lack access to expensive DNA sequencing facilities.

Highlights

  • Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype

  • The DNA of mutant libraries is sequenced, using a customised protocol, [e.g. transposon-directed insertion site sequencing (TraDIS), transposon sequencing (Tnseq), high-throughput insertion tracking by deep sequencing (HITS) and insertion sequencing (INSeq)], from the transposon reading into the adjacent genome to identify the location of transposon i­nsertions[5,9–11]

  • DNA was prepared for nucleotide sequencing using both the new LoRTIS method and the previously described TraDIS-Xpress protocols in duplicate, from DNA extraction to the generation of nucleotide sequence reads

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Summary

Introduction

Transposon insertion site sequencing (TIS) is a powerful method for associating genotype to phenotype. The DNA of mutant libraries is sequenced, using a customised protocol, [e.g. transposon-directed insertion site sequencing (TraDIS), transposon sequencing (Tnseq), high-throughput insertion tracking by deep sequencing (HITS) and insertion sequencing (INSeq)], from the transposon reading into the adjacent genome to identify the location of transposon i­nsertions[5,9–11]. These TIS methods use different transposon-genome junction enrichment techniques including restriction enzyme digestion, (e.g. MmeI), or the use of customised primer annealing followed by high-throughput sequencing of the resulting ­libraries[5,12].

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