LoRTIS: A long read method for analysis of large transposon mutant libraries. Long read transposon insertion site sequence (LoRTIS)

Transposon insertion sequencing (TIS) can be a powerful genomics tool for genotype-phenotype association experiments. However, all the TIS methods described to date use short nucleotide sequence reads which cannot determine uniquely the locations of transposon insertions within repeating genomic sequences whose repeat units are longer than the reads. To overcome this limitation, we have developed a TIS method using nanopore sequencing technology that uses long nucleotide sequence reads, called Long read Transposon Insertion-site Sequencing (LoRTIS). We sequenced large transposon mutant library of E. coli BW25113 using LoRTIS. It produced a range of reads from 300 basepairs to 14,000 basepairs that 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 the multicopy ribosomal RNA genes which are about 5 kilobases. The Oxford Nanopore sequencing device is cheaper than most other sequencing machines, small and easily portable. LoRTIS is also an efficient means of uniquely identifying transposon insertion sites within long repetitive genetic elements, is more cost effective than previous TIS methods, and can be easily transported to, and used in, laboratories that lack access to expensive DNA sequencing facilities. So, we demonstrate that LoRTIS is reproducible, gives comparable results to short read TIS methods for essential genes, and better resolution for repeat region of the genome, moreover, is amenable to multiplexing.