LoRTIS Software Suite: Transposon mutant analysis using long-read sequencing

Abstract To date transposon insertion sequencing (TIS) methodologies have used short-read nucleotide sequencing technology. However, short-read sequences are unlikely to be matched correctly within repeated genomic regions which are longer than the sequence read. This drawback may be overcome using long-read sequencing technology. We have developed a suite of new analysis tools, the “LoRTIS software suite” (LoRTIS-SS), that produce transposon insertion site mapping data for a reference genome using long-read nucleotide sequence data. Long-read nucleotide sequence data can be applied to TIS, this enables the unique mapping of transposon insertion sites within long genomic repeated sequences. Here we present long-read TIS analysis software, LoRTIS-SS, which uses the Snakemake framework to manage the workflow. A docker image is provided, complete with dependencies and ten scripts are included for experiment specific data processing before or after use of the main workflow. The workflow uses long-read nucleotide sequence data such as those generated by the MinION sequencer (Oxford Nanopore Technologies). The unique mapping properties of long-read sequence data were exemplified by reference to the ribosomal RNA genes of Escherichia coli strain BW25113, of which there are 7 copies of ∼4.9 kbases in length that are at least 99% similar. Of reads that matched within rRNA genes, approximately half matched uniquely. The software workflow outputs data compatible with the established Bio-TraDIS analysis toolkit allowing for existing workflows to be easily upgraded to support long-read sequencing.