Transposon Identification and Mapping Protocol

Sequencing reads were demultiplexed first by Illumina barcodes, and then by custom ‘inline index’ barcodes unique to each sample replicate. Barcodes were trimmed using the fastx barcode splitter and trimmer tools [106 (link)]. The transposon was identified in a two-step pattern matching process, allowing for 3 and 1 nucleotide mismatch respectively. Reads that did not contain the transposon sequence were discarded, while reads with successful transposon-matching were trimmed of the transposon sequence and mapped to the reference genome (CP025209.1) using bwa mem [107 (link)]. The reference genome was closed and annotated by microbesNG, using Prokka (v 1.12; [108 (link)]). The plasmid deposited with this reference sequence was found to be an artefact and subsequently discarded from our analysis. Transposon insertion sequencing reads that successfully mapped to the reference genome were sorted and manipulated using samtools and bedtools [109 (link)]. The first nucleotide at the 52 end of each read was counted as the transposon insertion site. Data were viewed using the Artemis genome browser [110 (link)]. FASTQ data are available at the European Nucleotide Archive for download (accession: PRJEB56349). The processed insertion data can be viewed online at: https://tradis-vault.qfab.org/