UNLABELLED: Transposon insertion sequencing is a high-throughput technique for assaying large libraries of otherwise isogenic transposon mutants providing insight into gene essentiality, gene function and genetic interactions. We previously developed the Transposon Directed Insertion Sequencing (TraDIS) protocol for this purpose, which utilizes shearing of genomic DNA followed by specific PCR amplification of transposon-containing fragments and Illumina sequencing. Here we describe an optimized high-yield library preparation and sequencing protocol for TraDIS experiments and a novel software pipeline for analysis of the resulting data. The Bio-Tradis analysis pipeline is implemented as an extensible Perl library which can either be used as is, or as a basis for the development of more advanced analysis tools. This article can serve as a general reference for the application of the TraDIS methodology. AVAILABILITY AND IMPLEMENTATION: The optimized sequencing protocol is included as supplementary information. The Bio-Tradis analysis pipeline is available under a GPL license at https://github.com/sanger-pathogens/Bio-Tradis CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

UNLABELLED: A typical prokaryote population sequencing study can now consist of hundreds or thousands of isolates. Interrogating these datasets can provide detailed insights into the genetic structure of prokaryotic genomes. We introduce Roary, a tool that rapidly builds large-scale pan genomes, identifying the core and accessory genes. Roary makes construction of the pan genome of thousands of prokaryote samples possible on a standard desktop without compromising on the accuracy of results. Using a single CPU Roary can produce a pan genome consisting of 1000 isolates in 4.5 hours using 13 GB of RAM, with further speedups possible using multiple processors. AVAILABILITY AND IMPLEMENTATION: Roary is implemented in Perl and is freely available under an open source GPLv3 license from http://sanger-pathogens.github.io/Roary CONTACT: [email protected] SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.