Identification of novel molecular targets for Weissella species-specific real-time PCR based on pangenome analysis

The multiplex capability and high yield of current day DNA-sequencing instruments has made bacterial whole genome sequencing a routine affair. The subsequent de novo assembly of reads into contigs has been well addressed. The final step of annotating all relevant genomic features on those contigs can be achieved slowly using existing web- and email-based systems, but these are not applicable for sensitive data or integrating into computational pipelines. Here we introduce Prokka, a command line software tool to fully annotate a draft bacterial genome in about 10 min on a typical desktop computer. It produces standards-compliant output files for further analysis or viewing in genome browsers. Prokka is implemented in Perl and is freely available under an open source GPLv2 license from http://vicbioinformatics.com/. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Summary: 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: roary@sanger.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

The 16S rRNA gene has been a mainstay of sequence-based bacterial analysis for decades. However, high-throughput sequencing of the full gene has only recently become a realistic prospect. Here, we use in silico and sequence-based experiments to critically re-evaluate the potential of the 16S gene to provide taxonomic resolution at species and strain level. We demonstrate that targeting of 16S variable regions with short-read sequencing platforms cannot achieve the taxonomic resolution afforded by sequencing the entire (~1500 bp) gene. We further demonstrate that full-length sequencing platforms are sufficiently accurate to resolve subtle nucleotide substitutions (but not insertions/deletions) that exist between intragenomic copies of the 16S gene. In consequence, we argue that modern analysis approaches must necessarily account for intragenomic variation between 16S gene copies. In particular, we demonstrate that appropriate treatment of full-length 16S intragenomic copy variants has the potential to provide taxonomic resolution of bacterial communities at species and strain level.