Computational protocol: Locating and Activating Molecular ‘Time Bombs’: Induction of Mycolata Prophages

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Protocol publication

[…] Bacterial and plasmid whole genome sequence (wgs) data were obtained from GenBank by searching for the genera of interest by name (Gordonia, Mycobacterium, Nocardia, Rhodococcus, and Tsukamurella), and those present are described in detail in and Tables. Putative prophage DNA sequence data were detected using PHAST [], which was preferred over other programs including Prophage Finder [], Prophinder [], and Phage finder [] because of its faster run times, ability to process a wider variety of file types (annotated or un-annotated) of differing completeness (draft or finished) and increased sensitivity []. [...] Phage DNA sequencing libraries were prepared using an Illumina Nextera XT sample preparation kit following the manufacturer’s instructions. The prepared DNA libraries were sequenced on an Illumina MiSeq as a 150-bp paired end run and sequence reads were assembled using CLC Workbench. Open reading frames (orfs) within the de novo assembled sequences were detected using Glimmer (v3.02), for orfs with a minimum size of 90 bp []. All predicted start codons were inspected manually for the presence of putative ribosomal binding sites, and corrected if required. Sequence similarity searches were performed against genome sequences in GenBank. The presence of tRNA and tmRNA was sought using ARAGORN [] and with tRNAScan-SE []. Transmembrane domains were predicted with the DAS transmembrane prediction server [], as described previously []. […]

Pipeline specifications

Software tools PHAST, Prophage Finder, Prophinder, Phage_Finder, Glimmer, ARAGORN, tRNAscan-SE
Databases tmRNA
Applications Genome annotation, WGS analysis
Organisms Tsukamurella paurometabola
Diseases Mycobacterium Infections
Chemicals Mycolic Acids, Mitomycin