Computational protocol: Cluster K Mycobacteriophages: Insights into the Evolutionary Origins of Mycobacteriophage TM4

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Protocol publication

[…] For phage Pixie, double-stranded DNA was phenol-extracted from dialyzed CsCl banded phage particles, then sequenced by 454 technology to 25-fold redundancy (∼4000 reads) at the University of Pittsburgh Genomics and Proteomics Core Laboratories as described previously . Shotgun sequencing for phages Anaya and Adephagia was done by the Joint Genome Institute using both 454 (∼30-fold redundancy) and Illumina (∼100-fold redundancy) technologies. Reads were assembled with Newbler ; Consed was used to assure quality control for the assemblies and identify the natures of the genome termini. To resolve remaining ambiguities, genome finishing was performed using targeted Sanger sequencing on phage genomic DNA templates. Finishing reads were incorporated into existing assemblies using PhredPhrap. The sequencing of TM4 , Angelica, and CrimD was described previously. The Genbank accession numbers for Phages TM4, Pixie, Anaya, Adephagia, Angelica and CrimD are AF068845, JF937104, JF704106, JF704105, HM152764, and HM152767 respectively.Finished sequences were analyzed and annotated in genome editors including DNAMaster (http://cobamide2.bio.pitt.edu), GBrowse , Apollo , and the University California Santa Cruz Genome Browser ; Glimmer , GeneMark , tRNA ScanSE , Aragorn , and Programmed Frameshift Finder were used to identify genome features. Genes were assigned to phams, and genome maps and phamily circle diagrams were drawn using Phamerator, database Mycobacteriophage_83 (S.G.C., R.W.H., G.F.H., manuscript submitted). The threshold parameters of 32.5% identity with ClustalW and a BlastP E-value of 10−50, are different to those used previously, and were derived by optimizing pham assembly over a range of possible values (S.G.C., R.W.H., G.F.H., manuscript submitted). DotPlots were made using Gepard . […]

Pipeline specifications

Software tools Newbler, Consed, TM4, GBrowse, Apollo, Glimmer, GeneMark, ARAGORN, Phamerator, Clustal W, BLASTP, Gepard
Applications Genome annotation, Genome data visualization
Organisms Mycolicibacterium smegmatis, Mycobacterium avium, Mycobacterium phage TM4
Diseases Tuberculosis