Computational protocol: Bacteriophages ϕMR299-2 and ϕNH-4 Can Eliminate Pseudomonas aeruginosa in the Murine Lung and on Cystic Fibrosis Lung Airway Cells

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Protocol publication

[…] The genomes of phages ϕNH-4 and ϕ299-2 were sequenced by Beckman Coulter Genomics (Sanger sequencing services; Beckman Coulter, Takeley, United Kingdom) on a 454 GS-FLX sequencer, and sequences were assembled into contigs using the Newbler program (Roche Applied Sciences). The quality of the sequence was assessed using Hawkeye (Amos) (). To confirm phage genome structure, primers were designed at contig ends for PCR amplification using Platinum PCR SuperMix (Invitrogen) followed by direct sequencing of the PCR products, and full phage genome assembly was performed using the Phred-Phrap-Consed package (, ). ORFs were predicted using GLIMMER 3.02 (). The resulting gene models were fed into GAMOLA () for annotation. Complementary annotation was provided using the RAST annotation server (). Data were manually curated using Artemis version 11 () where additional programs were then used, including BLASTp (), GATU () and RBS finder (). Comparative genomics with reference phages (ϕPaP3 and ϕLMA2) were analyzed using the Artemis comparison tool (ACT) () and the Mauve alignment tool (). […]

Pipeline specifications

Software tools Newbler, Hawkeye, Consed, Glimmer, GAMOLA, RAST, BLASTP, GATU, ACT
Applications Phylogenetics, De novo sequencing analysis
Organisms Pseudomonas aeruginosa, Mus musculus, Homo sapiens
Diseases Cystic Fibrosis, Infection, Lung Diseases, Pseudomonas Infections