Computational protocol: The rulB gene of plasmid pWW0 is a hotspot for the site-specific insertion of integron-like elements found in the chromosomes of environmental Pseudomonas fluorescens group bacteria

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Protocol publication

[…] PCR products were purified using QIAquick PCR purification kit (Qiagen, UK) and sequenced on the top strand directly from the forward primer of the reaction using Qiagen genomic services (Qiagen, Düsseldorf, Germany).The 10 kb region of pWW0::ILEFH1 in pFBA1001 was sequenced commercially (Qiagen, Germany) by Dye Terminator cycle sequencing (using a Model 3730XL automated DNA Analyser; Life Technologies) of pUC19-based shotgun clones to at least six times coverage and accuracy assured to at least 99.995%.The draft genomes of strains FH1, FH4 and FH5 were sequenced using the Illumina HiSeq platform (Illumina, Cambridge, UK). De novo assembly was performed using Velvet with settings selected using VelvetOptimiser (http://www.vicbioinformatics.com/software.velvetoptimiser.shtml). DNA (BLASTn) and protein (BLASTp) alignments, and ORF analysis (ORF Finder) were carried out using NCBI suite of facilities (http://www.ncbi.nlm.nih.gov). Multiple sequence alignments were performed and annotated using CLUSTALW (Thompson et al., ). Phylogenetic tree construction was carried out using the ‘One Click’ mode within the facilities found at http://www.phylogeny.fr (Dereeper et al., ; ). Graphical representations of DNA were performed manually or using SnapGene V1.4 software (http://www.snapgene.com). […]

Pipeline specifications

Software tools VelvetOptimiser, BLASTN, BLASTP, Open Reading Frame Finder, Clustal W, Phylogeny.fr
Application Phylogenetics
Organisms Pseudomonas fluorescens