Computational protocol: Genomic sequence and activity of KS10, a transposable phage of the Burkholderia cepacia complex

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Protocol publication

[…] KS10 was propagated on host B. ambifaria LMG 19467 for DNA extraction. KS10 DNA was isolated from bacteriophage lysate using the Wizard Lambda DNA purification system (Promega Corp., Madison, WI). The purified KS10 DNA was digested using the restriction enzymes SphI, EcoRI, and XhoI (Invitrogen Corp., Carlsbad, CA). To create cloned phage genomic DNA libraries, phage DNA fragments were purified using GeneClean II kit (QBiogene) and ligated into pUC19 or pGEM7Z. Ligated plasmids were transformed into chemically competent E. coli DH5α and the plasmids were purified for sequencing using a Qiaprep miniprep kit (Qiagen Inc., Mississauga, Ont.). Sequencing of the KS10 inserts was carried out using the ABI BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) with an ABI 3100 Gene Analyzer (Molecular Biology Service Unit, Biological Sciences, University of Alberta). Sequencing data was edited using EditView 1.0.1 (Applied Biosystems) and assembled using AutoAssembler (Applied Biosystems). GeneMark and NCBI's ORF Finder programs were used to detect possible open reading frames (ORFs) [,]. Where multiple start codons or ORFs were indicated, the presence of a potential ribosomal binding site (RBS) was used to help identify the most likely ORF. Each identified ORF was tested with BLASTX analysis to assign putative functions []. When BLASTX revealed no significant matches, PSI-BLAST (NCBI) was also used []. Comparisons of KS10 sequences with the B. cenocepacia J2315 genome sequence were carried out using the BLAST server at the Sanger Centre sequencing project web site ). Comparative maps were constructed using GenVision software (DNASTAR, Inc., Madison, Wis.) and Artemis Comparison Tool (Sanger Centre, UK) []. The complete annotated DNA sequence of bacteriophage KS10 can be found in GenBank under accession number EU822883. […]

Pipeline specifications

Software tools GeneMark, Open Reading Frame Finder, BLASTX, BLASTP, DNASTAR Molecular Biology Suite, ACT
Application Genome annotation
Organisms Burkholderia cepacia, Homo sapiens, Burkholderia cenocepacia, Burkholderia ambifaria
Diseases Cystic Fibrosis, Granulomatous Disease, Chronic