Computational protocol: Multiple Mechanisms Contribute to Lateral Transfer of an Organophosphate Degradation (opd) Island in Sphingobium fuliginis ATCC 27551

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Protocol publication

[…] All sequences were viewed and edited to remove vector sequences by using Chromas 2.13 software (www.technelysium.com.au/chromas). Sequences were assembled into contigs by using the program Contig Express of VectorNTI software (Invitrogen Technologies, USA). The assembled sequence of plasmid pPDL2 was annotated using Artemis sequence annotation tool (http://www.sanger.ac.uk/resources/software/artemis/) (). The open reading frames (ORFs) were identified by using the built-in tool of the Artemis software, and the start codons in the predicted ORFs were fixed with the help of BLAST searches. BLAST searches were made against the nonredundant database of NCBI using BLAST program (www.ncbi.nlm.nih.gov/BLAST). IS elements, transposons, and their repeat elements were identified by doing a pairwise alignment using BlastN program against the ISfinder database (http://www-is.biotoul.fr/). [...] For predicting attP sites, two independent approaches were used. In the first approach, sequences of all available plasmids having identical integrases were collected from the NCBI database. From bacteria having these plasmids, a dataset of all tRNA sequences along with their upstream and downstream sequences was created. Pairwise alignments were made between the plasmids and the tRNA sequences to identify the att sites.In a second approach, the genome sequences of bacteria having plasmids with integrase genes were collected from NCBI database. These genomic sequences were then used to predict genomic islands (GI) using Islandviewer software (http://www.pathogenomics.sfu.ca/islandviewer/query.php). The predicted GI sequences were then used to make pairwise alignments with sequences of tRNA genes of Sphingomonas wittichii and Sphingobium japonicum. Short sequence repeats that exactly matched the 3′ end of tRNA sequences were taken as putative att sites. The predicted att sites with a low E-value were considered as potential attP and attB sites. The att sites predicted in this manner were then used to align with plasmid pPDL2 sequence using BlastN to identify attP homologs. Alternatively, tRNA gene sequences taken from S.wittichii and S. japonicum were directly used to align with the pPDL2 sequence using BlastN to find short sequences that perfectly matched the 3′ end of tRNA genes. […]

Pipeline specifications

Software tools Chromas, BLASTN, IslandViewer
Databases ISfinder
Applications Genome annotation, Sanger sequencing
Organisms Escherichia coli, Sphingobium fuliginis ATCC 27551, Sphingobium japonicum