Computational protocol: Attributes of carbapenemase encoding conjugative plasmid pNDM-SAL from an extensively drug-resistant Salmonella enterica Serovar Senftenberg

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Protocol publication

[…] Plasmid DNA was prepared from the E. coli J53 tranconjugant using Qiagen Maxiprep kit (Qiagen). Paired-end libraries (300–500 bp fragments) were constructed by using the Illumina H TruSeqTM DNA Sample preparation kit (Illumina). Each library was deposited onto a HiSeq Flow Cell and sequenced using an Illumina HiSeq-2000 next-generation DNA sequencer. The distributions of “base quality” “base composition” and %GC of the plasmid were checked. Based on these distributions, the first 15 bases and last one base were trimmed to avoid specific sequence bias and poor quality bases. Errors were corrected in the sequence data using the HiTEC tool. Contig assembly and predicted gaps were then confirmed and filled by PCR-based gap closure, confirmed by DNA sequencing of the amplicons (Applied Biosystems). To assemble the contigs, ABySS and Edena software were used by varying the parameter ‘k’ and ‘overlap cut-off’, respectively. This step produced several contigs for each parameter setting. The contigs were then integrated using contig integrator for sequence assembly (CISA). A blastN search was made against the ‘nt’ database for each contig and two contigs were retained for further downstream analysis. The open reading frames (ORFs) from the contigs were generated by CISA using Glimmer-MG program. For all these ORFs, the nucleotide sequence and amino acid sequences were obtained and translated in the appropriate frame. The predicted ORFs were annotated using an in-house pipeline (CANoPI-Contig Annotator Pipeline) that also includes blastX search for each ORF sequence against ‘nr’ database provided by NCBI. ORF search and gene prediction was performed for the complete plasmid sequence with Lasergene software (DNASTAR, Inc., Madison, WI, USA) and pairwise alignment was analyzed by blastN and blastP homology search using the NCBI database (http://www.ncbi.nlm.nih.gov/blast). […]

Pipeline specifications

Software tools Edena, BLASTN, Glimmer-MG, BLASTX, BLASTP
Application Genome annotation
Organisms Salmonella enterica, Homo sapiens, Escherichia coli, Vibrio cholerae, Shigella flexneri 2a
Diseases Communicable Diseases
Chemicals Tetracycline