Computational protocol: Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis

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Protocol publication

[…] Genome sequencing was performed by the McGill University and Génome Québec Innovation Centre (Montreal, QC, Canada). Two sequencing technologies, Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000 PE100 were used for both samples. This strategy provided an opportunity to successfully close the genome sequences of these two samples and the necessary accuracy of base calls for the sequences. De novo assembly was done using DNASTAR’s SeqMan NGen12 software (DNASTAR, Inc., Wisconsin, USA). Assembly errors and poor quality data were manually trimmed where possible. The contigs were oriented and ordered according to the closed C. perfringens chromosome ATCC13124 (GenBank Accession number NC_008261) using progressiveMauve alignment software []. Subsequently, the complete chromosome sequences of JFP55 and JFP838 were annotated by the Prokaryotic Genome Annotation Pipeline (http://www.ncbi.nlm.nih.gov/genomes/static/Pipeline.html).Complete plasmid sequences of JFP55 and JFP838 were automatically annotated by MyRAST software, the next generation of Rapid Annotation using Subsystem Technology []. In addition, BLASTN and BLASTP analyzes [] were performed to compare the query plasmid sequences with the NCBI database of known sequences.The web-based server PHAST (PHAge Search Tool) [] was used to identify the prophage sequences within the sequenced genomes. [...] The Novel Region Finder of PanSeq software (http://lfz.corefacility.ca/panseq/) with a 500 bp cutoff was used to identify the unique chromosomal and plasmid nucleotide sequence of JFP55 and JFP838. The chromosomal unique regions were identified by comparison to three complete C. perfringens chromosome sequences: ATCC13124, strain 13 (GenBank NC_003366), and SM101 (GenBank CP000312).The unique regions of conjugative plasmids of JFP55 and JFP838 were determined by comparison to six complete C. perfringens conjugative-plasmids: pCW3 (GenBank NC_010937), pCP8533etx (GenBank NC_011412), pCPF5603 (GenBank NC_007773), pCPF4969 (Genbank NC_007772), pCPPB-1 (GenBank NC_015712), and pNetB-NE10 (GenBank NC_019688).In addition, BLASTN and BLASTP were used to determine the unique regions that were common to the two newly sequenced netF-positive C. perfringens isolates. To assign the putative function of the predicted CDSs (coding DNA sequence), significant similarity was defined as having an E value less than 10−20 and covering at least 80% of an CDS’s length available in GenBank. […]

Pipeline specifications

Software tools Mauve, PGAP, RAST, BLASTN, BLASTP, PHAST, Panseq
Applications Genome annotation, Nucleotide sequence alignment
Organisms Clostridium perfringens, Canis lupus familiaris