Computational protocol: Complete Genome Sequence and Annotation of a Campylobacter jejuni Strain, MTVDSCj20, Isolated from a Naturally Colonized Farm-Raised Chicken

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Protocol publication

[…] Campylobacter jejuni is a leading cause of bacterially derived human food-borne illness worldwide (, ). Human Campylobacter-derived gastroenteritis, campylobacteriosis, is predominantly caused by two species of Campylobacter, C. jejuni and C. coli (, ). Campylobacter infections are usually due to the consumption of contaminated poultry products, mainly chicken (). The colonization of broiler chickens with campylobacters usually occurs after 2 weeks of age and can persist until slaughter, with high levels of campylobacters isolated from the ceca (up to 109 CFU/g) ().In an effort to better understand the colonization mechanisms employed by campylobacters during a natural infection, Rainbow Ranger broiler chickens from a local farm in Dexter, MI, were tested for the presence of campylobacters. The cecal contents from 20 birds confirmed to be colonized with Campylobacter were harvested postslaughter, serially diluted in phosphate-buffered saline, and plated on Campylobacter-selective medium (Mueller-Hinton blood agar supplemented with vancomycin [40 µg/ml], cefoperazone [40 µg/ml], trimethoprim [10 µg/ml], and cycloheximide [100 µg/ml]). The colonies that arose on selective medium were confirmed to be Campylobacter by multiplex PCR using primers CJF/R (C. jejuni hipO), CCF/R (C. coli glyA), and 23SF/R (C. jejuni 23s rRNA) (). A single C. jejuni isolate from bird 20 (strain MTVDSCj20) was restreaked and used for whole-genome sequence analysis.Genome sequencing was performed using the shotgun reads obtained on an Illumina MiSeq desktop sequencer. A total of 3,437,992 reads with an average read length of 246 nucleotides (nt) were assembled de novo using the Roche Newbler assembler (version 2.6), resulting in 120 total contigs (>100 bp) and 70 large contigs (5 to 77 kb). A reference assembly against the C. jejuni NCTC 11168 genome was also performed within Newbler. The de novo large contigs and the contigs derived from the reference assembly were used to create a draft scaffold. The scaffold gaps were filled using the small-repeat de novo contigs and the Perl script Contig_extender3 (). The final genome sequence had a coverage of 512×. Homopolymeric GC tracts were characterized using the high-depth MiSeq reads.Protein-, rRNA- and tRNA-coding genes were identified as described previously (). The genome was annotated based on those of the C. jejuni strains NCTC 11168 and 81-176 (accession no. AL111168.1 and CP000538.1, respectively). Additional annotation was performed using Artemis (), the identification of Pfam domains (version 26.0 []), and BLASTp comparisons to proteins in the NCBI nonredundant database.The complete annotated genome sequence is 1.65 Mbp and contains 1,618 open reading frames. The MTVDSCj20 genome contains an additional 22 fragmented coding sequences (CDSs), identified as pseudogenes. BLASTp analysis against the proteins predicted to be encoded by the C. jejuni NCTC 11168 and 81-176 genomes indicated that 1,462 (90%) of the MTVDSCj20 CDSs have orthologs in either the 11168 or 81-176 genomes. Most of the variable genes in MTVDSCj20 were contained within five regions: two regions encoding putative type I restriction/modification systems, the lipooligosaccharide (LOS) and capsular biosynthetic regions, and a genomic island linked to an arginyl-tRNA. […]

Pipeline specifications

Software tools Newbler, BLASTP
Application WGS analysis
Organisms Campylobacter jejuni, Homo sapiens, Gallus gallus
Diseases Foodborne Diseases, Infection