Computational protocol: Closely related Campylobacter jejuni strains from different sources reveal a generalist rather than a specialist lifestyle

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Protocol publication

[…] 492 C. jejuni and C. coli strains from our collection (473 of which were isolates collected between 2006 and 2010, the others were collected earlier and included some reference strains) comprising strains from different sources in Germany were MLST-typed as described in Dingle et al., 2005 []. Briefly, genomic DNA was amplified with primer pairs for seven housekeeping genes, aspA, glnA, gltA, glyA, pgmA, tkt, and uncA. The PCR-products were purified as described above and sequenced in both orientations using the Sanger method. The MLST data were collected in Seqsphere (RIDOM GmbH, Wuerzburg, Germany; http://www.ridom.de/seqsphere/) and Bionumerics (Applied Maths, Sint Martens-Latem, Belgium). Phylogenetic relationships between the STs were calculated by MLST-based cluster analysis in Bionumerics. Minimal Spanning Trees were generated from the cluster analysis with 100 permutations (Bionumerics), using the seven MLST loci information.Based on the MLST analysis, 91 C. jejuni strains with representative STs were chosen for the PCR-based pathotyping. The presence or absence of selected putative virulence genes was detected by PCR as described above and in the results. For some genes with previously described sequence diversity, such as ggt and ansB, multiple primer combinations were tested to exclude false-negative results and to ensure that the absence of the gene is not due to the sequence diversity. The genes and primers are listed in Table . [...] Whole genomic DNA for 454 sequencing was isolated using Qiagen Genomic tip 100/G columns and the Genomic DNA Buffer Set (Qiagen). High quality genomic DNA of C. jejuni was then sheared according to a reproducible method by nebulization, quality-controlled on an Agilent bioanalyzer chip (DNA Chip7500, Agilent, Santa Clara, CA, U.S.A.), end-polished with T4 DNA polymerase and T4 polynucleotide kinase (New England Biolabs, Beverly MA, U.S.A.) []. DNA fragments were then subjected to bead coating and 454 sequencing in a Roche 454 sequencer, using FLX Titanium (Roche) chemistry according to the manufacturer's instructions.Subsequent to the pyrosequencing process, the Roche Assembler software was used for assembling the primary reads into contigs. Between 37 and 156 contigs were obtained for the five whole genome sequences (xy259: 68 contigs, estimated genome size: 1,7 Mbp; RB922; 156 contigs, estimated genome size 1.7 Mbp; 6399: 43 contigs, estimated genome size 1.5 Mbp; 04197: 83 contigs, estimated genome size 1.7 Mbp; 04199: 71 contigs, estimated genome size 1.8 Mbp). Further statistics for the sequencing process and additional quality controls are available upon request. The draft genome sequences are available in the ENA EMBL-Bank database with accession numbers CAFR01000001-68, CAFS01000001-156, CAFT01000001-43, CAFU01000001-83, CAFV01000001-71. Further, the genomes were annotated in the Kodon software (Applied Maths, Sint Martens-Latem, Belgium) using several publicly available complete C. jejuni genome sequences, and the contigs of the draft sequences were then ordered 1) by aligning all five draft sequences to each other, and 2) by aligning all five genome sequences using the finished genome of C. jejuni 11168 [] as a scaffold. Whole genome alignments were generated within the Kodon, GeneDoc (http://www.psc.edu/biomed/genedoc) and MAUVE [] softwares. All pairs of genomes (reference genome was 04199 for all pairwise comparisons) were analyzed using the Hidden Markov Model implemented in ClonalFrame [] which comprises two states: the "clonal" state in which only mutations separate the two genomes and the "recombined" state in which the polymorphism density is higher due to the import of unrelated DNA. This allowed to pinpoint the regions that had recombined, as well as to quantify the overall frequency and effect of recombination during the diversification of pairs of genomes. A similar approach was recently implemented by Kennemann et al []. […]

Pipeline specifications

Software tools BioNumerics, Mauve, ClonalFrame
Applications Phylogenetics, WGS analysis, Nucleotide sequence alignment
Organisms Campylobacter jejuni, Campylobacter coli, Homo sapiens, Gallus gallus, Bos taurus
Diseases Infection, Intestinal Diseases