Computational protocol: Comparative Genomics of the Staphylococcus intermedius Group of Animal Pathogens

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Protocol publication

[…] Whole genome sequencing of S. pseudintermedius ED99 was carried out as previously described (Ben Zakour et al., ). Genome sequencing of S. delphini 8086 and S. intermedius NCTC11048 was carried out using the Illumina 3G Genome Analyzer. For each strain, we generated a total of 4,087,613 and 3,879,139 paired-end reads, respectively, with a fixed length of 36 bp and an average insert size of 200 bp, corresponding to more than 58× and 50× genome coverage, respectively. De novo assembly was performed by using the Velvet short reads assembler program (Zerbino and Birney, ). For each genome, contigs were mapped against the completed whole genome of S. pseudintermedius ED99 using MauveAligner (Rissman et al., ) and manually inspected for potential mis-assemblies. To confirm the reliability of the sequences obtained by this de novo sequencing approach based only on very short reads, re-sequencing of S. pseudintermedius ED99 as an internal control was also performed in parallel to S. delphini 8086 and S. intermedius NCTC11048. An automatic annotation was then performed by the RAST annotation server to predict CDS and their putative functions (Aziz et al., ). Functional categories were assigned by searching all predicted proteins against the COG database (www.ncbi.nlm.nih.gov/COG). The software AlienHunter (Vernikos and Parkhill, ) was used to detect atypical genome regions corresponding to putative horizontal gene transfer, insertion sequence (IS) elements were identified by interrogation of the IS database (Siguier et al., ), and clustered regularly interspaced short palindromic repeat (CRISPR) elements were characterized by the CRISPRFinder web software (Grissa et al., ). The draft genome sequences of S. delphini 8086 and S. intermedius NCTC11048 have been deposited in the GenBank WGS database and have Genome Bioproject ID numbers PRJEA87011 and PRJEA87009, respectively. [...] Orthologous CDS between S. pseudintermedius ED99 (accession number CP002478), S. aureus Mu50 (accession number BA000017; Kuroda et al., ), Staphylococcus epidermidis RP62A (accession number CP000029; Gill et al., ), Staphylococcus haemolyticus JCSC1435 (accession number AP006716; Takeuchi et al., ), Staphylococcus saprophyticus ATCC15305 (accession number AP008934; Kuroda et al., ), Staphylococcus carnosus TM300 (accession number AM295250; Rosenstein et al., ), S. delphini 8086, and S. intermedius NCTC11048, were identified by reciprocal best hits using BLASTP, with a maximum e-value of 0.01, a minimum percentage identity of 40% and a minimum percentage sequence coverage of 80%. The average nucleotide identity (ANI) between the complete and draft genome sequences available for all staphylococcal species and the outgroup Macrococcus caseolyticus JCSC5402 (accession number AP009484; Baba et al., ), was determined by using the in silico DNA–DNA hybridization method of the JSpecies software using default parameters (Richter and Rossello-Mora, ). The distance matrix based on the ANI values obtained was then used in Splitstree to construct a Neighbor-Joining tree (Huson and Bryant, ). […]

Pipeline specifications

Software tools Velvet, RAST, CRISPRFinder, BLASTP, JSpeciesWS, SplitsTree
Applications Genome annotation, Phylogenetics, WGS analysis, Nucleotide sequence alignment
Organisms Canis lupus familiaris, Equus caballus, Homo sapiens, Neovison vison, Bacteroides fragilis, Staphylococcus pseudintermedius