Computational protocol: The Genome Sequence of Rickettsia felis Identifies the First Putative Conjugative Plasmid in an Obligate Intracellular Parasite

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Protocol publication

[…] We predicted protein-coding genes (ORFs) using SelfID [] as previously described []. tRNA genes were identified using tRNAscan-SE []. Database searches were performed using BLAST programs [] against Swiss-Prot/TrEMBL [], the NCBI CDD database [], and SMART []. The number of transposases, ankyrin/TPR-containing genes, autotransporter domains, and integrases were computed using PSI-BLAST with NCBI/CDD entries related to those domains with an E-value threshold of 10−5. Repeated DNA sequences were identified with the use of RepeatFinder [], by ignoring the sequence similarity between pRF and pRFδ. To identify Rickettsia palindromic elements, we used hidden Markov models [] based on the previously identified RPE sequences [].By taking advantage of genome colinearity, we identified orthologous relationships of genes in R. felis, R. conorii, R. sibirica, R. prowazekii, and R. typhi with the use of Genomeview (S. Audic, unpublished software). Based on the gene orthology, we defined R. felis–specific ORFs, which were of one of the following three classes: Class I ORFs exhibiting no homologous ORFs in the other four Rickettsia genomes; Class II ORFs exhibiting homologous ORFs but no orthologous ORFs in the other four Rickettsia genomes; and Class III ORFs exhibiting orthologous ORFs in some or all of the other four Rickettsia, all of which exhibit degraded (split or fragmented) genes relative to the R. felis ORF. Plasmid-encoded ORFs were by definition classified into Class I or II. A gene composed of more than one ORF was defined as “split gene.” A gene composed of a single ORF whose length is shorter than 50% of the longest ortholog was defined as a “fragmented” ORF. We used T-Coffee [] and MEGA [] for multiple sequence alignment and phylogenetic tree analyses, respectively. The analyses of horizontal gene transfer were performed by BLAST search against the Swiss-Prot/TrEMBL nonredundant database, excluding rickettsial sequences, as well as by methods based on nucleotide composition bias [,]. We obtained the minimum number of inversions to associate a pair of Rickettsia genomes using GRAPPA release 2.0 []. […]

Pipeline specifications

Software tools tRNAscan-SE, BLASTP, RepeatFinder, T-Coffee, MEGA, GRAPPA
Databases UniProt
Applications Genome annotation, Phylogenetics
Organisms Rickettsia felis, Homo sapiens