Computational protocol: "Candidatus Borrelia kalaharica" Detected from a Febrile Traveller Returning to Germany from Vacation in Southern Africa

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Protocol publication

[…] Slide microscopy was performed after standard Giemsa staining using a Zeiss Axioscope Microscope, objective 40X, ocular 10X. Microphotographs were obtained using a Canon 500D SLR connected to the same microscope and the 100x oil immersion objective.DNA extraction from 2 ml of EDTA-blood was performed using the MagNA Pure Compact System (Roche Diagnostics, Penzberg, Germany).Fragments of the 16S rRNA, flaB and glpQ were amplified using primers and PCR conditions as described previously [–] (). For uvrA the following primers were used as forward and reverse primers: uvrF1173 5´-GCGTTATCTTWCAACTGAATC-3’; uvrR2178 5'-TCTAGACTCTGGAAGCTT-3'. Sequencing was performed by GATC Biotech AG (Konstanz, Germany). Sequence alignment, genetic distance analyses and construction of phylogenetic trees was conducted in MEGA5 [, ]. Basic local alignment search tool (BLAST) [] searches in GenBank were conducted using standard settings. Genetic distance analyses were conducted using the Kimura 2-parameter model []. The evolutionary history was inferred by using the Maximum Likelihood method based on the General Time Reversible model []. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbour-Joining and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. To calculate node support values 1,000 bootstrap repeats were chosen. A discrete Gamma distribution was used to model evolutionary rate differences among sites [+G]. The rate variation model allowed for some sites to be evolutionarily invariable [+I]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. For all analyses, codon positions included were 1st+2nd+3rd+Noncoding for flaB sequences and uvrA sequences. All positions containing gaps and missing data were eliminated. Further information is given in the figure legend. […]

Pipeline specifications

Software tools MEGA, BLASTN
Application Phylogenetics
Organisms Homo sapiens, Borrelia parkeri, Borrelia duttonii, Borrelia hermsii, Borrelia crocidurae
Diseases Borrelia Infections, Relapsing Fever
Chemicals Doxycycline, Edetic Acid