Computational protocol: Isolation and characterization of Babesia pecorum sp. nov. from farmed red deer (Cervus elaphus)

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Protocol publication

[…] The sequences obtained were subjected to BLASTn identity searches in the GenBank database. Multiple sequence alignments were performed using the program Clustal W [] to highlight differences between the red deer 18S rRNA sequences and to perform comparisons with related sequences found in the NCBI database [].Two sets of phylogenetic trees were constructed. The first was constructed to place the new red deer isolates in the Babesia clade and define a suitable outgroup for a more refined analysis. For this purpose, 20 sequences representing the main Babesia sub-clades [,] were selected and aligned with the sequence of the main representative of the red deer isolate sequences. The alignment was truncated manually to the size of the shortest sequence. Sequences of Theileria annulata and Theileria buffeli were used as outgroups, based on recently published phylogenetic analyses [,]. Gaps were automatically removed during the analyses by MEGA5. The final alignment consisted in 1391 bp.A second set of alignments was performed using the 4 different complete red deer sequences as well as all the closely related sequences (5 other sequences from Babesia species isolated from Giraffe, sheep and roan antelope). B. divergens and B. bigemina 18S rDNA sequences were added as representative of other main sister clades. These sequences were also selected from the first set of alignment as they did not generate large indels, especially in the region where substitutions were described between the red deer and the closely related sequences. Following examination, the alignment was then manually optimized to remove indels. This optimization allowed the conservation of the 17 substitution positions described in the results section (Table ). The alignment used for phylogenetic analysis contained 13 sequences and 1461 aligned positions.The phylogenetic analyses were performed in MEGA5 [] using the Neighbor-Joining, the Maximum Likelihood and the Maximum Parsimony methods. Parameters used for each method are indicated in the Additional files with the corresponding trees [–]. The Bayesian phylogenetic analysis was conducted using MrBayes v3.2.2 [,]. The GTR model of nucleotide substitution, with gamma-distributed frequencies and invariant sites, was used. The first analysis was run for 300 000 generations, the second with 400 000 generations and trees in both cases were sampled every 200 generations. […]

Pipeline specifications

Software tools BLASTN, Clustal W, MEGA, MrBayes
Application Phylogenetics
Organisms Cervus elaphus, Ovis aries, Giraffa camelopardalis, Bos taurus
Diseases Babesiosis