Computational protocol: Molecular detection and identification of Wolbachia in three species of the genus Lutzomyia on the Colombian Caribbean coast

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Protocol publication

[…] The wsp gene obtained from Wolbachia were sent for sequencing (Macrogen, Korea) and the results were compared to previously identified sequences using the basic local alignment search tool (BLASTN) (https://www.ncbi.nlm.nih.gov/) and edited with Bioedit v.7.2.5 [] in order to obtain detected consensus sequence for every Lutzomyia species. This was also made with gene sequences of Wolbachia, which were available in the National Center for Biotechnology Information (NCBI) database and Wolbachia MLST database (http://pubmlst.org/wolbachia/). The nucleotide alignment reading framework reported by O'Neill (ftp://ftp.ebi.ac.uk/pub/databases/embl/align/; Access Number DS42468) was considered, which suggests starting the analysis by translating the sequences to amino acids as a guide to align the DNA sequences of the wsp gene [].Alignments of sequences of wsp genes obtained in Lutzomyia and reported in GenBank (Additional file ) were performed using the Clustal W and Muscle algorithms incorporated in MEGA 6. Verification of recombination events and the presence of chimeras was performed with RDP4 (Recombination Detection Program version 4) software, using all sequences of wsp obtained in this study in order to ensure the accuracy of nucleotide variability with respect to previously reported sequences in GenBank (Additional file ). Patterns of genetic divergence (nucleotide composition, number of haplotypes, variable sites) and K2P genetic distances were evaluated using Bioedit v.7.2.5 and DNAsp 5.0 software.All aligned sequences (= haplotypes) of wsp genes obtained in this study and reported in GenBank were exported using MEGA software. Description codes include the following abbreviations for species: Lev, Lutzomyia evansi; Lcy, Lutzomyia c. cayennensis and Luduv, Lutzomyia dubitans followed by the letters ov, which refer to the place where they were collected in Colombia (ov, municipality of Ovejas) and numbers corresponding to specimens with the same sequence.Subsequently, the identities and relationships of the Wolbachia strains obtained in our study was determined by performing a phylogenetic inference analysis using the Bayesian method (number of generations = 1,000,000) with the MrBayes 3.0 software under the substitution model GTR + G (number of estimated parameters k = 139; Akaike information criterion (AIC) = 7807.8819); with jModeltest 2.1.4 software []; and Phyml 3.0 software []. All of the sequences obtained in the present study (KR907869–KR907874) were submitted to GenBank (Additional file ). […]

Pipeline specifications

Software tools BLASTN, BioEdit, Clustal W, RDP4, DnaSP, MrBayes, jModelTest, PhyML
Databases PubMLST
Application Phylogenetics
Organisms Wolbachia endosymbiont of Armadillidium vulgare str. wVulC, Leishmania, Drosophila melanogaster
Diseases Bacterial Infections, Infection, Leishmaniasis