Computational protocol: Lutzomyia Sand Fly Diversity and Rates of Infection by Wolbachia and an Exotic Leishmania Species on Barro Colorado Island, Panama

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Protocol publication

[…] Sequences were examined using Sequencher 4.7 (Gene Codes Corporation, 2006). Ends were trimmed to clearly defined peaks. Forward and Reverse sequences for each sample were compared to correct dye blots and to ensure non-contamination. Sequences were aligned with Clustal W online implementation , and trimmed to equal length before analysis in PAUP 4.0 , ModelTest , and MrBayes ,.The phylogenetic relationships among Lutzomyia species were investigated by sequencing two fragments of the 18S gene (18SA 402bp, 18SB 482bp) from single Lutzomyia extracts. The two sequences were aligned separately using Clustal W2 ,; then the concatenated sequence was analyzed using ModelTest for AIC and hLTR best-fit models of evolution . The hLTR recommendation by ModelTest was a General Time Reversible model with invariant site (I) and gamma shape (G) parameter adjustments. The AIC recommendation was the Hasegawa-Kishino-Yano 1985 (HKN85) model, with I and G adjustments. These models were implemented in the Maximum Likelihood analysis in PAUP for tree construction, and gave identical topologies. The ML tree was bootstrapped using the HKN85 model. The concatenated sequences were also used for Bayesian Inference (BI) analysis with MrBayes ,. The HKN85 model was implemented in MrBayes so that BI posterior probabilities could be directly compared to bootstrap values in ML from PAUP. All trees were generated using Brumptomyia galindoi as an outgroup.Highly variable loop regions in 18S where alignment was difficult or non-existent were excised after alignment. No attempt was made to code gaps as binary or multi-state characters.CO1 barcode sequence was obtained from 49 individuals identified by morphology in 18 species. More than one specimen from each species was sequenced when available (). We aligned sequences manually as no insertions or deletions were present in the protein-coding fragment. Sequence reliability was determined by comparing the forward and reverse reads, as well as in silico translation using the invertebrate mitochondria genetic code to ensure nonsense codons were not present in the fragment. Aligned sequences were exported to PAUP* 4.0 where a neighbor-joining tree was generated using a Kimura 2-Parameter correction for DNA distances. […]

Pipeline specifications

Software tools Sequencher, Clustal W, PAUP*, ModelTest-NG, MrBayes
Application Phylogenetics
Organisms Drosophila melanogaster, Leishmania braziliensis, Homo sapiens
Diseases Brain Diseases, Infection, Leishmaniasis, Leishmaniasis, Cutaneous