Molecular evolution analysis workflow tutorial/for dummies

CAMPOS, MARÍA CLARA; PISCIOTTANO FRANCISCO; BRUQUE, CARLOS DAVID; SARAGÜETA, PATRICIA

Rosario

Congreso; XIII Congreso Argentino de Bioinformática y Biología Computacional, XIII SoIBio, III RiaBio; 2023

Asociación Argentina de Bioinformática y Biología Computacional

Creatinga workflow for molecular evolution analysis can be time-consuming andrequires significant resources, including bioinformatics expertiseand human involvement. To develop an effective workflow, it iscrucial to have experience with relevant tools. Wepresent a meticulously designed workflow for testing positiveselection hypotheses for the target protein in the taxonomic group ofinterest. This workflow exclusively employs freely available toolsand facilitates the assessment of conservation, as well as theidentification of specific sites responsible for the selectionsignatures within the protein's structure.Theworkflow consists of several stages: 1.Retrievethe coding sequences of the protein of interest's orthologs frompublic databases for the relevant species.2. UsingMEGAX, construct a multiple sequence alignment of the protein ofinterest by obtaining nucleotide sequences f2. Evaluate the degree ofconservation and sequence identity percentages using SIAS. 3.Reconstruct the gene phylogeny using maximum likelihood methods inMEGAX. 4.Calculate synonymous and non-synonymous substitution rates in orderto identify positive slection signatures throughsite-lineage-specific positive selection analysis using the codemlprogram from the PAML4 package implemented in multiPAML2.5, a toolcreated in our laboratory that automates PAML analysis. 5.Predict the three-dimensional structure of the protein usingAlphaFold2 via a command-line interface in ColabFold, which can beexecuted from a local computer. 6. Visualize and edit the top-rankedthree-dimensional model using Chimera. 7.Locate protein domains cross-referenced with specific literature, andadjust domain positions using secondary structures predicted byColabFold and visualized with PDBsum. In the case of glycosylatedproteins, also identify glycosylation sites using the UniProtdatabase.Byfollowing this simple workflow, researchers can easily analyzemolecular evolution for their protein of interest, test evolutionaryhypotheses in a given clade and eventually identify the positiveselected residues involved in their protein molecular adaptation orrapid evolution.p { line-height: 115%; margin-bottom: 0.1in; background: transparent }