11/3/2023 0 Comments Nucleotide sequence alignment tool![]() Generally, all of the protruding amino acids or nucleotides at either end of the alignment should be removed. In the aligned sequence, any position without an amino acid or nucleotide is represented by a hyphen "-"). They should then be edited or trimmed using an editing software, such as BioEdit, because phylogenetic tree construction by MEGA requires the sequences to be of equal length (including both amino acid abbreviations and hyphens. After the process is complete, the aligned sequences should be saved in the correct format for the next step. The alignment tool has many parameters that the user can adjust before starting the alignment, but the default parameters work well in most cases. The most widely used is Clustal Omega 6, 7 (), which can be used online or can be downloaded free of charge. Many different platforms such as Clustal W, Clustal X, Muscle, T-coffee, MAFFT, can be used for sequence alignment. It is worth noting that it is critical to collect high-quality sequences for the analysis, and only homologous sequences can be used to analyze phylogenetic relationships. As a result, DNA sequences are useful for comparison of homologous genes from closely related organisms, whereas amino acid sequences are appropriate for homologous genes from distantly related organisms 3.Ī phylogenetic analysis begins with the alignment of amino acid or nucleotide sequences 4 retrieved from an annotated genome sequencing database 5 listed in FASTA format, i.e., putative or expressed protein sequences, RNA sequences, or DNA sequences. In contrast, amino acid sequences are much more stable given that synonymous mutations in nucleotide sequences do not cause mutations in amino acid sequences. DNA sequences are more likely to be affected by changes during evolution. In the present study, a simple introduction of basic phylogenetic knowledge and an easy-to-learn protocol was provided for newcomers who need to construct a phylogenetic tree on their own.īoth DNA (nucleotide) and protein (amino acid) sequences can be used to infer phylogenetic relationships between homologous genes, organelles, or even organisms 2. Therefore, molecular information such as nucleotide sequences and amino acid sequences from related extant organisms are of value for exploring evolutionary relationships 1. However, many organisms, such as microorganisms, have little chance to form a fossil during long geologic ages. In evolutionary study, fossil evidence is very direct and convincing. In a sense, genetic information is more reliable than evaluating external phenotypes because the former is the basis for the latter. Two phylogenetic trees constructed using theoretically distinct methods have consistently suggested that the first eukaryotic SWEET gene might stem from the fusion of a bacterial SemiSWEET gene and an archaeal SemiSWEET gene. It is worth noting that one should be cautious to draw a conclusion based only on phylogenetic analysis, although it is useful to explain the underlying relationship between different taxa, which is difficult or even impossible to discern through experimental means.ĭNA or RNA sequences carry genetic information for underlying phenotypes that can be analyzed through physiological and biochemical methods or observed through morphological and fossil evidence. Interestingly, SemiSWEETs were found in prokaryotes, but SWEETs were found in eukaryotes. To investigate the origin of eukaryotic Sugars Will Eventually be Exported Transporters (SWEET) genes, 228 SWEETs including 35 SWEET proteins from unicellular eukaryotes and 57 SemiSWEET proteins from prokaryotes were analyzed. In this text, a detailed protocol is described for using the phylogenetic method, including amino acid sequence alignment using Clustal Omega, and subsequent phylogenetic tree construction using both Maximum Likelihood (ML) of Molecular Evolutionary Genetics Analysis (MEGA) and Bayesian Inference via MrBayes. Phylogenetic analysis can also be used to investigate domain relationships within an individual taxon, particularly for organisms that have undergone substantial change in morphology and physiology, but for which researchers lack fossil evidence due to the organisms' long evolutionary history or scarcity of fossilization. Phylogenetic analysis uses nucleotide or amino acid sequences or other parameters, such as domain sequences and three-dimensional structure, to construct a tree to show the evolutionary relationship among different taxa (classification units) at the molecular level.
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