Exploring the ancestral sequences is an extremely fascinating question in evolutionary biology, which allows us to understand what the given sequence was at various ancestral points in a phylogenetic tree. It is able to decipher the molecular functions of ancient genomes and to reveal the differences compared with their modern counterparts.

The phylogenetic reconstruction method of ancestral sequences is a powerful approach for studying the evolutionary relationships between protein sequence, structure, and function. This approach allows researchers to reconstruct or resurrect extinct proteins and study how they differ from modern proteins; identify important amino acid changes that, over evolutionary timescales, have altered protein function during evolution; and rank historical events in the evolution of protein function. Briefly, the prediction of ancestral genomes involves four general steps: 1) creating accurate multiple alignments of the existing orthologous sequences, thereby establishing the orthologous relationships between the nucleotides of each sequence; 2) performing an indel reconstruction that determines the most likely scenario of insertions and deletions that could have resulted in the existing sequences; 3) reconstructing the substitution history using a maximum likelihood approach; and 4) examining genome rearrangements (inversions, transpositions, translocations, duplications, and chromosome fusions, cleavages, and duplications). In the past, all of these steps were performed separately and required different computer tools and knowledge of different phylogenetic models. An important component of reconstructing phylogenetic sequences was knowledge of the phylogenetic relationships among the species being compared. Knowledge of the correct topology of the phylogenetic tree and estimation of the length of its branches are critical for accurate reconstruction, as well as for estimating the accuracy of that reconstruction through simulations. Over time, several automated software programs have been developed to provide user-friendly analysis of ancestral proteins. Many of them are online web portals with a user interface that greatly simplifies the reconstruction process and includes visual tools for ancestral analysis.

In this presentation, I will illustrate the process of reconstructing ancestral DNA sequences using the CFTR benchmark region from 12 mammals available in the NCBI database. The analysis will be performed using the user-friendly software MEGA 11.