Phylogenomic Tree Reconstruction from Bacterial Genomes.

The recent, rapid expansion of available prokaryotic genomes has fueled significant advancements in phylogenomics within microbial ecology and evolution. However, comprehensive protocols for reconstructing phylogenomic trees from bacterial genomes remain scarce. To address this gap, we present a series of essential bioinformatics steps. This protocol begins with setting up a Docker environment to ensure consistent implementation across different operating systems. We demonstrate phylogenomic tree reconstruction using the Genome Taxonomy Database and its toolkits with five test genomes, which were retrieved from the NCBI database, quality-checked with CheckM, aligned using GTDB-Tk, and analyzed for tree reconstruction with RAxML. Genome-level phylogenetic analyses are often complicated by gene duplication, horizontal gene transfer, and computational demands that increase with the number of taxa. We discuss practical strategies for addressing these issues and highlight the value of orthologous gene concatenation in generating accurate phylogenomic trees.