Due to a significant decrease in the cost of DNA sequencing, the number of sequences submitted to the public databases has dramatically increased in recent years. Efficient analysis of these data sets may lead to a significant understanding of the nature of pathogens such as bacteria, viruses, parasites, etc. However, this has raised questions about the efficacy of currently available algorithms for the study of pathogen evolution and construction of phylogenetic trees. While the advanced algorithms and corresponding programs are being developed, it is crucial to optimize the available ones in order to cope with the current need. The protocol presented in this study is optimized using a number of strategies currently being proposed for handling large-scale DNA sequence data sets, and offers a highly efficacious and accurate method for computing phylogenetic trees with limited computer resources. The protocol may take up to 36 h for construction and annotation of a final tree of about 20,000 sequences.