Impact of Phylogenetic Method Choice on Indel Analyses in HIV-1 Subtype B.

Insertions and deletions (indels) play a critical role in the evolutionary dynamics of genomes, yet their accurate detection and interpretation in phylogenetic studies remain challenging. Our study investigates the influence of different multiple sequence alignment (MSA) and ancestral sequence reconstruction (ASR) tools on indel pattern reconstruction, focusing on HIV-1 subtype B. We aim to understand how methodological choices affect the detection of indels, thereby emphasizing the importance of selecting appropriate tools for evolutionary analyses to improve phylogenetic accuracy. We conducted a comparative analysis using five MSA tools (MAFFT, PRANK+F, IndelMaP, ProPIP, and Historian) and five ASR tools (GRASP, FastML, IndelMaP, ARPIP, and Historian). By examining inferred indel events across all tool combinations, we evaluated their rates, lengths, and positions within the genome, specifically analyzing the env gene and its V1 variable loop. Even though each method tested was able to reconstruct known variable regions in the env gene, our results highlight that the choice of MSA tool significantly impacts indel conservation and interpretation, more so than the choice of ASR tool. This finding underscores the necessity of context-specific MSA tool selection in phylogenetic studies and provides crucial insights for improving the accuracy of indel detection and evolutionary inferences in phylogenetic studies of HIV-1 and other genomes.