Phylogenetic Tree-based Pipeline for Uncovering Mutational Patterns during Influenza Virus Evolution

Various computational and statistical approaches have been proposed to uncover the mutational patterns of rapidly evolving influenza viral genes. Nonetheless, the approaches mainly rely on sequence alignments which could potentially lead to spurious mutations obtained by comparing sequences from different clades that coexist during particular periods of time. To address this issue, we propose a phylogenetic tree-based pipeline that takes into account the evolutionary structure in the sequence data. Assuming that the sequences evolve progressively under a strict molecular clock, considering a competitive model that is based on a certain Markov model, and using a resampling approach to obtain robust estimates, we could capture statistically significant single-mutations and co-mutations during the sequence evolution. Moreover, by considering the results obtained from analyses that consider all paths and the longest path in the resampled trees, we can categorize the mutational sites and suggest their relevance. Here we applied the pipeline to investigate the 50 years of evolution of the HA sequences of influenza A/H3N2 viruses. In addition to confirming previous knowledge on the A/H3N2 HA evolution, we also demonstrate the use of the pipeline to classify mutational sites according to whether they are able to enhance antigenic drift, compensate other mutations that enhance antigenic drift, or both.