GI-16 lineage (624/I or Q1), there and back again: The history of one of the major threats for poultry farming of our era

Franzo, Giovanni; Cecchinato, Mattia; Tosi, Giovanni; Fiorentini, Laura; Faccin, Francesca; Tucciarone, Claudia Maria; Trogu, Tiziana; Barbieri, Ilaria; Massi, Paola; Moreno, Ana

The presence ofrecombinant strains was evaluated using RDP4 [29] on a dataset including the selected GI-16 like strains plus the reference ones. The RDP4 settings for each method were adjusted accounting for the dataset features according to the RDP manual recommendations. Only recombination events detected by more than 2 methods with a significance value lower than 10 −5 (p-value < 10 −5 ) and Bonferroni correction were accepted. After recombinants removal, the absence of residual breakpoints was confirmed with SBP implemented in HyPhy [30] . Only sequences with collection date and country were included in the study. Dedicated Python scripts were used to extract this information from the Genbank file or, if absent, original publications were consulted and the sequences were re-named accordingly. Finally, TempEst [31] was used to investigate the 'temporal signal' of the heterochronous sequences under investigation and the sequences whose sampling date was inconsistent with their genetic divergence and phylogenetic position were removed. Indeed, significant deviations from the expectation under the molecular clock hypothesis can be due to several factors including low sequencing quality, excessive passaging, recombination, etc. [31] . Xia test was performed for each codon position to exclude the presence of codon saturation and loss of phylogeny signal. In fact, when full saturation is reached, the phylogenetic signal is lost and the sequences are no longer informative about the underlying evolutionary processes.