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With the expansion of DNA sequencing technology, quantifying evolution during emerging viral outbreaks has become an important tool for scientists and public health officials. Although it is known that the degree of sequence divergence significantly affects the calculation of evolutionary metrics in viral outbreaks, the extent and duration of this effect during an actual outbreak remains unclear. We have analyzed how limited divergence time during an early viral outbreak affects the accuracy of molecular evolutionary metrics. Using sequence data from the first 25 months of the 2009 pandemic H1N1 (pH1N1) outbreak, we calculated each of three different standard evolutionary metrics—molecular clock rate (i.e., evolutionary rate), whole-gene dN/dS, and site-wise dN/dS—for hemagglutinin and neuraminidase, using increasingly longer time windows, from 1 month to 25 months. For the molecular clock rate, we …