Abstract
Proteins have played a fundamental role throughout life's history on Earth. Despite their biological importance, ancient origin, early function, and evolution of proteins are seldom able to be directly studied because few of these attributes are preservedacross geologic timescales. Ancestral sequence reconstruction (ASR) provides a method to infer ancestral amino acid sequences and determine the evolutionary predecessors of modern-day proteins using phylogenetic tools. Laboratory application of ASR allows ancient sequences to be deduced from genetic information available in extant organisms and then experimentally resurrected to elucidate ancestral characteristics. In this article, we provide a generalized, stepwise protocol that considers the major elements of a well-designed ASR study and details potential sources of reconstruction bias that can reduce the relevance of historical inferences. We underscore key stages in our approach so that it may be broadly utilized to reconstruct the evolutionary histories of proteins.
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