Vectors based on the adeno-associated virus (AAV) are attractive and versatile vehicles for in vivo gene transfer. The virus capsid is the primary interface with the cell that defines many pharmacological, immunological and molecular properties. Determinants of these interactions are often restricted to a limited number of capsid amino acids. In this study, a portfolio of novel AAV vectors was developed after a structure-function analysis of naturally occurring AAV capsid isolates. Singletons, which are particular residues on the AAV capsid that were variable in otherwise conserved amino acid positions, were found to impact on vector's ability to be manufactured or to transduce. Data for those residues that mapped to monomer-monomer interface regions on the particle structure suggested a role in particle assembly. The change of singleton residues to the conserved amino acid resulted in the rescue of many isolates that were defective on initial isolation. This led to the development of an AAV vector portfolio that encompasses six different clades and 3 other distinct AAV niches. Evaluation of the in vivo gene transfer efficiency of this portfolio after intravenous and intramuscular administration highlighted a clade-specific tropism. These studies further the design and selection of AAV capsids for gene therapy applications.