Sources of ambiguity in nucleic acid sequence alignment

The discussion of molecular sequence alignment is becoming more prominent in studies of molecular systematics and evolution. As the basis for initial homology statements, alignment is crucial to comparative molecular biology. Although fundamental, alignment is not a process which necessarily yields objective, precise results. Ambiguities can appear in alignment due to a number of factors. Three such sources of ambiguity are discussed here. These are ambiguity in the establishment of alignment parameters, pair-wise order and individual "path" variation. The first arises from the necessary but empirically untestable assumptions of gap costs and other factors which are required to align sequences objectively. The second is due to the possible existence of non-unique solutions to the same alignment parameters in heuristic and exact solutions. The third is a result of multiple optimal paths within single alignments, potentially generating huge numbers of equally costly but unique alignments. Some of the problems with and several possible solutions to the difficult situation of non-unique alignments are discussed.