I am aligning many similar sequences from a BLAST result and looking for mutations at certain positions. My inclination is that an MSA (Clustal Omega) is the best approach but my PI is worried about misalignments and believes that Pairwise alignments against a reference sequence would be the best approach. Assuming that all the sequences to be aligned are homologs, which method would be more accurate and why? I need to convince her that I am right i.e. more information will produce better alignments. Thanks!