When dealing with a coding sequence, specifically transcriptome data, can the alignment of the sequence modify the reading frame? This interests me because I'm trying to apply neutrality tests to these sequences, particularly those tests considering synonymous and nonsynonymous changes (like MK test), which requieres me to specify coding and noncondig regions as well as 1st, 2nd and/or 3rd codon positions. So, can I do this directly after aligning or do I have to do some checking to verify that the frame and therefore, the positions have not been displaced, and if so, how can I do the checking?