I guess I'll answer my own question....No, multiple sequence alignment programs do not flip the sequences to see if you have them in the correct/incorrect orientation; if you put some in the reverse/complement orientation, they will try and force the alignment that way. Yes, I flipped a few of the sequences using reverse/complement and they then aligned properly (better). I also took the likely opposing sequences and checked the 8-Forward primer and it came up on the left side on one sequence and toward the opposite end on the others. Interesting, I obtained them all from NCBI as Fasta (5'--> 3') and copy/pasted in a word document and used them in several different sequence alignment programs and they all had that ugly alignment in the image above. So some 16S rDNA seqs that you get are in the sense direction and others in the anti-sense. :-)

The alignment produces it towards the direction in which the sequences are.

What you could do is align everything against everything using BLAST. If all the sequences are in the correct direction in the BLAST result you should observe this, in the columns that indicate from which position to which position the alignment occurred. If you observe that some sequence the beginning of the alignment started in one of the final positions and ended in one of the initial positions, and in the rest of sequences the beginning began in the initial positions and ended in the final positions, then this means that there are sequences in both directions.

Peter M Muriana