I’m running classify.seqs with the latest version of mothur (1.24.1) and I get the following warning:
[WARNING]: mothur suspects some of your sequences may be reversed, please check ph1.combined.good.filter.unique.precluster.pick.bacteria.flip.accnos for the list of the sequences.
79 sequences from my dataset are listed in the .flip.accnos file as being suspect - I’m sequencing off the reverse primer and have used the flip=T from the trim.seqs step - I wonder how these 79 sequences could be in the reverse direction…? How exactly does mothur determine what to flag as a sequence in the reverse direction - is this based on the poor alignment?
Thanks very much for your help,