classify.seqs warning in mothur-1.24.1

Hi there,

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,

If you are classifying your sequences using the bayesian method, the classifier will now check for reverse sequences. Mothur does this by summing the probability that the kmers of your sequence will be in the reference, and summing the probability that the reverse compliment of your sequences kmers will be in the reference. If it is more probable that the reverse kmers are in the template, mothur reverses the sequence and classifies it. This results in a better classification of the sequence. Mothur reports the list of sequences we classified this way in case you would like to take a closer look.