Questions on the pre.cluster command

Commands in mothur
1

I’m studying the fungal community on decaying roots using 454 pyrosequencing data. To deal with the pyrosequencing error, I want to precluster my sequences before clustering. I have a few questions on the pre.cluster command in mothur:

  1. Can the pre.cluster command work on sequences that are not aligned ? (my sequences cannot be aligned since they are from universal fungi)

  2. Is there a mothur precluster command that is based on the distance matrix ? I have got the distance matrix (distances between unique sequences) by running unique.seqs and pairwise.seqs.

  3. On the mothur manual (the Pre.cluster page), given that one has already aligned,filtered and uniqued the sequences, why unique.seqs needs to be run again before running the pre.cluster command?

Thank you !

2
  1. Can the pre.cluster command work on sequences that are not aligned ? (my sequences cannot be aligned since they are from universal fungi)

No. They must be aligned.

  1. Is there a mothur precluster command that is based on the distance matrix ? I have got the distance matrix (distances between unique sequences) by running unique.seqs and pairwise.seqs.

No. pre.cluster uses aligned sequences.

  1. On the mothur manual (the Pre.cluster page), given that one has already aligned,filtered and uniqued the sequences, why unique.seqs needs to be run again before running the pre.cluster command?

To get the correct sequence frequencies once sequences have been filtered to cover the same alignment space.