Protozoa assignation

Hello guys!
Would like to ask if you would recommend doing protozoa assignations with Mothur (SILVA132 database).
I saw some comments in the forum but rise this question, because I was trying with a dataset. Instead of doing a reference alignment, I went for the pre.cluster (alignment=needleman) and after some cleaning (chimera and remove seqs), I asked for the pairwise.seqs to obtain the dist file. My problem is precisely in that last step. It is taking long and many times the process does not continue.

Any suggestion I would really appreciate it,



HI Daniel,

In theory it should work. Unfortuantely, I’m pretty out of my depths with protozoa and I don’t know how good the SILVA reference is. I’d say go for it, but be sure to double check the results to confirm that sure they make sense.


Hi Daniel,

I regularly use mothur for my 18S / eukaryote analyses. For that I normally follow the MiSeq SOP, including using the SILVA reference alignment. For the taxonomic classification, I use both SILVA and PR2, a curated protist database ( which is also available to be used with mothur. Normally, both lead to roughly the same results, sometimes SILVA is “better” (contains more sequences), sometimes PR2 (depending on the taxon and if somebody has curated it properly).
I think if you have problems with the more demanding mothur steps, like calculating distance matrices, then this has more to do with your data and not with analyzing protists per se.

Good luck!


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Hi Pat,
thank you for the reply. It is running again and hope is not taking long…

Hi René,
Thank you for your comments, for sure I will come back to them…