Query regarding 16S rRNA ITS sequencing

Dear Sir/Mam,

I have a simple question can I use mothur MiSeq SOP to analyze 16S rRNA ITS sequencing data which is used to identify the fungus community or is there any other method tutorial available?

Hi there - you can certainly use mothur to analyze ITS sequences, but I don’t have a protocol. There are a few adjustments you’ll need to make to the MiSeq SOP - namely because ITS sequences aren’t homologous, you can’t use a reference alignment with align.seqs. I would suggest using pairwise.seqs instead to generate a distance matrix for clustering


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Hi Dr. Schloss - does this mean one could not run through the MiSeq SOP with both 16S and ITS samples?

I recently received my sequencing data (both ITS and 16S on 16 samples) back from Zymo and wanted to run it through Mothur and compare their results from DADA.
(I have no experience with DADA and a teeny amount with Mothur (I ran through the MiSeq SOP in a class twice for a very small sample size and lab report).

I am unexperienced with coding. Is there a Mothur for dummies forum thread on here?


Hi - I would suggest analyzing the 16S and ITS separately. Alternatively, you could keep them together and use pairwise.seqs instead of align.seqs.


Thanks for your kind response, can you please tell me if it will be the same start and end in case of pcr.sec command? where we place for V3/V4 region of 16sRNA sequencing.

If you follow these instructions you should be able to find the coordinates:


I faced the same issue. I found that UNITE reference database is more suitable for ITS than SILVA; however, it gives an error when I use the command “align.seqs”!