ITS data processing

[vpandyar]

Hi everyone! I have a few questions about about ITS processing within qiime that I’m hoping someone would be able to answer. I have read through the ITSxpress and cutadapt method tutorials posted here and wanted to make sure I was doing everything correctly as I am having a lot of unidentified taxonomic sequences (anywhere from 33-98%).

1. Is there a difference to using ITSxpress vs cutadapt? Should I use one over the other or both sequentially?
2. When using cutadapt do I use the adapter and primer sequence of both my forward and reverse primers? The cutadapt ITS tutorial mentions using the reverse complement - do I need to do that also and if so how if I have 2 different adapters/primers.

I’m sure I’ll think of more questions but I figured I’d start with the basic stuff.

Thanks!

[Nicholas_Bokulich]

@vpandyar

• Is there a difference to using ITSxpress vs cutadapt? Should I use one over the other or both sequentially?

Yes, big difference. Check out the ITSx or ITSxpress papers for more details; basically ITSxpress will trim to the ITS region itself whereas cutadapt is just trimming away adapters.

Use one or the other on your data, no need to do both.

@vpandyar

When using cutadapt do I use the adapter and primer sequence of both my forward and reverse primers?

You can. Depends if you want to trim away one or both.

@vpandyar

The cutadapt ITS tutorial mentions using the reverse complement - do I need to do that also and if so how if I have 2 different adapters/primers.

The tutorial already gives you that answer:

@Nicholas_Bokulich

Note that we trim the forward primer (just in case it is still on the front of our sequences) and the reverse complement of the reverse primer from the forward reads. We trim the reverse primer and reverse complement of the forward primer from the reverse reads.

[vpandyar]

Thanks for the response! I ended up using the trimmed UNITE database file rather than the dynamic one with ITSxpress and it seemed to work better.