Testing ITS1 Pipeline: Finding mock communities or correcting ITSxpress error

General Discussion
1

I am attempting to verify my pipeline for ITS1 analysis with a mock community from mockrobiota (mockrobiota/data/mock-25 at master · caporaso-lab/mockrobiota · GitHub). Most of the pipeline seemed to work well but when I got to ITSxpress it removed all my reads. This seems odd because I didn't have this problem when I ran my own data.

The only thing I could think of is that my data is only the ITS1 region and the mock community contains the whole ITS region. However, when I've tried to modify the --p-region ( ALL, ITS1, ITS2) it still wasn't working properly.

I am wondering if anyone has any ideas how to either troubleshoot this or know of a different mock community specifically with the ITS1 region that I can use.

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2

Hi @mbode24 and welcome to the :qiime2: forum!

Could you share the full command you run for ITSxpress?

Best wishes,

Sergio

3

Hi Sergio,

This is the command I ran for ITSxpress. I've tried modifying the --p-region with each option (ALL and ITS2) but still no output.

qiime itsxpress trim-pair-output-unmerged
--i-per-sample-sequences sequences.qza
--p-region ITS1
--p-taxa F
--p-cluster-id 1
--p-threads 15
--o-trimmed trimmed_exact.qza

Best wishes,
Michelle

4

I think the region in this case is ITS2 (see the paper, PMC5118932).

Have you tried playing with this option? I don't think this will solve the issue (I normally use 1 and never had any problem), but it's worth trying. According to the documentation, you can use values in the range 0.99-1.0

Best,

Sergio

5

Hi Sergio,

Thanks for the quick response. Yeah when I read it was ITS2 I thought it might work if I switched it in my pipeline but was surprised when it didn't. When I opened the fasta file example it described multiple regions (mockrobiota/data/mock-25/source/expected-sequences.fasta at master · caporaso-lab/mockrobiota · GitHub) so I thought that might be why.

Unfortunately, I did try to modify p-cluster id as well with no luck.

Would you have any idea why this might happen? If you don't think it's fixable, would you have any suggestions for an alternative mock dataset to use?

Thanks for your time,
Michelle

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6

Not really, seems really strange to me! :thinking:

Which steps did you perform prior to ITSxpress? How many reads are you losing in each step?